Profiling the Effects of Process Changes on Residual Host Cell Proteins inBiotherapeutics by Mass Spectrometry

Matthew R. Schenauer, Gregory C. Flynn, and Andrew M. GoetzeDept. of Process and Product Development, Amgen Inc., Thousand Oaks, CA

DOI 10.1002/btpr.1748Published online May 21, 2013 in Wiley Online Library (wileyonlinelibrary.com)

An advanced liquid chromatography=mass spectrometry (MS) platform was used to iden-tify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance(DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles wereobserved among different biotherapeutic Pbs as well as one Pb purified via multiple proc-esses. The results can be rationally interpreted in terms of differences among the purificationprocesses, and demonstrate the power of this technique to sensitively monitor both the quan-tity and composition of residual HCPs in DS, where these may represent a safety risk topatients. The breadth of information obtained using MS is compared to traditional multi-product enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same sam-ples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCPcomposition of two upstream samples was also analyzed and used to demonstrate that HCPsthat carry through purification processes to be detectable in DS are not always among thosethat are the most abundant upstream. Compared to ELISA, we demonstrate that MS can pro-vide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitatingprocess development as well as more rationally assessing potential safety risks posed byindividual, identified HCPs. VC 2013 American Institute of Chemical Engineers Biotechnol.Prog., 29:951957, 2013Keywords: mass spectrometry, host cell proteins, biotherapeutics

Introduction

Modern recombinant biotherapeutics are typically pro-duced in non-human cell lines. Despite rigorous purification,low levels of host cell protein (HCP) impurities can remainin the final drug product (DP). Residual HCPs representpotential safety risks for patients, including immunogenic-ity,1 adjuvant activity,2,3 decreased product stability due toenzymatic activity,4 or, more theoretically, direct biologicalactivity.5 To reduce such concerns, clearance of HCPs tolevels deemed safe is required by regulatory agencies.6 Thiscould be considered even more important for todays high-dose therapeutics, such as antibody products, which are oftendosed at 100 mg, in contrast to the lower doses used forfirst-generation biotherapeutics such as insulin and growthhormone (10 mg=dose). With high-dose products, a patientmight receive impurity HCPs at levels comparable to theactive protein in first-generation biotherapeutics. Truly mean-ingful a priori evaluation of potential risks associated withresidual HCPs in DPs requires both identification and quanti-fication of the individual HCPs present, as individual pro-teins can vary widely with respect to attributes that mightgenerate safety concerns (e.g., immunogenicity). In addition,

total HCP levels may be less relevant than the amount ofspecific, high-risk protein(s). With HCP identification andindividual quantification, modern tools such as in silico or invitro prediction of immunogenicity could be used to helpassess safety risks; in addition, over time, clinical experiencewith common HCPs may become correlatable with levels ofspecific HCPs in DPs.

To date, HCP levels have been most commonly monitoredby multianalyte enzyme-linked immunosorbent assay(ELISA), using polyclonal antisera raised against large num-bers of HCPs present during an upstream process step.7,8

This type of assay provides a single numerical result repre-senting the totality of immunoreactive HCPs and is oftenused as a lot release specification test. However, it providesno HCP identification, and the accuracy of total HCP quanti-fication is questionable as: (a) it is difficult to obtain, anddemonstrate, proportional antibody coverage against allpotential HCPs and (b) the assay standard is unlikely tomatch the HCP composition (analyte) of the sample beinganalyzed, which is a fundamental requirement for quantita-tive analytical assays. Consequently, numerical results areantisera (and cell-line) specific, and, because most major bio-therapeutic companies develop their own proprietary anti-sera, not interchangeable between sponsors. Theseuncertainties likely contribute to the lack of a universal HCPspecification target, although with each sponsors uniqueELISA, values between 1 and 100 ppm (w=w) HCP areoften reported for approved products.7 However, in this para-digm, informed risk assessment, based on identification and

Current Address of Matthew R. Schenauer: 1 DNA Way, South SanFrancisco, CA 94080.

Additional Supporting Information may be found in the online versionof this article.

Correspondence concerning this article should be addressed to A.M.Goetze at goetzeam@amgen.com.

VC 2013 American Institute of Chemical Engineers 951

reliable quantification of individual HCP components in DPs,is not possible. Without sharing proprietary HCP ELISAreagents or manufacturing=purification procedures, compa-nies cannot meaningfully compare the HCP profiles of prod-ucts, including those of biosimilar and innovator products.Another significant drawback to lack of HCP identification isthat process improvements to reduce HCP levels must pro-ceed by trial and error, as rational process development thatexploits known differences in physiochemical propertiesbetween identified HCPs and product is not possible.

Mass spectrometry (MS) is now an indispensable tool forprotein characterization as well as for the characterization ofcomplex protein mixtures such as those used in proteomicsor biomarker discovery. Detecting ppm levels of HCPs in abiotherapeutic background presents major challenges to thedynamic range of such methods.9,10 One approach to miti-gate this challenge is to couple a high-resolution two-dimen-sional (2D) liquid chromatography (LC) separation withhigh-resolution MS. In one specific embodiment of thisapproach, 2D-LC=MSE, hereafter abbreviated as MSE, tryp-sin-digested samples are chromatographically separated by2D reversed-phase chromatography using high and low pHmobile phases in the first and second dimension, respec-tively, and the peptides are analyzed by MS.11,12 Data-inde-pendent acquisition methods such as MSE may offer higherduty cycles, improved chromatographic peak sampling, andmore reproducible mass spectra compared to traditional data-dependent acquisition methods.11,13,14 This approach hasdemonstrated the ability to identify, and quantify, individualHCPs present in biotherapeutic monoclonal antibodies(mAbs)9,10 and, importantly, to do so in an objective, antise-rum-independent manner.

Peptibodies (Pbs) are therapeutics in which bioactive pep-tides are fused to a human IgG1 Fc for improved stabilityand circulatory half-life.15 Pbs are typically expressed inEscherichia coli as inclusions bodies, which are subsequentlysolubilized (including complete reduction of all cysteines),refolded (with accurate disulfide bond pairing), and purified.We describe here a retrospective study using 2D-LC=MSE ofthe HCP content of one purified peptibody (Pb1) as a func-tion of process changes during development. Our focus wason the analysis of drug substance (DS), as this contains theHCPs at the stage at which they could pose a safety risk topatients. The breadth of information obtained by MSE is con-trasted to the single HCP ELISA number and is used to illus-trate the significant advantages provided by the MSE

information. The HCP profiles of two other purified Pbs,each with unique purification schemes, were also determinedand compared with those of Pb1. These results provideexamples of how both major and minor process changes canaffect the HCP profile in Pb DP. In addition, MSE was usedto compare the DS HCPs following extensive purificationwith their levels in two upstream samples. The type of

detailed information obtained retrospectively by MSE in thisstudy, could, in future applications, be used to more effi-ciently guide process development and, in principle, be usedas a starting point to better evaluate potential HCP-associ-ated safety risks.

Materials and Methods

The therapeutic peptibodies Pb1, Pb2, and Pb3 were pro-duced at Amgen (Thousand Oaks). Pb2 was produced usingan older, nonrepresentative process. Total E. coli HCP byELISA was determined using a multianalyte (and multiprod-uct) assay that was developed from commercial strain-appro-priate antisera raised against E. coli lysates and usesin-house E. coli lysate as assay standard and which was vali-dated according to ICH guidelines. DnaK was specificallyquantified using a polyclonal DnaK-specific ELISA devel-oped in house using commercial DnaK as both immunogenand assay standard and also fully validated. Identificationand quantification of HCPs by 2D-LC=MSE was performedessentially as previously described.10 Briefly, reduced, alky-lated tryptic protein digests were prepared for all samples.Chromatography was carried out on a Waters nanoAcquityultra-performance LC (UPLC) instrument with 2D technol-ogy. XBridge BEH 130 C18 (5 mm, 300 mm3 50 mm)Nanoease columns, Symmetry C18 (5 mm, 180 mm3 20mm) trap columns, and HSS T3 (1.8 mm, 75 mm3 150 mm)analytical columns (Waters Corporation, Milford, MA) wereused in all analyses. The first-dimension chromatographybuffers were 20 mM NH4HCO2, pH5 10, and acetonitrile,while second-dimension buffers were H2O and acetonitrile,respectively, each with 0.1% formic acid. MSE analyseswere carried out on a Synapt (G1) Q-IMS-TOF mass spec-trometer (Waters Corporation) operating in TOF V-mode.The eluate from the D2 column was sampled into the massspectrometer via a Z-spray nanosource (with lock mass)incorporating a universal sprayer and using PicoTip Emitters.Data were processed using ProteinLynx Global Server Ver-sion 2.4 (PLGS, Waters Corporation), and Microsoft Excel.As described previously, only high confidence (pass 1) pep-tides were used for quantitative DS analysis, and when highconfidence data on DS HCPs were available from additionalanalyses, these data were used to further restrict the quan-tity-indicating HCP peptides in DS to those ranking tenth orbetter in the higher confidence acquisitions.10

Optimal loading targets the HCPs to fall into the instru-ments dynamic range, often necessitating that the therapeu-tic be loaded at levels at least 1,000 times above that range.Changes in total protein loading across products and sampletypes were accommodated to facilitate optimal DS HCPdetection. For 10-fraction 2D-LC=MSE DS runs, 716 mgdigested protein were injected per run, while 2430 mg wereloaded in 20-fraction analyses. In all cases, restricted top 3peptide signals for identified HCPs were externally calibratedagainst the average response for 12 standard proteins spikedat various levels into therapeutic DS in both the 10- and 20-fraction modes.10 Pb1, Process 2, Unit Op A (see Table 1)eluate pool was analyzed with a 10-mg injection in a single10-fraction run. Null E. coli lysate (containing no therapeu-tics) was quantitatively analyzed with 390-ng injections inthree replicate 10-fraction runs to assess the 250 most abun-dant HCPs. One additional, nonquantitative 20-fraction runwas performed on the null material loaded at 7.8 mg to aidin identification of low-abundance proteins.

Table 1. Summary of Significant Differences Among Unit Operationsfor Pb1 Purification

Process: 1 2 3 4

Relative Refold Conc: 13 13 13 53Unit Op 1: Precipitation A A AUnit Op 2: A C C CUnit Op 3: B E EUnit Op 4: C D D D

AE5 unique separation modality, e.g., ion-exchange or hydrophobicinteraction chromatography.

952 Biotechnol. Prog., 2013, Vol. 29, No. 4

Criteria for identification of DS HCPs in this study variedfrom our previous study, and the stringency of criteria forconfident identification was somewhat relaxed to reflect thatthe method performance had largely been previously charac-terized10 and the substantial time requirements for excessivereplicate two-dimensional LC=MS analyses of many sam-ples. Criteria for DS HCP identification in this studyrequired that the HCP be identified in >50% of data acquisi-tions for a particular process. In cases in which only one setof data was obtained, the identification threshold was setwith a PLGS score of 500, a value we have previouslyfound to be reproducible in DS HCP analyses using PLGS2.4. Since only one analysis was performed on both the Pb1column pool 1 material and the high-sensitivity null lysateanalysis, the PLGS score 500 criteria were also applied.For the quantitative MSE null lysate analysis, HCPs musthave been identified in 2 of 3 runs to be consideredidentified.

Results and Discussion

Pb 1 HCP profiles

Table 1 provides a summary of the key differences amongfour distinct purification processes used during the develop-ment of Pb1. Process 1 was the initial purification processthat enabled first-in-human clinical studies; Process 2 wassubsequently developed to facilitate commercial-scale purifi-cation. A multiproduct E. coli-specific HCP ELISA showedlittle difference in total DS HCP levels between these proc-esses, yielding average values of 3 and 5 ppm (w=w, totalHCP=product) for Process 1 and Process 2 DS, respectively.In contrast, 2D-LC=MSE (MSE) identified significantly dif-ferent residual HCP impurity profiles between the samematerials. Two HCPs were identified in Process 1 DS, and11 HCPs were identified in Process 2 DS (Figure 1); onlyone of these (DnaK) was identified in common between bothprocesses. MSE quantification showed that this process

change had resulted in an increase in total detectable HCPsfrom 26 to 391 ppm (Table 2).

Levels of one specific HCP, DnaK, increased from 20 to96 ppm between the lots of Process 1 and 2 DS, as meas-ured by MSE. Identification of DnaK as a specific impuritywas confirmed by in-gel digestion and peptide map finger-printing of Process 2 DS. DnaK shares 53% sequence iden-tity with human HSP70, and its presence in a biotherapeuticat measurable levels was deemed a potential risk to induceanti-human HSP70 antibodies. MSE estimates for levels ofthis specific HCP were independently confirmed by DnaKELISA. With this ELISA, average values of 8 and 73 ppmDnaK were obtained for the lots of Process 1 and 2 DStested, respectively (Table 2).

Interestingly, the changes from Process 1 to Process 2, ini-tially monitored via the HCP ELISA to preserve a similarHCP impurity level, resulted in a highly distinct HCP profileby MSE. DnaK was the only common HCP across the twoprocesses, and its levels were significantly increased fromProcess 1 to Process 2. In this example, the MSE HCP pro-file of a DS sensitively reflected major process changes,whereas the HCP ELISA could not. The HCP ELISA valuesnot only did not reflect the increased amounts of DnaK inProcess 2, but also provided no indication that the HCP

Figure 1. Venn diagram showing distribution of identified DS HCPs among different Pb products and processes. Superscripts on thePb process numbers refer to the number of lots of DS analyzed for each process followed by the total number of MSE

acquisitions of those lots. For brevity, only the top 10 (previously identified) HCPs in Pb 2 are shown.10

Table 2. Average Quantification Results for DnaK and Total HCPby MSE and ELISA for Pb1 Processes

Pb1MSE ELISA

Process DnaK HCP DnaK HCP

1 20 26 8 32 96 391 73 53

composition and total HCP level had changed. Such an im-purity profile difference could represent an increased safetyrisk for patients, although no effort was made to assess this.In this case, MSE made this information available, whereasthe use of HCP ELISA did not.

Four Process 2 DS lots were analyzed quantitatively byMSE; they were chosen to represent the extremes in DnaKlevels as measured by ELISA. Nine of the eleven HCPs iden-tified for Pb1, Process 2 in Figure 1 were identified in allfour of these lots, while two were identified in three lots. Fig-ure 2 plots the MSE-determined concentrations of the ninemost abundant Process 2 HCPs against the DnaK ELISA-determined DnaK concentration, which itself shows verygood quantitative correlation with MSE.10 The other eightHCPs appear to fall into two classes: those whose concentra-tions scale in proportion to that of DnaK (MiaB, AsnA, andNarP) and those whose concentrations are approximately con-stant, independent of the DnaK levels (HsIU, YhbS, YdhR,and NfuA). These two groups of proteins did not exhibitobvious differences in pI or amino acid composition. It isconceivable that the proteins that scale similarly to DnaKmight be removed from the product with similar efficiency atthe same key chromatographic step(s) as DnaK, whereas theremainder might be cleared at step(s) that have less impacton DnaK concentration.

Process 3 was the result of adding a new purification stepto Process 2 (Table 1), with the specific goal of reducing re-sidual DnaK. Following identification of DnaK, differencesin physiochemical properties between DnaK and Pb1 werereadily exploited for DnaK removal with the addition of atailored chromatography step. As a result, DnaK levels werereduced from 73 ppm to 5 ppm by DnaK ELISA and from96 ppm to below detection by MSE. In contrast, the HCPELISA was unable to detect a significant HCP level differ-ence in Process 3 compared to Processes 1 or 2 (Table 2). Inaddition to greatly decreasing residual DnaK in Process 3,the spectrum of identified, non-DnaK HCPs was reduced toa smaller subset of those identified in Process 2 DP (Figure1). The latter observation is reasonable since Process 3 dif-fered from Process 2 only by the addition of a single unitoperation. With such a change, the quantity and spectrum ofHCPs might be expected to decrease, but no new HCP(s)

would be likely to be identified, as observed here. MSE

therefore not only confirmed that the targeted reduction inDnaK levels had been achieved in the augmented purifica-tion scheme, but also demonstrated that multiple other HCPshad been reduced in parallel. The latter is information thatthe HCP ELISA failed to provide and that a singly targetedELISA is fundamentally incapable of providing.

In a final change to Process 4, the protein concentrationduring the refolding step was increased 5-fold over that ofProcess 3. As measured by their respective ELISAs, neithertotal HCP nor DnaK levels changed significantly as a result.However, MSE provided more detailed information, demon-strating that the number of detectable HCPs had increased.Three more HCPs were detected in Process 4 DS comparedto Process 3 DS; two of these were also present in process 2DS but another (HinT) had not been previously observed inany Pb1 DS (Figure 1). The fact that Pb1, Process 4 DS wasonly analyzed once notwithstanding, one could easily con-ceive that a five fold higher protein concentration at a keyprocess step might result in a wider spectrum of residualHCPs carried though to DS, as observed here.

Pb2 and Pb3

The purified DS of two other peptibodies, Pb2 and Pb3,were also analyzed by MSE. Forty-three HCPs were confi-dently identified in Pb2 DS.10 Six HCPs were identified inPb3 DS. These results are summarized in Figure 1, which, forsimplicity, shows only the 10 most abundant Pb2 HCPs. Inthese Pbs, the presence of DnaK was nearly ubiquitous, beingidentified (by MSE) in the Pb1 DS resulting from two of fourpurification processes as well as in Pb2 and Pb3 DS, but alsosuggested to be present at lower concentrations in the othertwo Pb1 processes based on the DnaK ELISA. The proteinPhoP was the only other HCP found in more than one prod-uct, being detected in Pb1, Processes 2, 3, and 4, as well asin Pb3. However, the large majority of HCPs identified inPb2 and Pb3 DS were unique to their respective processes,which suggests that, for the most part, each therapeuticPb presents unique HCP clearance challenges, a findingperhaps not unexpected given the large number of HCPsinitially present, their tremendous diversity with respectto physicochemical properties, each Pbs distinct physico-chemical properties, and the unique purification schemesused.

Comparison of DS HCPs to those in upstream samples

To gain some quantitative insights into the carry-throughof E. coli HCPs into DS, two upstream samples were ana-lyzed for HCPs by MSE for comparison with HCPs in DS.The first sample was a null cell lysate of the cell line usedto produce Pb1, Pb2, and Pb3. In the absence of productexpression, the HCP expression profile has been shown to behighly similar to what would be observed for the productioncell line when producing product.16 Overall, 274 E. coli pro-teins were identified in at least 2 of 3 10-fraction runs of thenull E. coli lysate, while 1,539 proteins were identified in asingle 20-fraction run loaded with 20-fold more protein. Asecond sample consisted of the first column pool of Pb1,Process 2 (Table 1, Process 2, Unit Op 1 eluate pool). ByHCP ELISA, this pool contained 2,000 ppm total HCP.MSE identified 154 HCPs in this sample, the 50 mostabundant of which are listed in Supporting InformationTable 1.

Figure 2. Comparison of the MSE-determined concentrationsof the eight most abundant secondary DS HCPs inPb1, Process 2 lots with the primary HCP, DnaK.

954 Biotechnol. Prog., 2013, Vol. 29, No. 4

Table 3 lists a composite of all HCPs identified in Pb1and Pb3 DS, as well as the top 10 HCPs identified in the sig-nificantly less pure Pb2 DS. Each of the identified DS HCPswas then mapped back as to whether it was identified in ei-ther of the two analyzed upstream samples. Overall, 25 ofthe 26 DS HCPs were identified in the null cell lysate. How-ever, only 16 of these (62%) ranked among the 250 mostabundant proteins in the null cell lysate, indicating that pro-teins of relatively low abundance in upstream material mayalso persist into the DS. This finding has implications for thedevelopment of multicomponent HCP ELISA antisera, sincea proteins abundance in the null material could impactwhether the protein elicits a sufficient antibody response inthe immunized animal. Nevertheless, these initially low-abundance proteins, as shown here, can persist through puri-fication (and potentially even become concentrated in DS viacopurification). It is worth noting that MS quantifies basedon moles of peptide(s) detected, whereas ELISA resultsexpressed in ppm are mass-based. With MS, larger HCP pro-teins may have an inherently higher probability of beingdetected, based on generating a larger number of highly ion-izable, and detectable, peptides. However, for an equivalentweight-based HCP level (expressed in ppm), smaller proteinsare expected to be quantified with greater sensitivity, since alarger molar quantity of peptide(s) will be involved. Howthese competing factors play out and potentially influencethe comparison between ELISA and MS-based HCP quantifi-cation remains to be determined.

All DS HCPs identified in Pb1, Process 1, 2, or 3 werealso identified in the upstream Pb1 Unit Op 1 eluate pool,while only one HCP in the Process 4 DS was not identifiedin that same column pool. In contrast, six of the ten mostabundant HCPs in Pb2 DS and three of the six DS HCPs inPb3 were not detected in the Pb1 SM1 eluate pool, indicat-ing that divergence of HCP profiles may occur very early inthe purification process.

Because lower-abundance upstream HCPs were alsodetected in DS it is likely that for many DS HCPs, copurifi-cation can be attributed to sharing one or more physiochemi-cal properties with the product, thereby providing relativelylittle basis for chromatographic separation during purifica-tion. Ion-exchange forms a common modality for the purifi-cation of these Pbs and, in this regard, it is striking how, forthe most part, the isoelectric points (pI) of residual DS HCPscluster close to, or slightly below, that of their respectiveproduct (Figure 3). Only Pb3, with a pI of 7.91, containedDS HCPs with pI > 7. One HCP that may copurify due to adifferent mechanism is the common residual HCP DnaK, amolecular chaperone known to bind segments of unfoldedproteins,17 which could be copurifying with the Pb throughdirect binding to an unfolded peptide portion of the drug, assuggested by native gel western blotting (data not shown).When HCP copurifies due to sharing physiochemical proper-ties with product, increased resolution or greater orthogonal-ity among purification modalities may be most effective inlowering HCP levels, whereas when copurification occursdue to binding to product, more stringent wash steps ofresin-bound product could be considered.

Multiproduct HCP ELISAs most commonly use celllysates or supernatants as the immunogen, in an effort togenerate the broadest possible antibody coverage.7,18 How-ever, more process-specific strategies, in which the HCPpool originating from null cells has gone through one ormore mock purification steps, have also been less frequentlyused.8,18 Process-specific assays offer the potential forgreater accuracy, as the composition of the assay standard islikely to more closely match that of downstream samples.The present results show that HCPs partition to a significantextent at each stage of the purification process, as early asthe first chromatography step, the conditions for which willtypically vary among Pbs. Even mildly process-specific ELI-SAs, using HCPs carried through a first (mock) purification

Table 3. Compilation of all HCPs Identified in Pb1, Pb2 or Pb3 DS Along with Status of Each Proteins Identification in Upstream Samples

Entry Identified E coli Protein MWAll Null Lysate

ECPs (1539 Total)250 Most AbundantNull Lysate ECPs

Unit Op 1Pool of Pb1, Pr2

ASNA Aspartate ammonia ligase=Asparagine synthetase A 36651 Y N YCH60 60 kDa chaperonin=groL 57329 Y Y23 YCLPB Chaperone protein ClpB 95585 Y Y51 NDNAK Chaperone protein dnaK Heat shock protein 70 68984 Y Y8 YERPA Iron sulfur cluster insertion protein erpA 12101 Y N YFLMA Stable plasmid inheritance protein flmA 6108 N N NFNR Fumarate and nitrate reduction regulatory protein 27967 Y N NGRCA Autonomous glycyl radical cofactor 14284 Y Y240 NHINT HIT like protein hinT 13241 Y Y234 NHNS DNA binding protein H NS 15540 Y Y60 NHSLU ATP dependent protease ATPase subunit HslU 49594 Y N YIDH Isocitrate dehydrogenase NADP 45757 Y Y5 NMIAB Dimethylallyl adenosine tRNA methylthiotransferase miaB 53663 Y N YMPRA Transcriptional repressor mprA 20564 Y N NNARP Nitrate nitrite response regulator protein narP 23575 Y N YNFUA Fe S biogenesis protein nfuA 20998 Y Y171 NNIFU NifU like protein 13849 Y Y227 YPFLB Formate acetyltransferase 1 85357 Y Y49 YPHOP Transcriptional regulatory protein phoP 25535 Y Y224 YPTKB Galactitol specific phosphotransferase enzyme IIB 10222 Y Y145 YRIMM Ribosome maturation factor rimM 20605 Y Y45 YRL3 50S ribosomal protein L3 22244 Y Y95 YSUCC Succinyl CoA ligase ADP forming subunit beta 41393 Y Y24 NYBEL Uncharacterized protein ybeL 18797 Y N YYDHR Putative monooxygenase ydhR 11288 Y Y226 YYHBS Uncharacterized N acetyltransferase YhbS 18534 Y N Y

Superscript in column 250 Most Abundant Null gives rank among top 250 identified ECPs.

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step for one Pb, will not generally be applicable to othersthat utilize altered conditions for the first purification stepsince, at this step, a major portion of potential DS impurityHCPs may already have been removed. This observation ismade possible by the fact that analytical technologies havenow advanced to the levels of resolution and sensitivity toallow detection DS HCPs for comparison with upstreamsamples, thus facilitating a significantly enhanced under-standing of not only our purification processes, but also themethods we have traditionally relied upon to characterizethem.

Conclusions

This study represents, to our knowledge, only the seconddetailed MS characterization of the HCP content of a bio-therapeutic DS=DP (following10), and the first that correlatesresidual HCP content with changes in the purification pro-cess steps used. It demonstrates not only that MS has suffi-cient sensitivity to identify and quantify multiple residualHCPs in highly purified Pb biotherapeutics from E. coli, butalso that the composition of residual HCPs in a single thera-peutics DS may sensitively reflect purification processchanges. Significant differences in both composition andquantity of individual HCPs as a result of process changeswere monitored by MSE. The technique sheds light on over-all changes in HCP impurity profiles, while quantification ofa specific HCP, DnaK, using this technology was corrobo-rated by a quantitative DnaK-specific ELISA. In contrast, amultiproduct E. coli-specific HCP ELISA failed to detectsignificant changes in DnaK levels as well as major changes

in overall HCP composition and quantities. These observa-tions likely reflect the fact that a single analyte ELISA is ca-pable of good accuracy, whereas quantification with amultianalyte ELISA, like that for HCP in the present study,can be problematic and even fail to detect key componentsdepending on the initial immunoreactivity of HCPs in theimmunogen and complement of HCPs that eventually persistinto DS. If residual HCPs present safety risks, our studyindicates that patient safety may not always be adequatelyassured with sole reliance on a traditional HCP ELISA. Onepotential lesson is that whereas the biotechnology industrytypically uses multiple high-resolution bioanalytical methodsto analyze the DP itself, this rigor is conventionally notmatched by the assay methodologies used to monitor resid-ual HCP. In our study, a validated HCP ELISA was clearlyunable to detect DnaK, a HCP that presented possible safetyconcerns, as well as other HCPs. As demonstrated in thepresent study, high-resolution LC=MS methodologies arenow capable of providing more comprehensive, and accurate,DS HCP characterization, thereby facilitating rational assess-ment of potential safety risks posed by individual, identifiedHCPs. This information can also be used to accelerate andimprove process development by intelligently removingnewly identified HCPs of concern by exploiting each compo-nents hitherto unknown physiochemical properties. In con-clusion, the deeper understanding of product quality withregard to HCPs provided by a method such as MSE, not onlyaddresses the expectations of the Quality by Design initia-tive,19,20 but also provides a viable path forward in address-ing HCP comparability for biosimilars as well as in othermanufacturing changes.

Acknowledgments

The authors would like to acknowledge Ken Chen, HaiPan, and Gang Huang for the original identification of DnaKas an impurity in Pbs, Susan Callahan for DnaK ELISAdevelopment and support, and Amy Hu, Brian Williamson,and Oliver Kaltenbrunner for process development.

Notation

2D-LC=MSE = the specific high-resolution two-dimen-sionalLC separation with high-resolution MStechnique used in this study

DP = drug productDS = drug substance

ELISA = enzyme-linked immunosorbent assayHCP = host cell protein

LC = liquid chromatographymAb = monoclonal antibody

MS = mass spectrometryMSE = the specific high-resolution 2D-LC

separation with high-resolution MS tech-niqueused in this study

Pb = peptibodypI = isoelectric point

UPLC = ultra-performance LC

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