Upstream and Downstream of Recombinants Biomolecules to

BioMed Research International

Upstream and Downstream of Recombinants Biomolecules to Health Care Industry

Guest Editors Priscila G Mazzola Arthur Cavaco-Paulo Jorge G Fariacuteas and Jorge F B Pereira

Upstream and Downstream of RecombinantsBiomolecules to Health Care Industry



Guest Editors Priscila G Mazzola Arthur Cavaco-PauloJorge G Fariacuteas and Jorge F B Pereira

Copyright copy 2016 Hindawi Publishing Corporation All rights reserved

This is a special issue published in ldquoBioMed Research Internationalrdquo All articles are open access articles distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the originalwork is properly cited

Contents

Upstream and Downstream of Recombinants Biomolecules to Health Care IndustryPriscila G Mazzola Arthur Cavaco-Paulo Jorge G Fariacuteas and Jorge F B PereiraVolume 2016 Article ID 9374847 2 pages

Full-Length cDNA Prokaryotic Expression and Antimicrobial Activity of UuHb-F-I fromUrechis unicinctusRongli Niu and Xiang ChenVolume 2016 Article ID 5683026 8 pages

Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNASequencingSiyan Zhang Jason D Hughes Nicholas Murgolo Diane Levitan Janice Chen Zhong Liuand Shuangping ShiVolume 2016 Article ID 8356435 8 pages

Cloning and Expression of the 120574-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilisWB600Biaosheng Lin Zhijuan Li Huixia Zhang Jiangwen Wu and Maochun LuoVolume 2016 Article ID 3073949 7 pages

Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer FormulationsBrittany K Chavez Cyrus D Agarabi Erik K Read Michael T Boyne II Mansoor A Khanand Kurt A BrorsonVolume 2016 Article ID 2074149 8 pages

Azocasein Substrate for Determination of Proteolytic Activity Reexamining a Traditional MethodUsing Bromelain SamplesDiego F Coecirclho Thais Peron Saturnino Fernanda Freitas Fernandes Priscila Gava Mazzola Edgar Silveiraand Elias Basile TambourgiVolume 2016 Article ID 8409183 6 pages

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillussubtilis SCK6Shaista Bashir Saima Sadaf Sajjad Ahmad and Muhammad Waheed AkhtarVolume 2015 Article ID 636249 9 pages

One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of PhageDisplay Libraries Challenged to Cells Expressing Native Claudin-1Emanuele Sasso Rolando Paciello Francesco DrsquoAuria Gennaro Riccio Guendalina FroechlichRiccardo Cortese Alfredo Nicosia Claudia De Lorenzo and Nicola ZambranoVolume 2015 Article ID 703213 9 pages

EditorialUpstream and Downstream of RecombinantsBiomolecules to Health Care Industry

Priscila G Mazzola1 Arthur Cavaco-Paulo2 Jorge G Fariacuteas3 and Jorge F B Pereira4

1Faculty of Pharmaceutical Sciences University of Campinas (UNICAMP) 13083-859 Campinas SP Brazil2Departamento de Engenharia Biologica Universidade do Minho Campus de Gualtar 4710-057 Braga Portugal3Facultad de Ingenierıa y Ciencias Departamento de Ingenierıa Quımica Universidad de la Frontera Casilla 54-D Temuco Chile4School of Pharmaceutical Sciences Universidade Estadual Paulista (UNESP) 14800-903 Araraquara SP Brazil

Correspondence should be addressed to Priscila G Mazzola pmazzolafcmunicampbr

Received 7 June 2016 Accepted 7 June 2016

Copyright copy 2016 Priscila G Mazzola et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Biotechnology processes are the unique feasible way for theproduction of some pharmaceutical active principles Thusdevelopments in molecular biology recombinant techniquesseparation and purification methods have a primordialrole because of the innovative characteristic and economicimpact in obtaining these new drugs through biotechno-logical approaches This special issue compiles a series ofrelevant studies on different biotechnological fields and appli-cations reporting up-to-date developments on downstreamand upstream biopharmaceuticals

Summarizing the results reported in the manuscriptspublished here our readersmay find further insights througha series of fields from the most fundamental geneticapproaches to the general aspects of biological and biochem-ical engineering A complete study proposed by S Zhang etal applied next-generation RNA sequencing and developed amethod to analyse themutation rate of themRNA of Chinesehamster ovary producing monoclonal antibodies which arewidely used for the production of biological therapeuticsFollowing the concept of monoclonal antibodies E Sasso etal have presented a research study where they expanded theavailability of monoclonal antibodies interfering with hepati-tis C infection in hepatocytes The results of these authorsreport an effective sequencing approach for library screeningdemonstrating the successful conversion of recovered clonesto active immunoglobulinsThis novel approach allows rapidand cheap isolation of antibodies for virtually any native

antigen involved in human diseases for therapeutic andordiagnostic applications

On the other hand to clone and express 120574-polyglutamicacid (120574-PGA) synthetase gene in B subtilis B Lin et alhave constructed a plasmid which allowed the recombinantmicroorganism the synthesis of 120574-PGA into the fermentationbroth This approach has potential industrial applicationssince 120574-PGA is a new water-soluble biodegradable anionicpolypeptide and due to its interesting properties such asnontoxicity edibility adhesiveness film forming and mois-ture retention capability it can be a key compound for thehealth care industries Also R Niu and X Chen reported afull-length cDNA prokaryotic expression and antimicrobialactivity of cloned haemoglobin (Hb) fromUrechis unicinctusa marine spoon worm and economically important seafoodTheir results elucidate the structure and potential functionof Hb which may help to understand the immune defensemechanism of invertebrates and to give some new insightsinto antimicrobial peptides for drug discovery and diseasecontrol in U unicinctus aquaculture Following the sameconcept in ldquoEnhanced and Secretory Expression of HumanGranulocyte Colony Stimulating Factor by Bacillus subtilisSCK6rdquo S Bashir et al describe a simplified approach forenhanced expression and secretion of granulocyte colonystimulating factor (GCSF) a human cytokine in the culturesupernatant of B subtilis SCK6 cells Their results haveshown that after expression and purification the protein has

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9374847 2 pageshttpdxdoiorg10115520169374847

2 BioMed Research International

a biological activity similar to the commercial preparationof GCSF The last two works of this issue are aimed at theevaluation of stability of biomolecules and their accuratequantification respectively Formulating appropriate storageconditions for biopharmaceutical proteins is essential forensuring their stability and thereby their purity potencyand safety over their shelf life With that in mind B KChavez et al employed a model murine IgG3 produced in abioreactor and evaluated multiple formulation compositionsThese studies have evaluated the antibody stability in a seriesof conditions using an experimental design approach anoptimized formulation being identified in which the stabilitywas substantially improved under long-term storage condi-tions and after multiple freezethaw cycles The last work isfocused on the importance of proteases in the biotechno-logical and pharmaceutical industries and consequently thedetermination of optimum conditions and the developmentof a standard protocol are critical during selection of a reliablemethod to determine its bioactivity With that in mind D FCoelho et al employed a quality control theory to validate amodified version of a method proposed in 1947 presentinga validated protocol that offers a significant improvementgiven that subjective definitions are commonly used in theliterature and this simple mathematical approach makes itclear and concise

The quality of the results and protocols compiled in thisissue have caught our interest and we hope that these willhelp researchers and biotechnology-related professionals todevelop more exciting science regarding the improvementof the human health and the sustainability and safety of thebiotechnological industry

Priscila G MazzolaArthur Cavaco-Paulo

Jorge G FarıasJorge F B Pereira

Research ArticleFull-Length cDNA Prokaryotic Expression and AntimicrobialActivity of UuHb-F-I from Urechis unicinctus

Rongli Niu and Xiang Chen

Engineering Research Center of Molecular Medicine Ministry of Education Huaqiao University Xiamen 361021 China

Correspondence should be addressed to Rongli Niu niuronglihqueducn

Received 28 November 2015 Revised 1 May 2016 Accepted 10 May 2016

Academic Editor Jorge G Farıas

Copyright copy 2016 R Niu and X ChenThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hemoglobin which widely exists in all vertebrates and in some invertebrates is possibly a precursor of antimicrobial peptides(AMPs) However AMPs in the hemoglobin of invertebrates have been rarely investigated This study is the first to report thefull-length cDNA prokaryotic expression and antimicrobial activity of UuHb-F-I from Urechis unicinctus The full-length cDNAsequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids MALDI-TOF-MS suggestedthat the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 1516801 Da and its N-terminal aminoacid sequence was MGLTGAQIDAIK rUuHb-F-I exhibited different antimicrobial activities against microorganisms The lowestminimum inhibitory concentration against Micrococcus luteus was 278ndash463 120583M Our results may help elucidate the immunedefense mechanism of U unicinctus and may provide insights into new AMPs in drug discovery

1 Introduction

Hemoglobin (Hb) which widely exists in all vertebratesand in some invertebrates contains endogenous biologicallyactive proteins [1] exhibiting various properties includ-ing hormone release and immunomodulatory hematopoi-etic coronaroconstrictory antigonadotropic and opioid-likeactivities [2] Hb is also a possible precursor of antimicrobialpeptides (AMPs) [3ndash10]Thus far 30AMPs have been derivedfrom peptic Hb hydrolysates 24 peptides have been obtainedfrom the 120572 chain of Hb and 6 peptides have been obtainedfrom the 120573 chain of Hb [10 11] Intact Hb120572 or Hb120573 isalso a potent antibacterial protein [5] Hence Hb-associatedAMPs have been extensively investigated However few Hb-associated AMPs in invertebrates have been reported [12]

Urechis unicinctus (Uu) a marine spoon worm is eco-nomically important seafood mainly distributed through-out Russia Japan Korea and China Uu possesses a well-developed body cavity filled with coelomic fluid whichcontains cells with Hb In general AMPs are found in mostliving organisms and considered an essential component ofan organismrsquos innate immune system [13] Thus AMPs maybe found in the Hb or coelomic fluid of Uu AMPs mayalso play an important role in its innate immune system

However the Hb of Uu and its antimicrobial activity haveyet to be described Novel AMPs or antimicrobial substancesfrom the blood of Uu should be identified and isolated Inthis study the Hb of Uu was analyzed and its cDNA wascloned Recombinant expression and antimicrobial activityassay were then performed Our research on the structureand potential function of Hb may help elucidate the immunedefense mechanism of invertebrates This study may alsoprovide insights into new AMPs for drug discovery anddisease control in U unicinctus aquaculture

2 Materials and Methods

21 Cloning of the cDNA of UuHb-F-I Fragment Thecoelomic fluid of an adult fresh Uu (about 205 cm inlength and 305 g in mass) was collected and centrifuged at12000 rpm for 5min at 4∘C The precipitates were collectedand RNA was extracted by using a Trizol kit in accordancewith themanufacturerrsquos protocol (Shenggong BioengineeringCo Ltd China) First-strand cDNA was synthesized withM-MLV reverse transcriptase oligo dT dNTP mix and totalRNA Then PCR was conducted in 20 120583L reaction mixturecontaining 1 120583L of first-strand cDNA 05 120583L of each primer



Table 1 Primers used in this study

Name Sequences (51015840-31015840) Purpose

Adaptor primer (Ap) Containing the dT region designed by TaKaRa and adaptorprimer part 31015840-RACE cDNA

31015840-RACE outer primer TACCGTCGTTCCACTAGTGATTT 31015840-RACE31015840-RACE inner primer CGCGGATCCTCCACTAGTGATTTCACTATAGG 31015840-RACEGene-specific primer (GSP1) GGATATAGCGTTCTTTGACAAG 31015840-RACEGene-specific primer (GSP2) GCCCAGACTCTAACAGTTATCAGCTACTTGGAT 31015840-RACESMARTer IIA oligo primers 51015840-RACE cDNA51015840-RACE CDS primer A (T)25VN 51015840-RACE cDNA

10x universal primer Long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 51015840-RACE

AMix (UPM) Short CTAATACGACTCACTATAGGGC51015840-RACE outer primer CATGGCTACATGCTGACAGCCTA 51015840-RACE51015840-RACE inner primer GCGGATCCACAGCCTACTGATGATCAGTCGATG 51015840-RACEGene-specific primer (A1) CATCATTACAGACCAGACAATACG 51015840-RACEGene-specific primers (A2) CGCTTCAAGAGTTGTCCGAAATGCTTCGTGGTG 51015840-RACEPrimer P1 CAGGACGGAAGATATAGT cDNAPrimer P2 GTCGTTGTGATGTAGCAG cDNACDS-P1 GCGAGTCCATATG GGTCTTACTGGAGCTC Recombinant expressionCDS-P2 TATACTCGAGCTTCATGGCGGCCACCAGG Recombinant expression

(primers P1 and P2 Table 1) 10120583L of 2x Taq Master Mix(Omega Bio-Tek) and 8120583L of MilliQ H

2O Amplifications

were performed on PCR 3 Block Professional Thermocycler(Biometra) under the following conditions initial denatu-ration at 94∘C for 3min 30 cycles of denaturation at 94∘Cfor 30 s annealing at 48∘C for 30 s extension at 72∘C for50 s and final extension at 72∘C for 10min The obtainedcDNA was further purified with a SanPrep PCR productpurification kit (Shenggong Bioengineering Co Ltd China)and cloned into pUM-T vector Positive recombinants weretransformed into competent DH5120572 cells identified throughanti-Amp selection and verified through double digestionwith Sal I and BamH I (Thermo Scientific) Afterward thepositive clone was sequenced (Nanjin Jinsirui BiotechnologyLtd Co China)

22 Full-Length cDNA Sequence Determination

221 31015840-RACE 31015840-RACEwas performedusing 31015840-Full RACECore Set with PrimeScript RTase (TaKaRa) in accordancewithmanufacturerrsquos instructions Nested PCRwas conductedin 31015840-RACE outer primer and 31015840-RACE-GSP1 or 31015840-RACEinner primer and 31015840-RACE-GSP2 (Table 1)The first round ofPCR was performed using a reactionmixture containing 1 120583Lof the first-strand cDNA 05 120583L of each primer (10 120583M) 2 120583Lof 10x Trans TaqHiFi buffer 2120583L of dNTPs (25mM) 03 120583Lof Trans Taq HiFi DNA Polymerase (TransGen Biotech)and 137 120583L of MilliQ H

2O The second round of PCR was

conducted using a reaction mixture with 2 120583L of outer PCRpurified product 1 120583L of each primer (10 120583M) 5 120583L of 10xTrans Taq HiFi buffer 4 120583L of dNTPs (25mM) 05 120583L ofTrans Taq HiFi DNA polymerase and 365 120583L of MiliQ

H2O The amplifications of the first round were performed

with initial denaturation at 94∘C for 3min 30 cycles withdenaturation at 94∘C for 30 s annealing at 48∘C for 30 sextension at 72∘C for 50 s and the final extension step at 72∘Cfor 10min The second round was performed in the samemanner as that of the first round except annealing at 56∘CThe inner PCR product was ligated with pUM-T vector andfurther purified and transformed into DH5120572 The detailingprocess was the same as above The sequence was thendetermined (Nanjin Jinruisi Biotechnology Ltd Co China)

222 51015840-RACE 51015840-RACE was performed using 51015840-FullRACE kit with TAP (TaKaRa) in accordance with the man-ufacturerrsquos instructions Nested PCR was conducted with 51015840-RACE outer primer and 51015840-RACE-GSP1 or 51015840-RACE innerprimer and 51015840-RACE-GSP2 The PCR system in the firstround contained 2 120583L of reverse transcriptase 1 120583L of eachprimer 5120583L of 10x Trans Taq HiFi buffer 4 120583L of dNTP(25mM) 05 120583L of Trans Taq HiFi DNA polymerase and365 120583L of MilliQ H

2O The touchdown PCR profile was as

follows initial denaturation at 94∘C for 3min 30 cycles at94∘C for 30 s at 60∘C for 30 s (decreased by 05∘C in eachcycle) and at 72∘C for 1min 10 cycles at 94∘C for 30 sat 45∘C for 30 s and at 72∘C for 1min final extension at72∘C for 10min and being terminated at 15∘C The innerPCR was performed using 1 120583L of the purified outer PCRproduct 1 120583L of each primer 5120583L of 10x Trans Taq HiFibuffer 4 120583L of dNTPs (25mM) 05 120583L of Trans Taq HiFiDNApolymerase and 375 120583L ofMilliQH

2OThe touchdown

PCRwas performed using the following parameters 94∘C for3min 30 cycles at 94∘C for 30 s at 66∘C for 30 s (decreasedby 05∘C in each cycle) and at 72∘C for 40 s 10 cycles at 94∘C

BioMed Research International 3

for 30 s at 51∘C for 30 s and at 72∘C for 40 s final extensionat 72∘C for 10min and being terminated at 15∘C After theresults were verified through electrophoresis the product wassequenced to obtain the full length of UuHb-F-I cDNA

23 Bioinformatics Analysis Bioinformatics was conductedto predict the new gene and the conservation consistencyand structure of the mature peptide The homology ofnucleotide and protein sequences was blasted by using anonline tool at theNational Center for Biotechnology Informa-tion (httpblastncbinlmnihgovBlastcgi) The deducedamino acid sequence was analyzed by using a translate tool(httpwebexpasyorgtranslate) Clustal X and DNAmanwere used to perform multiple alignments of amino acidsequences The presence and location of a signal peptidewere predicted by using SignalP 41 Server online ProtScale(HphobKyte amp Doolittle) Sopma and Phyre2 online soft-ware were adopted to analyze possible amphiphytes andstructures

24 Expression and Purification of Recombinant UuHb-F-I

241 Construction of Recombinant UuHb-F-I The CDSsequence encoding mature peptide of UuHb-F-I was ampli-fied by a pair of primers (CDS-P1 and CDS-P2) The PCRproduct and pET-22b+ plasmids were double-digested withNde I and Xho I (Thermo Scientific) Afterward the puri-fied product was inserted into pET-22b+ vector by the T4ligation enzyme The ligation product was transformed intocompetent BL21(DE3) cells and sequenced to ensure in-frameinsertion Blank pET-22b+ plasmids were used as a negativecontrol

242 Expression and Determination of Recombinant Pro-tein BL21(DE3)pET-22b+ and BL21(DE3)pET22b-UuHb-F-I were inoculated in a TB medium with Amp (100 120583gmL)at 200 rpm and 37∘C until OD

600of 06ndash08 was reached

Isopropyl-120573-d-thiogalactosidase (IPTG 100mM) was addedto induce expression under the same conditions The cellswere harvested through centrifugation at 12000 rpm for1min Inducing conditions including the final IPTG concen-tration and induction time were optimized

Lactose instead of IPTG was used to induce proteinexpression The positive transformants of UuHb-F-I andthe negative control were incubated in an FML mediumcomposed of 15 gL tryptone 12 gL yeast extract 3 gLNaH2PO4sdot2H2O 7 gL K

2HPO4sdot3H2O 25 gL NaCl 02

glucose 21mM lactose 005 MgSO4sdot7H2O and 100 gmL

Amp at 37∘C with shaking at 180 rpm in accordance withthe procedure involving IPTG Lactose was added to induceexpression the cells were then harvestedThe induction timeobtained using lactose was compared with that recordedusing IPTG The quantities of the expressed proteins werecompared through SDS-PAGE

The recombinant protein of UuHb-F-I (rUuHb-F-I) wasfurther confirmed throughWestern blot analysis After SDS-PAGE was conducted the proteins were transferred fromthe gel to a PVDF film The film was blocked with 5

fat-free milk inoculated with His-Tag (27E8) mouse mAb(Cell Signaling) and peroxidase-conjugated AffiniPure goatanti-mouse IgG (H+L) (Shenggong Bioengineering Co LtdChina) and colored with a stable peroxide solution (A) anda luminolenhancer solution (B) Images were captured usingChemiDoc MP imaging system (Bio-Rad)

243 Purity and Renaturation of Recombinant ProteinsLactose was used to induce protein expression The recom-binant strain of pET-22b-UuHb-F-I was inoculated in anLB medium transferred to 100mL of FML in a 1 L flaskand cultivated for 16 h at 37∘C with 180 rpm The cultivationsolution was centrifuged at 10000 rpm for 10min The pelletwas solubilized with cell lysates (05M NaCl 50mM Tris-HCl 1mM EDTA and 05 Triton X-100 pH 74) Thesolutionwas sonicated for 20minwith 2 s ultrasonication and2 s intervals at 400W power and centrifuged at 10000 rpmand 4∘C for 20min The pellet contained inclusion bodieswhich were further washed with buffer I (05MNaCl 50mMTris-HCl 2M urea 05 Triton X-100 and 1mM EDTApH 74) and dissolved in buffer II (05M NaCl 50mM Tris-HCl 8M urea and 1mM EDTA pH 74) The supernatantwas prepared for column purification The samples fromeach step subjected to SDS-PAGE to determine the targetprotein rUuHb-F-I was purified with Ni+ affinity resinsunder denaturation conditions

The purified proteins were renatured through dialysisin the following gradient urea glycerol buffer (05M NaCl50mM Tris-HCl 1 glycine 10 glycerol 1mM EDTAand a gradient concentration of 4 2 and 1M urea in eachgradient pH 74 each gradient for 4 h) PBS for 4 h anddeionized water for 8 h The sample was cold-dried andanalyzed through SDS-PAGE

25 Determination of the Molecular Weight and AminoSequence of the Purified rUuHb-F-I The molecular weightof the purified rUuHb-F-I was confirmed by using an ABI5800MALDI-TOFTOF plusmass spectrometer (AB SCIEX)operated in a linear mode at Boyuan Bio-Tech Co (ShanghaiChina) MS and MSMS data were integrated and analyzedin GPS Explorer V36 (Applied Biosystems USA) withdefault parametersTheMSMS spectra revealed that proteinswere successfully obtained as indicated by ge95 confidenceinterval of their scores in MASCOT V23 search engine(Matrix Science Ltd London UK)

26 Antimicrobial Analysis The lyophilized protein was dis-solved in acetic acid (0025 VV) at different concen-trations 1 167 278 463 772 1286 214 357 595 and992 120583M The concentration of rUuHb-F-I was estimated byusing a BCA protein kit (Thermo Scientific) The antimi-crobial activities of eight microbial strains were measuredthree Gram-positive bacteria namely Staphylococcus aureusBacillus subtilis and Micrococcus luteus four Gram-negativebacteria namely Escherichia coli (ATCC8739) PseudomonasaeruginosaVibrio alginolyticus andVibrio parahaemolyticusand one fungus namely Pichia pastoris GS115 (China Gen-eral Microbiological Culture Collection Center (CGMCC


China)) V alginolyticus and P pastoris GS115 were culturedin TSB (17 gL tryptone 3 gL soytone 5 gL NaCl 25 gLglucose and 25 gL K

2HPO4) and YPD (2 (WV) tryptone

2 (WV) d-glucose and 1 (WV) yeast extract) at 30∘Cseparately Other bacteria were cultured in TSB at 37∘CAntibacterial activity was analyzed through a liquid phaseassay as described previously [14 15] The strains wereinitially adjusted to 103 CFUmL with LTM (1 agar in PBS)afterward 120 120583L of each strain was seeded into 96-well plateand each well contained 50 120583L of the protein sample Theplate was incubated for 3 h at 37∘C or 30∘C Subsequently125 120583L of the medium was added to each well and cultivatedfor another 12 h Then 100120583L sample from each well wasspread onto plates and cultivated for 24 hThe highest growthconcentration and the lowest inhibitory concentration wererecorded Minimum inhibitory concentration (MIC) wasdetermined by using the following equation 119886 minus 119887 where 119886is the highest protein concentration of bacterial growth and119887 is the lowest protein concentration that totally inhibitedbacterial growth Acetic acid (0025) was used as a negativecontrol Isopropanol (70) was used as a positive control forP pastoris GS115 Chloramphenicol solution (068mgmL)was utilized as a positive control for other bacteria Eachtreatment was repeated thrice

3 Results

31 cDNA Cloning and Sequence Analysis of UuHb-F-I Onthe basis of Urechis caupo F-I complete CDS (GI945055)we obtained the cDNA of U unicinctus The nucleotide anddeduced amino acid sequences are shown in Figure 1 and thesequence data were deposited in GenBank (KJ865621)

The full-length cDNA sequence of UuHb-F-I was 780 bpIt contains 95 bp 51015840-untranslated region (UTR) 256 bp 31015840-UTR and 429 bp open-reading frame (ORF) encoding 142amino acids (AA) The poly(A) tail was found in UuHb-F-Iand a canonical polyadenylation signal sequence (AATAAA)was detected The estimated molecular weight of matureUuHb-F-I was 1512067Da and the theoretical isoelectricpoint was 902 Moreover numerous 120572-helices were observedin the secondary structure of mature UuHb-F-I UuHb-F-I is amphiphilic as analyzed by HphobKyte amp Doolittlein ProtScale Signal peptide prediction revealed no signalsequences in UuHb-F-I Using Sopma and Phyre2 we couldfurther predict the secondary and tertiary structures of thisprotein (not shown in this study)

BLAST analysis revealed that the nucleotide acid anddeduced amino acid sequences ofUuHb-F-Imatched those ofUcHb-F-I with 82ndash87 and 79 similarities respectively[16] By contrast the sequence similarities to other organismswere relatively low and mainly conserved in the binding site(Figure 2) UuHb-F-I displayed 43 36 and 1379 aminoacid identities with Capitella teleta (GI443723524) Daphniamagna (GI322229317) [17] and human hemoglobin chain(GI3114508) respectively

32 Expression and Purification of Recombinant UuHb-F-IThe recombinant plasmids pET-22b-UuHb-F-I were trans-formed and expressed in E coli BL21(DE3) (Tianjin China)

Table 2 Antimicrobial activities and minimal growth inhibitionconcentrations (MIC) of the recombinant protein

Microorganisms MIC (120583M)G+

Staphylococcus aureus 772ndash1286Bacillus subtilis gt992Micrococcus luteus 278ndash463

Gminus

Escherichia coli 357ndash595Pseudomonas aeruginosa 357ndash595Vibrio alginolyticus gt992Vibrio parahaemolyticus 214ndash357

FungusPichia pastoris GS115 gt992

The results showed that the protein expression level of theinducing group was much higher than that of the noninduc-ing groupThe blank plasmid did not induce band expressionthis finding suggested that BL21(DE3)pET22b-UuHb-F-Iwas the actual strain that induced expression We furtheroptimized the IPTG inducing conditions and observed thatthe highest protein expression level was obtained at 1mMIPTG and 3 h induction time We also induced the proteinexpression by using lactose and found that the highest proteinexpression level was determined at 16 h induction time Theobtained protein expression level at 16 h was higher than thatrecorded at 8 or 12 h

After induction was completed the whole cell lysateand insoluble fraction were analyzed through SDS-PAGEThe results revealed that the recombinant UuHb-F-I wasmainly expressed as insoluble proteins and accumulated ininclusion bodies Western blot (Figure 3) demonstrated thatthe recombinant strain could produce recombinant proteinswith His-Tag after induction was completed This findingconfirmed that the obtained protein was indeed the targetprotein The target protein was purified using Ni+ affinitycolumn (Figure 4) dialyzed and cold-dried for antibacte-rial assay The purified rUuHb-F-I was further measuredby MALDI-TOF-MSMS The result showed that the purepeptide yielded an observed molecular mass of 1516801 Daand its N-terminal sequence was MGLTGAQIDAIK

33 Antimicrobial Activities of rUuHb-F-I The antibacterialactivities of rUuHb-F-I are described in Table 2 rUuHb-F-Iexhibited inhibitory activity against G+ and Gminus Among theobtained MICs the MIC against M luteus was the smallestwith 278ndash463120583M The MIC against S aureus was 772ndash1286 120583M The MIC of rUuHb-F-I against Gminus such as E coliand P aeruginosa was 357ndash595120583M which was higher thanthat of G+ This protein also elicited an inhibitory effect onV parahaemolyticus with MIC of 214ndash357 120583M By contrastthis protein did not affect V alginolyticus and P pastorisGS115


GAAAATCCTCATCTCGACTGCCTGATCGTCAGCAACCAGCTTGACA 4692

AGAATGGGTCTTACTGGAGCTCAGATCGACGCCATCAAGGGTCAT 137M G L T G A Q I D A I K G H 14

TG G TTTA CCA A CA TCA A G G G A CA TTTG CA G G CG G CA G G G G A TTCC 182W F T N I K G H L Q A A G D S 29

A TCTTCA TCA A G TA CCTCA TTA CTTA CCCA G G G G A TA TA G CG TTC 227I F I K Y L I T Y P G D I A F 44

TTTG A CA A G TTTTCCA CG G TCCCCA TCTA TG CCCTG CG A TCG A A C 272F D K F S T V P I Y A L R S N 59

G CA G CG TA CA A A G CCCA G A CTCTA A CA G TTA TCA G CTA CTTG G A T 317A A Y K A Q T L T V I S Y L D 74

A A A G TG A TTCA A G G TCTG G G CA G CG A TG CA G G TG CTTTG A TG A A A 362K V I Q G L G S D A G A L M K 89

GCCAAGGTCCCAAGTCACGAGGCTATGGGGATCACCACGAAGCAT 407A K V P S H EE A M G I T T K H 104

TTCGGACAACTCTTGAAGTTGGTGGGAGTTGTGTTCCAAGAACAG 452F G Q L L K L V G V V F Q E Q 119

TTTGGGGCATGCCCGGAAACTGTCGCTGCCTGGGGAGTCGCTGCT 497F G A C P E T V A A W G V A A 134

GGTGTCCTGGTGGCCGCCATGAAGTAAACCGAAAGACGCTGCTAC 542G V L V A A M K

GTCACGTTCCAAGAACTCGTGATTTAGGAACCGTTACCGCCTATG 587

TGACCTTATTAAGCACAATAATATGCAGTCATTAAATTTGGAGGC 632ATTTTGTTTTCAGCCGAAAATTCACATTTCGTATTGTCTGGTCTG 677TAATGATGTTGATGAAAATTTAACTCGAAAACTGATTCTTGTGAA 722A TTTG A TA TTTG G A G G CTTTTA TTTG A A TA A A A CG G A CA CTTA A A 767TTGAAAAAAAAAAA 780

lowast

TCTTAGCTTATCTCTTGATCACAAAATCCGGACGGAGAATATAGTC

Figure 1 Nucleotide and deduced amino acid sequences of F-I chain of hemoglobin from Urechis unicinctus The start codon (ATG) isboxedThe stop codon (TAA) is indicated by an asteriskThe polyadenylation signal motif (AATAAA) is in dotted lineThe protein sequenceof UuHb-F-I deduced from the nucleotide sequence is underlined The letters underlined with a curve line are the predicted combining siteof heme to protein The poly(A) is double-underlined Numbers on the right side of the sequence show the positions of the last nucleotide oramino acid on each line

UuHb-F-I 1 MGLT GAQI DAIKGHWFTNIKGHLQAAG DSIFIKYLITYPGD IAFF DKFSTVPI-YALRSN

UcHb-F-I 1 MGLT TAQI KAIQDHWFLNIKGCLQAAADSIFFKYLTAYPGD LAFF HKFSSVPL-YGLRSN

Ct-Hp 1 MGLT KAQI AAIQNNWAR-ISNN LQDFGDTLFMRYLTIYPGD LAFF PKFEHEG VGDH LRHN

UuHb-F-I 60 AAYK AQTL TVISYLDKVIQGLG--SDAGALMKAK VPSHEAMGITTKHFGQLLKLVGVVFQ

UcHb-F-I 60 PAYK AQTL TVINYLDKVVDALG--GNAGALMKAK VPSHDAMGITPKHFGQLLKLVGGVFQ

Ct-Hp 60 ADFQAQTL VVCQFLSKVIASLSDMDA AKAMLQERVRTHAPRGIAMA QFERLLDLLPRLVQ

UuHb-F-I 118 EQFGACPETVAAWGVAAGV LVAAMK------

UcHb-F-I 118 EEFSADPTTVAAWGDAAGV LVAAMK------

Ct-Hp 120 DASAASGP TADAWRVAVASLMPAMRQEFAKV

lowast lowast lowastlowast

lowast lowast lowastlowast lowast lowast

Figure 2 Multiple alignment of amino acid sequences of UuHb-F-I with other known globins Amino acid residues that are conserved inthe same sequences are shaded in black similar amino acids of at least 60 are shaded in gray Numbers on the right indicate the amino acidposition of the different sequences The heme-binding domains are marked with asterisk above the alignment The species and the GenBankaccession numbers are as follows UuHb-F-I (Urechis unicinctus hemoglobin F-I) UcHb-F-I (Urechis caupo hemoglobin F-I GI122733) andCt-Hp (Capitella teleta hypothetical protein GI443723524)


1 2 3

Recombinant protein

Figure 3 Result ofWestern blot for induced expression (1 negative2 IPTG induction 3 lactose induction)

M 1

70KD

40KD50KD

30KD

25KD

14KD

Figure 4 Purified recombinant protein (M marker 1 recombinantprotein)

4 Discussions

This study is the first to report the full-length cDNAprokaryotic expression and antimicrobial activity of UuHb-F-I from U unicinctus

Sequence analysis revealed that the mature peptide ofUuHb-F-I is a globin belonging to the heme protein familyUuHb-F-I contains many 120572-helices (7042) and heme-binding sites These properties are similar to those of Hbin other animals [14 16] The nucleotide acid and deducedamino acid sequences of UuHb-F-I exhibited 82ndash87 and79 similarities to those of UcHb-F-I respectively Thecombination sites of heme with UuHb-F-I are 31 (F) 41 (D)44 (F) 45 (F) 65 (Q) 68 (T) 94 (S) 95 (H) 105 (F) and108 (L) which are consistent with those of UcHb-F-I UcHb-F-I contains 137 (L) sites but UuHb-F-I does not consist ofthese sitesTherefore Uu and Uc were derived from the samedescendent and their Hb-F-I was the same

The mechanism of AMPs shows that positive chargesand amphiphilic 120572-helices are common molecular structureswhich accounted for their antimicrobial activity [18 19]Zhu et al [15] reported that 120572-helices in peptides andcharges are responsible for antimicrobial activities changesin amphiphilicity can affect antimicrobial properties Gian-gaspero et al [20] suggested that antimicrobial activities maybe decreased by reducing the positive charges or the number

of 120572-helices Our results showed that UuHb-F-I containsmany 120572-helices (7042) Therefore UuHb-F-I could exhibitantimicrobial activity Uu with a unique Hb can live in activepathogenic zones such asmuds and burrows in sand becauseof this property and thus protect themselves from othermicrobial invasions

As a strong inducer IPTG can induce high proteinproductivity at low doses In this study the expressionlevel increased as IPTG concentration increased within acertain range and the maximum product was obtained at1mM IPTG after 3 h of induction However IPTG mightbe replaced with lactose because of its high costs andtoxicity Lactose can produce the same or greater expressionlevel than that of IPTG [21ndash23] Our result indicated thatlactose could induce the expression of relatively pure pro-teins and thus simplify purification rUuHb-F-I was purifiedand further quantified through MALDI-TOF-MSMS Theresult revealed that the pure peptide yielded an observedmolecular mass of 1516801 Da and its N-terminal sequencewasMGLTGAQIDAIKTheother amino sequence fragmentsexhibited a theoretical molecular mass of 1512067 Da andthis finding is consistent with that of amino acid sequencessubjected to blast analysis Therefore rUuHb-F-I is the sameas UuHb-F-I With AMP prediction (CAMPR3 Collection ofAnti-Microbial Peptides httpwwwcampbicnirrhresinpredict chiiphp) many fragments in UuHb-F-I are pre-dicted as AMPs by the Support Vector Machine classifier Forexample GLTGAQIDAIKGHWFTNIKG in positions 2ndash21exhibits AMP probabilities of 10 (nucleotide acid sequence)and 0873 (peptide sequence) Nevertheless the hydrolysis ofrUuHb-F-I should be further investigated

In the current research G+ Gminus and fungus especiallycommon pathogenic species in aquaculture such as Valginolyticus and V parahaemolyticus may help elucidate theinnate immunity of Uu Bao et al [12] indicated that Tg-HbI(Hb dimer) from Tegillarca granosa is involved in immunedefense responses against microbial infection because themRNA expression of Tg-HbI (Hb dimer) is significantlyupregulated after T granosa is subjected to V parahaemolyti-cus challenge Thus our future work will conduct bacterialchallenge to investigate the relationship between Hb anddefense mechanisms of Uu

In general Hb and its fraction exhibit different antimi-crobial activities against microorganisms through recom-bination or isolation [5] Zhang et al [11] reported thatAJHb derived from Hb-120572 in Japanese eel exhibits a strongantibacterial activity against Edwardsiella tarda with anMICof 1130 120583M of MIC Srihongthong et al [24] found that theHbof alligatorHb exerts biological activity againstG+Bacillusspecies such as B amyloliquefaciens B subtilis and Bpumilus Belmonte et al [25] showed that the MICs of Hb98-114 against Cryptococcus neoformans and Candida tropicalisare 16 and 21120583M respectively Consistent with previousfindings our results revealed that rUuHb-F-I exhibits awide range of inhibitory activities and broad antibacterialspectrum against G+ and Gminus bacteria from nonaquatic andaquatic pathogenic species Our results also showed that theinhibitory effects of rUuHb-F-I were stronger against G+than against Gminus By comparison rUuHb-F-I did not affect


P pastorisGS115The lowestMICwas 278ndash463 120583MobservedinM luteusTherefore rUuHb-F-I is an antibacterial proteinor AMP precursor which may exhibit functional diversitiesor selective antimicrobial activitiesThe results also suggestedthat U unicinctus similar to other aquaculture animals maypossess an innate peptide-dependent host defense system toeradicate microbes as indicated by an MIC of 214ndash357 120583Magainst V parahaemolyticus Thus our study provided abasis for the development of potent therapeutics or agentsagainstU unicinctus disease Further studies on the digestionof rUuHb-F-I or its effects on other pathogens should beperformed to produce highly active AMPs

5 Conclusions

This study is the first to report the full-length cDNAprokaryotic expression and antimicrobial activity of UuHb-F-I from U unicinctus The full-length cDNA sequence was780 bp with an ORF of 429 bp encoding 142 AA The aminoacid sequence of the N-terminal chain of rUuHb-F-I wasMGLTGAQIDAIK with a molecular mass of 1516801 DaThis protein exhibited stronger inhibitory effects against G+than against Gminus By comparison this protein did not affectP pastoris GS115 The lowest MIC observed in M luteus was278ndash463 120583M

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Fujian Province OverseasStudies Program and Natural Science Foundation of FujianProvince (Grant no 2014J01365)

References

[1] V T Ivanov A A Karelin M M Philippova I V Nazimovand V Z Pletnev ldquoHemoglobin as a source of endogenousbioactive peptides the concept of tissue-specific peptide poolrdquoBiopolymersmdashPeptide Science Section vol 43 no 2 pp 171ndash1881997

[2] P Mak K Wojcik J Silberring and A Dubin ldquoAntimicrobialpeptides derived from heme-containing proteins hemocidinsrdquoAntonie van Leeuwenhoek vol 77 no 3 pp 197ndash207 2000

[3] D Hobson and J G Hirsh ldquoThe antibacterial activity ofhemoglobinrdquo Journal of Experimental Medicine vol 107 no 2pp 167ndash183 1958

[4] A C Fogaca P I da Silva Jr M T M Miranda et alldquoAntimicrobial activity of a bovine hemoglobin fragment in thetick Boophilus microplusrdquo The Journal of Biological Chemistryvol 274 no 36 pp 25330ndash25334 1999

[5] C A Parish H Jiang Y Tokiwa et al ldquoBroad-spectrumantimicrobial activity of hemoglobinrdquo Bioorganic amp MedicinalChemistry vol 9 no 2 pp 377ndash382 2001

[6] C Liepke S Baxmann C Heine N Breithaupt L Standkerand W-G Forssmann ldquoHuman hemoglobin-derived peptidesexhibit antimicrobial activity a class of host defense peptidesrdquo

Journal of Chromatography B Analytical Technologies in theBiomedical and Life Sciences vol 791 no 1-2 pp 345ndash356 2003

[7] P Mak K Wojcik Ł Wicherek P Suder and A DubinldquoAntibacterial hemoglobin peptides in human menstrualbloodrdquo Peptides vol 25 no 11 pp 1839ndash1847 2004

[8] J M O Fernandes and V J Smith ldquoPartial purificationof antibacterial proteinaceous factors from erythrocytes ofOncorhynchus mykissrdquo Fish amp Shellfish Immunology vol 16 no1 pp 1ndash9 2004

[9] N Nedjar-Arroume V Dubois-Delval K Miloudi et al ldquoIso-lation and characterization of four antibacterial peptides frombovine hemoglobinrdquo Peptides vol 27 no 9 pp 2082ndash20892006

[10] N Nedjar-Arroume V Dubois-Delval E Y Adje et al ldquoBovinehemoglobin an attractive source of antibacterial peptidesrdquoPeptides vol 29 no 6 pp 969ndash977 2008

[11] D L Zhang R Z Guan W S Huang and J Xiong ldquoIsolationand characterization of a novel antibacterial peptide derivedfrom hemoglobin alpha in the liver of Japanese eel Anguillajaponicardquo Fish and Shellfish Immunology vol 35 no 3 pp 625ndash631 2013

[12] Y B Bao QWang and Z Lin ldquoHemoglobin of the bloody clamTegillarca granosa (Tg-HbI) is involved in the immune responseagainst bacterial infectionrdquo Fish amp Shellfish Immunology vol 31no 4 pp 517ndash523 2011

[13] P H Mygind R L Fischer K M Schnorr et al ldquoPlectasin is apeptide antibiotic with therapeutic potential from a saprophyticfungusrdquo Nature vol 437 no 7061 pp 975ndash980 2005

[14] T Hasegawa F Shishikura and T Kuwada ldquoSide-necked turtle(Pleurodira Chelonia reptilia) hemoglobin cDNA-derivedprimary structures and X-ray crystal structures of Hb ArdquoIUBMB Life vol 63 no 3 pp 188ndash196 2011

[15] X Zhu N Dong Z Wang et al ldquoDesign of imperfectlyamphipathic 120572-helical antimicrobial peptides with enhancedcell selectivityrdquo Acta Biomaterialia vol 10 no 1 pp 244ndash2572014

[16] J R Garey and A F Riggs ldquoThe hemoglobin of Urechiscaupo The cDNA-derived amino acid sequencerdquo The Journalof Biological Chemistry vol 261 no 35 pp 16446ndash16450 1986

[17] O Simakov F Marletaz S-J Cho et al ldquoInsights into bilaterianevolution from three spiralian genomesrdquo Nature vol 493 no7433 pp 526ndash531 2013

[18] Q Y Zhao J M Piot V Gautier and G Cottenceau ldquoIsolationand characterization of a bacterial growth-stimulating peptidefrom a peptic bovine hemoglobin hydrolysaterdquo Applied Micro-biology and Biotechnology vol 45 no 6 pp 778ndash784 1996

[19] Y Shai ldquoMechanism of the binding insertion and desta-bilization of phospholipid bilayer membranes by 120572-helicalantimicrobial and cell non-selective membrane-lytic peptidesrdquoBiochimica et Biophysica ActamdashBiomembranes vol 1462 no 1-2 pp 55ndash70 1999

[20] A Giangaspero L Sandri and A Tossi ldquoAmphipathic 120572 helicalantimicrobial peptidesrdquo European Journal of Biochemistry vol268 no 21 pp 5589ndash5600 2001

[21] D Woyski and J R Cupp-Vickery ldquoEnhanced expression ofcytochrome P450s from lac-based plasmids using lactose as theinducerrdquo Archives of Biochemistry and Biophysics vol 388 no2 pp 276ndash280 2001

[22] B V Kilikian I D Suarez C W Liria and A K GombertldquoProcess strategies to improve heterologous protein productionin Escherichia coli under lactose or IPTG inductionrdquo ProcessBiochemistry vol 35 no 9 pp 1019ndash1025 2000


[23] E Dekel and U Alon ldquoOptimality and evolutionary tuning ofthe expression level of a proteinrdquo Nature vol 436 no 7050 pp588ndash592 2005

[24] S Srihongthong A Pakdeesuwan S Daduang T ArakiA Dhiravisit and S Thammasirirak ldquoComplete amino acidsequence of globin chains and biological activity of fragmentedcrocodile hemoglobin (Crocodylus siamensis)rdquo The ProteinJournal vol 31 no 6 pp 466ndash476 2012

[25] R Belmonte C E Cruz J R Pires and S Daffre ldquoPurifica-tion and characterization of Hb 98-114 a novel hemoglobin-derived antimicrobial peptide from themidgut ofRhipicephalus(Boophilus) microplusrdquo Peptides vol 37 no 1 pp 120ndash127 2012

Research ArticleMutation Detection in an Antibody-Producing ChineseHamster Ovary Cell Line by Targeted RNA Sequencing

Siyan Zhang1 Jason D Hughes2 Nicholas Murgolo3 Diane Levitan3

Janice Chen1 Zhong Liu1 and Shuangping Shi1

1Biologics amp Vaccines Merck Research Laboratories Kenilworth NJ 07033 USA2Biology amp Genetics Informatics Merck Research Labs IT Merck amp Co Boston MA 02115 USA3Discovery Pharmacogenomics Merck Research Laboratories Kenilworth NJ 07033 USA

Correspondence should be addressed to Shuangping Shi shuangpingshimerckcom

Received 18 November 2015 Revised 4 February 2016 Accepted 21 February 2016

Academic Editor Jorge F B Pereira

Copyright copy 2016 Siyan Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeuticsincluding monoclonal antibodies (mAb) The integrity of the gene of interest and the accuracy of the relay of genetic informationimpact product quality and patient safety Here we employed next-generation sequencing particularly RNA-seq and developed amethod to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb The effect of an extendedculturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culturewere also closely examined

1 Introduction

Thedevelopment of next-generation sequencing (NGS) tech-nologies has greatly improved the efficiency of sequencingand contributed to the understanding of dynamic changesin gene expression [1] With the maturation of NGS itsapplications in biomedical research and drug discoveryhave greatly advanced the identification of disease relatedmutations and the development of molecules targeting theaberrantly expressed gene products [2ndash6] Massively parallelcDNA sequencing (RNA-seq) has revolutionized transcrip-tomics studies compared to microarray technologies [7]RNA-seq allows both qualitative and quantitative analysis ofthe expressed gene product at messenger RNA (mRNA) levelwith wide dynamic ranges and superior sensitivity [8]

Mammalian cell lines such as the Chinese hamster ovary(CHO) cells have been widely used in the production ofrecombinant therapeutic product includingmonoclonal anti-bodies [9 10] These cell lines are propagated extensivelyto reach large-scale production vessel Production cell linesare generated by transfecting the host cells with a plasmidvector expressing the gene of interest (GOI) and a selectionmarker followed by drug treatment and clone selection

During a large-scale manufacturing process cells from afrozen bank need to be expanded multiple times to reach afinal volume as large as 20000 litersThe integrity of the GOIand the accurate flow of genetic information throughout thisprocess are crucial to product quality Traditionally proteinsequencing and mass spectrometry are used to characterizethe final product for its consistency and homogeneity at theprotein level [11] DNA sequencing based on the Sanger orpyrosequencing method has also been used for sequenceanalysis of themRNA (via cDNA) [12] Although thesemam-malian host cells have a proven track record in consistentlyproducing high-quality products a potential threat is posedto the quality of the final product by the drug selectionprocess cloning procedures and environmental stress overextended passaging conditions [13] Product variants includ-ing point mutations could develop during the life cycle ofthe production cells However the extent of this risk has notbeen fully understood due to the limitations of traditionalmolecular biology tools mentioned above

In this study we explored the use of RNA-seq technologyfor the characterization of the mutation rate in a stably trans-fected CHO cell line expressing a recombinant monoclonal



antibody (mAb) under extensive in vitro passaging The goalis to identify and quantify mutations in a cell population atthe transcript level under various culture conditions We firstcarried out a feasibility study by mixing two slightly differentmAb light chain cDNAs at different ratios and subjected themixture samples to RNA-seq analysis The detection limit ofthe mutation rate was determined by the feasibility studySince mutation rate is presumably related to the length ofpassaging and the presence of potentially mitogenic selectionreagents such as methotrexate (MTX) we next culturedthe CHO cell line continuously to reach an in vitro cellage of sim150 population doubling levels (PDLs) In parallelincreasing the dose of MTX was also evaluated for its impacton mutation rate The method we developed in this studywill be instrumental in defining the cell culture parametersto ensure consistent and reliable product quality


21 Feasibility Study by cDNAMixing Two cell clones (A andB) expressing a human IgG with different light chain (LC)sequences were thawed from frozen banks and cultured inalpha-MEM (Gibco Cat 12561) containing 10 dialyzed fetalbovine serum (FBS SAFC Cat 12015C) and 045 glucose(Sigma Cat G8769) Cells were passaged and expanded forRNA extraction RNA extraction was performed using theRNeasy kit (Qiagen Cat 74104) andRNAwas eluted in 50 120583LRNase-free water RNA concentrationwasmeasured onNan-oDrop Spectrophotometer (ND-1000 Thermo Scientific)

RT-PCR of IgG light chains was set up with 200 ng RNAper sample using the OneStep RT-PCR kit (Qiagen Cat210212) in 50 120583L reaction volume RT-PCR was run on theApplied Biosystems 2720 Thermal Cycler with incubationperiods of 30min at 50∘C and 15min at 95∘C 30 cyclesof 30-second denaturing at 94∘C 30-second annealing at62∘C and 2min extension at 72∘C followed by final 10minincubation at 72∘C cDNA was purified using the QiaquickPCR Purification Kit (Qiagen Cat 28106) and eluted in 30 120583LEB buffer (10mM Tris-Cl pH 85) cDNA concentrationswere measured on NanoDrop The cDNA of clone B wasmixed with cDNAof clone A atmixing ratios of 5 1 0501 005 and 001 Triplicate samples of pure cDNA ofclones A and B and each mixture were submitted to BGI forRNA-seq

See Supplementary Information in Supplementary Mate-rial available online at httpdxdoiorg10115520168356435for light chain and primer sequences

22 cDNA Preparation from Cell Line under Different CultureConditions (Main Study) Clone A derived from a singlecell was thawed from a frozen bank at about 14 PDLs sinceserum-free adaptation and cultured in Ex-cell ACF CHOmedium C5467 (SAFC Cat 86016C-1000mL) with 4mM L-glutamine (Gibco Cat 25030) 1x Trace Elements A (CellgroCat 99-182-C1) and 1x Trace Elements B (Cellgro Cat 99-175-C1) Cells after thawing were termed PDL 0 and around1 million cells were pelleted and resuspended in 350 120583L RLTbuffer with 1 beta-mercaptoethanol for RNA extraction

Cells were further passaged at 05millionmL every 3-4 daysin the presence of 0 20 or 80 nMMTX (Sigma Cat 8407) at37∘C and 75 CO

2

At PDLs 0 50 100 and 150 15 million cells were pelleteddivided into 3 aliquots upon lysis (except PDL 0 samplewhich was divided into replicates at RNA level) and RNAwas extracted following Qiagen protocol (Qiagen RNeasykit Cat 74104) Reverse transcription was performed with200 ng RNA using the AccuScript High Fidelity RT-PCR kits(Agilent Cat 600180) The thermal program includes 5minincubation at 65∘C and cooling to room temperature for5min followed by addition of 1 120583L of 100mM dithiothreitol(DTT) and 1 120583L of AccuScript Reverse Transcriptase Thereaction was further incubated at 42∘C for 30min and storedat 4∘C Three separate reverse transcription reactions wereperformed for PDL 0 RNA to create replicates cDNAs ofheavy chain (HC) light chain (LC) dihydrofolate reductase(DHFR) andGAPDHwere amplified via PCRusing PfuUltraHF DNA polymerase (Agilent Cat 600380) and the follow-ing thermal cycle program 1min at 95∘C 30 cycles of 30 sec-onds at 95∘C 30 seconds at 64∘C (62∘Cannealingwas used forDHFR) and 3min at 68∘C followed by a final 10min incuba-tion at 68∘C PCRproductswere purified usingQiaquick PCRPurification Kit (Qiagen Cat 28104) For each sample equal-molar ratios of HC LC DHFR and GAPDHwere mixed to atotal cDNAmass of 25 120583g and submitted for RNA-seq at BGIThe experimental procedure is outlined in Figure 1

For the feasibility study the amplified fragment for lightchain corresponded precisely to the target sequence In themain study a slightly larger region was amplified for eachtarget to ensure that the region of interest was outside therange of the PCR primers themselvesThe references used formapping were modified accordingly

23 RNA-Seq At BGI cDNA was fragmented to an averagefragment size of 170ndash180 bp using Covaris OnThermomixerthese fragments were subjected to end-repair and the 31015840end was adenylated Adaptors were ligated to the 31015840 endsThe ligation products were purified on TAE-agarose gel andsim14 rounds of PCR amplification were performed to enrichthe purified cDNA template For quality control the librarywas validated on the Agilent Technologies 2100 Bioanalyzerand the ABI StepOnePlus Real-Time PCR System Qualifiedlibraries were sequenced on Illumina HiSeq2000 and 100Mbclean sequence data were generated for each

See Supplementary Information for details on sequencesof primers and amplified regions Analysis was performedexcluding the regions corresponding to the PCR primers

3 Results

31 Feasibility Study cDNAs from two clones expressinglight chainwith closely related but slightly differing sequenceswere mixed in different ratios to assess the ability of NGS toquantitatively detect the fraction of mutant bases in a mixedpopulationThe sequences chosen for this were each 714 baseslong and differed in 46 positions The sequence alignment isshown in Figure S1


Cellisolation

RNAextraction

Dataanalysis

Reversetranscriptionand PCR of

specific genes

Equal-molarmixing and

submitting forsequencing

Figure 1 Experimental outline of RNA-seq studies of production CHO cell linesThe tested CHO cell lines expressing mAb were propagatedin suspension Cell pellets were isolated and RNA samples were subsequently extracted Reverse transcription was performed on the RNAsamples and certain genes of interest were amplified from cDNAs After library preparation the product was sequenced on IlluminaHiSeq2000 Details of data analysis are described in Section 3

Detecting the fraction of sequence reads from a mixtureof these clones is fundamentally different than detectingemerging mutations in cell culture in that one would notexpect to find so many mutations emerging at once In termsof the data analysis the main impact is on the ability to mapreads For example in the sequence between positions 80 and120 there are more than a dozen sequence differences Bydefault most short-readmappers will onlymap reads reliablywhen the error rate is less than around 5 If sequencesincluding mixtures of reads from clones A and B weremapped directly to clone A reference some reads from cloneBwould notmap at all to cloneA referenceThis would not beexpected to happen in the real case of an emerging mutationat a single position To address this issue for the feasibilitystudy we map reads to a reference sequence that includesboth clone A and clone B sequences using BWA (httpsgithubcomlh3bwa version 070 Li H and Durbin R(2009) Fast and accurate short read alignment with Burrows-Wheeler transform Bioinformatics 25 1754ndash1760 [PMID19451168]) BWA will output the single best alignment foreach read in SAM format For reads from regions whereclones A and B differ the alignment will indicate that themapping was specific to reference A or B For reads fromregions where clones A and B do not differ reads will berandomly assigned to one reference or the other In orderto obtain a mapping that is consistent with what we wouldexpect to find in the real study if any one of the 46 mutationshad occurred singly we modify the mappings obtained inthis way as follows We replace all occurrences of the cloneB sequence identifier in the SAM-formatted alignment fileswith the clone A identifier and we ignore the trailing tagfields Since there are no insertion or deletion differencesbetween the two clones the SAM file obtained in this wayis perfectly consistent with what would have been obtainedif the mutations had occurred separately This procedure isequivalent to mapping reads to each of the clone sequencesseparately determining which reference was a better fit and

then translating the clone B alignments to become cloneA alignments In this case that translation step is trivialsince the two sequences differ only by substitutions The keyadvantage of this approach over any single-referencemappingapproach is that it eliminates the possibility of any edgeeffects or incorrectly induced insertions or deletions in thealignments in regions where the clones A and B sequencesare significantly different Had we used a more exhaustiveapproach such as a Smith-Waterman alignment of all reads tothe clone A sequence for example the resulting alignmentsof reads from clone B that included significantly differingsections would have had small errors in alignment that wouldhave confounded the analysis Also it is important to notethat this modified alignment procedure is only relevant forthe initial validation portion of this study

Aside from this mapping difference the analysis for thefeasibility study is performed exactly as for the main studySequence data were received from BGI in FASTQ formatAdapters were removed using SeqPrep (httpsgithubcomjstjohnSeqPrep version 04 unpublished) and aligned tothe reference sequence using BWA Coverage across the lightchain sequence for all samples is shown in Figure S2 Theoverall mapping rate across all experiments was very highgenerally around 99 and the reads aligned with a very lowmismatch rate typically around 02 mismatches per 90 bpread This indicates that we had very little contamination inthe experiment

The SAMtools program ldquompileuprdquo (httpsgithubcomsamtoolssamtools version 0119 Li Hlowast Handsaker BlowastWysoker A Fennell T Ruan J Homer N Marth G Abeca-sis G andDurbin R and 1000Genome Project Data Process-ing Subgroup (2009) The Sequence alignmentmap (SAM)format and SAMtools Bioinformatics 25 2078-9 [PMID19505943]) was used along with custom scripts to extract foreach position in the target region the counts of each base of ACG andT aswell as the numbers of insertions and deletionsInsertions were counted according to the base immediately


preceding the insertion regardless of what sequence wasbeing inserted Similarly deletions were attributed to the basebeing deleted regardless of how many bases were spannedby the overall deletion These counts were stratified based onwhether they were found from reads aligned in the forwardor reverse directions Bases with quality scores less than15 were ignored in this analysis This cutoff was selectedto remove a minimum amount of data (typically 2ndash5 ofbases) while eliminating the lowest quality bases which aremainly those with reported base quality of two indicatingthat the sequencer failed to call the base at the positionWithin each experiment for each position in each targetsequence a preferred orientation was determined based onwhich orientation gave rise to higher overall coverage Onlydata from reads in the preferred orientation at each positionwas used to generate final results Overall this step has theimpact of removing a small portion of very-low-quality dataat the cost of ignoring just under half of the overall sequencedata which has little impact on most positions

This decision to use only data from reads in a preferredorientation is driven by the fact that some sequence contextsare problematic for sequencing (observed in a variety oftargeted sequencing experiments unpublished results) Theproblem may arise from any step in the process fromamplification to library prep to the sequencing itselfThe issueis often found in regions that are G-rich The reads on theG-rich strand will often have errors while the reads fromthe other C-rich strand do not In those cases we find thatthe ldquobetterrdquo strand usually has higher coverage presumablybecause the sequencer was unable to generate acceptablereads from that direction andor some of the base calls hadquality scores below the threshold of 15 By applying a cutoffbased on coverage we are able to identify the ldquobetterrdquo strandwithout explicitly biasing the analysis to lower-frequencyresults For consistency the strand choice is made once foreach unit of analysis the feasibility study and the main study

Once the data have been processed to the counts of A CG and T indels and deletions for each position we can deter-mine the consensus sequence and the rate of occurrence foreach possible alternate allele at each position If we considerthe data from the unmixed sample for clone A to be our ref-erence and any alternate allele observations to be errors wefind that the error rate across all possible positions measuredas the frequency of the most common alternate allele at eachposition ranges from less than 001 to a high of 027 with99of possible alternate alleles occurring at a rate of less than02 The full distribution is shown in Figure 2

To assess the reproducibility of the data we looked at theapparent error rates for each possiblemutation using replicateexperiments Figure S3 shows plots of error versus error fortwo of the 100 clone A reference samples versus the thirdThe plot has a point for each possible base at each positionincluding the reference baseThe reference base calls all hovernear 1 when there are consensus base calls that all fit into thesame pixel on the log-log plot In this way the plot focusesattention on the erroneous base callsThe red green and bluecurves correspond to a difference in apparentmutation rate of10 1 and 01 respectively Using these plots it is possibleto quickly identify any outliers that might correspond to true

minus45 minus40 minus35 minus30 minus25

Freq

uenc

y

Distribution of error rates (feasibility study)

0

50

100

150

200

250

300

log10 (frequency of major alt allele)

Figure 2 Distribution of error rates across all positions in lightchain from the feasibility study The most frequent alternate alleleat each position is used to populate the figure

mutations and to get an estimate of the overall noise level inthe experiment

For these samples there are a few points very close tothe blue 01 line but none that actually cross it in eithercomparison By contrast when there is a true signal in thedata set data points are expected to be well outside thisregion For example if we take two of the 01 spiked controlsand two of the 05 spiked controls and compare them to the0 reference we obtain the plots in Figure S4The points cor-responding to the true spiked-in mutations are colored red

We will take the signal for each mutation in each spiked-in sample to be the difference between the average alternateallele rate observed in each of the three replicate spike-insamples and the average alternate allele rate observed for thecorresponding mutation in the replicate reference samplesFor each of these possible mutations we will use a 119905-testto assess whether the difference between the two means isstatistically significant Given the small numbers of replicatesinvolved the 119905-test results will not be used aggressively butrather as a filter to weed out spurious results (uncorrected 119875value cutoff of 01)

The main results from the samples in the feasibility studyare shown in Figure 3 We find that the estimates of mixingratio are very accurateThemedian signals at positive controlsites for the 001 005 01 05 1 and 5 spike-in experiments were 0017 0057 011 057 11and 53 respectively The range of signals was typically asmuch as plusmn2x however Certain sites have consistently loweror higher signal estimates across different spike-in levelssuggesting that the variability may be sequence-dependentand may not be corrected by additional sequencing

All 46 true-positive mutations are observed with statis-tical significance for spike-in levels of 5 1 and 05At the 01 005 and 001 spike-in levels 4546 4246and 1046 of the mutations are observed Across all controlsites (true negative) 27 false positives were observed Theobserved signal was less than 001 in most of those cases


Feasibility study results

Mutation rate at each position

Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001

1e minus 011e minus 031e minus 051e minus 07

1

2

3

4

5

6

7

Figure 3 The seven horizontal bands of points correspond toexperiments with mixing ratios of 001 005 01 05 1 5and 100 There are points for each position in light chain for eachsample sequenced The 119909-axis corresponds to the apparent signalfor each spiked-in sample In order to include the negatives thatresult from this measurement on the log-scale plot they are plottedas their absolute values colored grey and offset just below theother points The points corresponding to the spiked-in mutationsare colored blue and offset just above the other points The lightblue points did not meet the threshold for statistical significanceTrue-negative mutations that did meet the criteria for statisticalsignificance are colored purple instead of black All points have hada small amount of vertical jitter addedThe jitter and offsets serve toallow visualization of the full distribution of points for the negativeand positive controls

and the highest signal observed was 003 By contrastfor the positive control sites at the 01 spike-in level thelowest observed excess signal was 00599 Based on theseobservations we set the following thresholds for mutationdetection in the main study excess mutation signal of morethan 005with a119875 value less than 01 In the feasibility studythese criteria would yield 4546 true positives at the 01spike-in level with no false positives The one false negativehad an apparent signal of 012 but just barely missed the 119875value cutoff with a value of 012 Therefore these settings aredesigned to be sufficient to detect (or rule out)mutationswitha true signal of more than 01

It is worth noting here that had we been interested onlyin mutations at higher levels the natural thresholds basedon this feasibility study would always be around one-half ofthe desired mutation detection rate That threshold wouldstill allow perfect sensitivity for all 46 tested mutations whileminimizing the false positive rate

32 Main Study We found that the error profile for the mainstudy was slightly different than that observed in the feasi-bility study Overall the error profile was better for the mainstudy with an average error rate over all possible substitutionsand indels of 011 versus 017 for the feasibility study

However while there were no mutations with a back-ground rate of more than 03 in the feasibility study therewere four such mutations in the main study including two

Error error comparison (main versus feasibility)

Error (feasibility study)

Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00

1e minus 061e minus 041e minus 021e + 00

Figure 4 Comparison of a baseline sample from the main studyversus a reference sample from the feasibility study showing therate of apparent error versus error for each possible alternate alleleat each position The dotted lines correspond to a mutation rate of03

PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501

Distribution of significant mutations from main study

0

20

40

60

80

Figure 5 Histogram of counts of mutations meeting the thresholdfor detection of mutations at the 01 level for each experimentalcondition tested Those mutations that also met the criteria for the05 level are highlighted in light grey

above the 1 level The overall correspondence betweenthe error rates was nevertheless quite good overall See theerror error plot in Figure 4 More importantly the errorprofiles for the main study samples compared to replicateswithin that study were very consistent See the error errorplots for the reference samples in Figure S5

We proceeded with the analysis as described Across allnine samples covering no MTX 20 nM MTX and 80 nMMTX at 50 100 and 150 PDLs 245 mutations met thecriteria established in the feasibility study for the 01 levelThese were unevenly distributed across the samples biasedstrongly toward samples with larger PDLs The distributionof mutations is shown in Figure 5 Also highlighted in this


Main study results (LC)


Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8

Main study results (HC)


Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80

Main study results (DHFR)


Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80

Main study results (GAPDH)


Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80

Figure 6 Four panels correspond to each of the four targets light chain heavy chain GAPDH and DHFR (clockwise from the top left)Each panel has points for each experimental condition stratified vertically exactly as done for the feasibility study (Figure 3) The coloringjittering and offsets for the points are also identical to Figure 3 except that there are no spike-in signals here and hence no blue pointsPositions meeting the criteria for significance (119905-test 119875 value lt01) are colored purple

figure are those mutations that would have met the criteriafor mutation detection at the 05 level In total there wereten signals detected at that level

The same analysis was performed with identical settingsfor the other three targets in the experiment The pattern ofmutations was very similar in each caseThe plots in Figure 6show the apparent rate of mutation for all possible mutationsin each of the four targets studied In this more quantitativeview it is possible to see the full distribution of error ratesacross the study While many mutations met the criteria forstatistical significance (119905-test 119875 value lt01 points coloredpurple) the vast majority of those have a very low apparentmutation rate Since we had only triplicate data it was notpossible to use a more stringent statistical cutoff However itis also possible to see some general trends in this view Acrossall four targets as the PDL increases the distribution ofapparent mutation rates shifts uniformly higher for examplePresumably this reflects small true shifts in the populationaccumulating over time though few mutations met ourcriteria for detection In terms of specific mutations meeting

the criteria established for detection at the 05 level thenumbers of signals observed in light chain heavy chainDHFR andGAPDHwere 10 17 4 and 0 respectively A tablewith all signals found across all four genes is included in theSupplementary Information

4 Discussion

Here we explored using RNA-seq technology for the detec-tion of emerging mutations in a CHO cell line producing arecombinant antibody during long-term culture In the feasi-bility study we established a high-confidence mutation leveldetection limit of 01 which is significantly more sensitivethan traditional molecular biology or protein characteriza-tion techniques The detection limit of mutation by SangerDNA sequencing is around 15ndash20 [14] When comparingthe feasibility study to the main study we noticed that thebackground error profile revealed by sequencing replicatesof the same biological sample can vary from batch to batchWithin each batch the error profile at each position (whether


arising from amplification library prep or sequencing itself)was very consistent Therefore a reference run should beincluded in each sequencing batch and used to assess vari-ation within each batch By considering each position tohave an independent error profile we can implicitly accountfor a variety of error sources without knowing exactly whatcontribution each source makes

In the main study we analyzed all three exogenous genesintroduced by the expression vector which were heavy chainand light chain of the mAb and the DHFR selection markerWe also analyzed the house-keeping gene GAPDH as arepresentative host endogenous gene As the study showsthe mutation rate displayed a clear increasing trend withextended culture passaging And in most cases the mutationrate also increased in the presence of selection pressure(MTX) In the actual cell culture manufacturing processthe cell inoculum typically needs to be passaged for at least30ndash40 PDLs starting from a frozen cell bank and often in thepresence of selection pressure such asMTXOur experimentswere designed to sufficiently cover this manufacturingwindow with respect to both process conditions In Figure 6there is a noticeable jump in the numbers of significantmuta-tions (above 01) starting at 150 PDLs At the same time upto 100 PDLs only the sample treated with 80 nMMTX exhib-ited detectable mutations higher than 05 No mutationabove 05was observed in the house-keeping gene GAPDHunder any of the culture conditions This indicates thatincreasing selection pressure and extending passaging periodmainly affect the stability of the transgenes but have minimaleffect on endogenous host genes presumably due to thedeleterious effect to the host It is noteworthy that mutationrate can be described in two ways The first is the numberof mutations above the 01 detection limit across theentire gene fragment And the second is the percentage ofpopulation that carries a specific point mutation Both repre-sentations showed similar trend in our study

On the molecular level mutations identified in mRNAcan be attributed to DNA template mutations [15] transcrip-tional errors [16 17] or posttranscriptionalmodifications [8]Understanding the mechanism behind individual mutationsrequires further characterization of all these possible factorsincluding DNA sequence analysis of the expression vectorinserted into the genome In addition mutations detected byRNA-seq require confirmation by protein sequence analysisto assess their impact on product quality

NGS technologies have played increasing roles in thedevelopment of cell culture production process and facilitatedthe understanding of the production cell line There has notbeen a report on applying RNA sequencing to systematicallyanalyze mutation rate during extended passaging of produc-tion CHO cells Production cell line stability with respectto sequence integrity is crucial for the biopharmaceuticalindustry because cell lines carrying the intended transgenesequences are essential for product quality and patient safetyHere we have demonstrated that RNA-seq can help to ensurethe accurate flowof genomic information to the final productAlthough CHO cell lines developed with DHFR as theselection system are used as a model system in this studyto characterize gene stability the methods developed in this

study should also be applicable for other production host celllines and selection methodologies The information gener-ated should further stimulate investigation on the molecularmechanisms behind sequence variations in mRNA

Competing Interests


Authorsrsquo Contributions

Siyan Zhang Jason D Hughes and Nicholas Murgolo con-tributed equally to this work

References

[1] M LMetzker ldquoSequencing technologiesmdashthe next generationrdquoNature Reviews Genetics vol 11 no 1 pp 31ndash46 2010

[2] S B Baylin and P A Jones ldquoA decade of exploring the cancerepigenomemdashbiological and translational implicationsrdquo NatureReviews Cancer vol 11 no 10 pp 726ndash734 2011

[3] E T Cirulli and D B Goldstein ldquoUncovering the roles of rarevariants in common disease through whole-genome sequenc-ingrdquo Nature Reviews Genetics vol 11 no 6 pp 415ndash425 2010

[4] Y-H Jiang R K C Yuen X Jin et al ldquoDetection of clinicallyrelevant genetic variants in autism spectrum disorder by whole-genome sequencingrdquo American Journal of Human Genetics vol93 no 2 pp 249ndash263 2013

[5] Z Kan H Zheng X Liu et al ldquoWhole-genome sequencingidentifies recurrent mutations in hepatocellular carcinomardquoGenome Research vol 23 no 9 pp 1422ndash1433 2013

[6] Y Song L Li Y Ou et al ldquoIdentification of genomic alterationsin oesophageal squamous cell cancerrdquoNature vol 508 no 7498pp 91ndash95 2014

[7] F Ozsolak and P M Milos ldquoRNA sequencing advanceschallenges and opportunitiesrdquo Nature Reviews Genetics vol 12no 2 pp 87ndash98 2011

[8] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[9] DMWuest SW Harcum and K H Lee ldquoGenomics inmam-malian cell culture bioprocessingrdquo Biotechnology Advances vol30 no 3 pp 629ndash638 2012

[10] X Xu H Nagarajan N E Lewis et al ldquoThe genomic sequenceof the Chinese hamster ovary (CHO)-K1 cell linerdquo NatureBiotechnology vol 29 no 8 pp 735ndash741 2011

[11] H Zhang W Cui and M L Gross ldquoMass spectrometryfor the biophysical characterization of therapeutic monoclonalantibodiesrdquo FEBS Letters vol 588 no 2 pp 308ndash317 2014

[12] F Cheung J Win J M Lang et al ldquoAnalysis of the Pythiumultimum transcriptome using Sanger and pyrosequencingapproachesrdquo BMC Genomics vol 9 pp 542ndash551 2008

[13] F M Wurm ldquoCHO quasispecies-implications for manufactur-ing processesrdquo Processes vol 1 no 3 pp 296ndash311 2013

[14] A C Tsiatis A Norris-Kirby R G Rich et al ldquoComparison ofSanger sequencing pyrosequencing andmelting curve analysisfor the detection of KRAS mutations diagnostic and clinicalimplicationsrdquo Journal ofMolecular Diagnostics vol 12 no 4 pp425ndash432 2010


[15] J A Stamatoyannopoulos I Adzhubei R E Thurman G VKryukov S M Mirkin and S R Sunyaev ldquoHuman mutationrate associated with DNA replication timingrdquo Nature Geneticsvol 41 no 4 pp 393ndash395 2009

[16] P Cui F Ding Q Lin et al ldquoDistinct contributions of repli-cation and transcription to mutation rate variation of humangenomesrdquo Genomics Proteomics amp Bioinformatics vol 10 no 1pp 4ndash10 2012

[17] P Green B Ewing W Miller P J Thomas and E DGreen ldquoTranscription-associated mutational asymmetry inmammalian evolutionrdquo Nature Genetics vol 33 no 4 pp 514ndash517 2003

Research ArticleCloning and Expression of the 120574-Polyglutamic Acid SynthetaseGene pgsBCA in Bacillus subtilis WB600

Biaosheng Lin12 Zhijuan Li1 Huixia Zhang1 Jiangwen Wu1 and Maochun Luo1

1College of Life Science Longyan University Longyan 364012 China2Fujian Provincial Key Laboratory of Preventive Veterinary Medicine and Veterinary BiotechnologyLongyan University Longyan 364012 China

Correspondence should be addressed to Maochun Luo 210414269qqcom

Received 1 December 2015 Revised 23 February 2016 Accepted 2 March 2016


Copyright copy 2016 Biaosheng Lin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To clone and express the 120574-polyglutamic acid (120574-PGA) synthetase gene pgsBCA in Bacillus subtilis a pWB980 plasmid was used toconstruct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilisWB600 PgsBCA was expressedunder the action of a P43 promoter in the pWB980 plasmid Our results showed that the recombinant bacteria had the capacity tosynthesize 120574-PGAThe expression product was secreted extracellularly into the fermentation broth with a product yield of 174 gLor higher 120574-PGA samples from the fermentation broth were purified and characterized Hydrolysates of 120574-PGA presented in singleform constituting simple glutamic acid only which matched the characteristics of the infrared spectra of the 120574-PGA standard andpresented asmultimolecular aggregates with amolecular weight within the range of 500ndash600 kDa Expressing the 120574-PGA synthetasegene pgsBCA in B subtilis system has potential industrial applications

1 Introduction

Gamma-polyglutamic acid (120574-PGA) is a new water-solublebiodegradable material It is an anionic polypeptide formedby the condensation of amide linkages between 120572-aminoand 120574-carboxylic acid groups of the D- andor L-glutamatein microorganisms It has nontoxic edible adhesive film-forming andmoisture retention properties [1] 120574-PGAand itsderivatives can be used as drug carriers andbioadhesivemate-rials that have beenwidely used in pharmaceutical cosmeticsfood agriculture and sewage treatment industries and havebecome one of the most interesting topics in biopolymerresearch [2]

Traditionally 120574-PGA is primarily produced throughmic-robial fermentation [3] Bacteria involved in 120574-PGA synthesisare mostly gram-positive (genus Bacillus class Bacilli) andare classified as glutamate-dependent or glutamate nonde-pendent types based on their needs for glutamate [4] Wild-type 120574-PGA-producing strains have unstable heritabilityeasily leading to a reduction or loss in the ability to synthe-size 120574-PGA during fermentation undergo 120574-PGA degrada-tion and produce extracellular polysaccharide by-products

thereby lowering product yield Compared to traditionalmutation breeding genetic engineering technologies havebeen expected to become an effective method to create 120574-PGA high-yield strains Ashiuchi et al [5] and Tarui et al [6]confirmed that pgsB pgsC and pgsA are three essential genesinvolved in 120574-PGA synthesis in glutamate-dependent strainsUrushibata et al [7] and Jiang et al [8] constructed recombi-nant plasmids containing the pgsBCA gene through differentmethods of fusion expression and further transformed theplasmids into Escherichia coli to obtain positive clones thatwere capable of producing 120574-PGA E coli a gram-negativebacterium has been reported as the primary host strain fortransforming the recombinant vector of the 120574-PGA synthasegene However its synthase gene is mainly derived fromBacillus subtilis (gram-positive bacteria) The membranestructures and protein secretion systems of both types of bac-teria vary which in turnmay result in poor positioning of therecombinant expressed 120574-PGA synthase system on the bac-terial cell membrane [9] Therefore the level of expressionof 120574-PGA in the host strain is lower and the amount of 120574-PGA obtained frompositive clones is only within the range of0024ndash0134 gL [10] B subtilis as a prokaryotic expression



host for food safety carries some excellent features in express-ing 120574-PGA that E coli does not possess For example B sub-tilis is capable of expressing the soluble and nonfusion pro-teins as well as preferentially expressing the nonpathogenicand nonapparent codons [11] In addition its expression of arecombinant plasmid after transformation is highThereforeits expression products have greater advantages and higherpotential in manufacturing biological engineering productsfor the food and pharmaceutical industries However therelevant study of cloning and expression of pgsBCA in Bsubtilis was comparatively scarce To date the expression ofthe 120574-PGA synthase gene pgsBCA still need D-xylose and L-arabinose induced generally with poor expression yield andlowmolecular weight (only 200ndash500 kDa) [12] indicating theneed to resolve this particular bottleneck Considering this inthis paper the recombinant plasmid expressing pgsBCA genewas reconstructed and highly expressed in B subtilis as toimprove the yield and molecular weight of 120574-PGA B subtilis168 has been widely used in the study of 120574-PGA regulationIt is one of the few bacterial strains that has a complete setof 120574-PGA synthase genes but does not produce 120574-PGA [13]The present study used the genomic DNA of B subtilis 168 asDNA template to amplify the 120574-PGA synthase gene pgsBCAand to further clone the pgsBCA gene into the B subtilisexpression vector pWB980 to transform into type strain Bsubtilis WB600 We constructed a recombinant B subtilisexpression system for 120574-PGA synthesis which may serve as afoundation for the high-yield industrial production of 120574-PGAbased on an engineered B subtilis expression system


21 Bacterial Strains and Plasmids B subtilis 168 and B sub-tilisWB600 were purchased from Shanghai Genemy BioTechCo Ltd (Shanghai China) E coli JM109 was prepared andpreserved at our laboratory and described in a previous studypMD19-T vector and B subtilis expression vector pWB980were purchased from TakaRa Biotechnology (Dalian) CoLtd (Dalian China)

22 Reagents All restriction endonucleases T4 DNA ligaseTaqDNA polymerase dNTPs DNA ladder marker and pro-tein molecular weight markers were purchased from TakaRaBiotechnology (Dalian) Co Ltd Plasmid extraction andagarose DNA extraction kits were purchased from TiangenBiotech (Beijing) Co Ltd (Beijing China) Bacterial geno-mic DNA extraction kits were purchased from and primerswere designed and synthesized by Sangon Biotech (Shanghai)Co Ltd (Shanghai China) Silica gel plates for thin layerchromatography (TLC) were purchased from Qingdao JiyidaSilica Reagent Factory (Model number 50 times 100 GF254Shandong China)

23 Culture Medium Lysogeny broth (LB) was preparedusing 10 gL tryptone 5 gL yeast extract and 10 gLNaCl (pH70) and 20 (WV) agar powder to solidify the medium Ecoli and B subtilis transformants were selected with 50 120583gmL

ampicillin (Ampr) and 30 120583gmL kanamycin (Kmr) respec-tively Fermentation broth for the genetically engineeredrecombinant bacteria contained 40 gL glucose 0ndash100 gLsodium glutamate 6 gL (NH

4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)

24 Primer Design With reference to the NCBI database theupstream and downstream pgsB pgsC and pgsA coding genesequences ofB subtilis 168were designed as follows BAC1 51015840-CGCGGATCCATGTGGTTACTCATFATAGCC-31015840 (restric-tion site of BamHI endonuclease is underlined) BAC251015840-CCCA AGCTTTTATTTAGATTTTAGTTTGTCA C-31015840(restriction site of HindIII endonuclease is underlined)

25 Cloning of 120574-PGA Synthetase Gene B subtilis 168 geno-micDNAwas used as template BAC1 andBAC2primerswereused to amplify the gene The PCR reaction system included2 120583L of DNA template 10 120583L of 5x buffer 2 120583L of dNTPs 2120583Lof individual primers of BAC1 and BAC2 05 120583L of 5x PrimerSTAR and sterile double-distilled water to prepare a finalvolume of 50 120583L Reaction conditions were as follows 94∘Cfor 3min followed by 30 cycles of 94∘C for 30 s 55∘C for 15 sand 72∘C for 3min and a final 72∘C extension for 10min Onepercent agarose gel electrophoresis was used to identify thePCR reaction products PCR products were recovered usinga DNA rapid recovery reagent and ligated into the pMD19-T vector which was followed by transformation into E coliJM109 competent cells using CaCl

2methods The selected

single colonies were inoculated into liquid LB to expandthe plasmid Intermediate vectors pMD-pgsBCA were thenobtained and identified using BamHI and HindIII doubledigestion as well as sequencing

26 Construction of B subtilis Expression Vector BamHI andHindIII double digestion was performed to cut the interme-diate vector pMD-T-pgsBCA and pWB980 plasmid followedby ligating these into the recombinant expression vectorpWB980-pgsBCA (Figure 1) Kanamycin resistance screeningwas performed to screen the recombinant plasmid followedby plasmid extraction and identification using restrictionenzyme digestion and sequencing to obtain the positiveclones of the bacterial strain

27 Induced Expression of pgsBCA Gene pWB980-pgsBCAplasmids were transformed into B subtilis WB600 to obtainrecombinant strains of BacillusWB600-pgsBCA which wereinoculated into 5mL of fresh liquid LB containing 30120583gmLkanamycin and incubated at 37∘C in a 200 rpm shaker over-night The next day a 2 inoculum of the culture suspensionwas further inoculated into 250mL flask with 100mL recom-binant fermentation medium containing kanamycin andincubated at 37∘C in a 200 rpm shaker for 36ndash48 h until thebacterial concentration stopped growing and fermentationwas terminated pWB980-pgsBCA contained a constitutiveP43 promoter Hence we did not add any inducers duringthe fermentation process Approximately 0ndash100 gL sodiumglutamate was added into the fermentation medium as asynthetic substrate for 120574-PGA to further study the impact of


BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase

Digest by BamHI and HindIII

Digest by BamHI and HindIII respectively

1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr

Figure 1 Construction of recombinant plasmid pWBb980-pgsBCA from Bacillus subtilis expression vector pWB600 and 120574-pgsBCA gene

different substrate concentrations on the synthetic yield of 120574-PGA

28 120574-PGA Isolation and Purification After adding the opti-mal substrate concentration and fermentation had ended thefermentationmediumwas centrifuged at 5000 rpm for 5minto collect the supernatant The supernatant was mixed with4 volumes of absolute ethanol and left to stand overnight at4∘C followed by centrifugation at 4000 rpm and then thesupernatant was discarded The pellet was redissolved in theappropriate amount of distilled water and further centrifugedat 5000 rpm to obtain the supernatant A 20mgmL solutionof proteinase K was added into the supernatant and dialyzedovernight using deionized water After the centrifugation asearlier described the supernatant was collected and freeze-dried to obtain the purified solid samples of 120574-PGA 120574-PGAsamples were stored at minus70∘C until analysis

29 Hydrolysis of 120574-PGA A 05 g purified 120574-PGA sample wasadded to 10mL of 6moLL HCl vacuumed for 10min andthen sealed The sample was then hydrolyzed at 110∘C for 12ndash24 h allowed to cool down and then filtered and redissolvedin 6moLL of NaOH to adjust the pH to 70 The aqueoussolutionwas transferred to a 100mLflask and the hydrolysatewas subjected to TLC using silica gel plates to analyze itsamino acid composition

210 Determination of 120574-PGA Contents and Properties 120574-PGA contents of fermentation broth were measured by high-performance liquid chromatography (HPLC) [14] The puri-fied 120574-PGA samples underwent infrared spectroscopy usingShimadzursquos IR Prestige-21 infrared spectrometer Shimadzu(China) Co Ltd (Beijing China) Potassium bromide (KBr)was used as reference material [15] The molecular weight of120574-PGA was determined by SDS-PAGE [16]


3 Results

31 PCR Amplification and Identification of 120574-PGA Syn-thetase Gene pgsBCA The target gene was amplified by PCRFigure 2 shows the PCR products that were separated andanalyzed using agarose gel electrophoresisThe observed sizeof the amplified pgsBCA fragment 28 kb was in agreementwith our expected results An agarose DNA extraction kitwas used to recover and purify the PCR products Afterconfirming with DNA sequencing the DNA sequence of thePCR products was determined to be 100 identical with thesequence of the reported gene of B subtilis 168

32 Identification of B subtilis ExpressionVectors After trans-forming the constructed recombinant expression vectorspWB980-pgsBCA into competent cells the plasmids werecollected and identified using BamHI andHindIII restrictionenzyme digestions Figure 3 shows that as shown in themap of double restriction enzyme digestions the size of thecleaved fragmentwas the same as that of the pgsBCAPCRpro-ducts thereby initially confirming the successful construc-tion of the recombinant expression vector pWB980-pgsBCA

33 Impact of Different Substrate Concentrations on the Syn-thetic Yield of 120574-PGA Figure 4 shows that with increasingamounts of the substrate glutamate the production of 120574-PGAwas enhanced However when glutamate concentration wasgt50 gL the synthetic yield of 120574-PGA declined This resultsuggested that pgsBCAwas secreted byB subtilisWB600Theexpressed product 120574-PGA could be secreted into extracel-lular fermentation broth Using a lower substrate concentra-tion we observed that the recombinant bacteria did not syn-thesize 120574-PGA indicating that an excess amount of substratewas necessary for the recombinant bacteria to synthesize 120574-PGATherefore from the perspective of economic efficiencywe identified that a substrate concentration of 50 gL wasoptimal to synthesize the highest possible amount of 120574-PGA(174 gL)

34 Characterization of Recombinant 120574-PGA in FermentationBroth Figure 5 shows the TLC results of the hydrolysatesamples observed under ultraviolet light wherein after theacid hydrolysis of 120574-PGA no other band was observed onthe silica gel plates but only single spots of uniform colorintensity Its retention (119877

119891) value was consistent with that of

the standard glutamate spots indicating that the hydrolysateshad no other amino acids and other protein impuritiesThesehydrolysateswere in single form solely consisting of pure glu-tamic acid Figure 6 shows the infrared (IR) spectroscopy of120574-PGAThe absorption peak at 3421 cmminus1 was the symmetricstretching vibration band of N-H and the absorption peak at1649 cmminus1 was the asymmetric stretching vibration band ofan amide group -CONHR Both peaks were themain indica-tors used in the identification of amides and for the presenceof amide groups in 120574-PGA molecules The absorption peakat 1408 cmminus1 was the symmetric stretching vibration band ofCOOH the absorption peak at 1076 cmminus1 was the hallmarkpeak representing the presence of aliphatic hydrocarbons

1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp

Figure 2 PCR product of pgsBCA gene Note Lane 1 pgsBCA PCRproduct Lane M DNA markerIII (Tiangen)

1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp

Figure 3 Map of electrophoresis of recombinant plasmid pWB980-pgsBCA after digestion Note Lane 1 pgsBCA PCR product Lane 2after double digestion of pWB980-pgsBCAwithBamHI andHindIIILane M DNA markerIII (Tiangen)

0

04

08

12

16

2

0 20 40 60 80 100 120Content of sodium glutamate (gL)

Prod

uctio

n of

120574-P

GA

(gL

)

Figure 4 Production of 120574-PGA in fermentation of recombinants(gL) As increasing amounts of the substrate glutamate the pro-duction of 120574-PGAwas enhanced However when glutamate concen-tration was gt50 gL the synthetic yield of 120574-PGA declined


1 2 3

Figure 5 The thin layer chromatography spectrums of samplehydrolysate Note Lane 1 standard sample of L-glutamic acid Lanes2 and 3 hydrolyzed sample of 120574-PGA

3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)

Figure 6 Analysis of FT-IR spectrum of the 120574-PGA sample Theabsorption peak at 3421 cmminus1 was the symmetric stretching vibra-tion band of N-H 1649 cmminus1 was the asymmetric stretching vibra-tion band of ndashCONHR 1408 cmminus1 was the symmetric stretchingvibration band of COOH 1076 cmminus1 was the hallmark peak repre-senting the presence of aliphatic hydrocarbons -CH

2or -CH

3(flex-

ural vibration) 1000 cmminus1ndash500 cmminus1 were caused by (CH2)119899(119899 gt 4)

planar rocking vibration as well as in-plane bending vibration

-CH2or -CH

3(flexural vibration) in themolecular structure

and the absorption peaks within the range of 1000 cmminus1ndash500 cmminus1 were caused by the (CH

2)119899(119899 gt 4) planar rocking

vibration as well as in-plane bending vibration The spectralcharacteristics of recombinant 120574-PGA in fermentation brothwas consistent with those of the standard 120574-PGArsquos IR spec-troscopy indicating that the sample obtained in the presentstudy contained the N-H and C=O functional groups as wellas the aliphatic hydrocarbon structure (CH

2)4of the 120574-PGA

[17] thereby confirming that the sample was 120574-PGA Themolecular weight of the 120574-PGA sample obtained after thefermentation isolation and separation of recombinant strain

M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa

Figure 7 SDS-PAGE analysis of product of pWB980-pgsBCANoteLane M marker high molecular weight standard protein (TakaRa)Lane 1 120574-PGA samples obtained and purified from the fermentationbroth Lane 2 control Bacillus subtilisWB600

Bacillus WB600-pgsBCA was determined using SDS-PAGEFigure 7 shows that the molecular weight of the 120574-PGA wasbetween 500 and 600 kDa and occurred as aggregates of amultimolecular mass but not of a single molecular composi-tion

4 Discussion and Conclusions

The present study evaluated the cloning and expression of 120574-PGA synthase gene pgsBCA in B subtilis and used plasmidpWB980 to construct the recombinant expression vectorpWB980-pgsBCA and to further transfer the recombinantexpression vector into B subtilis WB600 The P43 promoterof pWB980 induced the expression of pgsBCA then thehost cells of this expression vector showed a capacity tosynthesize 120574-PGA and the product yield of 120574-PGA reachedge174 gL The isolated and purified 120574-PGA sample from thefermentation broth was confirmed to have a single form ofhydrolysates that solely consisted of pure glutamic acid Thisresult matched the characteristics of the standard 120574-PGArsquos IRspectroscopy and showed the aggregates of a multimolecularmass with a molecular weight ranging between 500 and600 kDa

The present study used B subtilis as the expression hostand the pgsBCA gene originated and was expressed in Bsubtilis The 120574-PGA synthase system is better positioned inthe cell membrane (as shown in Section 1) Therefore thesynthetic yield and molecular weight of 120574-PGA produced inB subtilis were as high as ge174 gL and between 500 and600 kDa two features that are consistent with or even higherthan the expression system of E coli and B subtilis that hadpreviously been described to have high expression efficiency[18ndash20]Themolecular weight of 120574-PGA especially expressedin this host is the highest in the existing report [21ndash24]The recombinant expression vector pWB980-pgsBCA in thepresent study contained the P43 promoter Therefore the


costly use of isopropyl 120573-D-1-thiogalactopyranoside (IPTG)D-xylose and L-arabinose as an inducer to secrete thepgsBCA into the extracellular fermentation broth is circum-vented using themethodology developed in the present studyThis technique may also be potentially used in industrialproduction as it can increase the stability of products simplifythe purification work and have more obvious applicationpotential advantage

Although the constructed recombinant bacteria BacillusWB600-pgsBCA showed the capacity to synthesize 120574-PGAour results still could not match the highest synthetic yieldof 120574-PGA (40ndash50 gL) that is induced by the fermentationof mutated bacteria [25 26] Therefore our next researchstudywill focus on introducing hemoglobin other exogenousgenes or certain control sequences to efficiently synthesizeand express 120574-PGA and to increase the bacterial concen-tration oxygen uptake or endogenous synthase expressionthereby ultimately increasing 120574-PGA yield [27 28] Alter-natively we will knock out genes of degrading enzymes in120574-PGA-producing strains to reduce 120574-PGA degradationthereby increasing 120574-PGA yield [29] Therefore our futureresearch direction and goal will focus on establishing andmodifying our current engineered strains through geneticengineering to improve its performance and further increase120574-PGA yield thereby laying the foundation for the indus-trial production of high-yielding 120574-PGA engineered bacteriabased on the B subtilis expression system

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Student Innovation ampEntrepreneurship Training Program in Fujian Province (no201511312053) JK Project for the Department of Science andTechnology of Fujian Province (no JK2014051) and Scienceand Technology Planning Project of Longyan City (no2015LY32)

References

[1] R Bhatt P De Vries J Tulinsky et al ldquoSynthesis and in vivoantitumor activity of poly(l-glutamic acid) conjugates of 20(S)-camptothecinrdquo Journal ofMedicinal Chemistry vol 46 no 1 pp190ndash193 2003

[2] T Candela and A Fouet ldquoPoly-gamma-glutamate in bacteriardquoMolecular Microbiology vol 60 no 5 pp 1091ndash1098 2006

[3] P Dubruel L Dekie B Christiaens et al ldquoPoly-L-glutamic acidderivatives as multifunctional vectors for gene delivery part Bbiological evaluationrdquo Biomacromolecules vol 4 no 6 p 18682003

[4] A Richard and A Margaritis ldquoEmpirical modeling of batchfermentation kinetics for poly(glutamic acid) production andother microbial biopolymersrdquo Biotechnology and Bioengineer-ing vol 87 no 4 pp 501ndash515 2004

[5] M Ashiuchi C Nawa T Kamei et al ldquoPhysiological and bio-chemical characteristics of poly-120574-glutamate synthetase com-plex of Bacillus subtilisrdquo European Journal of Biochemistry vol268 no 20 pp 5321ndash5328 2001

[6] Y Tarui H Iida E Ono et al ldquoBiosynthesis of poly-120574-glutamicacid in plants transient expression of poly-120574-glutamate syn-thetase complex in tobacco leavesrdquo Journal of Bioscience andBioengineering vol 100 no 4 pp 443ndash448 2005

[7] Y Urushibata S Tokuyama and Y Tahara ldquoDifference in tran-scription levels of cap genes for 120574-polyglutamic acid productionbetweenBacillus subtilis IFO 16449 andMarburg 168rdquo Journal ofBioscience and Bioengineering vol 93 no 2 pp 252ndash254 2002

[8] H Jiang L Shang SHYoon S Y Lee andZYu ldquoOptimal pro-duction of poly-120574-glutamic acid by metabolically engineeredEscherichia colirdquo Biotechnology Letters vol 28 no 16 pp 1241ndash1246 2006

[9] J M Buescher and A Margaritis ldquoMicrobial biosynthesis ofpolyglutamic acid biopolymer and applications in the biophar-maceutical biomedical and food industriesrdquo Critical Reviews inBiotechnology vol 27 no 1 pp 1ndash19 2007

[10] S F Wang J He Y L Chen T Zheng Q R Shen and XY Yong ldquoClone and heterologous expression of the ploy-120574-glutamic acid synthesis gene pgsBCAF from Bacillus amyloliq-uefaciens C1rdquo Chinese Journal of Biotechnology Bulletin vol 31no 5 pp 158ndash166 2015

[11] L Vavrova K Muchova and I Barak ldquoComparison of differentBacillus subtilis expression systemsrdquo Research in Microbiologyvol 161 no 9 pp 791ndash797 2010

[12] M Ashiuchi K Shimanouchi T Horiuchi T Kamei and HMisono ldquoGenetically engineered poly-120574-glutamate producerfrom Bacillus subtilis ISW1214rdquo Bioscience Biotechnology ampBiochemistry vol 70 no 7 pp 1794ndash1797 2006

[13] T Yan and H S Xi ldquoProgresses of microbial synthesis of poly-120574-glutamic acid of related genes synthesis mechanism andfermentationrdquo Chinese Journal of Biotechnology Bulletin vol 31no 3 pp 25ndash34 2015 (Chinese)

[14] Q J Wang S W Chen J B Zhang M Sun Z D Liu and ZN Yu ldquoCo-producing lipopeptides and poly-120574-glutamic acid bysolid-state fermentation of Bacillus subtilis using soybean andsweet potato residues and its biocontrol and fertilizer synergis-tic effectsrdquo Bioresource Technology vol 99 no 8 pp 3318ndash33232008

[15] Y-G Liu Q-L Dai S-B Wang Q-J Deng W-G Wuand A-Z Chen ldquoPreparation and in vitro antitumor effectsof cytosine arabinoside-loaded genipin-poly-L-glutamic acid-modified bacterial magnetosomesrdquo International Journal ofNanomedicine vol 10 pp 1387ndash1397 2015

[16] G J Qiao C Wang Z H Zhou K Zhang and H CaildquoClone and expression of poly-glutamic acid synthase gene inEscherichia colirdquo Chinese Journal of Food and FermentationTechnology vol 49 no 1 pp 7ndash12 2013 (Chinese)

[17] M Ashiuchi and H Misono ldquoBiochemistry and moleculargenetics of poly-120574-glutamate synthesisrdquo Applied Microbiologyand Biotechnology vol 59 no 1 pp 9ndash14 2002

[18] M CaoW GengW Zhang et al ldquoEngineering of recombinantEscherichia coli cells co-expressing poly-120574-glutamic acid (120574-PGA) synthetase and glutamate racemase for differential yield-ing of 120574-PGArdquo Microbial Biotechnology vol 6 no 6 pp 675ndash684 2013

[19] M Ashiuchi K Soda andHMisono ldquoA poly-120574-glutamate syn-thetic system of Bacillus subtilis IFO 3336 gene cloning and bio-chemical analysis of poly-120574-glutamate produced by Escherichia


coli clone cellsrdquo Biochemical and Biophysical Research Commu-nications vol 263 no 1 pp 6ndash12 1999

[20] J Huang Y M Du G H Xu et al ldquoHigh yield and cost-effective production of poly(120574-glutamic acid) with Bacillussubtilisrdquo Engineering in Life Sciences vol 11 no 3 pp 291ndash2972011

[21] M Cao C Song Y Jin et al ldquoSynthesis of poly (120574-glutamicacid) and heterologous expression of pgsBCA genesrdquo Journalof Molecular Catalysis B Enzymatic vol 67 no 1-2 pp 111ndash1162010

[22] M CaoW Geng L Liu et al ldquoGlutamic acid independent pro-duction of poly-120574-glutamic acid by Bacillus amyloliquefaciensLL3 and cloning of pgsBCA genesrdquo Bioresource Technology vol102 no 5 pp 4251ndash4257 2011

[23] S B Da Silva V V Cantarelli and M A Z Ayub ldquoProductionand optimization of poly-120574-glutamic acid by Bacillus subtilisBL53 isolated from the Amazonian environmentrdquo Bioprocess ampBiosystems Engineering vol 37 no 3 pp 469ndash479 2014

[24] W Zhang W X Gao J Feng et al ldquoA markerless genereplacement method for B amyloliquefaciens LL3 and its usein genome reduction and improvement of poly-120574-glutamic acidproductionrdquo Applied Microbiology and Biotechnology vol 98no 21 pp 8963ndash8973 2014

[25] C S Qiao X Li L F Lan X Chen Z W Zheng and Z LildquoScreening of a high-yield of 120574-ployglutamic acid-producingstrain bymeans of bothUV light andHe-Ne laserrdquoChinese Jour-nal of Food Science vol 33 no 13 pp 183ndash186 2012 (Chinese)

[26] B S Lin F Y Qiu Q X Lin and Y P Hong ldquoOptimizationof breeding and cultivation conditions for mutant strain withhigh productivity of 120574-polyglutamic acidrdquo Journal of Pure andApplied Microbiology vol 7 no 4 pp 2479ndash2488 2013

[27] C-M Yeh J-P Wang S-C Lo W-C Chan and M-Y LinldquoChromosomal integration of a synthetic expression controlsequence achieves poly-120574-glutamate production in a Bacillussubtilis strainrdquo Biotechnology Progress vol 26 no 4 pp 1001ndash1007 2010

[28] Y Su X Li Q Liu et al ldquoImproved poly-120574-glutamic acidproduction by chromosomal integration of the Vitreoscillahemoglobin gene (vgb) in Bacillus subtilisrdquo Bioresource Technol-ogy vol 101 no 12 pp 4733ndash4736 2010

[29] J Feng W X Gao Y Y Gu et al ldquoFunctions of poly-gamma-glutamic acid (120574-PGA) degradation genes in 120574-PGA synthesisand cell morphology maintenancerdquo Applied Microbiology andBiotechnology vol 98 no 14 pp 6397ndash6407 2014

Research ArticleImproved Stability of a Model IgG3 by DoE-Based Evaluation ofBuffer Formulations

Brittany K Chavez1 Cyrus D Agarabi2 Erik K Read1 Michael T Boyne II3

Mansoor A Khan2 and Kurt A Brorson1

1Division II Office of Biotechnology Products OPQ CDER FDA Silver Spring MD 20903 USA2Division of Product Quality Research Office of Testing and Research OPQ CDER FDA Silver Spring MD 20903 USA3Division of Pharmaceutical Analysis Office of Testing and Research OPQ CDER FDA Silver Spring MD 20903 USA

Correspondence should be addressed to Kurt A Brorson kurtbrorsonfdahhsgov

Received 9 October 2015 Revised 20 November 2015 Accepted 29 November 2015

Academic Editor Priscila G Mazzola

Copyright copy 2016 Brittany K Chavez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby theirpurity potency and safety over their shelf-life Using a model murine IgG3 produced in a bioreactor system multiple formulationcompositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worstcase The stability of the antibody in each buffer formulation was assessed by UVVIS absorbance at 280 nm and 410 nm and sizeexclusion high performance liquid chromatography (SEC) to determine overall solubility opalescence and aggregate formationrespectively Upon preliminary testing acetate was eliminated as a potential storage buffer due to significant visible precipitateformation An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the bufferingcapacity of histidine From this final DoE an optimized formulation of 200mM arginine 50mM histidine and 100mMNaCl at apH of 65 was identified to substantially improve stability under long-term storage conditions and after multiple freezethaw cyclesThus our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibodymolecules

1 Introduction

The manufacturing of biotechnology products is a complexlogistical process that connects multiple unit operations andoften leads to lengthy in-process hold times or bulk drugsubstance storage Identification of appropriate storage con-ditions and optimized buffer systems for biopharmaceuticalproteins is essential in ensuring the stability of these productsand thereforemaintaining the purity potency safety and effi-cacy of these drug substances throughout the manufacturingprocess A typical purification scheme for monoclonal anti-bodies involves Protein A affinity chromatography followedby polishing chromatography and filtration steps with an endproduct of concentrated antibody in amild acid to neutral pHsolution prior to drug substance formulation Selection of asuitable buffer system that mitigates physical and chemicaldegredation of monoclonal antibodies especially one thatminimizes aggregate and particle formation is an important

consideration for efficient downstream fill-finish operationsand long-term stability [1] Parameters that are typicallystudied include solution pH buffering system inclusion ofsaccharides tonicity agents detergents and other excipients[2 3]

Regulatory guidance stipulates that antibodies intendedfor human subjects are tested both at lot release and in stabil-ity studies [4] for a variety of product attributes includingopalescence and degradation products such as aggregatesparticles or precipitate formation These undesirable degra-dation products may be associated with immune responses[5] and in extreme cases can lead to loss of significantmonomer content or protein insolubility impacting potencyand efficacy to the point where it is unacceptable to use inhumans

In this study we use a monoclonal antibody cell culturesystem that was developed by hybridoma technology and hasbeen used by several academic groups to evaluate different



aspects of manufacturing from cell culture to formulatedbulk drug substance [6ndash10] This model murine IgG3 whilenot a humanized antibody suitable for clinical use has noproprietary entanglements and can be successfully used asa model for bioreactor produced monoclonal antibodies Itsproduction system was previously adapted to serum-freesuspension bioreactor culture and used by several groupsto evaluate cell culture bioprocesses both in single runexperiments and in design of experiment (DoE) formats[11ndash13] We have subsequently found that certain aspectsof its biochemistry present a stringent challenge model forformulation development Acetate buffer can be used forother antibodies [2] but it seems to cause aggregation andprecipitation in the case where it is difficult to formulatemodel antibody

Prior experience with this antibody (data not shown)showed that it formed visible particulates over time atconcentrations above 5mgmL to the extent of noticeable lossof monomeric species over timeThe aggregation was furtherexacerbated by freezethaw cycles (data not shown) Whilethis drug substance model antibody has been stable enoughfor short-term storage in 50mM arginine and 100mMNaClpH 80 prior to use in drug product lyophilization studies[14] a stablemodel antibody solution is needed for long-termquality assessment and testing In addition by performingthis exercise with our model antibody we present a rigoroustest case for demonstrating the power of DoE approaches forliquid antibody formulation development

To this end we demonstrated the power of DoE basedstudies to quickly pinpoint suitable buffer formulations tomaximize the stability of this antibody We tested fourdifferent buffer systems that were chosen to possess a rangeof pH optima while also avoiding the antibodyrsquos knownisoelectric point (pI) range 84ndash88 The DoE approachenables comprehensive evaluations of relevant formulationparameters that can impact antibody stability


21 Reagents Buffers were prepared using componentscommonly employed to formulate antibodies L-Histidine(Sigma-Aldrich St Louis MO) Sodium Chloride (BDHRadnor PA) Hydrochloric Acid (Fisher Fairlawn NJ) andeither L(+)-Arginine (Acros Organics Waltham MA) orFreebase Arginine (Fisher) NuPAGE LDS Sample BufferNuPAGE Reducing Agent NuPAGE Antioxidant and NovexSharp Standard and MOPS were obtained from Invitrogen(Carlsbad CA) Brilliant Blue G-250 acetic acid and 2-propanol were obtained from Fisher Scientific Unless notedotherwise in the text reagents were as described in Read et al[7]

22 IgG Production A suspension adapted murinehybridoma that produces IgG3120581 antibody [15] was grownin a 75-liter Bioflo 110 bioreactor (New Brunswick ScientificEdison NJ) that contained 4 liters of media as describedin Read et al [7] Antibodies from the clarified cellculture fluid (CCF) were captured with a 25mL Prosep A

Table 1 Single buffer DoE composition ranges Levels for theindividual buffer 23 full factorial DoEs with center pointsEachvariable was assigned a high middle and low range before the fullfactorial was designed

Buffer Concentration (mM) pH NaCl (mM)Acetate 25 50 100 45 475 50

25 50 100Arginine 100 200 300 775 80 825Histidine 25 50 100 625 65 675

(Millipore Billerica MA) column run on an AKTA Avant(GE Healthcare Uppsala Sweden) and eluted with 1MArginine pH 40 [16] As described in other studies thiselution strategy results in two peaks an early peak containingmostly host cell proteins and a subsequent peak containinglargely intact antibody [13 16] Fractions that comprise thesecond elution peak were then tested by UV to confirmprotein content prior to pooling buffer exchange andanalytical methods described below

23 Preliminary Experimental Design An initial explorationof three common buffer systems was performed by a 23 fullfactorial DoE with a center point (Table 1) Experience withthe IgG3 antibody used in this study revealed that it wasa challenging model from the standpoint of stability andpropensity to precipitate (data not shown) Early attemptsto find a suitable single species buffer system (includingphosphate tris acetate histidine and citrate) encompassinga range of mildly acidic or neutral pH failed to produce asystem where opalescence or even gross precipitation didnot accumulate over time Given the need to establish asuitable buffer system for this model antibody we initiated acontrolled evaluation of commonly used single species buffersystems (acetate histidine and arginine) described in Table 1While arginine has limited buffering capacity in the neutralpH range it was chosen as a mild chaotropic agent that hasbeen reported to stabilize antibodies prone to aggregation[16] The following full factorial DoEs evaluated each bufferspecies while varying NaCl pH away from the antibodyisoelectric point and buffer species concentrationThe statis-tical design experimental randomization and analysis wereperformedon JMPversion 100 (SAS Institute Inc CaryNC)

24 Sample Analysis Plan To buffer exchange the 1M argi-nine stabilized antibody into the test single buffer speciesformulation buffers a 3mL aliquot of IgG3 at 2mgmL orabove was loaded into a 10 kDamolecular weight cutoff Slide-A-Lyzer cassette (Thermo Scientific Rockford IL) It wasdialyzed in the test formulation buffer overnight equivalentto an 18000-fold buffer exchange Dialyzed samples werecollected weighed to determine postdialysis volume andvisually inspected for the presence of gross precipitate andopalescence To monitor long-term stability SEC Protein Aand absorbance measurements at 280 nm (protein content)and 410 nm (opalescence) were performed at day 0 (T0) 30days in 4∘C (T30) and after three cycles of freezing (minus80∘Cheld for 2 hours) and thawing (FT) (37∘C for 10 minutes) for


Table 2 Full factorial DoE for dual buffer component (HisArg) formulations Detailed composition of each buffer tested in the 24 fullfactorial DoE

Pattern Arginine (mM) Histidine (mM) NaCl (mM) pHHR 1 ++minus+ 200 50 50 65HR 2 +minus+minus 200 25 100 60HR 3 ++minusminus 200 50 50 60HR 4 ++++ 200 50 100 65HR 5 minus minus minusminus 100 25 50 60HR 6 minus+++ 100 50 100 65HR 7 minus+minus+ 100 50 50 65HR 8 minus++minus 100 50 100 60HR 9 minusminus+minus 100 25 100 60HR 10 minus+minusminus 100 50 50 60HR 11 minus minus minus+ 100 25 50 65HR 12 +minus++ 200 25 100 65HR 13 +minusminus+ 200 25 50 65HR 14 +++minus 200 50 100 60HR 15 minusminus++ 100 25 100 65HR 16 +minus minus minus 200 25 50 60

the arginine and histidine buffer formulationsThe remaining9 acetate formulations were not fully tested based on initialanalytics indicating decreased stability of the antibody at T0

25 Experimentally Derived 24 Full Factorial DoE A 24full factorial combined arginine and histidine systems foran additional 16 buffer formulations Test articles from theHisArg (HR) DoE were analyzed by the same proceduresdescribed in Table 2

26 UVVIS (A280 nmA410 nm) Analyses A NanoDrop2000c system was blanked with the test buffer before mea-suring absorbance of the samples at 280 nm and 410 nmSamples were not centrifuged before these readings so as notto skew the 410 nm absorbance which accounts for opales-cencevisible particulates Tomake sure that the 280 nmmea-surement was within the instrument linear range sampleswere then diluted 10-fold and reanalyzed Any samples thatshowed an A410 reading of 02 or greater were consideredpoor candidates for further optimization and further analyt-ics were discontinued

27 SEC Analytical scale size exclusion chromatography(SEC) was performed with a TSKgel G3000SWxl column(Tosoh Bioscience Grove City OH) and Agilent 1200 HPLCsystem These data were used to determine the relativeproportion of aggregates with the antibody samples [7]Visible particulates were removed by centrifugation prior toHPLC analysis to prevent clogging of the frit

28 SDS-Page Gel (Reduced and Nonreduced) Samples(200120583L) were centrifuged at 17000timesg to create soluble

(supernatant) and insoluble (pellet) fractions The super-natant was recovered directly for analysis The pellet waswashedwith the corresponding test buffer formulation beforeit was resuspended in 20120583L of sterile water Both fractionswere mixed 1 1 with loading buffer (containing DTT forreduced samples) and held at 70∘C in a water bath for 10minutes 15 120583L of each sample was loaded onto a NovexNuPAGE (4ndash12) Bis-Tris Mini Gel (Invitrogen CarlsbadCA) in MOPS buffer NuPAGE Antioxidant was addedto the upper buffer chamber for reduced samples Afterelectrophoresis test article banding patterns were comparedto Novex Sharp Standards as a molecular weight reference

All gels were fixed using a solution of 25 acetic acid 10propanol for at least 20 minutes before staining with 0006Brilliant Blue G-250 in 10 acetic acid overnight Destainingwas achieved using 10 acetic acid replaced twice beforeimaging the gels

3 Results and Discussion

31 Preliminary DoE Results Our model IgG3 antibody hasbeen established over time to present a stringent challengemodel for formulation development Its amino acid sequence(Genbank protein sequence IDrsquos AKH40268 andAKH40269)establishes it as a murine IgG3120581 with V

1205814 and VH1-S121

regions To scout individual buffer species the IgG3 antibodywas formulated with variable NaCl concentration and pHranges and evaluated for gross stability of the antibody Singlebuffer species formulations were chosen based on historicalformulation experience and known acceptable pH rangesMany of these formulations were eliminated as candidatesbased on the T0 analytics that indicated decreased solubil-ity and decreased stability of the antibody Absorbance at410 nm (a surrogate for opalescence) and SEC proved to


Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)

Figure 1 Quantile graphs of the buffer formulations at all measurement points (a) Recorded absorbance of the samples at 410 nm and (b) thepercent aggregate as determined by SEC Histidine formulations showed gross precipitation so large that they are captured by a SEC-columnfrit during analysis and thismay have led to a false negative of percent aggregates (see Section 312)T0 denotes initial time pointT30 denotes30-day storage time point and FT denotes freezethaw

be sensitive measurement of solubility and stability of theantibody These data guided the 24 full factorial DoE basedon histidinearginine buffer formulations as described below

311 Acetate All acetate buffer formulations showed visibleprecipitation during the small scale buffer exchange processThis observationwas reflected in a highA410 reading coupledwith a decreased A280 This unusual result indicated that theantibody was becoming insoluble as the acetate formulationsreplaced the 1M arginine elution buffer during dialysis Thiswas verified in the SDS-PAGE showing heavy and light chainin the insoluble fraction of the buffer exchanged samples(Figure 2) All acetate formulations gave A410 readingsgreater than 05 (Figure 1(a) Table 3) and were thereforediscontinued from further study Although not a commonlot release test employed by manufacturers A410 actedas a measure for opalescence This test quickly ruled outless desirable formulations by quantifying particulates Forour model antibody insoluble aggregates in an abundancereflected in an A410 greater than 02 allowed us to focus ouranalytics on more promising buffer species After this initialprecipitation the antibodymaintained virtually 100percentmonomer as measured by SEC suggesting that componentsprone to nucleation precipitated completely leaving behindmonomer The high percent monomer remaining was notbeneficial enough to outweigh the solubility issues of acetatetherefore no further testing beyond a T0 time point wasconducted on these formulations

312 Arginine As expected arginine improved solubility AtT0 arginine buffer formulations showed minimal opales-cence reflected in generally lower A410 values The samplesseemed to fall into two categories moderate A410 around05 and undetectable A410 (Figure 1(a)) The A280 remainedstable after 30 days as well as after three freezethaw cyclesproving that antibody did not grossly precipitate to theextent seen when formulated in acetate Looking at all

Histidine

1

HC

LC

2 3 4

Acetate

Figure 2 Reduced SDS-PAGE HC denotes the heavy chain whileLC denotes the light chain of the antibody Lanes 1 and 3 representthe insoluble fraction immediately after dialysis into the respectivebuffer system while lanes 2 and 4 represent the supernatant

9 formulations there was decreased solubility at T30 ascompared to T0 leading to minimal opalescence in somebut not all formulations These findings suggest that thearginine was conferring a cytoprotective effect much likethat seen when lyophilizing antibodies in arginine solutions[17] The increased percent aggregates of the arginine bufferformulations as compared to acetate and histidine formu-lations (Figure 1(b) Table 3) arise from smaller aggregatesthat were not removed from the samples prior to runningHPLC Upon statistical analysis of the 9 formulations we


Table 31198790 analytic readout ranges for all DoEs for each buffer system the range of values for A410 A280 and percent aggregates is givenThisoverview of the range of values gives a snapshot of how the different buffer systems compare to each other lowastGross precipitation of largeraggregates that would have been centrifuged out of solution before SEC or trapped by the column frit may have led to an artifactual 0aggregate reading for antibody in the histidine formulations

Acetate Arginine Histidine Histidinearginine119860410 057ndash099 0ndash07 049ndash242 0ndash018119860280 211ndash37 223ndash293 222ndash888 136ndash224Percent aggregates 0ndash20 0ndash45 0

lowast 0ndash387

found that increased arginine concentration had the mostoverall positive effect on the antibody stability We usedthis information to create an additional DoE to narrow ourfocus on higher concentration arginine in combination witha different buffering system at a more typical pH used forformulating antibodies

313 Histidine Overall the histidine buffer system showedeven more extreme A410 versus acetate buffer at T0 whichtrended up by T30 as well as after the freezethaw procedureThis increase in opalescence over time was from the antibodybecoming less soluble and forming large aggregates thatcompletely fell out of solution indicating that the antibodywas increasingly unstable over time and after freezethawcycles These aggregates can be seen on the SDS-PAGE(Figure 2) andwere removed before SEC analysis leading to amisleading readout of 0 aggregate (Figure 1(b)) In additionthere was more variability in the A410 results with the lowerpH data points generally with lower opalescence (Table 3)Test formulations His 5 and His 6 both showed considerablylower absorbance at 410 nm as compared to the other buffersThis is likely due to the combination of high histidine(100mM) and high salt (100mM) Even after washing theinsoluble fraction the reduced SDS-PAGE of the histidinebuffer formulations at T0 shows that there was a substantialamount of heavy and light chain in the insoluble frac-tion after buffer exchanging the antibody (Figure 2) Theseresults indicated the particulates and precipitates formedwere the drug substance and not host cell proteins or otherinsoluble components A410 readings for histidine formula-tions were greater than 02 and discontinued from furtherstudy

314 Summary Histidine and acetate as single buffer sys-tems were eliminated in early rounds due to extensive opales-cence in allDoE test articles (see Figures 1(a) and 2) Arginineeven at a pH close to the antibody isoelectric point providedbetter results relative to the other two buffer systems andstability correlated with higher arginine concentrations Thisobservation argues that instability was not a pH effect but thatarginine was acting as a stabilizing agent Thus we furtheroptimized the formulation buffer by retaining the presumedstabilizing effect of the arginine while incorporating a secondparameter that could provide buffering capacity at a pH(625 plusmn 025) sufficiently lower than the reported antibodyisoelectric point (84ndash88) to help prevent self-association[15] Histidine even at lower concentrations would provide

this effect in combination with arginine It was further notedthat the stabilizing effect of NaCl wasmore pronouncedwhenNaCl was at a higher concentration across all three singlebuffer systems

32 Second Round DoE As described above in the singlespecies buffer experiments the antibody exhibited a modesttrend towards better solubility at lower pH and at higherarginine concentrations We hypothesized that a combinedhistidine and arginine (HisArg) DoE at a pH further awayfrom the antibody isoelectric point could further minimizeopalescence In this case histidine would buffer the pHbelow the pI of the antibody while arginine would promoteincreased solubility and protein integrity due to chaotropiceffects

After statistical analysis of the T0 data we found thatthere was a significant main effect for arginine buffer con-centration Lower arginine values (100mM) were associatedwith higher levels of A410 absorbance an undesirable indi-cation for product quality Additionally while not statis-tically significant but potentially biologically relevant theargininehistidine interaction (119875 = 005) and the histidineconcentrations (119875 = 00547) are markedly more impor-tant than the remaining factors when considering strate-gies for minimizing A410 absorbance Thus by adjustinghistidine concentration we could design an optimal bufferto achieve the goal of low opalescence while also mini-mizing arginine addition which could interfere in certainassays The increased solubility achieved in the HisArgDoE allowed us to select a final buffer formulation of200mMarginine 50mMhistidine and 100mMNaCl at a pHof 65

321 Buffer DoE Freeze-Thaw and Stability Bioprocessingusually occurs in separate drug substance and drug productfacilitiesThis approach requires drug substance and in somecases in-process material to undergo freezing and thawingto allow shipping between distant sites Regulatory agenciesrequire specific studies that support hold times these mayinclude shipping studies of materials between facilities andlong-term storage if not immediately processed into drugproduct [4] While freezethaw is usually performed onlyonce during shipping between drug substance and drugproduct sites manufacturers may also study the impact ofmultiple freezethaws on product stability to understand risksposed by potential temperature deviations and unanticipatedfreezing and thawing Poorly buffered formulations of other


Term Estimate Std errorArginine (100 200) 1675 038 441NaCl (50 100) 0725 038 191 01149pH (625 675) 0413 027 153 01855Histidine (25 50) 0575 038 151 01909

0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295

0012 027 005 09647ArgininelowastNaClArgininelowastHistidineArgininelowastpHHistidinelowastNaClHistidinelowastpHNaCllowastpH

00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl

ArgininelowastHistidineArgininelowastpH

HistidinelowastNaClHistidinelowastpHNaCllowastpH

00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003

Prob gt |t|Term EstimateArginine (100 200) 1437 025 575NaCl (50 100) 0513 025 205 00955Histidine (25 50) 0413 025 165 01597pH (625 675) 0275 018 156 01803

02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)

Figure 3 Significant effects on percent aggregate (a) shows that at T30 arginine concentration significantly reduced the aggregates (b) Afterfreezethaw arginine played a significant role in reducing aggregation

antibodies exposed to multiple freeze-thaw cycles have beenshown to be prone to aggregation subvisible particle forma-tion that can ultimately nucleate visible aggregation [18]Thiseffect has been hypothesized to lead to undesirable productimmunogenicity although to an unknown degree [19] Theycould also nucleate further aggregation during drug productfill operations [20] Therefore it is important to evaluate thedrug substance stability over multiple freeze-thaw cycles andfor extended hold times to evaluate the suitability of anybuffer system

To evaluate our HisArg formulations for cryoprotectionproperties and extended hold times we preformed thepreviously described analytics after 30 days of being held at4∘C as well as three freezethaw cycles Overall we foundthatA410 was consistently more favorable among all 16 bufferformulations The A410 of all the formulations from thecombined DoE were below 02AU (Figure 5) both over timeand after freezethaw cycles Not surprisingly the significanceof 200mM arginine for reducing A410 values continuedfrom the original T0 throughout the T30 and freeze-thawstudies This was also reflected in a significantly decreasedpercent aggregates (Figure 3(a)) However the importanceof the argininehistidine interaction became evident andstatistically significant (119875 = 00476 R2 = 097 119875 = 00355 R2= 096 resp) (Figure 4) This value was well below the A410

achieved by the histidine formulations alone and the 30-daystability in arginine formulations (Figure 1(a))

We also evaluated antibody freezethaw stability Uponthree freeze-thaw cycles arginine and the arginine-histidineinteraction was statistically significant (119875 lt 005 R2= 096) (Figures 3(b) and 4(b)) Histidine has previouslybeen shown to reduce mAb aggregation in a concentra-tion dependent manner under freezethaw conditions Ourresults of an optimal histidine concentration of 50mMcoincide with observations from Chen et al who foundthat 60mM histidine showed a minimum amount of aggre-gates after 3 cycles of freezethawing [21] It is often seenthat when excipients are combined the protective effectsconferred on the antibody may not necessary increase[22] The DoE format of our study allowed us to com-prehensively evaluate the interactions of our chosen bufferspecies

Overall our observations indicate that the dual buffersystem was improving the robustness and duration of thesolubility of the antibody An ArgHis interaction appearsto allow for a lower arginine concentration if the otherexcipients are carefully balanced The final buffer choiceconfers adequate solubility characteristics for short-termstorage to allow additional studies of this antibody This wasimportant for other studies that depend upon its stability


NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)

Figure 4 Significant interations on HR stability after both T30 (a) and freezethaw (b) and interactions between two variables lead to tosignificantly decreased A410 At T30 (a) NaCl concentration in combination with pH leads to a more desireable A410 After FT (b) theinteraction between arginine and histidine concentrations had a significant effect on A410

HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0

Figure 5 HisArg A410 at each measurement point Absorbanceat 410 nm of 16 histidinearginine buffer formulations as measuredafter the indicated time point

long enough to perform biochemical and physicochemicalanalysis

4 Conclusions

As an individual component in a larger manufacturingprocess bulk protein formulation choice is a critical step inantibody development The right selection strategy choicecan efficiently inform and assure that the best buffer choicewill be made that enables drug product process robustnessand ultimate product stability An organized and directedapproach can make the difference in determining if a bio-logical candidate has a future for clinical or commercial useClearly short-term long-term and freezethaw stability arecritical considerations for this decision as logistic constraintsand shipping requirements are an inevitable part of thebiotechnology manufacturing landscape As we show here

even the stability of difficult to formulate antibodies can bevastly improved by careful DoE-informed choice of bufferingspecies and pH as well as controlled inclusion of stabilizingchaotropic agentsWe also demonstrate that avoiding directlyoverlapping the antibody isoelectric point can minimizeopalescence and precipitation

Highlights

(i) We used 4 DoEs to test 43 buffer formulations forstability of a model IgG3

(ii) Arginine increased the solubility of the model anti-body

(iii) Combining 2 buffer systems arginine and histidineincreased stability

(iv) Shifts in pH were a critical attribute affecting solubil-ity of the antibody

Disclaimer

The findings and conclusions in this paper have not beenformally disseminated by the Food and Drug Administrationand should not be construed to represent any agency deter-mination or policy

Conflict of Interests


Acknowledgments

The authors acknowledge CDERrsquos Critical Path InitiativeGrant no 1500 for support of this project This project


was supported in part by an appointment to the ResearchParticipation Program at the CDEROffice of BiotechnologyProducts US Food and Drug Administration administeredby theOak Ridge Institute for Science and Education throughan interagency agreement between the US Department ofEnergy and FDAThe authors would also like to acknowledgeJuhong Liu and Audrey Jia for their careful comments in thepreparation of this paper

References

[1] J Y Zheng and L J Janis ldquoInfluence of pH buffer speciesand storage temperature on physicochemical stability of ahumanized monoclonal antibody LA298rdquo International Journalof Pharmaceutics vol 308 no 1-2 pp 46ndash51 2006

[2] S Uchiyama ldquoLiquid formulation for antibody drugsrdquoBiochim-ica et Biophysica Acta vol 1844 no 11 pp 2041ndash2052 2014

[3] A L Daugherty and R J Mrsny ldquoFormulation and deliveryissues for monoclonal antibody therapeuticsrdquo Advanced DrugDelivery Reviews vol 58 no 5-6 pp 686ndash706 2006

[4] ldquoSpecifications test procedures and acceptance criteria forbiotechnologicalbiological products Q6Brdquo in Proceedings ofthe International Conference on Harmonization of TechnicalRequirements for the Registration of Pharmaceuticals for HumanUse Geneva Switzerland 1999

[5] A S Rosenberg ldquoEffects of protein aggregates an immunologicperspectiverdquo The AAPS Journal vol 8 no 3 pp E501ndashE5072006

[6] B Kondragunta J L Drew K A Brorson A R Moreira andG Rao ldquoAdvances in clone selection using high-throughputbioreactorsrdquoBiotechnology Progress vol 26 no 4 pp 1095ndash11032010

[7] E K Read S A Bradley T A Smitka C D Agarabi S CLute and K A Brorson ldquoFermentanomics informed aminoacid supplementation of an antibody producing mammaliancell culturerdquo Biotechnology Progress vol 29 no 3 pp 745ndash7532013

[8] J R VallejosMMicheletti K A Brorson A RMoreira andGRao ldquoOptical sensor enabled rockingT-flasks as novel upstreambioprocessing toolsrdquo Biotechnology and Bioengineering vol 109no 9 pp 2295ndash2305 2012

[9] M A Hanson X Ge Y Kostov K A Brorson A R Moreiraand G Rao ldquoComparisons of optical pH and dissolved oxygensensors with traditional electrochemical probes during mam-malian cell culturerdquo Biotechnology and Bioengineering vol 97no 4 pp 833ndash841 2007

[10] A S Rathore S Kumar Singh M Pathak et al ldquoFermenta-nomics relating quality attributes of a monoclonal antibody tocell culture process variables and rawmaterials usingmultivari-ate data analysisrdquo Biotechnology Progress 2015

[11] B Kondragunta J Han B H Joshi et al ldquoGenomic analysis ofa hybridoma batch cell culture metabolic status in a standardlaboratory 5 L bioreactorrdquo Biotechnology Progress vol 28 no 5pp 1126ndash1137 2012

[12] J R Vallejos S Uplekar J F da Silva K A Brorson A RMoreira and G Rao ldquoA case study in converting disposableprocess scouting devices into disposable bioreactors as a futurebioprocessing toolrdquo Biotechnology and Bioengineering vol 109no 11 pp 2790ndash2797 2012

[13] C D Agarabi J E Schiel S C Lute et al ldquoBioreactor pro-cess parameter screening utilizing a plackettndashburman design

for a model monoclonal antibodyrdquo Journal of PharmaceuticalSciences vol 104 no 6 pp 1919ndash1928 2015

[14] D Awotwe-Otoo C Agarabi G K Wu et al ldquoQuality bydesign impact of formulation variables and their interactionson quality attributes of a lyophilized monoclonal antibodyrdquoInternational Journal of Pharmaceutics vol 438 no 1-2 pp 167ndash175 2012

[15] L J Rubinstein and K E Stein ldquoMurine immune response tothe Neisseria meningitidis group C capsular polysaccharide IISpecificityrdquoThe Journal of Immunology vol 141 no 12 pp 4357ndash4362 1988

[16] D Shukla L Zamolo C Cavallotti and B L Trout ldquoUnder-standing the role of arginine as an eluent in affinity chromatog-raphy via molecular computationsrdquo The Journal of PhysicalChemistry B vol 115 no 11 pp 2645ndash2654 2011

[17] F Tian C R Middaugh T Offerdahl E Munson S Saneand J H Rytting ldquoSpectroscopic evaluation of the stabilizationof humanized monoclonal antibodies in amino acid formula-tionsrdquo International Journal of Pharmaceutics vol 335 no 1-2pp 20ndash31 2007

[18] J G Barnard S Singh T W Randolph and J F CarpenterldquoSubvisible particle counting provides a sensitive method ofdetecting and quantifying aggregation of monoclonal antibodycaused by freeze-thawing insights into the roles of particlesin the protein aggregation pathwayrdquo Journal of PharmaceuticalSciences vol 100 no 2 pp 492ndash503 2011

[19] S K Singh N Afonina M Awwad et al ldquoAn industryperspective on themonitoring of subvisible particles as a qualityattribute for protein therapeuticsrdquo Journal of PharmaceuticalSciences vol 99 no 8 pp 3302ndash3321 2010

[20] J S Bee J L Stevenson B Mehta et al ldquoResponse of aconcentrated monoclonal antibody formulation to high shearrdquoBiotechnology and Bioengineering vol 103 no 5 pp 936ndash9432009

[21] B Chen R Bautista K Yu G A Zapata M G Mulkerrinand S M Chamow ldquoInfluence of histidine on the stability andphysical properties of a fully human antibody in aqueous andsolid formsrdquo Pharmaceutical Research vol 20 no 12 pp 1952ndash1960 2003

[22] D S Goldberg S M Bishop A U Shah and H A SathishldquoFormulation development of therapeutic monoclonal anti-bodies using high-throughput fluorescence and static lightscattering techniques role of conformational and colloidalstabilityrdquo Journal of Pharmaceutical Sciences vol 100 no 4 pp1306ndash1315 2011

Research ArticleAzocasein Substrate for Determination of Proteolytic ActivityReexamining a Traditional Method Using Bromelain Samples

Diego F Coecirclho1 Thais Peron Saturnino1 Fernanda Freitas Fernandes1

Priscila Gava Mazzola2 Edgar Silveira3 and Elias Basile Tambourgi1

1Chemical Engineering School Campinas State University (UNICAMP) Avenida Albert Einstein 500 13083-852 Campinas SP Brazil2Faculty of Pharmaceutical Sciences Campinas State University (UNICAMP) Rua Sergio Buarque de Holanda 25013083-859 Campinas SP Brazil3Biochemistry and Genetics Institute Federal University of Uberlandia (UFU) Avenida Getulio Vargas 230 Centro38700-128 Patos de Minas MG Brazil

Correspondence should be addressed to Diego F Coelho dfcoelhofequnicampbr

Received 26 November 2015 Accepted 12 January 2016

Academic Editor Pengjun Shi

Copyright copy 2016 Diego F Coelho et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Given the importance of proteasersquos worldwidemarket the determination of optimum conditions and the development of a standardprotocol are critical during selection of a reliable method to determine its bioactivity This paper uses quality control theory tovalidate a modified version of a method proposed by Charney and Tomarelli in 1947 The results obtained showed that usingazocasein substrate bromelain had its optimumat 45∘Cand pH9 (Glycine-NaOH 100mM)We also quantified the limit of detection(LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0072 and 0494mgsdotmLminus1 of azocasein resp) anda calibration curve that correlates optical density with the amount of substrate digested In all analysed samples we observed asignificant decrease in response after storage (around 17) which suggests its use must be immediately after preparation Thusthe protocol presented in this paper offers a significant improvement given that subjective definitions are commonly used in theliterature and this simple mathematical approach makes it clear and concise

1 Introduction

Because proteases represent the largest and most importantsegment in the industrial enzyme market [1] the consolida-tion of a reliable method to evaluate its quality is obviously ofextreme importance These enzymes are used in detergentsfood processing and leather industry as biocatalysts inorganic synthesis and among many other applications astherapeutics because their roles are involved in key deci-sions throughout an organism in several physiological andmetabolic processes [2]

The global market for industrial enzymes is expected toreach US $71 billion by 2018 [3] and is traditionally dividedinto three segments food technical and feed enzymes In2000 technical enzymes used in detergent leather textileand personal care industries accounted for 65 [4] of the totalsales (approximately US $15 billion [5]) while food enzymes

which include enzymes used in dairy brewing wine andjuices were valued at 25 and feed enzymes (used in animalfeeds) contributed with 10

Nearly 70 years ago Charney and Tomarelli [6] proposedthe use of an azoprotein (a protein coupled with diazotizedaryl amines) for the determination of proteolytic activityThe digestion of a solution with such proteins releases thechromophoric group which is soluble in trichloroacetic acidand gives it a red-orange colour

The method itself relies on the reaction between thesubstrate and an enzyme under its optimum temperaturepHfor a given time The solution colour intensity read at440 nm is a function of the amount of azoprotein digestedsince all proteins remaining precipitate after the addition oftrichloroacetic acid

The method is still one of the most reliable methods tostudy the proteolytic activity of enzymes [7 8] due its colour



stability and no need of chromogenic reagents Besides thesulphanilamide-azocaseinrsquos preparation is no longer neces-sary since it is now available widely in the market

However the available protocols that describe thoroughlythe method still are lacking in presenting the evaluation of itsanalytical parameters required for method validation Thusthis study aims to review and validate the azocasein methodto establish its detection and quantification limits in additionto reagent storage stability and a quantitative definition ofenzymatic activity


21 Bromelain Sample andOtherChemicals Bromelain (cata-logue B5144) and azocasein (catalogue A2765) obtained fromSigma-Aldrich (St Louis USA) were chosen as standardsfor these studies being used to prepare stock solutions atdifferent pH Unless specified all other reagents were alsoobtained from Sigma-Aldrich

22 Substrate Solution Given the nature of this study theamount of powdered substrate and buffer usedwill depend onthe concentration and pH of each experimentThe substratersquospH and concentration are part of the studied variables andare described in the following methods All pH buffers wereprepared following common protocols described elsewhere[9]

Basically 4mL of ethanol is added to the powderedsubstrate in a 120mL beaker and is stirred using a magneticstirrer to solubilise all aggregated protein and is then dilutedwith 96mL of appropriated buffer (100mM)

23 Bromelain Stock Solution Bromelain stock solution wasprepared following a modified version of a method describedby Hale et al [10] The 1mgsdotmLminus1 enzyme solution wasprepared using a 100mM buffer of different pH (since it wasalso under investigation) Concentration was chosen basedon its maximum solubility at experimental conditions

24 Enzymatic Assay The method consists in mixing equalvolumes of substrate and enzymatic sample at a given tem-perature and pH that corresponds to the optimum conditionsof the enzyme under investigation For practical reasons wechose 125 120583L as it is small enough to avoid wasting resourcesand does not compromise the methodrsquos precision

The kinetics of the digestion were studied during 420minutes using substratersquos concentration in a range from 01 to30 (ww) in order to determine a suitable time of digestion

The reaction was terminated adding 750 120583L of 5trichloroacetic acid (TCA) to the enzyme-substrate mixtureThe coagulated protein was removed by centrifugation at2000timesg for 10min at room temperatureThe obtained super-natant was then added to a 05N NaOH solution using a 1 1(vv) ratio and its absorbance was read at 440 nm

The blank was obtained by mixing the TCA to thesubstrate prior to the enzyme addition

25 Optimum pH and Temperature for Bromelain The opti-mum pH and temperature for assaying bromelainrsquos activity

were determined by performing a full factorial design ofexperiments using both variables in two levels and threecentral points The pH ranged from 6 to 8 and temperaturefrom 25∘C to 45∘C in the factorial design Temperature waskept constant during substrate digestion by using a Techne

Dri-Block heater model DB-3DThis design was extended to a central composite design

which had its variablersquos range adjusted based on the results ofthe first design All statistical datawas generated and analysedusing R [11] coupled with R-Studio [12] and using packagesakima [13] DoEbase [14] ggplot2 [15] and RColorBrewer[16]

26 Calibration Curve Using the curves of azocasein diges-tion obtained previously (as described in the topic EnzymaticAssay) a correlation between the colour intensity and thesubstrate concentration was created

The principle is simple if the enzymes digest the substratefor enough time we would achieve the solution maximumcolour intensity since all chromophoric groups had theirbonds to the protein broken and thus are soluble in TCAThissatisfies the assumptionmade in azocaseinrsquos original protocol[6] which states that a completely digested azocasein solutionhas the same colour intensity as an undigested sample

The calibration curve is obtained by plotting the opticaldensity measured when the time of digestion was 420minand the concentration of substrate at 119905 = 0

27 Detection and Quantification Limits The limit of detec-tion (LoD) and limit of quantification (LoQ) for the protocolwere based on the standard deviation of the response andthe slope of the mean of calibration curves following ICHlowastrsquosguidelines [17] and are given by the equations below

LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)

where 120590 is the standard deviation of the response and 119904 isthe slope of the calibration curve As described by ICH theresidual standard deviation of a regression line can be used asthe standard deviation during calculations

28 Stability Assays Stability assays followed the protocolsdescribed in a document provided by the US Department ofHealth and Human Services called Guidance for IndustryBioanalytical Method Validation [18]

Short-Term Temperature Stability Three aliquots of eachof the low and high concentrations were thawed at roomtemperature kept for 8 hours and then analysed

Long-Term Stability The storage time in a long-term stabilitywas evaluated within an interval of six weeks time usuallynecessary to perform a whole batch of our routine experi-ments Long-term stability was determined by storing threealiquots of each of the low and high concentrations at 5∘C To


80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)

Figure 1 Response contour of conditions optimisation for brome-lain solution

avoid contamination each sample was stored in its own vialand analysed on six separate occasions

Freeze and Thaw Stability Three aliquots at each of the lowand high concentrations were stored at minus20∘C for 24 hoursand thawed unassisted at room temperature When com-pletely thawed the samples were refrozen for 24 hours underthe same conditions The freeze-thaw cycle was repeated twomore times and then analysed on the third cycle


31 Optimum Conditions The study and determination ofbromelainrsquos biochemical properties have been studied exten-sively before through several methods but our interest wasto determine the optimum conditions specifically for thesubstrate under investigation to evaluate it at its best

Figure 1(a) corresponds to results obtained from the firstexperimental design and shows that at such variablersquos rangethe pH seems to have no influence over the enzyme activity

Then we modified the experimental design by increasingthe pHrsquos range in order to confirm the observation Howeverthe enzyme showed some increase in its activity at basic pH(Figure 1(b)) and served to establish the variables range forthe central composite design (CCD) shown in Table 1

Figure 1(c) shows clearly that bromelain has an impres-sively wide range of pH and temperature that can digestazocasein substrate with no apparent loss in its sensitivity Italso shows that bromelain is still active at moderately hightemperatures [19] Due to local operational reasons we chosepH 9 and 45∘C as the conditions to be used in the next steps

15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)

Azocasein concentration010025050075100

150200250300

0

Figure 2 Azocasein digestion curve at 45∘C and pH 9 usingbromelain 1mgmL with substrate concentration from 01 to 3(ww)

Table 1 Rotational central composite design used to study anddetermine assayrsquos optimum conditions shown in Figure 1(c)

Factor Temperature (∘C) pH

Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180

of this study For this case pH 9 Glycine-NaOH (100mM)buffer was used during substrate preparation

32 Calibration Curve Figure 2 shows the kinetics curvesobtained for each concentration of azocasein substrate usedAs expected curves with lower substrate concentration werecompletely digested in a matter of a few minutes whilesolution at 3 25 and 2 seems to be closer to such pointbut the enzymatic reaction would still be in process

By plotting the azocasein concentration against its corre-spondent optical density for all curves at 420min and usingthe assumptionmade byCharney andTomarelli [6] we obtaina calibration curve which creates a relationship between thesetwo variables (Figure 3)

The substrate concentration was converted easily frommass fraction to mgsdotmLminus1 by taking in account the solventsspecific mass and the volume retraction caused by theaddition of ethanol

The divergence between curves is mainly due the factthat reactions using substrate at 25 and 30 seem tohave significant amounts of undigested substrate and thusthe assumption becomes invalid Therefore the solid line(SL) curve represents the data series without these pointsResults from statistical analysis for both curves are presentedin Table 2


Table 2 Summary of statistical analysis results for both curves

Coefficients Std error 119905-value 119877

2

Solid line (SL) Intercept minus013561 004493 3018 09916Slope 147572 005533 26673

Dashed line (DL) Intercept minus02700 01161 2326 09687Slope 17441 01106 15764

000 025 050 075 100 125 150 175

Optical density (abs)

40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983

Figure 3 Calibration curves for azocasein concentration using 1sim20mgmL (solid line SL) and 1sim30mgmL (dashed line DL)

As the presented data suggests it is clear that removingthe points related to unfinished reactions put the correlationin a confidence level allowing it to be used as a calibrationcurve Consider

119862AZO (mgmL) = minus013561 + 147572 sdot Abs (2)

The limits of detection and quantification were calculatedusing (1) and their results are presented below Data was con-verted to mgsdotmLminus1 using (2) and coefficients obtained for SLConsider

LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)

One unit (U) of proteolytic activity was defined as the amountof enzyme capable of digesting 1mg of substrate per minuteas given in the equation below

119860

(U) =119862AZO sdot 119881

2

Total119905 sdot 119881ENZ

(4)

where 119862AZO is the concentration of azocasein obtained using(2) 119881Total is the sum of volumes of TCA substrate andenzyme solution (119881ENZ) used in the digestion and 119905 is thedigestion time (in minutes)

Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15

Azocasein concentration

Time (days)

Figure 4 Short-term stability results for azocasein substrate

33 Stability Assays Substratersquos storage stability is anotherimportant feature to be evaluated in order to establish a proto-col Short-term stability is important to evaluate whether thesubstrate can be kept at room temperature during a daylongset of experiments (Figure 4)

Results of time = 0 are relative to a substrate solution rightafter it was prepared while subsequent days showed resultsof each sample taken from the same stock solution left for 8hours at room temperature prior to analysis Results show asignificant loss of substrate response in both concentrations(around 10) when compared to the stock solution but that asimilar variation is observed within the time interval studied

Long-term stability is evaluated to check whether asolution can be stored and for how long without been frozen

While there was no observed formation of insolublesolids in the stock solution during storage the response ofsubstrate had a significant loss (around 17) after 14 days butthen it stabilized (Figure 5) This fact does not seem to createany interference in any step of the method but suggests thatthe substrate solutionwould offer amaximum response whenused right after preparation Further studies will be necessaryto understand the phenomena involved in the decrease ofresponse over time

The decrease in response for the substratersquos digestionalso occurred during freeze-thaw cycle (see Figure 6) whichreinforces the hypothesis that it is not caused by microbialactivity but somehow related to the substrate solubility Theobserved errors were lower than the ones observed during

BioMed Research International 5O

ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)

Figure 5 Long-term stability for azocasein substrate stored at 5∘C

Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles

Figure 6 Substrate stability after freeze-thaw cycles

long-term and short-term studies which make it the mostsuitable option for storage at the moment

4 Conclusion

The protocol described followed the main guidelines pre-sented by ICHlowast and establishes a reliable procedure to ana-lyse biological activity of proteolytic enzymes Besides themethod uses a mass correlation between the substrate usedand the optical density observed in the postdigestion sampleAlthough a simple and obvious idea it offers a significantimprovement given that subjective definitions are commonlyused in the literature Besides we ran a series of stabilityassays in order to evaluate the substrate and observed thata significant loss (10ndash20) occurred in all substrate sam-ples suggesting that substrate solution offers an enhancedresponse when prepared right after its use As the under-standing of the mechanism controlling the loss in substrateresponse was not part of this research further experimentswill be performed and analysed separately

Nomenclature

ICH International Conference onHarmonisation of Technical Requirementsfor Registration of Pharmaceuticals forHuman Use



Acknowledgments

The authors would like to acknowledge the financial supportof FAPESP (Sao Paulo Research Foundation) PROPP-UFU(Dean of Research and Graduate Studies at the FederalUniversity of Uberlandia) and CNPq (National Council forScientific and Technological Development) This Project hasbeen funded by grants from Sao Paulo Research FoundationFAPESP 201120733-7 and FAPESP 201214533-8

References

[1] O P Ward ldquo349mdashproteasesrdquo in Comprehensive BiotechnologyM-Y Murray Ed pp 571ndash582 Academic Press BurlingtonMass USA 2nd edition 2011

[2] H R Maurer ldquoBromelain biochemistry pharmacology andmedical userdquo Cellular and Molecular Life Sciences vol 58 no9 pp 1234ndash1245 2001

[3] S Cumming Global Market for Industrial Enzymes toReach Nearly $71 Billion by 2018 Detergent Enzyme Market toRecordMaximumGrowth BIO030H PRWeb 2014 httpwwwbccresearchcommarket-researchbiotechnologyenzymes-in-dustrial-applications-bio030hhtml

[4] J R Cherry and A L Fidantsef ldquoDirected evolution of indus-trial enzymes an updaterdquoCurrentOpinion in Biotechnology vol14 no 4 pp 438ndash443 2003

[5] M Mccoy ldquoNovozymes emergesrdquo Chemical amp EngineeringNews vol 79 no 8 pp 23ndash25 2001

[6] J Charney and R M Tomarelli ldquoA colorimetric method for thedetermination of the proteolytic activity of duodenal juicerdquoTheJournal of Biological Chemistry vol 171 no 2 pp 501ndash505 1947

[7] N S Leite A A B de Lima J C C Santana et al ldquoDeter-mination of optimal condition to obtain the bromelain frompineapple plants produced by micropropagationrdquo BrazilianArchives of Biology and Technology vol 55 no 5 pp 647ndash6522012

[8] L F Domingues R Giglioti K A Feitosa et al ldquoIn vitro and invivo evaluation of the activity of pineapple (Ananas comosus)on Haemonchus contortus in Santa Ines sheeprdquo VeterinaryParasitology vol 197 no 1-2 pp 263ndash270 2013

[9] C Mohan Buffers A Guide for the Preparation and Use of Buf-fers in Biological Systems Calbiochem-Behring Corporation LaJolla Calif USA 2008

[10] L P Hale P K Greer C T Trinh and C L James ldquoProteinaseactivity and stability of natural bromelain preparationsrdquo Inter-national Immunopharmacology vol 5 no 4 pp 783ndash793 2005

[11] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2015


[12] RStudio Inc RStudio Integrated Development Environment(IDE) for R vol 0981103 RStudio Inc Boston Mass USA2015

[13] A Gebhardt H Akima and T PetzoldtAkima Interpolation ofIrregularly Spaced Data R Package Version 05-11 2013

[14] UGroempingDoEbase Full Factorials Orthogonal Arrays andBase Utilities for DoE Packages R Package Version 027 2015

[15] H Wickham ggplot2 Elegant Graphics for Data AnalysisSpringer New York NY USA 2009

[16] E Neuwirth R Color Brewer Color Brewer Palettes R PackageVersion 11-2 2014

[17] ICH Expert Working Group Validation of Analytical Proce-dures Text andMethodology Q2 (R1) ICHHT-ICHHarmonisedTripartite Guideline 2005

[18] Food and Drug Administration Draft Guidance for IndustryBioanalytical Method Validation US Food and Drug Adminis-tration Rockville Md USA 1999

[19] B C Martins R Rescolino D F Coelho B Zanchetta EB Tambourgi and E Silveira ldquoCharacterization of bromelainfrom ananas comosus agroindustrial residues purified by eth-anol factional precipitationrdquo Chemical Engineering Transac-tions vol 37 pp 781ndash786 2014

Research ArticleEnhanced and Secretory Expression of Human GranulocyteColony Stimulating Factor by Bacillus subtilis SCK6

Shaista Bashir1 Saima Sadaf2 Sajjad Ahmad1 and Muhammad Waheed Akhtar1

1School of Biological Sciences University of the Punjab Lahore 54590 Pakistan2Institute of Biochemistry and Biotechnology University of the Punjab Lahore 54590 Pakistan

Correspondence should be addressed to Saima Sadaf sasadafhotmailcom andMuhammadWaheedAkhtar mwasbspuedupk

Received 2 October 2015 Revised 8 December 2015 Accepted 8 December 2015


Copyright copy 2015 Shaista Bashir et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokinethat is granulocyte colony stimulating factor (GCSF) in the culture supernatant of Bacillus subtilis SCK6 cells Codon optimizedGCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generatemultimeric plasmid DNA which was used directly to transform B subtilis SCK6 supercompetent cells Expression of GCSF wasmonitored in the culture supernatant for 120 hours The highest expression which corresponded to 17 of the total secretoryprotein was observed at 72 hours of growth Following ammonium sulphate precipitation GCSF was purified to near homogeneityby fast protein liquid chromatography on aQFF anion exchange column Circular dichroism spectroscopic analysis showed that thesecondary structure contents of the purified GCSF are similar to the commercially available GCSF Biological activity as revealedby the regeneration of neutrophils in mice treated with ifosfamine was also similar to the commercial preparation of GCSF Thisto our knowledge is the first study that reports secretory expression of human GCSF in B subtilis SCK6 with final recovery of upto 96mgL of the culture supernatant without involvement of any chemical inducer

1 Introduction

The development of efficient systems for the production ofbiosimilars is one of the key targets of the biotechnologyindustry Escherichia coli by far is regarded as one of theconvenient hosts for the recombinant production of thera-peutically important and commercially relevant proteins [1ndash3] However overexpression of many recombinant proteinsin this host leads to the accumulation of desired productin the form of inclusion bodies (IBs) which are biologicallyinactive Whereas the additional steps required in the recov-ery of bioactive protein from the IBs result in an overall lowyield the presence of lipopolysaccharides (endotoxins) in theoutermembrane ofE coli further complicates the purificationscheme and hence limits the usefulness of this system ([4ndash7]and references therein)

Targeting expression of heterologous proteins in theculture medium may be an attractive choice as it may reducethe downstream processing cost [8] In this regard Gram-positive bacterium Bacillus subtilis which directly exports

proteins into the extracellular medium may be exploitedwell [6 9] B subtilis owing to its naturally high secretoryability provides better folding conditions and thus preventsformation of IBs as opposed to the E coli based expressionsystems [10 11] Its Sec-dependent secretary pathway isinvolved in the formation of secretory preproteins complexwith the chaperons that bind to the secretory translocaseand help in translocation across the cytoplasmic membraneThe protein is released from translocase after removal ofsignal peptide refolded and crosses the cell wall [8 10 1213] Low protein yield abundant secretion of proteases andplasmid instability however are some bottlenecks whichmaysometime limit the application potential of B subtilis ([9] andreferences therein)

Neutropenia that is decreased count of neutrophils isone of themost common side effects of chemotherapy andorbone marrow transplantation Human granulocyte colonystimulating factor (GCSF) is an important biosimilar thatplays important role in survival proliferation and activationof neutrophils and thus reduces morbidity rate in patients



[14 15] It is amongst the few cytokines that have been usedin clinical trials with diverse applications that is the stem cellmobilization treatment of central nervous system disorderslike cerebral ischemia and stroke regeneration of hepatictissues and so forth [16ndash18] Cloning and expression of thistherapeutically important cytokine (sim19 kDa protein) havebeen reported by several research groups in E coli but in theform of IBs [14 19 20] AchievingGCSF expression in native-like biologically active form however is a more attractiveoption

The present study was designed with an objective to gen-erate a vector-host system that may be exploited for the cost-effective production of human GCSF in soluble and bioactiveform B subtilis expression host which is ldquogenerally regardedas saferdquo by the US Food and Drug Administration has beenutilized in combination with pNWPH vector that contains astrong promoter (PHbaII) and SpymwC signal sequence forimproved secretion of GCSF into the culture medium Asimplified approach for simultaneous amplification of thevector and the insert DNAs followed by direct transformationof the multimeric recombinant DNA into the expressionhost is also described here This to our knowledge is thefirst report that explains multimeric cloning enhanced andsecretory cost-effective production of human GCSF in Bsubtilis SCK6 The study is likely to contribute to developingbiosimilars by the biopharmaceutical companies for diverseapplications and analysis


21 Chemicals Kits Plasmids and Bacterial Strains Allchemicals and kits used in the present study were of highestpurity grade commercially available Pfu DNA polymerasedNTPs DNA and protein size markers were purchased fromThermo Scientific (USA)Thedesigned oligonucleotides usedin POE-PCR were acquired from Oligo Macrogen (USA)

Plasmid pNWPH and the B subtilis SCK6 (httpwwwbgscorgviewdetailphpbgscid=1A976ampSearch=sck) bacte-rial strain used in this study were a kind gift from DrX-Z Zhang [21] Virginia Polytechnic Institute and StateUniversity Blacksburg VA 24061 USA Media used for thegrowth of B subtilis were Luria-Bertani [LB (1 tryptone05 yeast extract 1 NaCl and pH 7)] and the modified 2xL-Mal medium (2 tryptone 1 yeast extract 1 NaCl 75maltose hydrate and 75120583gmL MnSO

4) Chloramphenicol

and erythromycin at a final concentration of 5 and 1120583gmLrespectively were used as selection antibiotics

22 Recombinant Plasmid Construction Plasmid pNWPH-mini-scaf [22] containing chloramphenicol resistance genea strong PHpaII promoter and SPymwC signal sequence wasused for the construction of pNWPH-GCSF (Figure 1) Theprimers used for the multimer cloning were comprised of50 nucleotides (nt) each having 25 nt overlapping regionof the insert and 25 nt of the vector (Table 1) The codonoptimized gene of human GCSF (KT326155) was amplifiedfrom pGCSF-08 construct of our lab (unpublished data) byusing IFIR primer pair while the vector (pNWPH) backbonewas linearizedamplified using VFVR primer pair

PCR reactions were performed in a mixture containingcodon optimized GCSF gene as template 1x HF buffer02mM dNTPs 05 120583M of each forward and reverse primerand 5 units of Pfu DNA polymerase The conditions usedfor high-fidelity PCR used for amplification are 98∘C denatu-ration 1 minute 30 cycles of 98∘C denaturation 10 s 64∘Cannealing 20 s and 72∘C extension 75 s followed by 72∘Cextension for 5 minutes The multimerization process ofpurified PCR products of the linearized vector and GCSFwas performed through prolongeded overlap extension PCRessentially as described by You et al [23] using high-fidelity Pfu DNA polymerase (004U) dNTPs (02mM foreach) PCR-GCSF (2 ng120583L) and PCR-linearized pNWPH(2 ng120583L) The cycling profile was initial 98∘C denaturation(30 sec) and then 20 cycles of 98∘C denaturation (10 sec)58∘Cannealing (30 sec) and 72∘Cextension (3min) followedby 15 cycles of 98∘C denaturation (10 seconds) and 72∘Cannealing and extension (6min) with final 72∘C extension for10min (Figure 1)

B subtilis SCK6 supercompetent cells were preparedessentially as described by X-Z Zhang and Y-H P Zhang[21] Briefly LB medium (5mL) containing 1 120583gmL ery-thromycin was inoculated with the B subtilis SCK6 andgrown overnight at 37∘C with constant shaking at 200 rpmThe overnight culture was diluted with fresh LB mediumcontaining 2 (wv) xylose to A

600of 10 and grown for

another two hours B subtilis SCK6 strain contains additionalcopy of the comK gene inserted downstream of the xylosepromoter Xylose when added during the exponential phaseof the SCK6 cells acts as an inducer of the comK geneexpression which adds up to the competency of cells Theresultant supercompetent cells were either used directly forthe transformation or stored at minus80∘C as 10 (vv) glycerolstocks

For transformation plasmidmultimers (1120583L)weremixedwith 100 120583L supercompetent cells and incubated at 37∘Cfor 90min with constant shaking at 200 rpm The positivetransformants were selected on LB agar plates containing5 120583gmL chloramphenicol following incubation at 37∘C for14 hours Modified alkaline lysis method [24] involving thetreatment of cell pellet with lysozyme to break up the cellwall was used to isolate the plasmid from two well-isolatedpositive colonies Restriction digestion with HindIII andNdeI restriction endonucleases was performed to confirm thepresence of insert in the isolated plasmids

23 Expression in Bacillus subtilis Transformed B subtilisSCK6 cells containing the recombinant human GCSF weregrown in two different media LB and 2x L-Mal at 37∘C at200 rpm in baffled Erlenmeyer flasks For secretory expres-sion the cells were grown at low temperature that is 30∘Cfor a total of 120 hours 1mL sample aliquots were taken outat regular intervals of 12 hours until 120 hours and changein growth was monitored spectrophotometrically (OD

600)

Culture supernatant was examined for secretory expressionof GCSF after centrifugation (6500timesg 4∘C 20min) andprecipitation through a modified TCA-acetone precipitationmethod Briefly to 1mL of protein solution 150 120583L TCA(100) was added placed at minus20∘C for 10 minutes and


Table 1 Sequence of oligonucleotides used to amplify insert (IFIR) and vector (VFVR) DNAs during prolonged overlap extension (POE)PCRlowast

Primer Sequence 51015840-31015840

VF CCTTGCCCAGCCCTGATAGAAGCTTGGATCCGGAGTCGAACCATAAAAGCVR TGGCAGGGCCCAGGGGGGTCATATGAGCTGATGCCGAATACGTAAAGGTAIF TACCTTTACGTATTCGGCATCAGCTCATATGACACCTCTGGGCCCTGCCAIR GCTTTTATGGTTCGACTCCGGATCCAAGCTTCTATCAGGGCTGGGCAAGGlowastPrimers were designed using online available software (httpwwwxiaozhouzhangcom) AAGCTT andCATATG (underlined sequences) are the recognitionsites for the HindIII and NdeI restriction endonucleases respectively

---ATG CCCACC -------- CAG CCC TGA------TAC GGGTGG -------- GTC ACTGGG ------ M PT -------- Q lowastP ---

pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n

PCR amplification of vector and

insert(30 cycles)

Multimericexpression

plasmid

Denaturation

Annealing

Extension

Denaturation annealing and extension

Denaturation annealing and extensionDimer

Monomer

Multimer

1stcycle

2ndcycle

Further cycles

Circularization by bacterial host

GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat

Figure 1 Construction of the pNWPH-GCSF expression plasmid using prolonged overlap extension PCRmultimeric cloning strategySimple PCR generated 31015840 and 51015840 overhangs of insert (GCSF) and vector (pNWPH)These overhangs acted as primers during the formation ofmultimers Circular plasmid pNWPH-GCSF was thereafter generated in B subtilis by direct transformation of multimers containing GCSFgene repB replication protein B Cat chloramphenicol transferase gene PHbaII promoter SDgsiB Shine-Dalgarno sequence of the gsiB geneSPywmC signal sequence


then centrifuged at 14000timesg for 5 minutes Supernatant wasdiscarded and the pellet was washed with 700120583L of 100ice-cold acetone to remove the residual TCA The solutionwas placed at minus20∘C for 10 minutes prior to centrifugationSecond washing was done with 70 acetone and the pelletwas dissolved in 50mMTris-Cl for use in subsequent analysisby 13 (wv) SDS-polyacrylamide gel electrophoresis

Bradford assay [25] and UV absorption method wereused to measure the total secretory protein contents andpurified recombinant GCSF concentration Densitometricanalyses of the SDS-gels were also used to determine the ofexpression andor the purity level of GCSF in different samplepreparations

24 Purification of Recombinant Human GCSF For purifica-tion of rhGCSF the culture supernatants of 72ndash80-hour frac-tions were subjected to salting out by ammonium sulphateprecipitation Ammonium sulphate was added slowly withconstant stirring at 4∘C to saturation of 65ndash80 The pre-cipitates were collected by centrifugation at 6500timesg 10minand dialyzed against 50mM Tris-Cl (pH 85) buffer Theprotein was subsequently purified on anion-exchange FPLCsystem using 1mLHiTrapQFF column (GEHealthcare)Thecolumn was preequilibrated with 50mM Tris-Cl (pH 85)After sample injection the columnwaswashedwith 2 columnvolumes of 50mMTris-Cl (pH85) and the proteinwas elutedusing linear gradient of 0 to 1M NaCl in 50mM Tris-Cl (pH85)

25 Circular Dichroism Spectroscopy Circular dichroism(CD) data of purified rhGCSF were collected on a Chiras-canPlus CD spectrophotometer (Applied Photophysics UK)equipped with a peltier thermal-controlled cuvette holderFor comparative purposes CD spectra of the commerciallyavailable preparations of human GCSF (Filgrastim) were alsoobtained Calibration was done with an aqueous solution of1S-(+)-10-camphorsulfonic acid The protein solution con-taining 156 120583gmL in 10mM Tris-Cl (pH 85) was scannedover wavelength 185 nmndash260 nm at 2∘C using a quartz cell of05mmpath length Eachwavelength spectrumwas the resultof averaging of two consecutive scans with a bandwidth of10 nm The wavelength spectra were refined by subtractinga blank spectrum with buffer only The secondary structurecontent of protein was calculated using the CD spectrumdeconvolution software CDNN [26] which calculates thesecondary structure of the peptide by comparison with a CDdatabase of known protein structures

26 Biological Activity Assessment Male mice each weighing20ndash24 g were divided into two sets of 3 groups each groupconsisting of four animals They were fed ad libitum andmaintained under controlled conditions of temperature (24ndash28∘C) relative humidity (sim65) and artificial illumination(12 h per day) One set of three groups was used for admin-istration of the drug One of the groups was given in-houseprepared rhGCSF second group was given commerciallyavailableGCSF (Filgrastim SigmaUSA) and the third groupwas given 01BSA in 1x PBS (pH 74)The second set of three

groups was treated in the same way except that the drug wasadministered through intraperitoneal route

All the animals were given a single dose of ifosfamine(43mg05mL) either through subcutaneous or intraperi-toneal route to each animal of respective group to introduceneutropenia Both the in-house produced rhGCSF and thecommercial preparation were diluted to the concentrationsof 15 and 40 120583gmL in 1x PBS (pH 74) containing 01BSA The drug injections (1-2 120583g per gram of mouse weight)were administered one day after the injection of ifosfamineand continued daily for the next four days Six hours afterthe last dose peripheral blood samples were collected fromorbital venous sinus Glass slide smears were stained withMay-Grunwald-Giemsa (Sigma) and the total number ofneutrophils as well as the white blood cells was counted usinga blood cell counter

The percentage of neutrophils was calculated by takingmean plusmn SD of four animals for both routes of administrationBy using GraphPad Prism Program (Version 40) one-way analysis of variance (ANOVA) followed by Bonferronirsquosposttest was performed to check the statistical significance ofthe data 119875 values were considered as significant when le 005

3 Results

31 Secretory Expression of rhGCSF in B subtilis The strategyfor producing the pNWPH-GCSF vector used for the secre-tory expression ofGCSF in B subtilis is described in Figure 1As shown the codon optimized gene of GCSF is placed underthe regulation of a strong PHbaII promoter and the YwmCsignal peptide encoding sequence (SPywmC) of B subtilisNucleotides (sim25) present at 51015840 and 31015840 termini of the insertand the vector generated during PCR amplification served asprimers for each other and led to the formation of dimers dur-ing the first round of multimeric PCR The dimers increasedin number with each round of PCR cycle and finally themultimers were formed with repeated insert-vector-insert-vector sequences The multimeric cloning strategy used inthe present study involved the direct transformation of Bsubtilis SCK6 supercompetent cells with the plasmid multi-mers which is unlike the conventional cloning approach thatincludes additional steps of restriction digestion and ligationprior to the transformation step

Positive transformants were selected using chloram-phenicol as selection antibiotic while the presence and in-frame cloning of GCSF in pNWPH vector were confirmedthrough restriction digestion Two bands that is sim33 kb ofpNWPH vector and the sim05 kb GCSF insert could be seenon 1 agarose gel following digestion of the recombinantplasmid with NdeI and HindIII (Figure 2(a)) TransformedB subtilis SCK6 cells were grown in 2x L-Mal medium for120 hours Cell growth (OD

600) was recorded (Figure 2(c))

and the secretory expression of GCSF at different stageswas monitored by analysis of the sample aliquots of culturesupernatant (Figures 2(b) and 2(d))

When analyzed by SDS-PAGE the culture supernatantof transformed B subtilis SCK6 displayed a prominent bandof sim19 kDa at 60 hours of growth which increased gradu-ally with the passage of time Maximum expression level


(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)

Figure 2 (a) Restriction analysis of pNWPH-GCSF expression plasmid resolved on 1 agarose gel M 1 kb DNA size marker Lane 1undigested pNWPH-GCSF Lane 2 pNWPH-GCSF after double digestion with NdeI and HindIII restriction endonucleases (b) 13 SDS-PAGE analysis of TCA-acetone precipitated culture supernatant of transformed B subtilis SCK6 Lane M represents protein size markerLanes 1ndash7 sample fractions collected at 24 36 48 60 72 84 and 96 hours of cell growth (c) Growth of recombinant B subtilis harboringpNWPH-GCSF in 2x L-Mal medium 119909-axis shows time in hours while primary 119910-axis reflects the concentration of GCSF (120583gmL) in culturesupernatant and secondary 119910-axis shows cell growth monitored by absorbance measurement at 600 nm

corresponding to sim17 of the total secretory protein wasattained at 72 hours which remained constant until 96 hoursThereafter a sharp decline in cell growth was observed witha resultant drop in the levels of recombinant protein in theculture supernatant (Figures 2(c) and 2(d))

32 Purification of rhGCSF Secretion of recombinant pro-teins into the extracellular medium facilitates early down-stream processing For purification of GCSF the culturesupernatant was clarified by centrifugation and precipitatedwith 65ndash80 ammonium sulphate saturation While verylittle amount got precipitated at 65 highest amount could berecovered at 80 ammonium sulphate saturation with puritylevel of 75 (Table 2)

The collected fractions were dialyzed against 50mMTris-Cl to remove ammonium salt and the partially purified GCSFwas purified to near homogeneity through anion exchangechromatography on FPLC as described in Section 2 The

Table 2 Purification and recovery of human GCSF expressed in Bsubtilis Culture supernatant of transformed cells grown in 1 liter of2x L-MALmedium for 72 hours at 30∘CwithOD

60060 was clarified

by centrifugation and used for the purification of recombinantGCSF

Steps TSPlowast GCSF Recovery Purity(mg) (mg) () ()

Culture supernatant 720 122 100 17Ammonium sulphate precipitation 235 115 94 49Dialysis 212 110 90 52FPLC purification (QFF) 107 96 78 90lowastTSP total secretory protein

protein of interest eluted at sim03M NaCl gradient as shownin second peak of the chromatogram (Figure 3(a))TheGCSFpurity level attained following two steps of purification was


(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000

Fraction volume (mL)

0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)

Figure 3 (a) Purification of recombinant human GCSF by FPLC on QFF column Inset shows the purified GCSF eluted with 03M NaClconcentration gradient Blue and red colors show absorbance at A

280and A

260 respectively (b) CD spectrum of the recombinant in-house

produced GCSF (solid line) and the commercially available GCSF preparation that is Filgrastim (dotted line) scanned over 185ndash260 nmrange

sim90 with a final recovery of 96mg per liter of the culturesupernatant (Table 2)

33 CD Spectrometry Analysis CD spectrum of recombinantGCSF at 20∘C showed double negative minima at 209 and222 nm (Figure 3(b)) Analysis of the secondary structureusing the CDNN software showed the presence of 578 120572-helices and 43 parallel and 42 antiparallel 120573-sheetsThese secondary structure values are typical of a proteincontaining a large proportion of 120572-helical structure andare in coherence with the commercially available GCSFpreparation Since GCSF belongs to cytokine superfamilymembers containing 120572-helices and lack 120573-sheets our datasupports that recombinant GCSF produced in B subtilis is ina properly folded conformation

34 Biological Activity Assessment The biological activity ofrecombinant in-house produced GCSF was assessed in an invivo model of neutropenia Mice treated with single dose ofifosfamine to induce neutropenia were given recombinantGCSF and the percentage of neutrophils was monitored(Figures 4(a) and 4(b)) Amongst the two routes of drugadministration tested in this study that is intraperitoneal andsubcutaneous the former delivery route of biosimilar wasfound to be more effective than the latter route (data notshown)

Statistically significant dose-dependent increase in neu-trophil count (119875 value lt 0001) was observed in the micegroup treated with in-house produced GCSF The trendwas similar to what we observed in the group treated withcommercially available Filgrastim (119875 value lt 0001) At15 120583gmL GCSF concentration the increase in neutrophilcount was up to 50 but improved further to a level of60 with an increase in GCSF injection dose to 40 120583gmL(Figure 4(b)) Overall the effect of in-house produced GCSF

and the commercially available filgrastim preparation on thetwo treated mice groups was statistically indistinguishable

4 Discussion

Chemotherapy in addition to killing cancer cells oftendamages the rapidly dividing normal cells including theleukocyte producing bone marrow cells Since leukocytesmore specifically neutrophils play central role in defenseagainst invadingmicrobes their reduced levels in response tochemotherapy or as a result of bone marrow transplantationmake the body more susceptible to various life-threateninginfections and sepsis [15 27] The injections of GCSF eitherglycosylated or nonglycosylated are therefore recommendedand have been approved by US FDA for the treatment ofchemotherapy-induced neutropenia neutropenia caused bybone marrow transplantation and neutropenia associatedwith the mylodysplatic syndrome or aplastic anemia [28]Besides its applications in the treatment of neutropeniaGCSF has been found to have role in the treatment ofcentral nervous system disorders like cerebral ischemia andstrokes regeneration of hepatic tissues and so forth [16ndash18] Therefore biopharmaceutical companies following theexpiration of recombinant first-generationGCSF areworkingon the production of new bioactive GCSF biosimilars

We in the present study were able to produce native-like biologically active form of human GCSF in the culturemedium by using a combination of pNWPH-GCSF expres-sion vector and B subtilis SCK6 host system Multimericcloning approach which involves the use of POE-PCR wasopted for the construction of expression of plasmid pNWPH-GCSF (containing sim05 kb GCSF gene downstream of thePHbaII promoter)This technique originally described by Youet al [23] is relatively new but is simple and cost-effectiveand has certain advantages over the conventional cloning


(a)

Control 15 4015 40

Concentration (120583gmL)

ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)

Figure 4 (a) GCSF biological activity assay Leftmice being injectedwithGCSF by subcutaneous route rightmicroscopic analysis of Giemsastained slides wherein the encircled cells represent the neutrophil counts (b) Mice in the sample and the control group received two differentdoses of GCSF (15 and 40120583gmLmouse) The control group was treated with 01 BSA in PBSThe abbreviations cGCSF and rhGCSF standfor commercially available GCSF and in-house produced recombinant human GCSF respectively

strategies in particular the direct transformation of hostwithout additional steps of restriction digestion and DNAligation [22]

Amongst the commonly available expression hosts forthe recombinant production of therapeutic proteins namelyChinese Hamster Ovary (CHO) cells Human EmbryonicKidney (HEK) 293 cells Pichia pastoris [29ndash32] and E colithe latter has widely been used to produce GCSF with highyields of up to 15mgL in shake-flask cultures [14 33 34] Ofnote the expression of GCSF in E coli reported in almostall the studies was in the form of IBs which demands useof denaturant (strong or mild) for solubilization and thenremoval of the denaturant as a prerequisite of refoldingscheme [31 32]

Earlier we cloned and expressed the GCSF in E coliBL21 (DE3) cytoplasm at levels corresponding to sim35 oftotal E coli cellular proteins but in the form of IBs Theapproaches used to improve the solubility of GCSF in Ecoli transformants that is growth of transformed cells atlow temperature (16ndash25∘C) targeting of GCSF into the Ecoli periplasm by attaching pelB leader sequence of the pET

system and the coexpression of GCSF with M tuberculosisheat shock protein (Hspx) met with only limited success(unpublished results) However use of B subtilis as expres-sion host in the present study resulted in enhanced andsecretory expression of human GCSF with almost 6-foldhigher yields than reported previously ([33] and referencestherein)

SPywmC one of the powerful Sec-type peptides of theB subtilis general secretory pathway (Sec pathway) was usedfor secretory expression of GCSF as used for heterologousexpression of esterase previously [35] When grown in 2x-LMAL nutrient enriched model medium [36ndash38] the cellgrowth increased gradually until the 50 hours and reachedplateau afterward The GCSF secretion however reachedto maximum level (17) at 72 hours that is during thestationary phase of cell growth (Figures 2(c) and 2(d)) Theseresults are in good agreement with the nonclassical secretionof recombinant proteins in B subtilis as reported previously[39] Secretory expression facilitated rhGCSF downstreamprocessing By using ammonium sulphate precipitation andsingle FPLC column chromatography gt90 purity levels of


recombinant protein were achieved Purified GCSF injectedin mice to assess its biological activity showed similar effectas commercially available Filgrastim without any side effectson mice Commercially available Filgrastim preparationswere used to confirm the secondary structure of rhGCSFby circular dichroism High 120572-helical content showed typicalcharacteristic of cytokines [40] In conclusion this studyreports for the first time the secretory expression of biologi-cally active rhGCSF in B subtilis SCK6 strain with minimumdownstream processing steps and much higher yield thanreported previously using the E coli based expression system[33]

5 Conclusion

In conclusion this study reports for the first time the secre-tory expression of biologically active rhGCSF in B subtilisSCK6 strain with minimum downstream processing stepsand much higher yield than reported previously using theE coli based expression system Our results showed that Bsubtilis SCK6 with twofold advantages of convenient down-stream processing and cost-effective high yield productionof heterologous proteins (no inducer is required) may beexploited as an alternate expression system for the productionof GCSF biosimilars


The authors of this paper declare no conflict of interests

Acknowledgment

This study was supported by a grant from Pakistan Academyof Sciences Pakistan

References

[1] S Sadaf M A Khan D B Wilson and M W AkhtarldquoMolecular cloning characterization and expression studiesof water buffalo (Bubalus bubalis) somatotropinrdquo Biochemistryvol 72 no 2 pp 162ndash169 2007

[2] L Westers H Westers and W J Quax ldquoBacillus subtilis ascell factory for pharmaceutical proteins a biotechnologicalapproach to optimize the host organismrdquoBiochimica et Biophys-ica ActamdashMolecular Cell Research vol 1694 no 1ndash3 pp 299ndash310 2004

[3] QMChen YQGeng J Ni G FWang andR Z Jiang ldquoStudyon Bacillus pumilus as a recipient strain for genetic engineeringof Bacillusrdquo Acta Genetica Sinica vol 16 no 3 pp 206ndash2121989

[4] S Sadaf H Arshad and M W Akhtar ldquoA non-ionic surfactantreduces the induction time and enhances expression levels ofbubaline somatotropin in Pichia pastorisrdquo Molecular BiologyReports vol 41 no 2 pp 855ndash863 2014

[5] L Bredmose S Madsen A Vrang et al ldquoDevelopment of aheterologous gene expression system for use in Lactococcuslactisrdquo in Recombinant Protein Production with Prokaryotic andEukaryotic Cells A Comparative View on Host Physiology pp269ndash275 Springer 2001

[6] D Petsch and F B Anspach ldquoEndotoxin removal from proteinsolutionsrdquo Journal of Biotechnology vol 76 no 2-3 pp 97ndash1192000

[7] R V Datar T Cartwright and C G Rosen ldquoProcess economicsof animal cell and bacterial fermentations a case study analysisof tissue plasminogen activatorrdquo Nature Biotechnology vol 11no 3 pp 349ndash357 1993

[8] F G Durrani R Gul S Sadaf and M W Akhtar ldquoExpressionand rapid purification of recombinant biologically active ovinegrowth hormone with DsbA targeting to Escherichia coli innermembranerdquoAppliedMicrobiology andBiotechnology vol 99 no16 pp 6791ndash6801 2015

[9] W Li X Zhou and P Lu ldquoBottlenecks in the expression andsecretion of heterologous proteins in Bacillus subtilisrdquo Researchin Microbiology vol 155 no 8 pp 605ndash610 2004

[10] M Simonen and I Palva ldquoProtein secretion in Bacillus speciesrdquoMicrobiological Reviews vol 57 no 1 pp 109ndash137 1993

[11] T Moks L Abrahmsen E Holmgren et al ldquoExpression ofhuman insulin-like growth factor I in bacteria use of optimizedgene fusion vectors to facilitate protein purificationrdquo Biochem-istry vol 26 no 17 pp 5239ndash5244 1987

[12] L L Fu Z R Xu W F Li J B Shuai P Lu and C X HuldquoProtein secretion pathways in Bacillus subtilis implication foroptimization of heterologous protein secretionrdquo BiotechnologyAdvances vol 25 no 1 pp 1ndash12 2007

[13] K H M V WelyThe general protein secretion pathway of Bacil-lus subtilis [PhD thesis] University of Groningen GroningenThe Netherlands 2000

[14] A L S Vanz G RenardM S Palma et al ldquoHuman granulocytecolony stimulating factor (hG-CSF) cloning overexpressionpurification and characterizationrdquoMicrobial Cell Factories vol7 article 13 2008

[15] D R Barreda P C Hanington and M Belosevic ldquoRegulationof myeloid development and function by colony stimulatingfactorsrdquo Developmental and Comparative Immunology vol 28no 5 pp 509ndash554 2004

[16] L J Bendall and K F Bradstock ldquoG-CSF from granulopoieticstimulant to bone marrow stem cell mobilizing agentrdquo Cytokineand Growth Factor Reviews vol 25 no 4 pp 355ndash367 2014

[17] A Schneider C Kruger T Steigleder et al ldquoThe hematopoieticfactor G-CSF is a neuronal ligand that counteracts programmedcell death and drives neurogenesisrdquo The Journal of ClinicalInvestigation vol 115 no 8 pp 2083ndash2098 2005

[18] S Sell ldquoHeterogeneity and plasticity of hepatocyte lineage cellsrdquoHepatology vol 33 no 3 pp 738ndash750 2001

[19] C K Kim C H Lee S-B Lee and J-W Oh ldquoSimpli-fied large-scale refolding purification and characterization ofrecombinant human granulocyte-colony stimulating factor inEscherichia colirdquo PLoS ONE vol 8 no 11 Article ID e801092013

[20] S A Dehaghani V Babaeipour M R Mofid A Divsalar andF Faraji ldquoAn efficient purification method for high recoveryof recombinant human granulocyte colony stimulating factorfrom recombinant E colirdquo International Journal of Environmen-tal Science and Development vol 1 no 2 pp 111ndash114 2010

[21] X-Z Zhang and Y-H P Zhang ldquoSimple fast and high-efficiency transformation system for directed evolution ofcellulase in Bacillus subtilisrdquoMicrobial Biotechnology vol 4 no1 pp 98ndash105 2011

[22] S Ahmad H Ma M W Akhtar Y-H P Zhang and X-ZZhang ldquoDirected evolution ofClostridium phytofermentans gly-coside hydrolase family 9 endoglucanase for enhanced specific


activity on solid cellulosic substraterdquo Bioenergy Research vol 7no 1 pp 381ndash388 2014

[23] C You X-Z Zhang N Sathitsuksanoh L R Lynd and Y-HPercival Zhang ldquoEnhanced microbial utilization of recalcitrantcellulose by an ex vivo cellulosome-microbe complexrdquo Appliedand Environmental Microbiology vol 78 no 5 pp 1437ndash14442012

[24] J Sambrook and D W RusselMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor NY USA 3rd edition 2001

[25] M M Bradford ldquoRapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[26] G Bohm R Muhr and R Jaenicke ldquoQuantitative analysis ofprotein far UV circular dichroism spectra by neural networksrdquoProtein Engineering vol 5 no 3 pp 191ndash195 1992

[27] B Wittman J Horan and G H Lyman ldquoProphylactic colony-stimulating factors in children receiving myelosuppressivechemotherapy a meta-analysis of randomized controlled tri-alsrdquoCancer Treatment Reviews vol 32 no 4 pp 289ndash303 2006

[28] R Garcıa-Carbonero J I Mayordomo M V Tornamira etal ldquoGranulocyte colony-stimulating factor in the treatment ofhigh-risk febrile neutropenia a multicenter randomized trialrdquoJournal of the National Cancer Institute vol 93 no 1 pp 31ndash382001

[29] A Apte-Deshpande S Somani G Mandal S Soorapaneniand S Padmanabhan ldquoOver expression and analysis of O-glycosylated recombinant human granulocyte colony stimulat-ing factor in Pichia pastoris using Agilent 2100 BioanalyzerrdquoJournal of Biotechnology vol 143 no 1 pp 44ndash50 2009

[30] A Saeedinia M Shamsara A Bahrami et al ldquoHeterologousexpression of human granulocyte-colony stimulating factor inPichia pastorisrdquo Biotechnology vol 7 no 3 pp 569ndash573 2008

[31] M A Lasnik V G Porekar and A Stalc ldquoHuman granulo-cyte colony stimulating factor (hG-CSF) expressed by methy-lotrophic yeast Pichia pastorisrdquo Pflugers Archiv vol 442 no 6pp R184ndashR186 2001

[32] N Kubota T Orita K Hattori M Oh-Eda N Ochi and TYamazaki ldquoStructural characterization of natural and recombi-nant human granulocyte colony-stimulating factorsrdquo Journal ofBiochemistry vol 107 no 3 pp 486ndash492 1990

[33] F R Gomes A C Maluenda J O Tapias et al ldquoExpressionof recombinant human mutant granulocyte colony stimulatingfactor (Nartograstim) in Escherichia colirdquo World Journal ofMicrobiology and Biotechnology vol 28 no 7 pp 2593ndash26002012

[34] P Gascon ldquoPresently available biosimilars in hematology-oncology G-CSFrdquo Targeted Oncology vol 7 supplement 1 ppS29ndashS34 2012

[35] U BrockmeierNewStrategies toOptimize the SecretionCapacityfor Heterologous Proteins in Bacillus Subtilis Biowissenschaftender Ruhr-Universitat Bochum 2006

[36] K Manabe Y Kageyama M Tohata K Ara K Ozaki and NOgasawara ldquoHigh external pH enables more efficient secretionof alkaline 120572-amylase AmyK38 by Bacillus subtilisrdquo MicrobialCell Factories vol 11 article 74 2012

[37] TMorimoto R Kadoya K Endo et al ldquoEnhanced recombinantprotein productivity by genome reduction in Bacillus subtilisrdquoDNA Research vol 15 no 2 pp 73ndash81 2008

[38] K Ara K Ozaki K Nakamura K Yamane J Sekiguchi andN Ogasawara ldquoBacillus minimum genome factory effectiveutilization of microbial genome informationrdquo Biotechnologyand Applied Biochemistry vol 46 no 3 pp 169ndash178 2007

[39] C-K Yang H E Ewis X Zhang et al ldquoNonclassical proteinsecretion by Bacillus subtilis in the stationary phase is not dueto cell lysisrdquo Journal of Bacteriology vol 193 no 20 pp 5607ndash5615 2011

[40] D A Parry E Minasian and S J Leach ldquoConformationalhomologies among cytokines interleukins and colony stimu-lating factorsrdquo Journal of Molecular Recognition vol 1 no 3 pp107ndash110 1988

Research ArticleOne-Step Recovery of scFv Clones from High-ThroughputSequencing-Based Screening of Phage Display LibrariesChallenged to Cells Expressing Native Claudin-1

Emanuele Sasso123 Rolando Paciello12 Francesco DrsquoAuria12

Gennaro Riccio12 Guendalina Froechlich12 Riccardo Cortese2 Alfredo Nicosia12

Claudia De Lorenzo12 and Nicola Zambrano123

1Dipartimento di Medicina Molecolare e Biotecnologie Mediche Universita degli Studi di Napoli Federico II Via S Pansini 580131 Napoli Italy2CEINGE Biotecnologie Avanzate SC a RL Via G Salvatore 486 80145 Napoli Italy3Associazione Culturale DiSciMuS RFC 80026 Casoria Italy

Correspondence should be addressed to Alfredo Nicosia anicosianouscomcomClaudia De Lorenzo claudiadelorenzouninait and Nicola Zambrano zambranouninait

Received 4 August 2015 Accepted 5 October 2015


Copyright copy 2015 Emanuele Sasso et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active fieldof investigation within medical biotechnologies to prevent graft reinfection in patients subjected to liver transplantation and toovercome resistances elicited by novel antiviral drugs In this paper we describe a complete pipeline for screening of phage displaylibraries of human scFvs against native Claudin-1 a tight-junction protein involved in hepatitis C virus infection expressed on thecell surface of human hepatocytes To this aim we implemented a high-throughput sequencing approach for library screeningfollowed by a simple and effective strategy to recover active binder clones from enriched sublibraries The recovered cloneswere successfully converted to active immunoglobulins thus demonstrating the effectiveness of the whole procedure This novelapproach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases fortherapeutic andor diagnostic applications

1 Introduction

Monoclonal antibodies (mAbs) represent valuable tools inbiological treatments for a variety of clinical conditionsincluding viral infections and cancer Screening of antibodylibraries by phage display allows for rapid selection of single-chain variable fragments (scFvs) from which to isolatethe sequences of variable heavy (VH) and variable light(VL) chains for mAb conversion Thus avoiding animalimmunization it is possible to obtain antibodies against toxicor highly conserved antigens or against plasma membraneproteins or receptors in their native conformation [1 2]This possibility is of relevance for isolation of antibodiesto interfere with viral infections In the paradigm of viralhepatitis mAbs have been generated preventing hepatitis

C virus (HCV) infection of hepatocytes HCV utilizes a setof different cell membrane receptors to infect liver cellsCD81 SR-BI and the tight junction proteins CLDN1 andOCLN [1 3ndash6] CD81 and SR-BI mAbs actually inhibit HCVinfection both in vitro and in vivo [7] Non-human orchimeric anti-CLDN1 antibodies were shown to be effectiveagainst HCV infection in vitro and in vivo [8ndash11] So farno fully human anti-CLDN1 or OCLN mAbs are availableStill generation of novel mAbs is a relevant issue eventhough antiviral drugs such as boceprevir and telaprevirare currently in clinical use However besides their toxicside effects their use may be limited by the occurrenceof drug-resistant phenotypes [12ndash16] Furthermore theseantiviral drugs are not as effective to prevent graft rein-fection in patients subjected to liver transplantation since



the treatment is delayed until several months from surgery[17]

High-throughput sequencing (HTS) was successfullyapplied to phage display technology to get full advantagefrom screening of phage display libraries [18 19] It allows usto rapidly identify the potential binders of a given antigenbased on the counts of the corresponding scFv fragmentswithin a cycle and on the kinetic of their enrichments withinconsecutive cycles that may provide useful information onthe whole screening After their identification the clonesof interest need to be recovered from the DNA library ofthe relevant selection cycle for validation of binding HTS-based selection of phage display libraries should providerapid information on the screening progression and a com-prehensive set of scFv clones since it limits the possibility toloose potential good binders during the repetitive handlingof clones which is required during a classical screeningThe bottleneck of a HTS-based screening is however therecovery of scFv clones of interest The availability of a set ofalternative strategies to recover rapidly the clones of interestwould allow us to overcome the limiting step in HTS-basedscreening of phage display libraries [19] In this paper wetested the whole procedure of a HTS-based screening toisolate binders of native CLDN1 protein expressed on thecell surface of mammalian cells We successfully identifieda set of 75 potential binders of CLDN1 from which novelhuman antibodies could be isolated possessing the ability tointerfere with HCV infection We also implemented a rapidand effective method for one-step recovery of scFv clonesfrom the enriched population of fragments This methodwas applied to some scFv fragments characterized by heavy-chain complementarity determining regions 3 (HCDR3) ofdifferent length to demonstrate its effectiveness in the gener-ation of complete and functional monoclonal antibodies


21 Cell Cultures TheHuman Embryonic Kidney HEK 293Tcells were cultured in standard conditions using DulbeccorsquosModified Eaglersquos medium (DMEM Life Technologies IncPaisley UK) with the addition of nonessential amino acidsolution (Gibco Life Technologies Inc) The HEK 293Tcells transduced with the gene encoding CLDN1 [1] weregrown in DMEM containing Blasticidin (2120583gmL) (GibcoLife Technologies Inc) Media were supplemented with 10FBS 50 unitsmL penicillin and 50 120583gmL streptomycin (allfrom Gibco Life Technologies Inc)

22 Selection of scFv Phage on Living Cells The phagelibrary was grown in 2xTY medium containing 100 120583gmLof Ampicillin and 1 glucose up to an optical density at600 nm (OD600) of 05 Subsequently 1times 109 plaque-formingunits of M13-K07 helper phage encoding trypsin-cleavablepIII protein were added to 25mL of culture and were grownfor 1 hour The bacterial cells were then pelleted throughcentrifugation for 15 minutes at 4000 rpm and then resus-pended and grown overnight in 500mL of 2xTY containing100 120583gmL of Ampicillin and 25 120583gmL of Kanamycin at

30∘C Phages were collected by two steps of precipitationwith polyethylene glycol (PEG) and resuspended in PBSThetheoretical diversity of naıve library was about 1 times 1010

Both HEK 293T cells mock and transduced with CLDN1cDNA were detached by using cell dissociation solution(Sigma-Aldrich Saint Louis USA) and washed with PBSPhages (1013 pfu) were blocked with 5milk powder (Sigma-Aldrich) in PBS for 15 minutes and submitted to two roundsof negative selection by incubation with HEK 293T mockcells (5 times 106) for 2 hours at 4∘C The unbound phages wererecovered from supernatant after centrifugation at 1200 rpmfor 10 minutes and then were used for the positive selectionperformed on CLDN-1 transduced HEK 293T (1 times 106) byincubation for 16 hours at 4∘C Cells were recovered bycentrifugation at 1200 rpm for 10 minutes and washed twicewith PBS Boundphages fromeach selectionwere eluted fromCLDN-1 transduced HEK 293T with a solution of 1 120583gmLof Trypsin (Sigma-Aldrich) which was then inhibited byEDTA-free protease inhibitor cocktail (Roche DiagnosticMannheim Germany) The recovered phages were amplifiedby infectingE coliTG1 cells to prepare phage for the followinground of selection Four whole cycles of selection wereperformed

23 VH Extraction and Purification The double strand DNAplasmids containing the scFvs were isolated from each cycleof selection from a culture of superinfected E coli TG1 cellsusing GenElute HP Plasmid Maxiprep Kit (Sigma-Aldrich)The VHs were excised by double digestion with restrictionenzymes NcoI and XhoI (New England Biolabs) and thenpurified from a 12 agarose gel (Figure 1(a))

24High-Throughput Sequencing Library preparations of thefragments sequencing reactions and preliminary analysisof the data were performed at the Center for TranslationalGenomics and Bioinformatics Hospital San RaffaeleMilanoItaly Briefly for the preparation of the bar-coded librariesTruSeq ChIP sample prep kit (Illumina) was used on theVHDNA samples isolated from cycles 1ndash4 A complementaryscheme for bar-coding was implemented in order to performsequencing reactions frommixtures of subcycles 1 and 4 (run1) and of subcycles 2 and 3 (run 2) The bar-coded sampleswere diluted to a final concentration of 10 pM and sequencedwith 2 times 300 nt SBS kit v3 on an Illumina MiSeq apparatus

25 scFv Recovery from the Enriched Sublibrary The threeselected clones were isolated from the population of scFv atcycle 3 The QuickChange II XL Site-Directed MutagenesisKit (Agilent Technologies) was used to perform extensionreactions with overlapping primers designed within thecorresponding HCDR3 regions

The extension reactions were assembled as follows 50ndash250 ng of template 25 120583L QuickSolution reagent 1 120583L PfuUltra High Fidelity DNA polymerase (25U120583L) 5 120583L 10xreaction buffer 1120583LdNTPmix 125 ng forward primer 125 ngreverse primer H

2O to a final volume of 50 120583L


SM Cycle 1 Cycle 4Cycle 3Cycle 2

VH

(a)

80

70

60

50

40

Cycle 1 Cycle 4Cycle 3Cycle 2

Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000

Cycle 1 Cycle 2 Cycle 3 Cycle 4

(d)

Figure 1 Library screening and analysis of sequences (a) The panel shows the DNA fragments gel-purified from sublibraries after theindicated selection cycles The corresponding plasmid preparations were digested with NcoI and XhoI restriction endonucleases to releasethe DNA fragments encoding for the VH regions of the scFv fragments The fragments were bar-coded and subjected to high-throughputsequencing as described in the text SM sizemarker (b)The chart reports the entropy values for the populations of fragments originating fromthe indicated selection cycles after sequencing (c)The reported values indicate the total number of clones and the relative representation ofthe most abundant clone within the corresponding selection cycles (d) The chart indicates the relative distribution of clones according tothe number of counts observed within the indicated ranges for each of the 4 selection cycles Cycles 3 and 4 show similar distributions

The primers used were

3 2 forward 51015840-GAGTTATTATCCATTTGACTA-CT-31015840 3 2 reverse 51015840-AGTAGTCAAATGGAT-AATAACTC-31015840 3 5 forward 51015840-CGAGAGACT-ACTACGGACTTGACTACTG-31015840 3 5 reverse 51015840-CAGTAGTCAAGTCCGTAGTAGTCTCTCG-310158403 67 forward 51015840-CGCGTGGGGCAGGAGGAG-CCTTTGACTACTG-31015840 3 67 reverse 51015840-CAGTAG-TCAAAGGCTCCTCCTGCCCCACGCG-31015840

The template DNA was removed by restriction with1 120583L of DpnI enzyme as suggested by the kit provider Anappropriate amount of reaction was used to transform XL10-GOLDULTRACOMPETENTCELLS (Agilent Technologies)

and then plated on LBagar containing 100120583gmLAmpicillinSome colonies were picked and the screen success wasevaluated by double digestion and sequencing

26 Preparation of Phage Particles Electrocompetent TG1cells were transformed with dsDNA plasmid of rescuedclones and grown in 100 120583L of 2xTY medium containing1 glucose 25120583gmL Kanamycin and 100 120583gmL Ampicillinfor 18 hours at 37∘C Then TG1 cells were infected withthe M13-K07 helper phage The culture was centrifugedat 1200 rpm for 30min to pellet bacteria and recover thescFv phage containing supernatant useful for ELISA PEGprecipitation was used as previously described to concentratephage particles


27 Antibody Production and Purification For the conver-sion of the selected scFvs into whole IgG4 the VHs andVLs were amplified by PCR and purified by agarose gelThen In-Fusion HD cloning kit (Clontech LaboratoriesMountain View CA USA) was used to insert the variablefragments in vectors expressing the constant antibody heavyand light chains The VHs were cloned in the linearized(BamHIBssHII) Peu 82 vector and the VLs were cloned inlinearized (ApaLIAvrII) Peu 42 vector Stellar CompetentCells (Clontech Laboratories IncMountainView CAUSA)were transformed with obtained vectors and the colonieswere screened by digestion and sequence analysisThe correctpreps were cotransfected in HEK293-EBNA by using Lipo-fectamine Transfection Reagent (Life Technologies Inc) andgrown up for about 10 days at 37∘C in serum-free CD CHOmedium (Gibco Life Technologies Inc) in 6-well platesTheconditioned media were collected and the antibodies werepurified by using Protein A HP SpinTrap (GE HealthcareLife Sciences New York USA) The primers used were thefollowing

For VH

3 2 3 5 3 67 51015840-CTCTCCACAGGCGCGCACTCC-GAGGTGCAGCTGTTGGAGT

Rev VH

3 2 3 5 51015840-GGCATTGGGTGGATCCGTCGACAG-GACTCACCACTCGAGACGGTGACCATTGTC-CC

3 67 51015840-GGCATTGGGTGGATCCGTCGACAG-GACTCACCACTCGAGACGGTGACCGTGGTC-CC

For VL

3 67 51015840-CTCCACAGGCGTGCACTCCCAGTC-TGTGTTGACGCAGCCG

3 2 51015840-CTCCACAGGCGTGCACTCCCTTAATTT-TATGCAGACTCAGCCCC

3 5 51015840-CTCCACAGGCGTGCACTCCCAATCTGC-CCTGACTCAGCCT

Rev VL

3 2 3 5 3 67 51015840-TTCTGACTCACCTAGGACGGT-CAGCTTGGTCCCTCC

28 ELISA To confirm the binding specificity for CLDN1of the selected scFv phages or purified mAbs cell ELISAwere performed by using HEK293 T CLDN-1 positive andmock cells The cells were detached with nonenzymatic celldissociation solution (Sigma-Aldrich) and washed with PBSand then resuspended in PBSBSA 6 in 96 multiwell plates(2 times 105cellswell) The phages or mAbs were added toplate and incubated for 30 minutes at RT The following

antibodies were used to reveal binding of phage-scFvs orof the corresponding antibodies mouse HRP-conjugatedanti-M13 mAb (GE Healthcare Bio-Sciences AB UppsalaSweden) goat HRP-conjugated anti-human IgG (PromegaCorporation Madison USA) After 3 washes cells wereresuspended and incubated for 2 minutes in 50120583L of TMBreagent (Sigma-Aldrich) After the incubation the reactionwas stopped through addition of 50 120583L of 1N HCl and theabsorbance (A450) was measured

3 Results

31 HTS-Based Screening of a Phage Display Library onCLDN1 Expressing Cells For isolation of CLDN1 scFvs thephage display library was subjected to 4 selection cycles eachcycle consisted of a subtractive step on HEK-293 cells notexpressing the antigen on the cell membrane followed bypanning onHEK-293 cells transducedwith CLDN1 construct[1] In order to maximize the exposure of proteins on thecell membrane panning and the subtractive steps were per-formed on suspension cultures Phages from each selectionstep were collected and amplified for recovery of dsDNAphagemid DNA preparations were digested with NcoI andXhoI restriction endonucleases to excise the subcollectionsof VH fragments (Figure 1(a)) The isolation of the VHfragments (350 bp on average) was preferred to the isolationof the whole scFv fragments (about 750 bp in length) in orderto get full sequencing coverage of the most variable HCDR1HCDR2 and HCDR3 regions In order to minimize loss ofrepresentation of clones we preferred excision of the VHfragments by restriction enzyme digestion rather than theiramplification by PCR Thus the unique amplification stepof the whole procedure was implemented for bar-coding ofthe sublibraries The bar-coded VH fragments from the fourselection cycles were finally sequenced on a MiSeq Illuminaplatform (see Section 2) We also combined cycles 1 and 4in a run and cycles 2 and 3 in an additional run to test thepossibility to further reduce the costs of the analyses Theaim of analysis was to reveal the most abundant clones aswell as their enrichment profiles throughout the selectionrounds

As a parameter of complexity of 4 sublibraries we initiallyexplored the number and the diversity of HCDR3s fromeach selection cycle through evaluation of the entropy (Fig-ure 1(b)) a strong decrease of entropy occurred throughoutthe 4 cycles of selection Accordingly the relative represen-tation of the most abundant clone inside each sublibrarywas progressively increasing over cycles (maximal relativerepresentation from 076 to 2549) while the complexity(ie the number of different clones) was accordingly decreas-ing over more than one order of magnitude (Figure 1(c))Finally as detailed in Figure 1(d) during the selection cycleswe observed that an increasing percentage of sublibrarieswas occupied by VH fragments with high counts untilcycle 3 cycle 4 showed distributions of counts similar tothose observed in cycle 3 thus indicating that selectionof CLDN1 binders was bona fide completed after threecycles



Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3

FR1 FR2 FR3 FR4 Linker VL

(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)

Figure 2 Selection of scFv clones and strategy for recovery (a) The chart reports the relative enrichments within the indicated selectioncycles for 75 scFv clones The threshold for inclusion was arbitrary set to a relative representation value of 1 times 10minus3 (01) Most clones weremaximally enriched at cycle 3 Compared to cycle 4 cycle 3 also showed the highest number of different clones with a relative representationgt1 times 10minus3 (75 versus 63) Cycle 3 was accordingly selected for recovery of scFv clones (b)The cartoon describes the strategy implemented forrecovery of scFv clonesThemethylated template DNA from cycle 3 sublibrary was copied by PfuDNA polymerase from overlapping primers(block arrows) corresponding to specific sequences within HCDR3 region of VH The dashed lines represent the newly synthesized DNAnonmethylated since it was generated in vitro After DpnI digestion methylated and hemimethylated DNAs are removed so that the nickedDNA originating from template copy is able to transform competent E coli cells The originating colonies thus represent the recovered bonafide scFv clones (c)The panel shows the products of the extension reactions carried out on template from selection cycle 3 with overlappingprimers for HCDR3 regions of clones 3 2 3 5 and 3 67The upper bands correspond to the supercoiled methylated template the lower bandsrepresent the primer-extended nicked products SM size marker T template DNA

32 Recovery of scFv Clones from Sublibraries As shown inFigure 1(d) cycles 3 and 4 show similar distributions of clonescharacterized by high counts For further analysis we focusedon clones for which relative representation was above 1 times10minus3 Cycle 3 gave the highest number of clones above suchthreshold 75 versus 63 clones from cycle 4

Thus we analyzed the enrichment profiles for each of the75 clones from selection cycle 3 as shown in Figure 2(a)most clones were already enriched from cycle 1 to cycle2 some clones (30 in number) reached their maximal

enrichment at cycle 2 while 37 clones were still increasingtheir representation over cycle 3 The remaining clones (8 innumber) showed comparable enrichment values from cycle 2to cycle 3 Considering cycles 3 and 4 19 clones were showingincreasing enrichments while 49 actually showed decreasedrepresentation at cycle 4The remaining clones (7 in number)did not show relevant changes from cycle 3 to cycle 4 Thusmost scFv clones reach the maximal enrichment at cycle 3

We then selected cycle 3 for recovery of the scFv clones Tothis aimwe took advantage of an approachwhich is routinely


used in molecular biology labs for site-directed mutagenesis(Figure 2(b)) The system allows us to obtain nicked plasmidDNA by enzymatic copy of a template the in vitro generatedDNA is then suitable for E coli transformation and isolationof the clones of interest Thus we subjected DNA templatesfrom cycle 3 to enzymatic copy with pairs of overlappingprimersThe oligonucleotide sequences were designed insidethe HCDR3 regions since they represent the most variable(thus selective in terms of DNA sequence) regions in theantibody repertoire The range in HCDR3 lengths for the 75clones was from 10 to 24 amino acidsWe selected the scFvs of3 different VH fragments characterized by CDR3 regions ofdifferent lengths the shortest (10 aa clone 3 5) was selectedsince it provides the tightest constraint in the design ofspecific primers The additional CDR3s were 13 aa- (clone3 67) or 17 aa-long (clone 3 2) Clones 3 2 and 3 5 werehighly enriched within cycle 3 (corresponding frequencieswere resp 8 times 10minus2 and 5 times 10minus2) while clone 3 67 was closeto the lowest enrichment (frequence was 1 times 10minus3) among the75 selected

As shown in Figure 2(c) nicked DNA was generated foreach of the selected clones In order to remove the librarytemplate which could give rise to undesired scFv clonesthe samples were digested with DpnI which cleaves themethylated and hemimethylated templates while preservingthe fully in vitro generated nonmethylated DNA The result-ing DNAs were transformed in E coli to obtain bona fidephagemidDNAs corresponding to the selected VHsThe cor-responding constructs were isolated from the transformationreactions with occasional retrieval of undesired constructsSanger sequencing of the recovered 3 2 3 5 and 3 67 clonesconfirmed 100 identity of the VH regions to the HTS datafor each of the three clones Sanger analysis also allowed us toidentify their corresponding VL sequences

33 Validation of Binding for scFv Fragments and ConvertedAntibodies Purified phage particles for clones 3 2 3 5 and3 67 were generated and tested by cell ELISA to validatetheir binding Two out of the three tested clones (3 5 and3 67) showed a specific binding to CLDN1 expressing cells(Figure 3(a)) Clone 3 2 instead revealed binding to bothcell cultures Thus we focused on clones 3 5 and 3 67 forfurther experiments They were converted into human IgG4antibodies Figure 3(b) shows that the isolated VH and VLregions of these clones actually generate full antibodiesThey were also tested in ELISA to validate their binding toCLDN1 exposed on the surface of HEK-293 cells Figure 3(c)shows that the corresponding antibodies actually maintainthe ability to bind specifically CLDN1 expressing HEK-293cells as for the corresponding scFvs from which they weregenerated

4 Discussion

In this paper we report a complete workflow for HTS-based isolation of scFv phagemid clones binding to nativeCLDN1 a cell surface protein involved in HCV infectionHTS-based screening of phage display libraries starts to

become a useful method to isolate putative scFvs for antigensinvolved in diseases ranging from viral infections to cancerThis approach may have some advantages compared to theclassical screening schemes such as the possibility to compar-atively evaluate the complexities of the sublibraries from eachselection cycle and the corresponding enrichments of phageclones from which to derive functional antibodies against agiven antigen This allows us for instance to decide whetherto stop or to continue the screening for a given antigen In ourcase the screening strategy was composed of four selectioncycles each one characterized by progressive decreases inentropy Cycle 3 however showed maximal enrichments formost clones since the majority of the 75 scFv constructsselected for further analysis dropped their relative repre-sentation during selection cycle 4 The main interpretationfor this occurrence is that cycle 4 represents a plateau forour selection thus rendering ineffective additional selectioncycles

During classical screening procedures much effort isdedicated to repetitive tests isolation and sequencing ofclones at completion of multiple downstream selectioncycles Following a HTS-based screening instead each ofthe enriched clones is known in advance and then tested forbinding at a single occurrence Thus HTS-based screeningwill reveal the widest possible set of enriched clones limitingthe possibility to lose good binders during repetitive isolationand characterization of active scFvs Our experimental setupalso provides a sustainable alternative to classical screeningsince HTS costs are kept to the lowest combining multiplesamples in a single sequencing run Accordingly after havingperformed 4 cycles of selection we combined cycles 1 and4 in a run and cycles 2 and 3 in an additional run TheIllumina MiSeq platform was used demonstrating its properadaptability to a screening approach The versatility and thecheap costs (on average 1000 USD per run in the interna-tional market) of our approach may expand the applicabilityof such HTS-based screening to the selection of scFv clonesfor multiple targets

There is however a disadvantage in the use of HTS-based screening compared to classical approachesThe latterin fact allows for direct isolation of phagemid DNA forbiochemical validation of binding via production of solublescFv protein fragments On the contrary there is the needonce the enriched clones have been identified to recoverthem from DNA preparations of the enriched sublibrariesSome methods have been developed to overcome the prob-lem of recovering selected clones one of them was based onoverlapping PCR reactionsThese allowed the reconstructionof full scFvs from 2 PCR products corresponding to VHand VL [18] An additional method provides single-stepisolation of complete phagemid DNA via a thermostableDNA polymerase and DNA ligase using an inverse PCRapplication with 51015840-phosphate oligonucleotides [20 21] Thelattermethod is like the one implemented in this paper basedon single-step recovery It was shown to be highly effectiveallowing recovery of a single scFv clone spiked into a libraryand represented to 00025 of the total DNA [21] It was alsoeffective in the recovery of scFv clones bearing short HCD3sequences due to the design of one oligonucleotide primer


0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4

Heavylight chain dimer

Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)

Figure 3 Evaluation of binding for scFv phages and IgG4 (a) The panel shows the binding of scFv phages clones 3 2 3 5 and 3 67 toHEK293 cells (gray bars) and to cells transduced with CLDN1 vector (HEK293-CLDN1 black bars) Clone 3 2 was discarded because ofnonspecific binding to HEK293-CLDN1 cells (b) SDS-PAGE analysis of IgG4 converted from the scFv clones 3 5 and 3 67 as indicatedSamples in lanes 1 and 3 were run under nonreducing conditions so that the whole IgG4 and the heavy-chainlight chain IgG4 dimers wereaccordingly visualized Under reducing conditions the IgG4 preparations showed the fully denatured light and heavy chains as indicated(c) The panels show the binding of the IgG4s converted from scFv fragments 3 5 and 3 67 to HEK293 (gray lines) and to HEK293-CLDN1(black lines) cells at increasing antibody concentrations

at the boundary between HCD3 and FR4 region and of thesecond primer within FR4 Our approach was fully validatedwithin an experimental screening for CLDN1 antibodies Wewere indeed able to identify 75 potential binders from whichwe decided to isolate 3 representative scFv clones selectedaccording to the length of the corresponding HCDR3 regions(range 10ndash17 amino acids) and within an experimentallyvalidated range of frequencies (from 8 down to 01 of thepopulation of clones represented within cycle 3) Its prelim-inary implementation by Zhang and coworkers [19] was not

fully exploited since these authors focused on hybridization-mediated capture of the selected clones via hybridizationwith biotinylated oligonucleotides designed within HCDR3regions Since the identification of an effective mAb againstSR-BI [1 3 7] our groups are actively isolating novel mAbsagainst cellular proteins involved in HCV infection [22]accordingly a wide search and characterization of novelantibodies preventing viral entry through the tight-junctionprotein CLDN1 is in progress In the present work two outof the three selected clones showed good binding specificities


to CLDN1 expressing cells 3 5 and 3 67 clone 3-2 howeveralthough highly enriched did not generate a specific bindingIts positive selection could represent a combination of abiological advantage and of the peculiarity of the system usedfor screening consisting of native CLDN1 expressed on thecell surface The epitope selected by clone 3 2 may representa very abundant protein expressed on HEK-293 cells so thatthe corresponding scFv is not efficiently removed during thenegative selections During a classical screening such clonewould have been selected and discarded at each cycle afterrepeated testing the HTS-based screening instead allows usto discard it at the firstunique characterization of binding

The validity of the whole procedure from screeningto antibody production was verified since the antibodiesgenerated by scFv conversions of clones 3 5 and 3 67 wererapidly and efficiently obtained they fully recapitulated thebinding properties of the scFv from which they were derivedAs a whole the application of the complete pipeline proposedwithin this work characterized by low costs and high effec-tiveness may guarantee rapid sustainable and successfulisolation of antibodies for multiple proteins against nativeantigens involved in human diseases

5 Conclusions

In this paper we screened scFv ldquophage displayrdquo library onsuspension cultures of HEK-293 cells expressing CLDN1on plasma membrane from which we successfully isolatedspecific CLDN1 binders The optimized high-throughputsequencing approach followed by a single-step recovery ofrepresentative full scFv constructs and their conversion toIgG4 antibodies demonstrated the versatility and scalabilityof the procedure to obtain rapid and cheap isolation ofantibodies for virtually any native antigen involved in humandiseases

Abbreviations

CLDN1 Claudin-1dsDNA Double-stranded DNAHCDR Heavy-chain complementarity

determining regionHTS High-throughput sequencingmAb Monoclonal antibodyscFv Single-chain fragment variableVH Heavy chain variable regionVL Light chain variable region



Acknowledgments

This work was supported by the EU FP7 Grant ldquoHepaMAbrdquo(305600) and POR ldquoRete delle Biotecnologie in CampaniardquomdashProgetto MOVIE The authors wish to thank Dr MT

Catanese for HEK 293-CLDN-1 cells and D Lazarevic and DCittaro (HSR) for optimization of sequencing and bioinfor-matic analysis

References

[1] M T Catanese R Graziani T von Hahn et al ldquoHigh-aviditymonoclonal antibodies against the human scavenger class Btype I receptor efficiently block hepatitis C virus infection inthe presence of high-density lipoproteinrdquo Journal of Virologyvol 81 no 15 pp 8063ndash8071 2007

[2] C De Lorenzo D B Palmer R Piccoli M A Ritter and GA DrsquoAlessio ldquoA new human antitumor immunoreagent specificfor ErbB2rdquo Clinical Cancer Research vol 8 no 6 pp 1710ndash17192002

[3] E Scarselli H Ansuini R Cerino et al ldquoThe human scavengerreceptor class B type I is a novel candidate receptor for thehepatitis C virusrdquo The EMBO Journal vol 21 no 19 pp 5017ndash5025 2002

[4] B Bartosch A Vitelli C Granier et al ldquoCell entry of hepatitisC virus requires a set of co-receptors that include the CD81tetraspanin and the SR-B1 scavenger receptorrdquo The Journal ofBiological Chemistry vol 278 no 43 pp 41624ndash41630 2003

[5] M J Evans T von Hahn D M Tscherne et al ldquoClaudin-1 is ahepatitis C virus co-receptor required for a late step in entryrdquoNature Letters vol 446 pp 801ndash805 2007

[6] A Ploss M J Evans V A Gaysinskaya et al ldquoHuman occludinis a hepatitis C virus entry factor required for infection ofmousecellsrdquo Nature vol 457 no 7231 pp 882ndash886 2009

[7] P Meuleman M T Catanese L Verhoye et al ldquoA humanmonoclonal antibody targeting scavenger receptor class B typeI precludes hepatitis C virus infection and viral spread in vitroand in vivordquo Hepatology vol 55 no 2 pp 364ndash372 2012

[8] M Yamashita M Iida M Tada et al ldquoDiscovery of anti-claudin-1 antibodies as candidate therapeutics against hepatitisC virusrdquo Journal of Pharmacology and Experimental Therapeu-tics vol 353 no 1 pp 112ndash118 2015

[9] I Fofana S E Krieger F Grunert et al ldquoMonoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection ofprimary human hepatocytesrdquo Gastroenterology vol 139 no 3pp 953ndash964 2010

[10] M Fukasawa S Nagase Y Shirasago et al ldquoMonoclonal anti-bodies against extracellular domains of claudin-1 block hepatitisC virus infection in a mouse modelrdquo Journal of Virology vol 89no 9 pp 4866ndash4879 2015

[11] L Mailly F Xiao J Lupberger et al ldquoClearance of persistenthepatitis C virus infection in humanized mice using a claudin-1-targetingmonoclonal antibodyrdquoNature Biotechnology vol 33no 5 pp 549ndash554 2015

[12] C Welsch F S Domingues S Susser et al ldquoMolecular basis oftelaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virusrdquoGenome Biology vol 9 no1 article R16 2008

[13] J-M Pawlotsky ldquoTreatment failure and resistance with direct-acting antiviral drugs against hepatitis C virusrdquoHepatology vol53 no 5 pp 1742ndash1751 2011

[14] N M Dabbouseh and D M Jensen ldquoFuture therapies forchronic hepatitis Crdquo Nature Reviews Gastroenterology andHepatology vol 10 no 5 pp 268ndash276 2013

[15] T J Liang and M G Ghany ldquoCurrent and future therapiesfor hepatitis C virus infectionrdquo The New England Journal ofMedicine vol 368 no 20 pp 1907ndash1917 2013


[16] R T Chung andT F Baumert ldquoCuring chronic hepatitis Cmdashthearc of amedical triumphrdquoTheNewEngland Journal ofMedicinevol 370 no 17 pp 1576ndash1578 2014

[17] R S Brown Jr ldquoHepatitis C and liver transplantationrdquo Naturevol 436 no 7053 pp 973ndash978 2005

[18] U Ravn F Gueneau L Baerlocher et al ldquoBy-passing in vitroscreeningmdashnext generation sequencing technologies applied toantibody display and in silico candidate selectionrdquoNucleic AcidsResearch vol 38 no 21 article e193 2010

[19] H Zhang A Torkamani T M Jones D I Ruiz J Ponsand R A Lerner ldquoPhenotype-information-phenotype cyclefor deconvolution of combinatorial antibody libraries selectedagainst complex systemsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 108 no 33 pp13456ndash13461 2011

[20] S DrsquoAngelo S Kumar L Naranjo F Ferrara C Kiss and AR Bradbury ldquoFrom deep sequencing to actual clonesrdquo ProteinEngineering Design and Selection vol 27 no 10 pp 301ndash3072014

[21] A Spiliotopoulos J Owen B Maddison I Dreveny H Reesand K Gough ldquoSensitive recovery of recombinant antibodyclones after their in silico identification within NGS datasetsrdquoJournal of Immunological Methods vol 420 pp 50ndash55 2015

[22] R Paciello R A Urbanowicz G Riccio et al ldquoNovel humananti-Claudin 1 monoclonal antibodies inhibit HCV infectionand may synergize with anti-SRB1 mAbrdquo Journal of GeneralVirology 2015







Contents

































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References





























Contents

































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References


























Contents

































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References
























Contents

































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References
















































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References





































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References
































1 Introduction















2O Amplifications











2OThe touchdown
























3 Results








Gminus




















lowast
















1 2 3

Recombinant protein


M 1

70KD

40KD50KD

30KD

25KD

14KD


4 Discussions










5 Conclusions


Competing Interests


Acknowledgments


References





































1 Introduction














2





3 Results



Cellisolation

RNAextraction

Dataanalysis


specific genes














Freq

uenc

y


0

50

100

150

200

250

300











Vary

ing

mix

ing

ratio

s

100

5

1

05

01

005

001


1

2

3

4

5

6

7








Erro

r (m

ain

study

)

1e minus 06

1e minus 04

1e minus 02

1e + 00



PDL0

5000

MTX

PDL0

5020

MTX

PDL0

5080

MTX

PDL1

0000

MTX

PDL1

0020

MTX

PDL1

0080

MTX

PDL1

5000

MTX

PDL1

5020

MTX

PDL1

5080

MTX

0501


0

20

40

60

80







Vary

ing

PDL

MTX

trea

tmen

t

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80


2

4

6

8



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80



Vary

ing

PDL

MTX

trea

tmen

t


2

4

6

8

PDL50MTX = 0

PDL50MTX = 20

PDL50MTX = 80

PDL100MTX = 0

PDL100MTX = 20

PDL100MTX = 80

PDL150MTX = 0

PDL150MTX = 20

PDL150MTX = 80





4 Discussion








Competing Interests




References



























1 Introduction












4)2SO4 2 gL K

2HPO4 and

02 gL MgSO4(pH 75)








BamHI

BamHI

BamHI

BamHI

BamHI

HindIII

HindIII

HindIII HindIII

HindIII

PCR Amplification

pgsB pgsC and pgsA

pgsB pgsC and pgsA

T4 DNA ligase

T4 DNA ligase



1

pWB980

P43 promoterrep

ble

NheI

NheI

KpnI

KpnI

1

pWB980-pgsBCA

P43 promoter

rep

ble

pgsBCA

1

pMD19-T

LacZ

ori

1

pMD19-T-pgsBCA

LacZ

ori 55kb

27 kb

66 kb

38kb

28 kb

Kmr

Kmr

Ampr

Ampr







3 Results







1 M

4500bp3000 bp2000 bp

1200 bp800 bp

500bp

200bp


1 M2

4500bp3000 bp2000 bp1200 bp800 bp

500bp

200bp


0

04

08

12

16

2


Prod

uctio

n of

120574-P

GA

(gL

)



1 2 3


3421

1649

1408

1076

540

4000 3500 3000 2500 2000 1500 1000 500 00

10

20

30

40

50

60

70

80

T (

)

n (cmminus1)


2or -CH

3(flex-



-CH2or -CH







M 1 2

600kDa

440kDa

230kDa

140kDa

67kDa









Competing Interests


Acknowledgments


References









































1 Introduction































1205814 and VH1-S121



Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

A41

0 (A

u)

3

2

1

0

05

35

25

15

T0

T0T0

T30

T30

(a)

Acet

ate

Arg

inin

e F

T

Arg

inin

e

Arg

inin

e

Hist

idin

e F

T

Hist

idin

e

Hist

idin

e

0

1

2

3

4

5

SEC

aggr

egat

es (

)

T0

T0

T0

T30

T30

(b)





Histidine

1

HC

LC

2 3 4

Acetate






lowast 0ndash387










0263 027 098 03737015 027 056 06010015 027 056 06010

027 08945027 09295


00070lowast

t ratiot ratio

minus014minus004

minus003 minus009

Prob gt |t|

(a)

Std error

ArgininelowastNaCl



00022lowast

t ratiot ratio

minus011 minus064

minus035

minus014

minus006

minus003


02 018 113 030900188 018 106 03371015 018 085 04347

018 05523018 07380018 08930

(b)








NaCl

pH

NaC

lpH

50 60 70 80 90 100 110 63 64 65 66 67 68

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

(a)

Arginine

Histidine

Arg

inin

eH

istid

ine

0

005

01

015

02

A41

0

0

005

01

015

02

A41

0

160140 220120 180 200100 40 453025 50 5535(b)


HisArg FT0

005

01

015

02

A41

0

HisArg T30HisArg T0



4 Conclusions



Highlights





Disclaimer




Acknowledgments




References

































1 Introduction




























LoD = 33 sdot 120590119904

LoQ = 10 sdot 120590119904

(1)






80

75

70

65

60

pH

25 30 35 40 45

09

07

05

03

Opt

ical

den

sity

Temperature (∘C)

(abs

)

(a)

10987654

pH

25 30 35 40 45

10

08

06

04

02

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(b)

10

11

9

8

7

pH

40 42 44 46 48 50

100

090

080

070

Temperature (∘C)

Opt

ical

den

sity

(abs

)

(c)









15

10

05

00

Opt

ical

den

sity

(abs

)

50 100 150 200 250 300 350 400

Time (min)


150200250300

0




Levels

minus1414 3793 617minus1000 4000 7000000 4500 9001000 5000 11001414 5207 1180









2



000 025 050 075 100 125 150 175


40

30

20

10

0

Azo

case

in co

ncen

trat

ion

(mg

mL)

102255

51

7631014

1514

2008

2498

2983





LoD = 33 sdot 120590119904

=

33 sdot (006295)

147572

= 01407686Abs

= 0072mgmL

LoQ = 10 sdot 120590119904

=

10 sdot (006295)

147572

= 04265714Abs

= 0494mgmL

(3)


119860

(U) =119862AZO sdot 119881

2


(4)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Time (days)








ptic

al d

ensit

y (a

bs)

00

05

10

100

200

300

20 30 40

15


Time (days)


Opt

ical

den

sity

(abs

)

00

05

10

1

200

300

2 3

15


Freeze-thaw cycles



4 Conclusion


Nomenclature




Acknowledgments


References





























1 Introduction












4) Chloramphenicol









600)







pNWPH

VR

VF

Prolonged overlap

extension PCR

(35 cycles)

1st PCR 2nd PCR

n


insert(30 cycles)


plasmid

Denaturation

Annealing

Extension



Monomer

Multimer

1stcycle

2ndcycle

Further cycles


GCSFIFIR

pGCSF-08

pNWPH-GCSF

SDgsiB

SPymwC

(sim38 kb)

3422bp572bp

PHbaII

repB

HindIIIGCSFNdeI

Cat











3 Results







(sim33 kb)

(sim05 kb)

(kb) 21M

050

100

300

Insert

Vector

(a)

(kDa) 7654321M

10

15

203035

4050

200

(b)

20 40 60 80 100 1200Time (hours)

0

1

2

3

4

5

6

7

OD600

(c)

0

20

40

60

80

100

120

140

GCS

F (120583

gm

L)

20 40 60 80 100 1200Time (hours)

(d)






60060 was clarified






(kDa) M GCSF200

5040302520

15

100

200

400

600

800

1000

1200

Abso

rban

ceA280

(mAU

)

1501005000


0

20

40

60

80

100

NaC

l gra

dien

t (0

-1M

)

B

(a)

GCSF

195 205 215 225 235 245 255185Wavelength (nm)

minus15

minus10

minus5

0

5

10

15

20

Circ

ular

dic

hroi

sm (m

deg)

Filgrastim

(b)


280and A








4 Discussion




(a)

Control 15 4015 40


ControlcGCSFrhGCSF

0

10

20

30

40

50

60

70

Neu

troph

ils (

)

(b)









5 Conclusion




Acknowledgment


References





















































1 Introduction



















VH

(a)

80

70

60

50

40


Entropy for CDR3

(b)

Cycle 1 2 3 4

Maximalrelative

enrichment076 324 930 2549

Total number of

clones

151013 57545 40881 11835

(c)

142834

7443

592

91

53

47372

9733

298

62

66

12

2

33256

741413627

32

6

4

6

9656

201510022

32

5

5

100

()

80

60

40

20

0

gt20000 501ndash1000

101ndash500

2ndash100

11001ndash5000

5001ndash10000

10001ndash20000


(d)









For VH


Rev VH



For VL




Rev VL




3 Results





Rel

ativ

e en

rich

men

t

1

01

001

0001

00001

000001

0000001

(a)

CH3

CH3

CH3

CH3

CH 3 CDR1 CDR2 CDR3


(b)SM T

Supercoiled

template

Ext product

3_2 3_5 3_67

(c)










4 Discussion






0

02

04

06

08

1

12

HEK293

HEK293-CLDN1

3_23_53_67

Ab

sorb

ance

450

nm

(a)

Mature IgG4


Light chain

Heavy chain

1 2 3 4

3_53_67

140kDa

50kDa

25kDa

(b)

Concentration (nM)

00

02

04

06

08

10

12

0 20 40 60

3_67

Ab

sorb

ance

450

nm

0 50 100 150 200 250

Concentration (nM)

3_5

00

02

04

06

08

10

12

Ab

sorb

ance

450

nm

HEK293

HEK293-CLDN1

HEK293

HEK293-CLDN1

(c)







5 Conclusions


Abbreviations





Acknowledgments



References
























Documents

Upstream and Downstream of Recombinants Biomolecules to