A Novel Sample Preparation for Shotgun Proteomics Characterization

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A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies Lihua Huang, Ning Wang, Charles E Mitchell, Tammy J Brownlee, Steven R Maple, and Michael R De Felippis Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00304 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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Analytical Chemistry

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Lihua Huang, Ning Wang, Charles E Mitchell, Tammy Brownlee, Steven R Maple and Michael R De Felippis

Running title: Novel method for mAb HCP characterization

Corresponding Author: Lihua Huang, Ph.D., BioProduct Research and Development (317-277-1561) [email protected]

ACS Paragon Plus Environment


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Abstract Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection and quantitation purposes is most commonly used to monitor HCP removal during process development but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm HCP with molecular weight > 60 kD, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2DUPLC/IM/MSE method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.


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Introduction Host cell proteins (HCP) are process-related impurities of biopharmaceuticals derived from cell culture and may cause immunogenic reactions, adjuvant activity, product instability, and result in protein-specific biological activity [1-9] after injection. Thus, the downstream purification process must be designed to achieve robust removal of these impurities. An enzyme-linked immunosorbent assay (ELISA) with polyclonal anti-HCP antibodies used for capture, detection and quantitation purposes is most commonly used to monitor HCP removal during process development [10-11]. Accurate monitoring and measuring of residual HCPs in biopharmaceuticals by ELISA is highly dependent on the procedures used to generate the polyclonal antibodies and analytical standards. Generally, the antigens used for producing polyclonal antibody reagents and analytical HCP standards are obtained from a blank-run fermentation (or null strain) imitating the production run but lacking the specific coding gene for the protein product of interest. There are thousands of HCPs present in a null strain sample but not every host protein will produce antibodies for ELISA detection. HCP profiles in a null strain are very different from process samples, which may cause inaccurate quantitation by ELISA. Furthermore, ELISA provides no capability to identify specific host proteins. This information is essential to assess HCP immunogenicity risks of the biopharmaceutical [7] and potential effects on stability [8-9]. Application of mass spectrometry for protein identification using shotgun proteomics is now routine. However, if host proteins co-elute with the biopharmaceutical, a mass spectrometer with up to six orders of magnitude dynamic range is required to directly detect 1 to 100 ppm HCPs in the sample, which is out of range of current mass spectrometers. There are several ways to overcome the dynamic range issues for HCP characterization. One way is to resolve coeluting peptides before MS/MS analysis with data dependent acquisition (DDA) or data independent acquisition (DIA) by increasing the separation time [12-17], adding another dimension separation [6, 18-23], such as 2D-HPLC, or using ion mobility [24]. The other way is to enrich residual HCPs either by removing the therapeutic protein from the sample with affinity purification, such as using Protein A or G for an antibody [25-26], or by capturing residual HCPs with polyclonal antibodies [27]. In one study, near single digit ppm detection sensitivity [28] was reported for HCPs in biopharmaceutical samples using LC-MS/MS with DIA and a pre-



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established library comprising accurate masses, retention times and fragment ions for each peptide derived from HCPs in the null strain runs. The existing methods are powerful tools for detecting HCPs in biopharmaceutical samples but they also have some limitations. Generally, one dimensional or even two dimensional HPLC MS/MS is only capable of detecting 10 to 50 ppm HCP and the cycle times are very long, especially for 2D-HPLC (e.g., 1 to 2 samples/day). These techniques are not sensitive enough to detect very low level HCPs, which may be present in sufficient amount to cause product instability [9]. LC-MS/MS with DIA and a pre-established library is sensitive and has rapid throughput; however, the method may miss detection of HCPs which are only co-expressed with specific products. Furthermore, the technique is only suitable for a specific mass spectrometer and still suffers from the dynamic range issue for ion trap instruments when peptides are coeluted. In this paper, we describe a simple and powerful method to characterize HCPs in monoclonal antibodies (mAbs) or related products by combining a novel tryptic peptide sample preparation with traditional shotgun proteomics. Antibody samples are directly treated with trypsin, pH adjusted and incubated overnight. Undigested antibody is precipitated with heat treatment, membrane filtered or kept in the solution before shotgun proteomics analysis. Following this procedure, the antibody is not digested or only minimally digested while residual HCPs in the sample are digested. The dynamic range of the mass spectrometer required for detection of HCPs is found to be one or two orders of magnitude less than the same LC/MS method but with a traditional (i.e., denatured first) sample preparation for the tryptic digest.

Experimental Materials: All chemicals were reagent grade or higher purity and obtained from commercial sources. Chromatographic solvents were LC-MS grade. CHO null strains, monoclonal IgG1, IgG4 and five CHO recombinant proteins, rLPLA2, rPLBL2, rLAL, rPPT1, rIAH1, as well as recombinant human PCSK9 and bovine trypsin were produced at Eli Lilly and Company (Indianapolis, IN). Dithiothreitol (DTT) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Alcohol dehydrogenase from Bakers yeast was obtained from Sigma-


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Aldrich Co. (St. Louis, MO) and monoclonal antibody standard RM 8670 was from National Institute of Standards and Technology (NIST). LC/MS analysis for IgG1 and IgG4 antibodies without or with treatment of trypsin An aliquot containing 1 mg antibody of IgG4 or IgG1 solution was mixed with 5 µL of 1 M tris-HCl buffer, pH 8 and water to 195 µL and then treated with 5 µL of 0.5 mg/mL recombinant bovine trypsin, 0.05 mg/mL dehydrogenase (ADH) from Bakers yeast, and 0.02 mg/mL recombinant human PCSK9 (hPCSK9) solution at 37 ºC for overnight.

Each digest was mixed

with 2 µL of 50 mg/mL DTT solution, heated at 90 ºC for 5-10 minutes and then centrifuged at 15000 g for 2 minutes. The supernatant was transferred for LC/MS analysis. At each step, a 5 µL aliquot of each solution and 20 µL of pure water were transferred into HPLC vial for LC/MS analysis. A 0.5 µL injection of each sample was analyzed by LC-MS. A Waters Acquity UPLC (Milford, MA USA) coupled to Waters SYNAPT G2-S mass spectrometer (Manchester, UK) was applied for intact analysis. Separations were performed on a Varian PLRP-S reversed-phase column (1 × 50 mm, 1000Å, 5 µm) at 80 ⁰C using 0.05% TFA in water as mobile phase A and 0.04% TFA in acetonitrile as mobile phase B. Each peak eluted from the column was analyzed using an electrospray ionization (ESI) source operating at positive, resolution model, scan range of 400 to 4000 amu, spray voltage of 3.0 kV, cone voltage of 100V, source offset of 120 V, source temperature of 120 ºC, desolvation temperature of 400 ºC and desolvation gas of 900 (L/h). Tryptic digests of CHO null strain solutions without or with denaturation Aliquots containing 200 µg CHO proteins from each of five CHO null strain solutions were mixed with 5 µL of 1 M tris-HCl buffer, pH 8 and Barnstead purified water to 198.4 µL or dried using a speed-vacuum. Dried samples were reconstituted with 15 µL of 7 M guanidine•HCl, 0.625 mM tris-HCl buffer, pH 8 and 2 µL of 50 mg/mL DTT solution, incubated at 37 ºC for 30 minutes, and then mixed with 3 µL of 100 mg/mL iodoacetamide solution and purified water to 198.4 µL. Each solution was then treated with 1.6 µL of 2.5 mg/mL r-bovine trypsin solution and incubated at 37 ºC overnight (>16 hours). Each tryptic digest without denaturation was mixed with 2 µL of 50 mg/mL DTT solution and then incubated at 90 ºC for 10 minutes. The



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digests were centrifuged at 13000 g for 2 minutes and each supernatant was mixed with 5 µL of 10% formic acid in water before LC/MS/MS analysis. Tryptic Digest of NIST monoclonal antibody standard RM 8670 and control antibody A100 µL aliquot of 10 mg/mL NIST standard or control antibody solution was mixed with 5 µL of 1 M tris-HCl buffer, pH 8, and 90 µL of pure water. To these solutions was added 5 µL of 0.5 mg/mL recombinant bovine trypsin, 0.05 mg/mL ADH1, and 0.02 mg/mL hPCSK9 solution and the preparations were incubated at 37 ºC for overnight. Each sample was mixed with 2 µL of 50 mg/mL DTT solution and then heated at 90 ⁰C for 10 minutes. Each sample was centrifuged at 13000g for 2 minutes and the supernatant was transferred into HPLC vials and acidified with 5 µL of 10% FA in H2O before LC/MS analysis. Tryptic Digest of IgG1 and IgG4 samples Spiked with CHO Null strain or CHO proteins IgG1 and IgG4 solutions were mixed with CHO null strain to produce 0, 0.1, 0.2, 0.5, 1, 2, 5, or 10 k ppm spiked samples or spiked with 0, 0.1, 0.2, 0.3, 0.5, 1, 2, 5, 10, 20, 50 and 100 ppm each of five recombinant CHO proteins: lyophospholipase A2 (rLPLA2), phospholipase B-like 2 (rPLBL2), lysosomal acid lipase (rLAL), palmitoyl-protein thioesterase 1 (rPPT1) and isoamyl acetate-hydrolyzing esterase 1 homolog (rIAH1). Each spiked sample containing 1 or 2.5 mg IgG1 or IgG4 antibody was mixed with 5 µL of 1 M tris-HCl buffer, pH 8 and water to 195 µL. The samples were then subjected to the trypsin digestion procedure described above. UPLC-MS/MS analysis of tryptic digests The prepared tryptic peptides were analyzed using UPLC-MS/MS. Samples were directly injected onto a Waters Acquity UPLC CSH C18 (Milford, MA USA) (2.1×50 mm, 1.7 µm particle size) at a volume of 50 µL. The column was heated to 60 ºC during analysis. Separation was performed on a Waters Acquity UPLC system with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile with equilibrating at 0% mobile phase B for 2 minute at 200 µL/min, linearly increasing from 0% to 10% over 23 minutes, to 20% B over 57 minutes, to 30% over 30 minutes at a flow rate of 50 µL/min, followed with multiple zig-zag wash cycles at a flow rate of 400 µL/min. Mass


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spectrometric analysis was performed on a Thermo Scientific Q Exactive Plus mass spectrometer (Bremen, Germany). Data-dependent MS/MS was performed as follows: the first event was the survey positive mass scan (m/z range of 230 – 1500) followed by 10 HCD events (28% NCE) on the 10 most abundant ions from the first event. Ions were generated using a sheath gas flow rate of 15, an auxiliary gas flow rate of 5, a spray voltage of 4 kV, a capillary temperature of 320 ºC, and an S-Lens RF level of 50. Resolution was set at 35,000 (AGC target of 5E6) and 17,500 (AGC target of 5E4) for survey scans and MS/MS events, respectively. The maximum ion injection time was 250 ms for survey scan, 300 ms for the other scans. The dynamic exclusion duration of 60 s was used with a single repeat count. Protein identification and quantification A customized protein database composed of sequences obtained from the CHOK1_refseq_2014.Protein.fasta database (downloaded 08/23/2014 from http://www.chogenome.org) was developed to predict the identities of HCPs from the MS/MS data. The MS/MS data was searched with a mass tolerance of 10 ppm and 0.02 Da, and a strict false discovery rate (FDR) ≤ 1% against this database using the Proteome Discoverer software package, version 1.4 (Thermo Scientific, Bremen, Germany) with Sequest HT searching. Further peptide/protein filtering was performed by eliminating peptides that had scored 0 and single spectrum hit, or single spectrum hit and ≥ 10 ppm. Protein area from the top 3 peptides (if possible) for each HCP and the areas for the three spiked proteins, r-trypsin, PCSK9 and ADH1 were used to calculate individual HCP concentration (ppm or ng HCP/mg mAb).

Results and Discussion Host cell proteins (HCPs) are process-related impurities in biopharmaceutical products derived from cell culture. Application of sensitive analytical methods to monitor HCPs throughout product development is expected by worldwide regulatory authorities. While ELISA remains the industry standard method for HCP detection a shotgun proteomics approach using mass spectrometry is becoming more generally applied as an additional characterization tool. One problem for mass spectrometry detection of HCPs in biopharmaceutical products is the dynamic range, which requires 5 to 6 orders of magnitude to detect 10 to 1 ppm of individual



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HCPs if their peptides are co-eluted. This dynamic range is outside the capability of current mass spectrometers. In general, antibodies are very stable and resistant to trypsin digestion in their native state. Data for an IgG1 and an IgG4 monoclonal antibodies treated with trypsin overnight is shown in Figure 1. For IgG4, the UV profiles are very similar without (Figure 1a) or with (Figure 1b) trypsin treatment followed by overnight incubation. For IgG1, the UV profile after treatment with trypsin (Figure 1e) is very different from that of the untreated sample (Figure 1d). The two new peaks eluting earlier than the IgG1 intact peak (8.1 min) are Fab (7.6 min) and (Fc)2 (7.4 min) due to the cleavage in the hinge region (•••CDK/THT•••), which is a sensitive site for trypsin and Lys-C. However, for either IgG4 or IgG1 antibody samples, addition of the reducing reagent, DTT, followed by heating results in precipitation of undigested antibody or generated Fab and Fc domains which can be removed by centrifugation. Even without the precipitation and removal of the IgG proteins, they will be eluted later than most of the tryptic peptides of HCPs and will not complicate HCP detection. If CHO HCPs can be digested or partially digested with trypsin under the same conditions, the required dynamic range to detect HCPs is only 3 to 4 orders of magnitude, which is the range of current mass spectrometers. Additional experiments were conducted to confirm whether this type of sample preparation is sufficient to enable detection and quantitation of trace amounts of residual HCPs in monoclonal antibody preparations.

CHO protein identification for null strain samples We first wanted to determine whether our sample preparation method is as effective at digesting host cell proteins compared to the traditional method, which involves tryptic digestion following denaturing and reduction/alkylation. A sample containing the protein pool produced from a null CHO cell line fermentation (i.e., cells do not contain the gene for antibody production) was used as an example to evaluate our method against the traditional procedure. Typically, we observed greater than 1000 HCPs in null strain samples treated by both sample preparation methods (Table 1). For the top 500 HCPs, 18 to 24 unique proteins were identified with the traditional method while 3 to 13 unique HCPs were identified with our procedure. Careful analysis of the unique HCPs related to each sample preparation revealed a total of 10 HCPs, detected with the traditional sample preparation procedure but not found in any of the five


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null strain samples examined using our method for sample preparation. These HCPs generally contain a high content of Cys residues. For example, granulins, an HCP only identified with the traditional sample preparation, contains 94 Cys residues out of a total of 610 amino acid residues. Another possibility for missed HCP detection using our method would involve a specific and small secreted host cell protein that is an antigen of the expressed monoclonal antibody (mAb) and its epitope is conformational. Although a rare case, such a situation may exist. Regardless, less than 2% of HCPs for the top 500 HCPs detected in the CHO null strain could not be identified with our method. This small difference is less than typical run to run variability. Other differences between the two sample preparations were observed in the protein sequence recovery, score and unique peptides for each identified protein. For example, the large, highly abundant basement membrane-specific heparan sulfate proteoglycan core protein, yielded the following results: score 414.2, protein sequence recovery 35.3 and number of unique peptides, 102 by the traditional sample preparation while values of 228.4, 19.5 and 55, respectively, were obtained for our method. In general, proteins identified with the traditional sample preparation produced slightly higher scores, and protein sequence recovery. The average score, protein sequence recovery and unique peptides for top 500 proteins identified in the five null strain samples are 38.7±2.6, 30.8±2.0 and 10.0±0.9 with the traditional sample preparation and 32.8±4.1, 25.4±1.8 and 7.8±0.4 with our method. The ability to identify CHO proteins in the null strain samples and data for native antibody demonstrate that direct treatment of the sample with trypsin followed by overnight incubation causes minimal degradation of the antibody while most CHO HCPs are digested. The procedure we devised achieves a major improvement in the dynamic range required for mass spectrometry detection which is 1 to 2 orders of magnitude lower than what is required using the traditional sample preparation method. Another advantage with having less digested antibody is the RPHPLC column has more capacity to separate HCP peptides from antibody peptides thereby increasing the HCP identification capability. HCP Detection with Spiked Samples A series of experiments were conducted to evaluate whether our method is effective for detecting HCPs spiked into samples. In one experiment, preparations containing IgG1 or IgG4



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antibodies were spiked with varying amounts (0 to 10000 ppm) of a CHO null strain sample. The results from this experiment are presented in Table 2. At low level of spiking, more HCPs were detected in the spiked IgG samples than the null strain sample while the opposite was observed at the very high level of spiking. This result may be related to there being more HCPs injected for the spiked IgG samples as compared to the null strain sample due to the trace amount of residual HCPs in the non-spiked antibody samples. In contrast, at the high spiked level, the minimally digested antibody peptides diminished HCP detection due to the selected peptide competition for MS/MS analysis. Regardless of the spiking level, application of our sample preparation procedure achieved ≥ 85% detection of spiked null strain proteins. In another experiment, five recombinant CHO proteins were spiked into samples to evaluate the detection sensitivity of our method. These proteins were spiked into preparations containing 5 mg/mL antibody and subjected to our sample preparation procedure followed by mass spectrometry analysis. As shown in Table 3, all five proteins spiked at 1 ppm each were detected in the IgG4 preparations while three of the proteins were detected in the IgG1 containing samples. When the proteins were spiked at ≥ 2 ppm, all five were identified for both the IgG1 and IgG4 containing preparations. A similar number (Table 2) of HCPs was detected for the spiking experiments with a CHO null strain sample demonstrating that antibody precipitation did not co-precipitate HCP peptides. The fact that HCP peptides were preserved during the precipitation was also confirmed by the purified CHO protein spiking experiments, which were all linear between 2 - 100 ppm spiking range (Table 3). One advantage of our method is much higher protein concentration can be used for tryptic digestion without experiencing precipitation. With the traditional tryptic digestion approach, it is difficult to keep antibodies in solution when they are denatured at high protein concentration without including high concentration of denaturing agents, such as urea or guanidine. Hence, in order to increase the detection sensitivity of HCP, concentrations of antibody greater than 5 mg/mL can be used. For example, using our method to analyze a sample containing 12.5 mg/mL antibody, the five CHO proteins could be detected at a spiked level of 0.5 ppm.


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The applicable antibody concentration for our method is not limited to 12.5 mg/mL. We have analyzed samples with concentrations ≥ 150 mg/mL when instrumentation with lower sensitivity than the Thermo Q-Exactive plus mass spectrometry was used, or to detect very low ( 5 and < 20 mg/mL. For higher concentration samples, a simple dilution works well. HCP Quantitation Protein sequence recovery with our method is generally lower than that obtained with the traditional sample preparation procedure. Consequently, protein quantitation with Hi3 peptide intensities [30, 31] may be different for our method compared with the traditional sample preparation approaches. To achieve more consistent quantitation, instead of using one internal standard as in the original Hi3 method, three standards were chosen here including two spiked proteins, recombinant human PCSK9 (hPCSK9) and alcohol dehydrogenase (ADH1) from Baker’s yeast, as well as recombinant bovine trypsin (i.e., the enzyme used for digestion). Individual HCP titers can be calculated based on the top 3 peptides of each spiked protein to give three HCP titers. These values are then averaged to yield the final reported number. The recombinant bovine trypsin and human PCSK9 (hPCSK9) were selected because no trypsin or PCSK9 were detected in the CHO null strains. Users of our method can apply other purified proteins as standards for quantitation. Using this approach, the calculated recoveries for IgG1 and IgG4 preparations spiked with CHO null strain and the five recombinant proteins are shown in Tables 2 and 3. The recovery of CHO null strain is between 98 to 169% or 112 to 139% for IgG1 or IgG4, respectively, when spiked at ≥ 500 ppm. For the spiked recombinant proteins, the recovery of each protein is different. rLPLA2, rLAL and rPPT1 showed ≥ 100% recovery; rIAH1 < 100%; and rPLBL2 approximately 100% for levels between 2 to 100 ppm spiked into preparations containing 5 mg/mL digest concentration of IgG1 or IgG4. Although an HCP standard is certainly required for accurate measurement of each HCP, quantitation using the Hi3 method versus the three protein standards is sufficient to support purification process development.



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Method Robustness A separate monoclonal antibody control sample is simultaneously analyzed in each experiment to ensure run-to-run consistency. During a 450 day time interval, the control was analyzed 70 times. The calculated concentrations (ppm) for the predicted HCPs in the control sample are presented in Figure 2a. Average values for HCPs are 919 and 788 ppm with relative standard deviation of approximately16 and 17% accounting for every HCP detected or only accounting for those HCPs ≥ 10 ppm per HCP, respectively. The control antibody also contains more than 10 HCPs at levels ≥ 10 ppm per HCP and detected in every run. The relative standard deviation is between 20 to 40% for the 70 runs over the 450 day period of time. Figure 2b shows the estimated concentrations for five HCPs which are present at levels from ~ 10 to > 200 ppm. These data demonstrate that our method is robust and capable of consistently detecting HCPs at levels ≥ 10 ppm. HCPs in NIST Monoclonal Antibody Standard RM 8670 Many powerful methods have been published for characterization of HCPs in biopharmaceutical samples but most have been only applied for specific products that are not available for analysis by other researchers. For this reason, it is not possible to directly compare results across the different methods. Doneanu, et al. applied their 2D-HPLC/IM/MSE method in three independent laboratories [24] to analyze the NIST antibody standard RM 8670 and identified a combined 34 HCPs. Fourteen of the 34 HCPs were detected by all three laboratories and one, mouse peroxiredoxin 5, was present at a level of about 1 ppm or 1 ng/mg antibody. For comparison, we applied our method to characterize the HCPs in the NIST RM 8670 standard. More than one hundred mouse proteins were identified for each of three LC/MS/MS injection replicates processed using Thermo Scientific Proteome Discoverer 1.4 against the Uniprot mouse protein database and a false positive detection rate (FDR) ≤ 1%. If we only consider those HCPs with at least two unique peptides per protein and detected in all three injections, fifty-nine mouse HCPs were identified (see Table 4). Thirteen of the fourteen HCPs detected with 2D-HPLC/IM/MSE procedure were identified by our method. The missing protein, beta-2-microglobulin, was actually detected but only with one unique peptide for each of the three injections. The tandem mass spectrum of the unique peptide identified demonstrated


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excellent peptide sequence coverage as shown in Figure 1S in the Supporting Information. While similar trends were observed, the quantitation data for HCPs obtained with our method was not in good agreement with reported data for several HCPs. Forty-six mouse HCPs in the NIST antibody standard RM 8670 identified with our method were not reported in the study using 2D-HPLC/IM/MSE. Fourteen of the 46 mouse HCPs have tandem mass spectra for five or more unique peptides, seventeen for ≥ 4 unique peptides and twenty-eight for ≥ 3 unique peptides. Most of the 46 HCPs were at levels less than 10 ppm or ng HCP/mg antibody and only three HCPs, protein disulfide-isomerase A6 (132 ppm), hepatocyte growth factor-like protein (14 ppm), and protein ABHD11 (10 ppm), were ≥ 10 ppm. Generally, the quality of the tandem mass spectrum data obtained for the identified peptides was very good even for HCPs present at ≤ 1 ppm (Figure 2S in the Supporting Information). Why did our method identify many more HCPs than 2D-HPLC/IM/MSE? Under native conditions, the NIST antibody standard treated with trypsin overnight only showed five major peaks and the other peaks related to antibody are relatively low (Figure 3). For a 250 µg antibody (calculated) injection, the intensities of the five major peaks are much lower and peak widths much narrower than the tryptic peptide peaks obtained with the traditional method. Comparing 1D-UPLC (our method run time: 2 hours) to 2D-UPLC (total run time: ~10 hours), the resolution of separation was increased five times assuming that the correction of the resolution and HPLC run time is linear. However, our method only digests a few percent of the antibody, effectively increasing the resolution of separation more than 10 times. Furthermore, with our procedure, the dynamic range of mass spectral detection is 1 to 2 orders of magnitude lower than the traditional sample preparation. Interestingly, the NIST antibody standard RM 8670 is the only sample that did not precipitate after the trypsin-treated sample was heated with DTT at 90 ºC for 10 min out of more than sixty molecules of different types including: IgG1, IgG1-null, IgG2, IgG4 antibodies, bispecific antibodies, Fc-fusion peptides and proteins, Fab, and PEG-Fab analyzed with our method.. However, HCP detection is still possible even if the antibody does not precipitate because the reduced antibody is eluted later than most of the HCP tryptic peptides and does not interfere with detection. In fact, removal of undigested antibody is not necessary for detection of HCPs using our method. The only benefit to removing undigested antibody is to increase the column lifetime



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or shorten the HPLC run cycle. Without removal, 250 µg digested or undigested (major form) antibody will be injected on the column. The column will lose resolution after several injections without using a prolonged column wash step. With the precipitation step, the column resolution will be persevered after several hundred injections with moderate column washing.

Conclusions We developed a simple and powerful methodology for HCP identification and quantitation in antibody preparations by combining a shotgun proteomics approach with a novel sample preparation procedure utilizing trypsin digestion under non-denaturing conditions. Under these conditions, the therapeutic derived from the CHO cell line is maintained largely intact while HCPs are digested. Spike recovery experiments demonstrate that the method is very sensitive and robust. The method consistently detected recombinant proteins spiked as low as 0.5 ppm in preparations having antibody concentrations at 12.5 mg/mL, and always detected HCP when its level is ≥ 10 ppm in preparations having antibody concentrations at 5 mg/mL. Our method also detected a greater number of HCPs in the NIST monoclonal antibody standard RM 8670 from compared to results obtained using 2D-HPLC to analyze samples prepared with the traditional tryptic digest procedure. More importantly, our methodology has an overall shorter cycle time compared to the 2D-HPLC method making it possible to achieve higher throughput sample analysis to support purification process development.

ACKNOWLEDGEMENTS We thank Troii Hall and Stephanie Sandefur for preparing purified proteins used in this work.

Reference

1. Bierich, J.R. Acta Pediatr. Scand. Suppl. 1986, 325, 13-18. 2. Rosenberg, A.S.; Worobec, A.S. BioPharm. Int. 2004, 17, 34-42.


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3. Romer, T.; Peter, F.; Saenger, P.; Starzyk, J.; Koehler, B.; Korman, E.; Walczak, M.; Wasik, R.; Ginalska-Malinowska, M.; Solyom, E.; and Berghout, A.; J. Endocrinol. Invest. 2007, 30, 578-589. 4. Zuch de Zafra, C.L.; Quarmby, V.; Francissen, K.; Vanderlaan, M.; and Zhu-Shimoni, J.; Biotechnol. Bioeng. 2015, 112, 2284-2291. 5. Bracewell, D.G.; Francis, R.; Smales, C.M. Biotechnol Bioeng, 2015, 112, 1727-37. 6. Jawa, V.; Joubert, M.K.; Zhang, Q.; Deshpande, M.; Hapuarachchi, S.; Hall, M.P.; Flynn, G.C. AAPS J., 2016, 18, 1439-1452. 7. Fischer, S.K.; Cheu, M.; Peng, K.; Lowe, J.; Araujo, J.; Murray, E.; McClintock, D.; Matthews, J.; Siguenza, P.; and Song, A. AAPS J., 2016, oct 13, 1-10. 8. Dixit, N.; Salamak-Miller, N.; Salinas, P.A.; Taylor, K.D.; Basu, S.K. J. Pharma. Sci. 2016, 105, 1657-1666. 9. Hall, T.; Sandefur, S; Frye, C.C.; Tuley, T.; Huang, L., J. Pharma. Sci. 2016, 105, 16331642. 10. Krawitz, D.C.; Forrest, W.; Moreno, G.T.; Kittleson, J.; Champion, K.M. Proteomics, 2006, 6, 94–110. 11. Hogwood, C.E.M.; Bracewell, D.G.; Smales, C.M. Bioengineered, 2013, 4, 1–4. 12. Rogers R.; Bell A.; Kowski T.; Bailey R. 61st ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, June 9−13, 2013. 13. Yu, C. 10th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry, Boston, MA, September 23−26, 2013. 14. Joucla, G.; Le Senechal, C.; Begorre, M.; Garbay, B.; Santarelli, X.; Cabanne, C. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2013, 942−943, 126. 15. Reisinger, V.; Toll, H.; Mayer, R. E.; Visser, J.; Wolschin, F. Anal. Biochem. 2014, 463, 1. 16. Xiao, G.; Ren, D.; Bondarenko, P. 62nd ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD, June 15−19, 2014. 17. Sperry J. B. First Annual BEBPA Workshop on Host Cell Proteins Assays, Dubrovnik, Croatia, May 16, 2014. 18. Doneanu, C.; Xenopoulos, A.; Fadgen, K.; Murphy, J.; Skilton, S.J.; Prentice, H.; Stapels, M.; Chen, W. mAbs 2012, 4, 24−44. 19. Schenauer, M. R.; Flynn, G. C.; Goetze, A. M. Anal. Biochem. 2012, 428, 150. 20. Schenauer, M. R.; Flynn, G. C.; Goetze, A. M. Biotechnol. Prog. 2013, 29, 951. 21. Tscheliessnig, A. L.; Konrath, J.; Bates, R.; Jungbauer, A. Biotechnol. J. 2013, 8, 655. 22. Zhang, Q.; Goetze, A. M.; Cui, H.; Wylie, J.; Trimble, S.; Hewig, A.; Flynn, G. C. mAbs 2014, 6, 659−670. 23. Farrell, A.; Mittermayr, S.; Morrissey, B.; Mc Loughlin, N.; Iglesias, N.N. Marison, I.W.; Bones, J. Anal. Chem. 2015, 87, 9186−9193. 24. Doneanu, C.E., Anderson, M., Williams, B.J., Lauber, M.A., Chakraborty, A., Chen, W. Anal. Chem. 2015, 87, 10283−10291. 25. Thompson, J.H., Chung, W.K., Zhu, M., Tie, L., Lu, Y., Aboulaich, N., Strouse, R., Mo, W. Rapid Commun. Mass Spectrom. 2014, 28, 855–860. 26. Madsen, J.A.; Farutin, V.; Carbeau, T.; Wudyka, S.; Yin, Y.; Smith, S.; Anderson, J.; Capila, I. mAbs 2015, 7, 1128-1137. 27. Bishop E. BEBPA HCP conference, Lispon, Portugal, May 17-19, 2016. 28. Johansen E. BEBPA HCP conference, Lispon, Portugal, May 17-19, 2016. 29. Ecker, D.M.; Jones, S.D.; Levine, H.L. mAbs 2014, 7, 9-14.



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30. Silva, J. C.; Gorenstein, M.V.; Li, G. J.; Vissers, J.P.C.; Geromanos, S.J. Mol. Cell. Proteomics 2006, 1, 144. 31. Silva, J. C.; Denny, R.; Dorschel, C.; Gorenstein, M.; Li, G. J.; Richardson, K.; Wahl, D.; Geromanos, S. J. Mol. Cell. Proteomics 2006, 5, 589.


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Table 1. Number of HCPs Detected by Shotgun Proteomics for the Tryptic Digests of CHO Null Strains (NS) with the Traditional or Novel Method and Number of Unique HCPs Associated with Sample Preparations Are Reported for the Top 500 HCPs Detected

Null Strain NS 1 NS 2 NS 3 NS 4 NS 5 NS 5, 10% Injected NS 5, 20% Injected NS 5, 50% Injected

HCPs Detected Traditional Novel 1159 1199 1179 1165 1147 1211 1113 1049 1077 1134 871 959 1084 1074 1175 1176

For Top 500 HCPs, Unique HCPs with Traditional Novel 18 7 19 3 20 7 18 4 24 13 33 18 26 11 15 11

Table 2. Number of HCPs Detected and HCP Concentration (ppm) Quantified by the Novel Method for IgG1 and IgG4 Preparations Spiked with Various Amounts of the Null Strain Null Strain Spiked (ppm)* 0 100 200 500 1000 2000 5000 10000

Null Strain without Antibody No. HCP HCP Conc. (ppm) 0 0 6 1 21 28 36 92 84 239 207 735 438 3643 594 9589

IgG1 Spiked with Null Strain No. HCP HCP Conc. (ppm) 11 31 34 101 56 209 103 636 156 1289 223 1969 388 7728 502 16872

IgG4 Spiked with Null Strain No. HCP HCP Conc. (ppm) 7 9 33 56 75 208 135 559 221 1278 343 2773 481 6524 575 13169

* ppm (or ng HCPs/mg antibody) based on antibody concentration.



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Table 3. Recovery of Recombinant Proteins Spiked into IgG1 or IgG4 Samples by Shotgun Proteomics with the Novel Method

Spiked Protein

0* 11

Recovery of Spiked Protein Concentration, ppm (No. unique peptides) IgG1 Spiked with Proteins (ppm) 0.3* 0.5* 0.75*1 1.0 1 2.0 1 5.0 1 10 1 20 1 50 1 14 (5)

40 (7)

2.6 (2) 4.4 (1)

15 (5)

33 (6)

76 (7)

171 (9)

2.2 (2) 6.1 (4)

18 (6)

39 (6)

112 (9)

211 (12)

ND

2.6 (2) 3.1 (2) 6.9 (8)

12 (7)

27 (11)

70 (13)

129 (14)

ND

0.6 (1) 1.4 (2) 2.3 (4)

8.4 (8)

20 (10)

56 (13)

95 (13)

IgG4 Spiked with Proteins (ppm) 0.75*1 1.0 1 2.0 1 5.0 1 10 1

20 1

50 1

100 1

rLPLA2

-

ND

ND

ND

rLAL

-

ND

ND

ND

-

rPPT1

-

ND

ND

ND

-

rPLBL2

-

ND

ND

rIAH1

-

ND

ND

Spiked Protein

0* 11

0.3*

0.5*

100 1

2.1 (3) 3.8 (2) 7.2 (3)

113 (10) 227 (12)

rLPLA2

-

-

0.8 (1) 1.8 (2) 1.5 (3) 3.1 (4) 9.7 (8)

21 (10)

44 (13) 105 (16) 226 (18)

rLAL

-

-

1.2 (1) 1.4 (2) 1.4 (1) 2.4 (4) 9.8 (7)

18 (8)

37 (7)

rPPT1

-

0.4 (1) 0.6 (2) 0.7 (3) 0.6 (1) 1.3 (4) 8.4 (7)

16 (7)

43 (10) 102 (12) 214 (13)

rPLBL2

-

0.0 (1) 0.5 (4) 0.8 (5) 0.5 (2) 1.9 (4) 3.4 (4)

10 (7)

25 (9)

62 (14)

124 (22)

rIAH1

-

0.3 (1) 0.5 (2) 0.6 (3) 0.4 (1) 1.2 (5) 4.0 (8)

7.4 (8)

16 (10)

40 (11)

95 (14)

91 (9)

191 (10)

Note: ND = not determine; - = not detected; * mAb concentration of the final tryptic digest is 12.5 mg/mL and all the other are 5 mg/mL.


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Table 4. HCPs Detected in the National Institute of Standards and Technology (NIST) Monoclonal Antibody Standard, RM 8670, by the Novel Method No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Accession P05064 P05063 P06745 Q91YR9 P40142 Q99KN9 Q9CZ44 P08101 Q923D2 P99029 Q8BL97 Q9WTP6 Q60864 P01887 Q9Z0X1 Q8C7U7 Q8K4F5 Q62179 O88569 Q9EPX2 Q9ER00 Q6PB93 P49312 Q03173 Q922R8 P26928 Q9D2M8 P10126 Q3UEB3 P40124 Q9D8B3 Q68FL6 Q8BGD9 Q6KCD5 Q68FF6 P45878 P11680 Q8BND5 Q62165 Q9DBP5 P35700 P21460 Q3TLH4 Q8CGC7 Q80WJ7 O70305 Q8K4Z5 P32020 Q9QUR8 O55131 P18242 P09041 P03975 Q9CQF3 P34902 P53996 P08249 Q6PDM2 Q6PGH2 P19157

Description Fructose-bisphosphate aldolase A Fructose-bisphosphate aldolase C Glucose-6-phosphate isomerase Prostaglandin reductase 1 Transketolase Clathrin interactor 1 NSFL1 cofactor p47 Low affinity immunoglobulin gamma Fc region receptor II Flavin reductase (NADPH) Peroxiredoxin-5, mitochondrial Serine/arginine-rich splicing factor 7 Adenylate kinase 2, mitochondrial Stress-induced-phosphoprotein 1 Beta-2-microglobulin Apoptosis-inducing factor 1, mitochondrial Polypeptide N-acetylgalactosaminyltransferase 6 Protein ABHD11 Semaphorin-4B Heterogeneous nuclear ribonucleoproteins A2/B1 Papilin Syntaxin-12 Polypeptide N-acetylgalactosaminyltransferase 2 Heterogeneous nuclear ribonucleoprotein A1 Protein enabled homolog Protein disulfide-isomerase A6 Hepatocyte growth factor-like protein Ubiquitin-conjugating enzyme E2 variant 2 Elongation factor 1-alpha 1 Poly(U)-binding-splicing factor PUF60 Adenylyl cyclase-associated protein 1 Charged multivesicular body protein 4b Methionine--tRNA ligase, cytoplasmic Eukaryotic translation initiation factor 4B Nipped-B-like protein ARF GTPase-activating protein GIT1 Peptidyl-prolyl cis-trans isomerase FKBP2 Properdin Sulfhydryl oxidase 1 Dystroglycan UMP-CMP kinase Peroxiredoxin-1 Cystatin-C Protein PRRC2C Bifunctional glutamate/proline--tRNA ligase Protein LYRIC Ataxin-2 Splicing factor 3A subunit 1 Non-specific lipid-transfer protein Semaphorin-7A Septin-7 Cathepsin D Phosphoglycerate kinase 2 IgE-binding protein Cleavage and polyadenylation specificity factor subunit 5 Cytokine receptor common subunit gamma Cellular nucleic acid-binding protein Malate dehydrogenase, mitochondrial Serine/arginine-rich splicing factor 1 Hematological and neurological expressed 1-like protein Glutathione S-transferase P 1

Unique Pep 22 19 15 8 5 4 4 4 4 4 3 3 2 1 11 10 7 7 7 7 7 7 6 6 5 5 5 5 4 4 4 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Inj No 1 222 61 19 5 2 10 3 3 3 1 2 2 7 12 4 5 10 5 6 6 4 3 3 3 132 14 4 2 3 0.8 1 6 5 3 3 2 1 2 2 1 1 < 0.5 7 3 3 2 1 1 1 1 1 1 1 1 1 1 < 0.5 < 0.5 < 0.5 < 0.5

Measured HCP (ppm) Inj No 2 Inj No 3 222 221 54 50 20 20 5 4 1 1 12 11 3 4 4 3 2 3 1 1 2 2 2 2 7 7 12 12 6 4 7 6 10 9 10 5 6 6 7 5 5 3 3 3 4 4 3 3 131 132 11 16 4 4 2 2 3 3 0.8 0.8 1 1 6 8 5 4 4 4 3 2 3 2 2 1 2 2 1 1 1 1 1 1 1 < 0.5 10 7 3 4 2 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 < 0.5 < 0.5 1 < 0.5 < 0.5 < 0.5 1 < 0.5 < 0.5 < 0.5 < 0.5

Ave 222 55 20 5 1 11 3 3 3 1 2 2 7 12 5 6 10 6 6 6 4 3 4 3 132 14 4 2 3 0.8 1 7 5 4 3 2 2 2 1 1 1

A Novel Sample Preparation for Shotgun Proteomics Characterization

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