How stable are new biologics?Insights into the stress profiles of an antibody-based biopharmaceutical drawing on the example

of Adalimumab

Bettina Katterle

Eurofins BioPharma Product Testing Munich GmbH, Munich

Correspondence: Dr. Bettina Katterle, Eurofins BioPharma Product TestingMunich GmbH, Behringstr. 6/8, 82152 Planegg/Munich, Germany

n ABSTRACTThe interest in biopharmaceuticals, mostly recombinantproteins and/or peptides, is increasing worldwide in healthcare[1]. To ensure long-term stability and safety of proteins in thedrug life-time from release through to product shelf life andfinally patient use, the drug formulations were developed bybiopharmaceutical companies to keep drug properties stableeven after unexpected periods of storage temperature varia-tions. In comparison to most chemically synthesised products,biopharmaceuticals are particularly sensitive to changes intemperature andhumidity, light and oxygen exposure, and shearforces. If a therapeutic protein cannot be stabilized adequately, itwill partially or totally lose its therapeutic properties or evencause immunogenic reactions thus potentially further endan-gering patients’ health. In order to understand and monitorchanges in the complex structure of biopharmaceuticals duringtheir production and lifetime, various validated physicochem-ical, biochemical and immunochemical analytical test methodsshould be employed.

n ZUSAMMENFASSUNGWie stabil sind biologische Wirkstoffe? Stressprofile vonAntikörperwirkstoffen am Beispiel AdalimumabBiopharmazeutika, meist rekombinante Proteine und/oderPeptide, sind weltweit von steigendem Interesse für dasGesundheitswesen [1]. Um die Langzeitstabilität und Sicher-heit der biopharmazeutischen Arzneimittel von der Freigabeüber die Produktlagerung bis hin zum Patientengebrauch zugewährleisten, werden Arzneimittelformulierungen von bio-pharmazeutischen Unternehmen speziell entwickelt, um dieEigenschaften auch nach unerwarteten Lagertemperatur-schwankungen stabil zu halten.Im Vergleich zu den meisten chemisch synthetisiertenProdukten sind Biopharmazeutika besonders empfindlich

gegenüber Tempera-tur- und Feuchtig-keitsschwankungen,Licht- und Sauer-stoffbelastung sowieScherkräften. Wennein therapeutischesProtein nicht ausrei-chend stabilisiertwerden kann, ver-

liert es seine therapeutischen Eigenschaften ganz oderteilweise oder verursacht sogar immunogene Reaktionen, diedie Gesundheit der Patienten weiter gefährden können. UmVeränderungen in der komplexen Struktur von Biopharma-zeutika während ihrer Herstellung und Lebensdauer zuverstehen und zu überwachen, sollten verschiedene validiertephysikalisch-chemische, biochemische und immunochemi-sche analytische Testmethoden eingesetzt werden.

1. Introduction

This article describes the exposure of the commerciallyavailable therapeutic antibody Humira® (Adalimumab)to the various stress conditions in order to detect anypossible change of the monoclonal antibody sequenceand structure (monoclonal antibody – mAB).

Quality control (QC) testing of protein biologics isone of the critical parts of biopharmaceutical manufac-turing. The analytical results of various QC testing meth-ods help to define and monitor the batch to batch varia-tions [2]. Any profound structural changes in the proteinwill affect activity and safety. In this case, actions haveto be taken. Formulated biopharmaceuticals, the finaldrug product, are developed to preserve the drug sub-stance stability and the formulation has become a keyaspect of the pharmaceutical development.

In general, stability studies must be performed onboth the drug substance and the final drug product. Thegoal of a stability study is to ensure that the productquality remains the same over a certain time period.This is used, among other things, to establish recom-mended storage conditions and shelf-lives. Correspon-dent International Conference on Harmonisation (ICH)guidelines define the types of studies, analytical testingsand their specifications [2–5].

The ICH guideline Q1A(R2), chapter 2.1.2, emphasizesthat the stress testing on the drug substance should beemployed for the assessment of parent drug stability, theinherent stability characteristic and the setting of thedrug shelf life. This includes the development of test stu-dies to ascertain the stability of the drug substance withthe suitable stability-indicating analytical procedures.

n KEY WORDS

• Stability of Biopharmaceuticals• Stress Conditions• Analytical Testing• Chromatographic and Electro-phoretic Methods

Pharm. Ind. 80, Nr. 11, 1557–1563 (2018)

Wissenschaft und Technik

Originale

Pharm. Ind. 80, Nr. 11, 1557–1563 (2018)© ECV • Editio Cantor Verlag, Aulendorf (Germany) Katterle • New Biologics 1557

Therefore, during the development of the analyticalprocedures as well as their validation, it is critical toshow that the methods are capable of detecting modi-fied protein forms or degradation products of drug prod-uct samples resulting from product instability. In casethat degradation standards (impurity standards) are notavailable, chapter 1.2.2 of the ICH guideline Q2 (R1) re-commends including samples in the validation programthat are stored under relevant stress conditions such aslight, heat, humidity, acid/base hydrolysis and oxidation.Therefore, controlled force-degraded experiments undervarious conditions have to be set up in order to show ifthe analytical independent, orthogonal methods are ca-pable of detecting those formed impurities.

Commonly, even an unstressed recombinant (monoclo-nal) antibody displays already a complex heterogeneousprofile caused by its sequence and structure and the possi-bility of post translational modifications, for instance C-terminal lysine processing, aggregates and N-linked glyco-sylation deamidation and oxidation sites ( fig. 1). The sitesof possible post translational modifications are known forwell characterized antibodies. Here, the characterizationwork of the different modifications is predominately per-formed by using highly advanced mass spectrometrymethods. The different modifications are regarded asproduct-related substances or impurities (terminology de-pends on their effects on activity and safety). Table 1shows the selected physicochemical analytical techniquesused for assessing the product–related substances or im-purities in the quality control process.

Various and independent (orthogonal) analyticalmethods as listed in Table 1 are typically used for releaseand stability testing in order to determine identity andimpurity. They are mainly chromatographic and electro-phoretic separation techniques. Activity testing usingcell-based bioassay and/or binding assays completes therelease and stability testing panel.

In short-term accelerated studies, typically a proteinsample is exposed to elevated temperatures to acceleratethe degradation (truncation) and/or aggregation. Acidicor basic pH can promote aggregation, hydrolytic degrada-

tion or deamidation. Peroxide or other oxidizing agentsmight be used to induce oxidation of methionine [8]. This“worst case” approach reflects sample alterations thatmight be detected in the drug substance and drug prod-uct samples during long-term and short-term storage.

The ICH guideline Q2 (R1) describes the validation ofanalytical procedures. The validation of product-relatedsubstances or impurities aims to validate either quantifi-cation or % limit [3 and table therein]. If the % limit isevaluated, parameters such as specificity precision (in-termediate precision, repeatability) and limit of detec-tion should be included in the validation. The collectedvalidated data ensure ruggedness or robustness and re-liability of the state-of-the-art analytical methods.

A stress study of Adalimumab, a commercially avail-able formulated monoclonal antibody, has been de-signed to demonstrate that the independent, state-of-the-art sensitive testing methods are able to detect thevarious modifications to the structure of the protein.Stress testing is desirable as “worst case scenario” be-cause degradation pathways and reaction kinetics willbe able to be identified or at least degradation productswill be able to be characterized. Different stress condi-tions were chosen to induce degradation and/or aggre-

n Table 1

Assessment of the purity / impurity profile of a monoclonal antibody by a combination of orthogonalmethods.

Attributes Aim of the test Analytical technique

Structure/post-translational modi-fications

Deamidation/oxidationN/C-terminal modifications

Peptide mapping high-performance liquid chromatog-raphy (HPLC) / ultra high performance liquid chro-matography (UPLC) (UV, MS)Assessment of disulfide bridges

Intact LC/HC, HMW/LMW CE-SDS/HPLC (UPLC) (UV; MS)

Product related substances and im-purities

Charge heterogeneity/Isoforms iCIEF (UV)

Aggregates SE-HPLC/UPLC (UV)

Carbohydrate profiling HPLC/UPLC (FL, MS)

Figure1:General structureofanantibodywith450 to550aminoacids.The heavy chain (HC), the light chain (LC) aswell as the common sitesprone to posttranslational modifications are depicted (Source:Eurofins BioPharma Product Testing Munich GmbH [10]).

Wissenschaft und TechnikOriginale

1558 Katterle • New BiologicsPharm. Ind. 80, Nr. 11, 1557–1563 (2018)

© ECV • Editio Cantor Verlag, Aulendorf (Germany)

gation as well as post translational modifications, e.g.,oxidation of methionines or deamidation.

2. Materials and Methods

2.1 Stability and degradation at acceleratedconditionsThe commercially available recombinant monoclonal antibody was sub-jected to various stress conditions. For this series of stress experimentsfor this publication it was decided to perform only one experimental setup per condition. The reason is that robustness validation is generallyperformed with a similar set-up. The aim was to monitor new or disap-pearing peaks in order to understand which kind of effects can be ex-pected if the protein had been stressed. Hence, evaluation of precision(repeatability and intermediate precision) was not in the scope of theexperiments presented here. The chromatographic and electrophoreticruns are only evaluated semi-quantitatively (e.g., in (limit) %) for decreas-ing or new peak area(s). The resulting qualitative stress profiles are regu-larly observed in client-specific validations of monoclonal antibodies.

Here, the impact of thermal stress, 55 °C for a time period of up to72 h, acidic (0,1 N HCl), basic (0,1 N NaOH) and oxidative stress(3 % H2O2) was investigated. The acidic and basic stress conditionscould not be measured since the protein was denatured and could notbe subjected to the different analytical methods. The remaining sam-ples were analyzed by two different capillary electrophoresis methods(CE – SDS), imaged capillary isoelectric focusing (iCIEF), and by sizeexclusion chromatography (SEC). Aggregation amount, charge var-iants, level of high-molecular-weigth (HMW) and low-molecular-weight (LMW) variants were evaluated. The obtained data were com-pared to the data of the unstressed antibody.

Adalimumab drug product samples (50 mg/mL) were subjected tothermal stress by incubating at 55 °C for a time period of 24 h, 48 h and72 h. Prior to analysis, samples were diluted with water to 10 mg/mL.

In order to subject Adalimumab to oxidative stress, H2O2 in a finalconcentration of 3 % (v/v) was added to 50 mg/mL drug product sam-ples. The samples were then diluted to a concentration of 40 mg/mLby adding purified water and incubated for 24 h at room temperature.

Finally, samples were diluted to 10 mg/mL with purified water beforeanalysis. The unstressed samples were processed in the same way, butthe stress agent was excluded. They were analyzed in parallel. Thestressed samples were aliquoted and analyzed by the techniques de-scribed below. Only one chromatographic and electrophoretic run perstress condition has been evaluated.

2.2 System suitability of the chromatographicand capillary electrophoretic methodsInternally validated methods were employed to detect degradationprofiles of Adalimumab drug product samples. Defined system suit-ability criteria were applied. For the electrophoretic and chromatog-raphic methods described below, the relative standard deviation (RSD)should be less than 1 % for retention/migration time and for peak areaevaluated for controls/marker proteins.

2.3 Reduced and non-reduced CapillaryElectrophoresis – Sodium Dodecyl SulfateAll experiments were performed on the PA 800 Plus PharmaceuticalAnalysis System (SCIEX) controlled by Empower3 ChromatographyData Software (Waters). A bare fused-silica capillary of 50 μm i.d. x360 μm o.d. x 30.2 cm total length were used for the separation.

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) was usedfor separation of denatured protein size variants under non-reduced orreduced conditions. For non-reduced CE-SDS (nrCE-SDS), samples weredenatured with sodium dodecyl sulfate at 70 °C for 5 min in the presenceof iodoacetamide (Sigma). For reduced CE-SDS (rCE-SDS), 2-mercap-toethanol (Bio-Rad) was added to the protein denaturation step to reducethe disulfide bonds. After denaturation, both non-reduced and reducedsamples were injected into a bare fused-silica capillary (Beckman Coulter,Brea, CA, USA) and separated based on hydrodynamic size resulting froman applied electric field in which migration of smaller sized proteins isinversely related to overall size. Analytes were monitored by UV absor-bance at 220 nm. Purity was evaluated by determining the corrected peakarea (CPA) of each species as a percentage of the total peak area. A10 kDamarker protein (Beckman Coulter, Brea, CA, USA) was used.

2.4 Imaged capillaryisoelectric focusingCharge profiles/isoelectric points (pI) of theanalytes were determined by imaged capil-lary isoelectric focusing (icIEF) analysis usingan iCE3 system (ProteinSimple) equippedwith a 5 cm x 100 µM ID fluorocarbon coatedcapillary. Samples were separated accordingto their pI and detected by using a whole col-umn UV absorption detector (280 nm) thatavoids disturbing focused protein zones. TheiCE CFR Software (ProteinSimple) were usedfor automated pI calibration and data con-version. The sample peaks were integratedwith Empower 3 Software (Waters) in orderto determine the distribution between acidic,basic and neutral forms in percent. pI mark-ers with pI 8.18 and pI 9.46 were used.

2.5 Size Exclusion HighPerformance LiquidChromatography with UVdetectionAggregates were analyzed by size exclusionchromatography (SEC) with UV absorbance(280 nm). SEC measurements were made on

Figure 2: Analysis of mAB Monomer, aggregates (HMW) and degradation products (LMW) bySizeExclusionChromatographybeforeandafter thermalstress;mABincubatedfor72 hat55 °C(black line),untreatedMABcontrol sample (blue line);AU(280 nm)absorbanceunits. Inset:Theevaluation of% total peak area of themonomer (main peak) and the HMWand LMWspecies isshown. The decrease in monomer and increase in LMW and HMWwith time (h) was detected.

Pharm. Ind. 80, Nr. 11, 1557–1563 (2018)© ECV • Editio Cantor Verlag, Aulendorf (Germany) Katterle • New Biologics 1559

a Waters Acquity H-class Bio UPLC system with a Tosoh Biosciences(Minato, Tokyo, Japan) TSK-GEL G3000SWXL column (5 µm, 7.8 x300 mm). 100 µg samples were injected into the UPLC system. The lev-els of high molecular weight (HMW) species, monomer and low molec-ular weight species (LMW) were detected and their distribution inpercent of total peak area evaluated.

3. Results

3.1 Formation of aggregates and degradedproducts detected by SE chromatography undernative conditionsAggregates of monoclonal antibodies can be already in-duced during the manufacturing process due to manyparameters such as pH, temperature, reactive oxygenspecies or shear forces and contribute to the heteroge-neity profile of the drug substance and drug product.They are regarded as product-related impurities sincethey are known to affect drug activity and safety. In theauthors’ experience, the acceptance criteria (specifica-tions) for release and stability are set to around 2 % oreven less in quality control testing . Protein aggregatescan be categorized in soluble/insoluble, covalent/non-covalent, reversible/non-reversible and native/dena-tured [6]. Non-covalent aggregates of the monoclonalantibody can be separated and detected under nativeconditions by size exclusion chromatography. As seen infig. 2 the monomeric peak of the antibody of 147 kD (re-tention time16 min) and ag-gregates (HMW)as well as thedegraded frag-ments (LMW)were separatedand quantifiedas % of mono-mer peak area.

FormulatedAdalimumabshowed a slightlyincreased forma-tion of higher or-der aggregates at55 °C over time(maximum 72 h)than the un-stressed formu-lation ( fig. 2) butnot higher than1–2 % in total.This is expectedsince the formu-lated antibodywas stressed.However, theharsh conditions

led to a pronounced degradation (inset of fig. 2) of themonomer (decrease of the main peak at the retention timeof 16 min, increase of LMW fraction).

3.2 Formation of aggregates and degradedproducts detected by CE electrophoresis underdenaturing conditionsProduct-related substances and impurities including dif-ferent size or modified variants can be analyzed by re-duced capillary electrophoresis (rCE) and non-reducedcapillary electrophoresis (nrCE; fig. 3 and 4) to identifycovalently associated aggregates and incomplete disulfide– linked fragments. Fragment species are considered acritical quality attribute and are routinely monitored toassess the antibody integrity. The unstressed antibodyshows the glycosylated heavy chain (HC) and the lightchain (LC) as well as a low level of non-glycosylated heavychain (NG) under reduced conditions ( fig. 3). After ther-mal stress the proteins starts to degrade as already de-tected by SEC and peaks with lower molecular weight asHC were detected. The amount of HC decreases whereasthe impurities increase. The amount of light chain (LC)was not significantly affected (inset of fig. 3).

The nrCE shows the arising degradation profile underthermal stress more clearly ( fig. 4). The fragments (con-sidered as impurities) can be separated from the IgGmonomer. The IgG monomer decreased to around 60 %.

Figure 3: Analysis of thermal stressed mAB by reduced CE-SDS; mAB incubated for 72 h at 55 °C (blue line),untreated MAB control sample (black line); AU (220 nm) absorbance units; NG – non-glycosylated species; LC –light chain, HC – heavy chain; 10 kDa marker. Inset: The evaluation of % total peak area of the heavy chainpeak (HC), the light chain (LC) and impurities is shown. The decrease in HC and increase in impurities withtime (h) was detected.

Wissenschaft und TechnikOriginale

1560 Katterle • New BiologicsPharm. Ind. 80, Nr. 11, 1557–1563 (2018)

© ECV • Editio Cantor Verlag, Aulendorf (Germany)

The amount of monomer or LC/HC has been evaluatedin % of total corrected peak area. The corrected peakarea is calculated by peak area multiplied by the capil-lary length; the result is divided by the migration time.

Altogether, the results contribute to the understand-ing of potential impurity profiles of a monoclonal anti-body after exposure to the stress conditions.

3.3 Charge variants detected by imaged capillaryisoelectric focusingImaged capillary isoelectric focusing (iCIEF) for theanalysis of therapeutic antibodies and antibody drugconjugates provides a fast separation and resolution ofthe acidic (A), neutral (MI) and basic (B) variants of anantibody as shown in fig. 5 and 6). The dominant neutralmajor isoform (MI) does not contain structures with C-terminal lysine(s). It consists primarily of the heavychain domain glycosylated via the asparagine with bian-tennary structures with 0, 1 or 2 terminal galactoses (seegeneral antibody structure in fig. 1).

The charged isoforms are caused by heterogeneityin the glycosylation, i.e., content of sialic acids, trun-cation, mannose but also by common post transla-tional modifications such as deamidation, oxidationand fragmentation. For instance, methionine oxida-tion may be a post translational modification thatcan impact the bioactivity of the antibody and poten-tially induce an immunogenic response [8, 9]. MostIgG1 antibodies have two conserved heavy chain (HC)

methionine re-sidues locatedat the FcRnbinding inter-face of the con-stant heavychain domains.It was reportedthat oxidationof these twomethionine re-sidues de-creased thermalstability, pro-tein A binding,FcRn binding,and circulationhalf-life of IgG1antibodies [8].

Table 2 sum-marizes exam-ples of chemicalchanges for for-mation of acidicand basic var-iants [6]. Duringstress condi-

tions the variants can be formed. The yielding iCIEF pro-file can be used for the control during release and stabil-ity testing. In fig. 5 it is shown that the analyte is sepa-rated according to its pI, a low pI compared to the mainpeak comprises the acidic variants while a high pI com-pared to the main peak represents the basic variants( fig. 4, control). Due to the thermal stress (55 °C, 72 h)the basic variants increase. Here, mainly the inducedfragmentation as already shown by the capillary electro-phoresis can be assumed to be the reason. The acidicvariants are only slightly increased.

Figure 6 shows the separation of a sample treated with3 % hydrogen peroxide. Under oxidative stress, the acidicspecies increased predominately whereas the major iso-form and the basic isoform decreased. This is different tothe results of the thermal stress. Here, the increase of theacidic forms may be due to fragmentation of the oxidizedprotein. It has been described that the variety of acidicforms may be based on multiple modifications [6].

4. Conclusion

State-of-the-art analytical testing methods for assessingthe heterogeneity profile of proteins have been used inorder to analyze the main quality attributes of antibodyproducts such as aggregation amount, charge variants,level of low-molecular-weight (LMW) variants. For in-stance, the phenomenon of protein aggregation (e.g.,covalent/non-covalent) is a common issue that compro-

Figure 4: Analysis of thermally stressed mAB by non-reduced CE-SDS; mAB incubated for 72 h at 55 °C (blue line),untreatedMAB control sample (black line), IgGmonomer (main peak) at 28 min; AU (220 nm) absorbance units; NG –non-glycosylated species; 10 kDamarker. Inset:Theevaluationof%totalpeakareaof the IgGmonomerand impurities isshown. The decrease in IgG monomer and increase in impurities with time (h) was detected.

Pharm. Ind. 80, Nr. 11, 1557–1563 (2018)© ECV • Editio Cantor Verlag, Aulendorf (Germany) Katterle • New Biologics 1561

mises the qual-ity, safety, andefficacy of anti-bodies and canhappen at dif-ferent steps ofthe manufactur-ing process, in-cluding fermen-tation, purifica-tion, final for-mulation, andstorage.

The stressstudy performedon a commer-cially availableformulated anti-body yielded indifferent impu-rity profiles thatwere monitoredwith size exclu-sion chromatog-raphy (SEC), re-duced and non-reduced capil-lary electro-phoresis (rCEand nrCE) andimaged capillaryisoelectric fo-cusing (iCIEF).It showed thatthe protein de-grades underthermal and oxi-dative stress ex-hibiting trunca-tions and differ-ent charge dis-tribution as theunstressed ref-erence samples.The results indi-cate the impor-tance of a stressstudy as de-scribed in theICH guidelineQ2 (R1) in orderto confirm theability of themethod to de-tect differentdrug modifica-

Figure 5: Charge profiles of thermally stressed mAB measured by imaged capillary isoelectric focusing; mABincubated for 72 h at 55 °C (blue line), untreated mAB control sample (black line); pI markers at 8.18 and 9.46;AU (280 nm) absorbance units; pI – isoelectric points. Inset: The evaluation of % total peak area of the acidicspecies (A), the major isoform (MI) and the basic species (B) is shown. Predominately, the percentage of thebasic species increased, and the major isoform decreased whereas the acidic percentage is mainly unaffected.

Figure 6: Charge profiles of mAB after oxidative stress measured by imaged capillary isoelectric focusing; mABincubated for 24 h with 3 % H2O2 (blue line), untreated MAB control sample (black line); pI markers at 8.18 and9.46; AU (280 nm) absorbance units; pI – isoelectric points. Inset: The evaluation of % total peak area of theacidic species (A), the major isoform (MI) and the basic species (B) is shown. The percentage of the basicspecies and the major isoform decreased whereas the acidic increased.

Wissenschaft und TechnikOriginale

1562 Katterle • New BiologicsPharm. Ind. 80, Nr. 11, 1557–1563 (2018)

© ECV • Editio Cantor Verlag, Aulendorf (Germany)

tions as well as to assess the robustness of the used ana-lytical technique.

n REFERENCES[1] Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges

for the next generation of therapeutic antibodies. Nat Rev Immu-nol. 2010 May;10(5):345–52.

[2] Q6B: Harmonized Tripartite Guideline. Specifications: Test pro-cedures and acceptance criteria for biotechnological/biologicalproducts. 1999.

[3] Q2(R1): Validation of Analytical Proce-dures: Methodology, Guidance for In-dustry. U.S. Department of Health andHuman Services, FDA, CDER, CBER. No-vember 1996.

[4] Q1A(R2): Stability Testing of New DrugSubstances and Products. In: Proceed-ings of the International Conference onHarmonisation, IFPMA. Geneva, 2003.

[5] Q5C: Quality of Biotechnology Products.Stability Testing of Biotechnological/Biological Product.

[6] Torashvand F, Vaziri B. Main Quality At-tributes of Monoclonal Antibodies andEffect of Cell Culture Components. IranBiomed J. 2017 May;21(3):131–41.

[7] Dada OO, Jaya N, Valliere-Douglass J, Sa-las-Solano O. Characterization of acidic

and basic variants of IgG1 therapeutic monoclonal antibodiesbased on non-denaturing IEF fractionation. Electrophoresis. 2015Nov;36(21–22):2695–702.

[8] Mo J, Yan Q, So CK, Soden T, Lewis MJ, Hu P. Understanding theImpact of Methionine Oxidation on the Biological Functions ofIgG1 Antibodies Using Hydrogen/Deuterium Exchange MassSpectrometry. Anal Chem. 2016 Oct 4;88(19):9495–502.

[9] Hermeling S, Crommelin DJ, Schellekens H, Jiskoot W. Structure-immunogenicity relationships of therapeutic proteins. PharmRes. 2004 Jun;21(6):897–903.

[10] General Presentation of Eurofins BioPharma Product TestingMunich GmbH.

n Table 2

Various post-translational modifications that may result in chargeheterogeneity. Examples are listed in [6].

Acidic Variants Basic Variants

de-amidation (Asn residue) C-terminal Lys

fragments methionine oxidation

reduced disulfide bonds incomplete disulfide bonds

glycosylation – e.g., sialic acids; mannoses de-glycosylation

non-classical disulfide linkage fragments

ECV · Editio Cantor Verlag

BestellungTel. +49 (0)711-6672-1924 · Fax +49(0)711-6672-1974eMail svk@svk.de · Webshop, Leseproben und Inhaltsverzeichnisse

Auslieferung und Rechnungsstellung unserer Produkte erfolgt durch unseren Vertragspartner Stuttgarter Verlagskontor SVK GmbH. www.ecv.de

Beschrieben werden die Stabilitätsprüfungen für synthetische und biotechnologische Wirk-stoffe sowie für Phytopharmaka. Exemplarisch wird gezeigt, wie man auf rationellem Weg vom Wirkstoff zur erfolgreichen Zulassung kommt.

Schwerpunkte der Darstellung sind:• Packmittelwesen• Definitionen• Die naturwissenschaftlichen Grundlagen

(z.B. Veränderungen des Wirkstoffes,Untersuchungsmethoden, Reaktionskinetik)

• Stabilitätsprüfungen in der Praxis(ein schließlich biotechnologischer Zubereitungen und Phytopharmaka),auch Organisationsformen der Prüfungen

Zielgruppen

• Pharmazeutische Industrie• Zulieferindustrie• Auftragslabore• Behörden• Apotheker in Klinik und Offizin• Hochschulen / Fachhochschulen

Stabilitätsprüfung in der PharmazieTheorie und PraxisGrimm W, Harnischfeger G, Tegtmeier M

ISBN 978-3-87193-408-7• 192,00 €• 3., vollständig überarbeitete und erweiterte Auflage• 17 x 24 cm, Hardcover, 579 Seiten

• Ausführliche Kommentierungen von behördlichen Vorgaben auf internationalerwie auf nationaler Ebene, v. a. von den einschlägigen ICH Guidelines einschließlichder Biotech-Guidelines

• Beurteilung der Haltbarkeit in Offizin undKrankenhausapotheke

• Berücksichtigung der unterschiedlichenKlimazonen