Asparagine Deamidation in a Complementarity Determining Region of

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Asparagine deamidation in a complementarity determining region of a recombinant monoclonal antibody in complex with antigen Christine Nowak, Ashish Tiwari, and Hongcheng Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01322 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Analytical Chemistry

Asparagine deamidation in a complementarity determining region of a recombinant monoclonal antibody in complex with antigen

Christine Nowak, Ashish Tiwari, and Hongcheng Liu* Product characterization, Alexion Pharmaceuticals, 100 Colleague Street, New Haven, CT06510

* Corresponding author Email: [email protected] Telephone: 203-271-8354

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Analytical Chemistry 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

Asparagine deamidation in the complementarity determining regions of recombinant monoclonal antibodies has been extensively studied and shown to have a negative impact on antigen binding. Those asparagine residues are typically exposed and thus have a higher tendency towards deamidation, depending also on local structure and environmental factors such as temperature and pH. Deamidation rates and products of a susceptible asparagine residue in the complementarity determining regions of a recombinant monoclonal antibody free in solution or in antibody-antigen complex were studied. The results demonstrated that incubation of the antibody or its antigen complex generated a similar amount of aspartate. The expected amount of isoaspartate product was detected in free antibody, but it was completely lacking in the antibody-antigen complex.


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Asparagine (Asn) deamidation through the β-elimination mechanism has been established as a ubiquitous protein degradation pathway, which under neutral and slightly basic conditions to form isoaspartate (isoAsp) and aspartate (Asp) in an approximately 3:1 molar ratio1,2. The overall reaction is shown in Figure 1. Specifically, the main chain nitrogen exerts a nucleophilic attack of the side chain carbonyl carbon of the preceding Asn residue to form the thermodynamically favored five member ring structure of succinimide. The succinimide is unstable under physiological conditions and can be readily hydrolyzed to form isoAsp or Asp. IsoAsp is formed when a water molecule attacks the main chain carbonyl-carbon. Asp is formed when a water molecule attacks the side chain carbonyl carbon. Deamidation has been a concern for the development of recombinant monoclonal antibody therapeutics because of the potential immunogenicity and negative impact on efficacy. Animal studies demonstrated that isoAsp triggered an immune response not only towards the isoAsp containing peptide, but also to the native peptide containing the original Asn residue3-5. Asn deamidation causing decreased antigen binding affinity has been demonstrated in several studies, including deamidation in light chain complementarity-determining region 1(CDR1)6-8, light chain CDR3 9 heavy chain CDR110, and heavy chain CDR211. Deamidation of Asn in CDRs8,10-12 and Asn residue in the conserved Fc region13 have been reported to continue occurring in recombinant mAbs in vivo. Deamidation at the conserved region also occurs in human endogenous IgGs13.

Asn deamidation is highly dependent on neighboring amino acids and protein structures. The presence of a glycine (Gly) residue following an Asn residue exerts the most profound effect on to acceleration of Asn deamidation, while amino acids prior to the Asn have little impact14 2,15. Secondary 16

and tertiary 17,18 structure have significant impact on deamidation, most often, stabilizing Asn against

deamidation. The structural impact has been observed in mAbs6 19 6,20. For example, one study demonstrated that, although, two mAbs contained Asn followed by Gly in heavy chain CDR2, only one was susceptible to deamidation 20. In addition to impacting deamidation rates, protein structure also



influences the ratio of the two deamidation products, resulting in IsoAsp and Asp deviating from the expected 3:1 ratio obtained from using peptides1,2,21,22. We hypothesized that antigen binding to a mAb could induce local conformational changes, which could change the microenvironment of the otherwise susceptible Asn residue in CDRs. As a result, the deamidation propensity and the deamidation products may be different compared to free mAb without binding to antigen. Experimental data presented in this study showed that the formation of antibody-antigen complex not only decreased the level of deamidation, it also changed the deamidation product profiles.

Materials and methods Materials The recombinant monoclonal IgG1 antibody was expressed in a Chinese hamster ovary (CHO) cell line and purified at Alexion (New Haven, CT). Acetonitrile, bovine serum albumin, dithiothreitol, guanidine hydrochloride, iodoacetic acid, sodium sulfate and trifluoroacetic acid (TFA) were purchased from Sigma (St,Louis, MO). Trypsin was purchased from Promega (Madison, WI). Lys-C was purchased from Wako (Richmond, VA).

Hydrophobic interaction chromatography Hydrophobic interaction chromatography was performed using a Waters Alliance high performance liquid chromatography (HPLC) system and a ProPac HIC-10 hydrophobic interaction chromatography (HIC) column (4.6 x 150 mm, Thermo Scientific, Sunnyvale, CA). Mab samples were injected at 50% mobile phase A (1 M sodium sulfate in 1x PBS) and 50% mobile phase B (1xPBS). After 5 minutes, mAb variants were eluted off the column by increasing mobile phase B to 90% within 20 minutes. The column was washed using 90% mobile phase B for 5 minutes and then equilibrated using


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the loading condition before the next injection. The flow-rate was set at 0.5 mL/min for the entire run. Proteins eluted off the column were monitored using UV absorption at 280 nm. MAb variants in each peak were manually collected under UV280 guidance and then concentrated using Amicon Ultra centrifuge device with a molecular weight cut-off or 30 kDa (Millipore). The concentrated samples were extensively buffer exchanged into phosphate buffered saline (PBS).

Size exclusion chromatography Size exclusion chromatography (SEC) was performed using a Waters Alliance HPLC system and a TSKgel G3000SWxL column (7.8 mm I.D. x 30 cm, Tosoh Bioscience, King of Prussia, PA). An isocratic method with a mobile phase of 20 mM sodium phosphate and 150 mM sodium chloride at a flow-rate of 1 mL/minute for 15 minutes was used to analyze antibody, antigen and antibody-antigen complex. The proteins eluting off the column were monitored using UV280nm.

In vitro deamidation The main peak separated by HIC containing the monoclonal antibody without deamidation was used for in vitro deamidation studies. The antibody at a concentration of 0.5 mg/mL in PBS at pH 7.4 was either incubated alone, or after mixing with either its specific antigen at 3:1 antigen to antibody ratio or with bovine serum albumin (BSA) at a similar molar concentration as the antigen. The samples were then incubated at 37 °C for 10 days. The samples were analyzed before and after incubation by SEC to ensure the formation of antibody-antigen complex. After trypsin digestion, the same set of samples was analyzed liquid chromatography and mass spectrometry to determine the level of deamidation.

Trypsin digestion The T=0 sample, and the samples after incubation including the mAb alone, mAb-antigen complex, and mAb with an un-related protein were denatured and reduced using 6 M guanidine



hydrochloride in 20 mM Tris, pH 7.8 and 10 mM dithiothreitol (DTT) at 37 °C for 30 minutes. The fully denatured and reduced samples were alkylated using iodoacetic acid at a final concentration of 25 mM at 37 °C for 30 minutes. The sample pH was adjusted immediately after the addition of iodoacetic acid to approximately 7.8 using sodium hydroxide. The samples were buffer exchanged into 20 mM Tris, pH 7.8 using Zeba columns (ThermoScientific, Rockford, IL). The samples were digested using trypsin at an enzyme to protein ratio of 1:5 (w:w) at 37 °C for 2 hours.

LC-MS analysis of peptides A Maxis 4 G mass spectrometer (Bruker, Billerica, MA), an ultra-performance liquid chromatography (UPLC) system (Waters) and a Proto 200 C18 column (1.0 x 250 mm, Higgins Analytical. Inc) were used for peptide analysis. The samples were loaded at 98% mobile phase A (0.1% TFA in water) and 2% mobile phase B (0.1% TFA in acetonitrile). A gradient of increasing mobile phase B from 5% to 35% within 165 minutes and then to 60% within 15 minutes was used to elute the peptides. The column was then washed and equilibrated. The column was heated at 60 °C and the flow-rate was set at 50 µL/min. The mass spectrometer was run in the positive scan mode with m/z in the range of 1503000. Capillary voltage was set at 4500 V. Dry gas was set at 10 L/min. Dry temperature was set at 220 °C and the nebulizer was set at 2.0 bar.

Results and Discussion Characterization of HIC peaks Ion exchange chromatography (IEX) or isoelectric focusing electrophoresis (IEF) have been the methods of choice to analyze mAb charge variants, where various modifications contribute to the


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formation of acidic or basic peaks23,24. In addition, hydrophobic interaction chromatography (HIC) has shown some unique or complementary separations to charge based methods because modifications causing acidic or basic species may or may not cause hydrophobicity differences 24-26. For the recombinant monoclonal antibody used in the current study, similar peak profiles were obtained from analysis by an imaged capillary isoelectric focusing electrophoresis (icIEF) method and by HIC (data not shown), indicating the same modifications drive separation by both charged or hydrophobicity based separation. HIC was used in the current study because it not only allowed charge variant separation but also fraction collection. The recombinant monoclonal antibody, when analyzed by HIC, showed three peaks at retention times of 5, 8 or 12 minutes respectively (Figure 2). The major peak at the retention time of 12 minutes was assigned as peak 1, while the two peaks in front were assigned as peak 2 and peak 3. Fractions corresponding to peaks 1-3 were collected and analyzed by LC-MS. No molecular weight differences (data not shown) were observed between the collected peaks when intact molecule or the reduced light chain and heavy chain were analyzed, indicating that the potential modification caused no or minimal molecular weight differences such as Asp isomerization or Asn deamidation. The collected fractions were further analyzed by LC-MS after trypsin digestion. The only difference among the three peaks observed is between the retention times of 150 and 159 minutes, where the tryptic peptide containing the light chain CDR3 was eluted. Only one peak was observed in HIC peak 1 fraction (Figure 3A), where additional peaks were observed in HIC peak 2 (Figure 3B) and HIC peak 3 (Figure 3C) fractions. The observed molecular weight of the only peak in HIC peak 1 fraction is 4588.1Da which is in good agreement with the calculated molecular weight of 4588.1 Da based on the known amino acid sequence, confirming the peak identity. The middle peak in the HIC peak 2 (Figure 3B) fraction also has a molecular weight of 4588.1 which is in agreement with the calculated molecular weight. However, both the earlier and later HIC peaks have an observed molecular weight that is approximately 1 Da



higher than the calculated molecular weight. Because this tryptic peptide contains an Asn residue, the 1 Da increase was likely caused by deamidation. Although the peak areas cannot be accurately integrated because of peak overlapping, the relatively larger first peak and a smaller later peak also supported the deamidation hypothesis because Asn deamidation resulted in the formation of isoAsp and Asp roughly in a 3:1 ratio1,2. In HIC peak 3, the isoAsp and Asp peaks are much higher than that of the original Asn peak, indicating a higher level of deamidation. The HIC peaks were assigned based on the relative amounts of isoAsp, Asn and Asp shown in Figure 3. HIC peak 1 corresponded to the antibody with Asn on both light chains. HIC peak 2 corresponded to the antibody with Asn on one heavy chain and isoAsp or Asp on the other heavy chain. HIC peak 3 corresponded to the antibody with either isoAsp or Asp on both light chains. The remaining Asn detected in peak 3 (Figure 3C) was probably caused by the fact that those variants were not baseline separated by HIC. It is worthwhile to mention that, although molecular weight differences caused by deamidation at intact and reduced light chain and heavy chains were not detected here, previous studies have demonstrated that small and consistent molecular weight increases detected at intact or subunit levels are a good indication of deamidation27,28. Additionally, a high throughput enzymatic method has been established to detect the total amount of isoAsp 29,30. However, for site-specific identification and quantitation, LC-MS peptide mapping is still the method of choice.

Deamidation in the antibody-antigen complex Because HIC peak 1 contained non-deamidated mAb, it was collected, analyzed by the HIC method to confirm sufficient purity and then used to study the deamidation propensity of the antibody in complex with its specific antigen. The collected peak 1 was incubated either alone, as a mixture with this mAb’s specific antigen or with a bovine serum albumin. Bovine serum albumin was chosen because its molecular weight is close to the antigen monomeric molecular weight. The samples were then


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incubated in PBS at pH 7.4 at 37 °C for 10 days. The samples were analyzed before and after the 10 day incubation to confirm the formation of antibody-antigen complex. A single peak was observed when the antibody was analyzed by SEC (Figure 4A), as expected. On the other hand, a much more complicated chromatogram (Figure 4B) was observed from analyzing the antigen because it exists as trimer or oligomers. Nevertheless, when the antibody-antigen complex was analyzed by SEC, no peak at the retention time of the free antibody was observed (Figure 4C), supporting the formation of antibodyantigen complex. The peak profile of the antibody-antigen is also different from that of the antigen alone, which further confirmed the formation of antibody-antigen complex. After incubation, the samples were also analyzed by SEC. As shown in Figure 4D, no free antibody was observed in the sample containing antibody-antigen complex. The lack of free antibody indicated that the antibody remained in complex with the antigen during incubation. In the samples containing the antibody alone or in the mixture with BSA, only free antibody was detected, confirming the antibody remained in solution (data not shown). Next, the incubated samples were analyzed using the same peptide mapping procedure. The extracted ion chromatograms of the tryptic peptides are shown in Figure 5. Similar peak profiles were observed for the antibody alone and the antibody mixed with BSA (Figure 5A and 5B, respectively). The respective spectrum shows three peaks corresponding to the peptide containing isoAsp, Asn or Asp. Interestingly, the peak corresponding to the peptide containing isoAsp is missing in the antibody-antigen complex (Figure 5C), although a similar amount of Asp was generated for all three samples judged from similar Asp peak area. Considering the total amount of deamidation products including isoAsp and Asp, much less of the products were observed in the sample containing antibody-antigen complex. Deamidation through the common β-elimination mechanism forms the succinimide intermediate. For small peptides lacking structural protection, hydrolysis of the succinimide forms



isoAsp and Asp in approximately 3:1 ratio. However, within the native protein structure, nucleophilic attack on the carbonyl carbon could be restricted, causing isoAsp or Asp in a ratio deviated from 3:1. The absence of isoAsp in the antibody-antigen complex indicated that main chain carbonyl carbon is protected from a water attack likely caused by a conformational change. The shielding effect of protein structure has been demonstrated using a mutant histidine containing protein, and while hydrolysis of a succinimide only forms Asp, elimination of a glutamate (Glu) residue in close proximity within the structure results in the formation of both isoAsp and Asp21. The authors hypothesized that one of the Ƴ-carboxyl oxygens of this Glu prevents the nucleophilic attach of the main chain carbonyl-carbon. The impact of antibody-antigen complex formation on the level and product ratio of Asn deamidation adds another case highlighting the critical role of protein structure on deamidation

Conclusions Deamidation of asparagine in recombinant monoclonal antibodies has been extensively studied because it is a common degradation pathway and has a negative impact on potency. The current study compared the deamidation propensity of a susceptible asparagine residue in the light chain complementarity-determining region of an antibody free in solution, in antibody-antigen complex or in a mixture containing an irrelevant protein. The results demonstrated that the formation of antibodyantigen complex not only decreased the level of deamidation, but also altered the deamidation products. More studies are warranted to fully understand the significance of this observation because the ratio of free antibody and antibody-antigen complex is expected to vary depending on dosing of therapeutic antibodies and the levels of their specific antigens in vivo.


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12. Bults, P.; Bischoff, R.; Bakker, H.; Gietema, J. A.; van de Merbel, N. C. LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma. Anal. Chem. 2016, 88 (3), 1871-1877. 13. Liu, Y. D.; van Enk, J. Z.; Flynn, G. C. Human antibody Fc deamidation in vivo. Biologicals 2009, 37 (5), 313-322. 14. Patel, K.; Borchardt, R. T. Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides. Pharm. Res. 1990, 7 (8), 787-793. 15. Robinson, N. E.; Robinson, A. B. Molecular clocks. Proc. Natl. Acad. Sci. U. S. A 2001, 98 (3), 944949. 16. Xie, M.; Schowen, R. L. Secondary structure and protein deamidation. J. Pharm. Sci. 1999, 88 (1), 8-13. 17. Robinson, N. E. Protein deamidation. Proc. Natl. Acad. Sci. U. S. A 2002, 99 (8), 5283-5288. 18. Clarke, S. Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int. J. Pept. Protein Res. 1987, 30 (6), 808-821. 19. Sydow, J. F.; Lipsmeier, F.; Larraillet, V.; Hilger, M.; Mautz, B.; Molhoj, M.; Kuentzer, J.; Klostermann, S.; Schoch, J.; Voelger, H. R.; Regula, J. T.; Cramer, P.; Papadimitriou, A.; Kettenberger, H. Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions. PLoS. One. 2014, 9 (6), e100736. 20. Phillips, J. J.; Buchanan, A.; Andrews, J.; Chodorge, M.; Sridharan, S.; Mitchell, L.; Burmeister, N.; Kippen, A. D.; Vaughan, T. J.; Higazi, D. R.; Lowe, D. Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues. Anal. Chem. 2017, 89 (4), 2361-2368. 21. Athmer, L.; Kindrachuk, J.; Georges, F.; Napper, S. The influence of protein structure on the products emerging from succinimide hydrolysis. J. Biol. Chem. 2002, 277 (34), 3050230507. 22. Wright, H. T. Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Critical Reviews in Biochemistry and Molecular Biology 1991, (26), 1-52. 23. Du, Y.; Walsh, A.; Ehrick, R.; Xu, W.; May, K.; Liu, H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs. 2012, 4 (5), 578-585. 24. Vlasak, J.; Ionescu, R. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Curr. Pharm. Biotechnol. 2008, 9 (6), 468-481. 25. Haverick, M.; Mengisen, S.; Shameem, M.; Ambrogelly, A. Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications. MAbs. 2014, 6 (4), 852-858.


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Figure legend Figure 1. Diagram showing Asn deamidation reaction. Nucleophilic attack of the carbonyl carbon of an Asn residue by the following main chain nitrogen results in the formation of succinimide. The unstable succinimide can be readily hydrolyzed when water molecules attach carbonyl-carbon. Attacking the main chain carbonyl-carbon leads to the formation of isoaspartate. Attacking the side chain carbonylcarbon leads to the formation of aspartate.



Figure 2. HIC chromatogram of the recombinant monoclonal antibody. The peaks were assigned as peaks 1, 2, or 3 based on the abundance. Peaks 2 and 3 are formed due to deamidation of a light chain CDR Asn residue as shown by the cartoons. Figure 3. Extracted ion chromatograms of the tryptic peptide containing light chain CDR2. The single peak detected in HIC peak 1 has a molecular weight in agreement with the calculated molecular weight (A). Two additional peaks with a molecular weight increase of 1 Da were observed before and after the Asn containing peak in HIC peak 2 (B). Higher levels of the two additional peaks were observed in HIC peak 3 (C). Amino acid sequence and the calculated molecular weight are shown on top of this figure. The additional peaks were assigned as containing either isoAsp or Asp based on their relative retention time and abundance. Figure 4. SEC chromatograms of antibody (A), antigen (B) and antibody-antigen complex before (C) or after (D) incubation. A single peak was observed for the antibody as expected. Multiple antigen peaks were observed because the antigen exists as trimer or oligomers (B). Peaks at the antibody retention time of 8 min were detected at a minimal level in the antibody-antigen complex before (C) and after (D) incubation, confirming the formation of antibody-antigen complex. Different amounts of samples were injected for each chromatogram because each sample has different protein components. The shift of peak retention times indicated the formation of mAb-antigen complex. Figure 5. Extracted ion chromatograms of the tryptic peptide containing light chain CDR2. The expected three peaks corresponding to the peptide containing isoAsp, Asn or Asp were observed in the samples of free antibody (A) or antibody mixed with BSA (B). The peak corresponding to the peptide containing isoAsp was not detected in the antibody-antigen complex sample (C).


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



For ToC


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Asparagine Deamidation in a Complementarity Determining Region of

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