Accurate Determination of Succinimide Degradation Products Using

Jun 21, 2011 - This method minimizes artifacts caused by the sample preparation process ..... With this hypothesis in mind, we developed a high fideli...
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Accurate Determination of Succinimide Degradation Products Using High Fidelity Trypsin Digestion Peptide Map Analysis X. Christopher Yu,* Koman Joe, Yu Zhang, Andrea Adriano, Yaning Wang, Helene Gazzano-Santoro, Rodney G. Keck, Galahad Deperalta, and Victor Ling Protein Analytical Chemistry and Biological Technologies, Genentech, a Member of the Roche Group, South San Francisco, California 94080-4990, United States ABSTRACT: We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.

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uccinimide formation in therapeutic protein products is an important and common degradation event. Conditions used during manufacturing processes such as cell culture, recovery, and formulation as well as those used during product storage can all influence the extent of degradation. Extensive studies have been conducted and published regarding the origin, stability, and reaction pathways of succinimide.1,2 Formation of succinimide at asparaginyl residues occurs when the backbone amide on the carboxyl side attacks the side chain carbonyl group, resulting in the loss of an ammonia molecule. Reaction at aspartyl residues requires a protonated side chain carboxyl group and is accompanied with the loss of a water molecule. In therapeutic proteins, succinimide has been observed to form at significantly faster rates in mildly acidic pH conditions.3 5 The rate of succinimide formation depends on the equilibrium concentration of the anionic form of the backbone amide nitrogen, which can be influenced by the inductive effect of the side chain group on the carboxyl side as well as the local and three-dimensional conformation of the protein molecule.2,6 8 For monoclonal antibody protein therapeutics, succinimide formation in regions of relative solvent accessibility and functional importance can have detrimental effects on biological activity and requires sensitive detection and accurate characterization.5,9 Detection of the succinimide-containing variant form has been achieved using various separation techniques such as ion exchange chromatography,5,9,10 hydrophobic interaction chromatography,11 13 and electrophoresis.8,14 Upon initial observation of a minor variant form, direct identification of the amino acid residue at r 2011 American Chemical Society

which succinimide formation occurs typically requires the use of peptide map analysis in conjunction with analysis by mass spectrometry and/or other protein chemistry techniques. A significant technical challenge lies in the fact that succinimide is relatively unstable and quickly hydrolyzes under alkaline pH and denaturing conditions. Conventional peptide map analysis is typically performed with protein denaturation and cysteine reduction and alkylation at alkaline pH as well as with significant exposure to alkaline pH during enzymatic digestion. The highly ubiquitous and often mandated use of such sample preparation conditions has been recognized as one of the most significant challenges in the sensitive and accurate detection of succinimide.5,9 The relative inability to correctly identify and quantify succinimide and its hydrolysis products can lead to limited or inaccurate understanding of protein stability and degradation pathways.8 Taking advantage of the tendency of succinimide to hydrolyze in water and the superior resolution of modern mass spectrometry, researchers have reported efforts to fully hydrolyze succinimide in O18-labeled water and to detect the now 2 Da heavier peptides as surrogates for succinimide formation.3,7,15 Another approach relies on direct detection of succinimide in proteolytic peptides generated with attention to minimizing exposure to alkaline pH and accelerating the enzymatic digestion.4,5,10 Although the use of surfactant allows a faster digestion, the Received: March 24, 2011 Accepted: June 21, 2011 Published: June 21, 2011 5912

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Analytical Chemistry approach requires an additional acid treatment step at low pH after the digestion and subsequent centrifugation step to remove the degraded surfactant. These further manipulations could introduce modifications to acid labile groups in the protein sample and lead to variable recovery of hydrophobic peptides. In this article, we report the use of a modified protein reduction and trypsin digestion procedure that is simple, fast, and accurate with minimal hydrolysis of succinimide and artifactual deamidation. The proteolysis using porcine trypsin is completed in 1 h at pH 7.0, without the use of surfactant or exceptional enzyme to substrate ratio that may introduce artifacts. Supported by the use of this improved peptide map analysis, we report the identification of a stable succinimide that is correlated to significantly lower biological activity as measured in a cell based potency assay. The availability of our high fidelity trypsin digestion peptide map analysis as a generic method also provides new opportunities for screening succinimide and other deamidation and isomerization “hot” spots in support of protein engineering and formulation development, in advance of the often timeconsuming and costly product-specific method development.

’ MATERIALS AND METHODS Materials. Recombinant immunoglobulin G subclass 1 (IgG1) monoclonal antibody (mAb) samples were expressed in Chinese hamster ovary cells and purified at Genentech (South San Francisco, CA). MAb1, MAb2, and MAb3 samples analyzed and described in this work were investigational monoclonal antibody products in development for potential therapeutic uses. Stressed materials of MAb1 were prepared by subjecting samples to various pH and temperature storage conditions for prolonged periods. Chemical Reagents. TRIS base, TRIS HCl, trifluoroacetic acid, calcium chloride, dithiothreitol (DTT), (2-(N-Morpholino)ethanesulfonic acid (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), and iodoacetic acid were purchased from Sigma (St. Louis, MO). Guanidine hydrochloride, trifluoroacetic acid (TFA), and tris(2-carboxyethyl) phosphine (TCEP) were from Thermo Fisher (Rockford, IL). Ethylenediaminetetraacetic acid (EDTA, disodium, dihydrate) and acetonitrile (HPLC grade) were purchased from J. T. Baker (Phillipsburg, NJ). Sequencing grade modified trypsin (porcine, both lyophilized and frozen) was from Promega (Madison, WI). PD-10 desalting columns were purchased from GE Healthcare (Uppsala, Sweden). Carboxypeptidase B (DFP treated) was purchased from Roche Applied Science (Indianapolis, IN). Imaged Capillary Isoelectric Focusing. Charge variant distribution was assessed by using an iCE280 analyzer (Convergent Bioscience, Toronto, Canada) with a fluorocarbon-coated capillary cartridge (100 μm  5 cm). The ampholyte solution consisted of a mixture of 0.35% methyl cellulose (MC), 2% 5 8 carrier ampholytes, 3% 8 10.5 carrier ampholytes, and 0.3% pI markers 6.61 and 9.22 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.1% MC. Samples were diluted, mixed with the ampholyte solution, and then focused by introducing a potential of 1500 V for 1 min, followed by a potential of 3000 V for 10 min. An image of the focused charge variants was obtained by passing 280 nm ultraviolet light through the capillary and into the lens of a charge-coupled device digital camera. In order to remove heavy chain C-terminal lysine residue, carboxypeptidase B was added to

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each sample at the dilution step at an enzyme-to-substrate ratio of 1:100 (w/w) followed by incubation at 37 °C for 20 min. Cation Exchange Chromatography for Basic Variant Isolation. A basic variant was isolated using cation exchange chromatography performed with an Alliance 2695 HPLC from Waters (Milford, MA) and ProPac WCX-10 column (4.6 mm  250 mm) from Dionex (Sunnyvale, CA). Mobile phase A was 20 mM MES at pH 6.1 and mobile phase B was 200 mM sodium chloride in mobile phase A. A gradient of 25% to 40% B in 30 min was used for separation, with a flow rate of 0.5 mL/min and a column temperature of 40 °C. Measurement of Biological Activity. The biological activity assay is based upon the ability of MAb1 to inhibit cell proliferation mediated by the interaction between the ligand and cell surface receptor. Cells transfected with the targeted receptor were seeded in a 96-well cell culture plate and incubated for 1 2 h at 37 °C, 5% CO2 to allow for cell settlement. MAb1 standards, reference control, and samples were added into the plate followed by incubation at 37 °C with 5% CO2 for 3 days. Cell viability fluorescent dye was added into the wells for another overnight incubation at 37 °C with 5% CO2. The fluorescence unit was read by a fluorescence plate reader. The results, expressed in relative fluorescence units (RFU), were plotted against MAb1 concentrations, and a four-parameter curve-fitting program was used to estimate the sample activity relative to the reference standard. Reference standard was kept in frozen conditions during storage to minimize potential degradation over time. Results were reported as percent specific activity with the reference standard assigned as 100%. Reduction of MAb1 and Molecular Mass Determination of Reduced Heavy and Light Chains. The MAb1 samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) using an Applied Biosystems/MDS Sciex QStar Elite mass spectrometer (Foster City, CA). After reduction of the disulfide bonds with TCEP, the samples were desalted by reversed-phase high-performance liquid chromatography (RP-HPLC) for direct online MS analysis. Spectra for reduced samples were derived from multiply charged ions and deconvoluted using the Analyst QS 2.0/BioAnalyst 2.0 software package (Applied Biosystems/ MDS Sciex, Foster City, CA). Conventional Trypsin Digestion Sample Preparation. A total of 1 mg of mAb sample was reduced by incubation with 20 mM DTT for 60 min at 37 °C in 6 M guanidine hydrochloride, 360 mM Tris, and 2 mM EDTA at pH 8.6. After cooling to room temperature, iodoacetic acid was added to a final concentration of 50 mM and reaction was allowed to occur in the dark for 15 min. The reaction mixture was buffer exchanged into digestion buffer (25 mM Tris, 2 mM CaCl2, pH 8.2) using PD-10 desalting columns. Protein concentration was determined by absorbance at 280 nm and then adjusted to ∼0.4 mg/mL with digestion buffer. Samples were digested with trypsin for 5 h at 37 °C using an enzyme to substrate ratio of 1:40 (w/w). The digestion was stopped by adjusting the pH to 2 with addition of 10% TFA solution. The digested samples were stored at 2 8 °C until injection onto the column. High Fidelity Trypsin Digestion Sample Preparation. A total of 1 mg of mAb sample was reduced by incubation with 10 mM TCEP for 15 min at 37 °C in 6 M guanidine hydrochloride and 300 mM sodium acetate at pH 5.0. The reaction mixture was buffer exchanged into digestion buffer (20 mM MOPS, 0.5 mM TCEP, pH 7.0) using PD-10 desalting columns. Protein concentration was determined by absorbance at 280 nm 5913

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Analytical Chemistry and then adjusted to ∼0.4 mg/mL with digestion buffer. Samples were digested with porcine trypsin for 1 h at 37 °C using an enzyme to substrate ratio of 1:40 (w/w). The digestion was stopped by adjusting the pH to 2 with addition of 10% TFA solution. The digested samples were stored at 2 8 °C until injection onto the column. Reversed Phase HPLC and Online LC MS/MS Analysis. Peptide map analysis was performed with an Agilent (Santa Clara, CA) 1100/1200 HPLC using a Phenomenex (Torrance, CA) Jupiter C18 column (2.0 mm  250 mm, 5 μm, 300 Å). Mobile phase A was water with 0.1% TFA, and mobile phase B was 90% acetonitrile in water with 0.09% TFA. A linear gradient from 0 to 40% B in 160 min was used, with 40 μg of digested protein injected for each analysis. The flow rate was 0.25 mL/ min, with a column temperature of 55 °C. LC MS and LC MS/MS experiments were performed with Thermo Fisher Scientific LTQ XL and LTQ-Orbitrap XL (San Jose, CA) mass spectrometers equipped with electrospray ionization sources. Mass spectrometers were operated in the positive ionization mode with the following source conditions: 4.5 kV spray voltage, 300 °C capillary temperature, and 35 and 9 L/min sheath and auxiliary gas flows, respectively. Collision induced dissociation (CID) experiments were performed with a 4 atomic mass unit (amu) isolation width and a normalized collision energy of 30%. Instruments were tuned and calibrated according to the manufacturer’s recommendations prior to data acquisition. High-resolution mass determination was performed with an LTQ-Orbitrap XL instrument using a full-MS survey scan with the resolution set at 30 000 at m/z 400, followed by ion trap MS2 scans for ions of interest. For estimation of the relative levels of each species, extracted ion chromatograms were generated using the monoisotopic m/z ( 2 amu for LTQ XL results and monoisotopic m/z ( 0.1 amu for LTQ-Orbitrap data.

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Figure 1. Imaged capillary isoelectric focusing electropherograms of MAb1 samples that were stored frozen (blue) and at 40 °C for 4 weeks (black); each shown in duplicate.

’ RESULTS AND DISCUSSION Thermal Degradation as Indicated by Changes in Charge, Molecular Mass, and Biological Activity. As part of manufac-

turing process development for an IgG1 monoclonal antibody molecule MAb1, both pH- and thermal-stressed materials were analyzed by several analytical methods. Analysis of charge distribution profile by imaged capillary isoelectric focusing (icIEF) showed an increase in species that are more basic than the main peak (with higher apparent pI values) at relatively lower pH or higher temperature conditions. Figure 1 shows an overlay of the electropherograms of material that was stored at 40 °C for 4 weeks and one that was stored frozen at 70 °C. While practically no changes were detected in regions more acidic than the main peak, the basic variants increased dramatically and accounted for all of the relative peak area reduction in the main peak. Determination of light chain and heavy chain molecular masses were carried out using TCEP-reduced MAb1 samples and online reversed phase LC MS analysis. Examination of the measured light chain molecular masses of two samples stored at pH 5.0 for 4 weeks (Sample A at 5 °C and Sample B at 40 °C) revealed a significant increase in Sample B of a molecular species that was 18 Da smaller than the theoretical light chain molecular weight (Figure 2). The heavy chain masses showed minimal differences between these two samples (data not shown). Among several possible explanations for this apparent mass reduction, we hypothesized that a likely cause was the formation of succinimide at an Asp residue (loss of water molecule, 18 Da) or at an Asn

Figure 2. Deconvoluted light chain mass spectra of MAb1 samples that were stored at 5 °C (blue, Sample A) and at 40 °C for 4 weeks (red, Sample B).

residue (loss of ammonia molecule, 17 Da). Molecules with succinimide formation at an Asp residue would be expected to be more basic due to the loss of the carboxylic acid group, and this could be consistent with the observed increase in basic variants by icIEF. In fact, we observed good correlation between increases in 18 Da light chain species and increases of basic variants by icIEF among a panel of pH and thermal stressed samples (linear regression, R2 = 0.93). The stressed samples were also analyzed for their biological activity in a cell based potency assay relative to a reference control that was stored frozen. The assay is designed to reflect the ability of MAb1 to prevent a ligand from binding to the cell surface receptor. It is expected to be reflective of the mechanism of action of the IgG1 molecule and is sensitive to physicochemical changes or degradation in the complementarity determining region (CDR). Sensitivity to thermal degradation in MAb1 was clearly illustrated with a measured activity of 93% for Sample A (4 weeks at 5 °C) and 54% for Sample B (4 weeks at 40 °C). The relative activities reported here are the mean of duplicate measurements, with observed percent differences of 4% and 7% for Sample A and Sample B, respectively. In general, lower pH samples correlated with higher levels of basic variants, higher levels of 18 Da species, and lower activities. These results 5914

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Figure 3. Partial UV chromatograms and extracted ion chromatograms of conventional peptide map analysis of MAb1 samples that were stored at 5 °C (black, Sample A) and at 40 °C for 4 weeks (red, Sample B): (3a) UV for Sample A; (3b) UV for Sample B; (3c) extracted ion chromatogram of all forms of peptide T3L for Sample A; (3d) extracted ion chromatogram of all forms of peptide T3L for Sample B.

Figure 4. Partial UV chromatograms and extracted ion chromatograms of high fidelity peptide map analysis of MAb1 samples that were stored at 5 °C (black, Sample A) and at 40 °C for 4 weeks (red, Sample B): (4a) UV for Sample A; (4b) UV for Sample B; (4c) extracted ion chromatogram of all forms of peptide T3L for Sample A; (4d) extracted ion chromatogram of all forms of peptide T3L for Sample B.

are consistent with reported studies indicating that succinimide forms more readily (or is more stable) at around pH 5 as compared to higher pH formulations.3,5 On the basis of the apparent reduced biological activity, we believed that there was likely degradation in one or more CDR regions in the light chain which negatively impacted the antigen binding properties. Detection of iso-Asp Formation at Asp30 in Light Chain by Conventional Tryptic Map. Samples A and B (pH 5.0, 4 weeks, at 5 and 40 °C, respectively) were both analyzed under conventional tryptic peptide map conditions with online LC MS/MS analysis.16,17 Comparison of the UV absorbance profiles of the two samples (Figure 3a,b) indicated that in Sample B, peak P1 (at 75.0 min) is relatively decreased and a new peak P2 has appeared at 70.9 min. Both P1 and P2 had the same observed m/z of 742.3 (2+ charge) and the same MS/MS fragmentation pattern (data not shown) that are consistent with those of expected tryptic peptide T3L with the amino acid sequence of ASQD28VD30TSLAWYK. Both D28 and D30 residues are located within the light chain CDR1 region of MAb1. We did not observe peptides with masses that are 18 Da less than the expected, which might suggest the presence of succinimide. Extracted ion chromatograms (Figure 3c,d) show only the presence of ions at m/z 742.3 at retention times of 75.0 and ∼71 min and none at m/z 733.3 (expected 2+ m/z if 18 Da species was present). HPLC fractions corresponding to both P1 and P2 were collected and subjected to amino acid sequencing analysis by Edman degradation. Results confirmed that fraction P1 contained the expected tryptic peptide T3L, while the sequencing for fraction P2 stopped at D30 after the first five Edman cycles (ASQD28V). Our interpretation of these results was that fraction P2 from Sample B consisted of a degraded form of peptide T3L with iso-Asp at the D30 position. We hypothesized that succinimide was present at the D30 position in a large amount in Sample B and the iso-Asp observed at this position had come from the hydrolysis of succinimide during the sample preparation of peptide map analysis. It is

well documented that the alkaline pH conditions used during sample preparation and digestion can lead to significant or complete hydrolysis of the succinimide and result in the artificial generation of iso-Asp and Asp.11,18 Using light chain molecular mass determination alone, one is not able to discern whether isoAsp was present prior to digestion, as its mass is isobaric to that of Asp residue. In fact there are known instances where iso-Asp was determined to be present in mAb molecules, either at the point of product generation or as a degradation product from storage or stress conditions.22 Since succinimide and iso-Asp may have different implications on the product structure and activity, we felt it is critical that we determine correctly and accurately the nature and identity of degradation products in MAb1. Identification of Succinimide Formation at Light Chain Asp30 by High Fidelity Trypsin Digestion Peptide Map Analysis. On the basis of the evidence that light chain molecular mass showed a significant amount of 18 Da species in Sample B, we hypothesized that succinimide formed at Asp30 residue under stressed storage conditions was converted to iso-Asp as an assay artifact during the conventional peptide map sample preparation and digestion. With this hypothesis in mind, we developed a high fidelity trypsin digestion procedure that minimized the sample exposure to alkaline pH conditions. Sample reduction with DTT at pH 8.5 was replaced with TCEP reduction at pH 5.0, and alkylation of the reduced cysteine residues with iodoacetic acid at pH 8.5 was eliminated. Reduced sample was buffer-exchanged into a digestion buffer that consisted of 20 mM MOPS at pH 7.0 with 0.5 mM TCEP. Trypsin digestion was then carried out for 1 h at pH 7.0, instead of 5 h at pH 8.2 in the conventional digestion procedure. We note that this modified procedure produced comparable and complete digestion using porcine trypsin from three different manufacturers (Promega Sequencing grade, Roche Recombinant Proteomics grade, and Serva Modified Sequencing grade). In contrast, the use of bovine trypsin (Sigma Modified Sequencing grade, Roche Modified 5915

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Figure 5. Tandem mass spectra of P1 at m/z 742 (top) and P3 with succinimide at m/z 733 (bottom). The y and b series fragment ions highlighted in red are used to support the localization of succinimide ( 18 Da mass shift) to the Asp30 residue.

Sequencing grade, and Worthington TPCK-Treated Sequencing grade) resulted in various degrees of incomplete digestion (data not shown). Samples A and B were subjected to these optimized sample preparation conditions, and the resulting digests were analyzed by online LC MS/MS. As shown in parts b and d of Figure 4, a dominant new peak P3 with an observed m/z of 733.3 was found in Sample B at retention time ∼81 min. This mass is 18 Da smaller than the mass of peptide P1 and would be consistent with the presence of succinimide at Asp30. Fragmentation of the P3 peptide and examination of product ions in the MS/MS spectrum provided additional evidence that succinimide formation and the reduction of 18 Da in mass was located at Asp30 indeed (Figure 5). Comparing Figure 4d to Figure 3d, we noticed only a trace amount of P2 (T3L peptide containing isoAsp at D30) present in Sample B under the high fidelity trypsin digestion conditions. If we assume that column recoveries and electrospray ionization efficiencies are similar among the three versions of peptide T3L (iso-Asp, Asp, and succinimide (Asu) at Asp30), the relative amount of each can be estimated using the integrated area of each peak in the extracted ion chromatogram. The relative level of the succinimide form can be determined accurately by light chain molecular mass measurement since the sample was never exposed to alkaline pH conditions and therefore no method related hydrolysis and production of iso-Asp is expected. However, light chain mass measurement would not detect the possible presence of the iso-Asp residue as its mass is identical to an Asp residue. As seen in Table 1, the percent succinimide derived from high fidelity trypsin digestion peptide map analysis and from light chain mass measurement was in good agreement for both Samples

Table 1. Quantification of Succinimide (Asu), Asp, and isoAsp Forms of T3L Peptide by LC MSa peptide map

Sample A Sample B a

LC mass

% iso-Asp

% Asp

% Asu

% Asu

conventional high fidelity

3 ND

97 94

ND 6

8

conventional

49

51

ND

69

high fidelity

2

28

70

Relative quantification is estimated to have a % RSD of e20%.

A and B. We concluded that hydrolysis of succinimide was minimal under the high fidelity trypsin digestion conditions. The low level of iso-Asp detected in Sample B could have been formed during storage and present prior to the samples being analyzed. Taking together the results described in previous sections, we concluded that the great majority of the degradation product in Sample B was succinimide at Asp30 in the light chain. The development and utilization of high fidelity trypsin digestion conditions played an essential role in this determination. In contrast, we show that the conventional tryptic map conditions were not suitable for analysis in this study, and this point should be considered carefully when succinimide degradation is suspected or possible. Relative quantification using specific peak area in the extracted ion chromatograms was performed to reveal the significant differences in the results obtained with conventional and high fidelity trypsin digestion peptide map analyses. The similarity between relative succinimide levels obtained by high fidelity 5916

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Figure 6. Cation exchange chromatograms of MAb1 samples: (6a) frozen bulk; (6b) thermal stress at 40 °C for 4 weeks; (6c) main peak fraction from the frozen bulk; (6d) basic peak fraction from the thermal stress sample.

Figure 7. Co-crystal structure and model of MAb1 and its antigen in CDR L1 region: (7a) favorable interaction between the S30 residue hydroxyl group in pre-MAb1 and an Arg residue side chain group in the antigen molecule as shown by the dotted line; (7b) favorable interaction between the D30 residue hydroxyl group in MAb1 and the same Arg residue side chain group in the antigen molecule.

trypsin digestion and those by light chain molecular mass measurement further demonstrate the accurate nature of high fidelity trypsin digestion peptide map analysis in succinimide identification and characterization. A systematic evaluation was not performed in this study to fully characterize the precision of the relative quantification in general and especially when performed under interday assay and multiple analyst conditions. On the basis of our observations in this and previous work, we estimate a percent relative standard deviation (% RSD) of e20% when analysis is performed by the same analyst in the same day using the same HPLC and MS instruments. Further Evidence that Succinimide Formation at LC Asp30 Results in Reduced Activity of MAb1. It was considered possible that degradation in other regions of the MAb1 molecule caused the apparent reduction in biological activity, and our characterization could have failed to reveal these modification events. However, the observed increases in light chain 18 Da and basic variant species in the pH and thermal stressed samples correlated well with the reduced activity (linear regression R2 = 0.98 and 0.87, respectively). This strongly suggested that the formation of succinimide at Asp30 was likely the direct cause of the reduced activity. We sought to obtain further evidence to support this hypothesis by charge variant isolation with cation exchange chromatography (CEX) and characterization of the isolated basic variant. Using a thermally stressed sample as the starting material, we isolated a basic variant fraction (Figure 6) that was found to have 54% succinimide by light chain molecular mass determination, with 67% relative activity. This was compared with a control sample using the main peak fraction of frozen bulk (Figure 6) which showed ∼7% succinimide in the light chain and 97% activity. Binding kinetics experiments were performed with the basic variant fraction displaying more than 60% reduction of the ON rate of the frozen bulk, while the OFF rate remained relatively unchanged (data not shown). Similar loss of binding was reported when succinimide was formed at Asn55 in the CDR H2 region of an IgG1 molecule.5 These characterization results on the isolated basic variant are consistent with those obtained using pH and thermal stressed materials and support the conclusion that the succinimide formation in the light chain is primarily responsible for the observed activity loss in the MAb1 molecule. Structural Basis of the Correlation between Succinimide Formation and Reduced Activity. We were intrigued by the possibility that the apparent correlation between succinimide

formation in the light chain CDR1 region and loss of activity in the MAb1 molecule can be explained at the molecular structure level. As part of the research activities, the Fab fragment of an earlier generation of the MAb1 molecule prior to affinity maturation (Pre-MAb1) was crystallized as a complex with the extracellular region of the antigen molecule (a cell surface receptor). The X-ray structure of the cocrystal complex was determined at 2.1 Å resolution and was examined specifically in this study for understanding the molecular basis of the apparent reduction in activity upon succinimide formation. Among several other differences in the primary sequence, Ser30 in the light chain of the Pre-MAb1 was replaced by Asp30 in the affinity matured MAb1 molecule. In the crystal structure, Ser30 is seen to form a salt bridge between its hydroxyl group and the side chain of an Arg residue on the antigen (Figure 7a). This water molecule mediated favorable interaction between the antibody and antigen is believed to remain or possibly strengthen with the Asp30 carboxylic group in MAb1 having replaced the Ser30 hydroxyl moiety in Pre-MAb1 (modeled in Figure 7b). Upon thermal stress, formation of succinimide at Asp30 would result in the loss of the negatively charged carboxylic acid group and substantially reduce the favorable binding with the Arg side chain on the antigen molecule. Although it is estimated that the majority of the specific binding affinity of mAb molecules often comes from interactions at CDR H3 and H2,23 the loss of these interactions could typically result in 10- to 100-fold reductions of binding affinity. A singular degradation event in the form of succinimide formation at Asn55 in CDR H2 was reported to result in a loss of ∼50% binding affinity,5 consistent with our observations described above. Conformation-Dependent Succinimide Hydrolysis. Similar to what we observed here in MAb1, several others studies have shown that succinimide can accumulate readily at mildly acidic pH and thermal stress conditions3,5 but is generally considered an unstable intermediate and difficult to detect at physiological pH conditions. It is believed that the optimal pH for succinimide formation is at about pH 5.3 Upon hydrolysis, the distribution ratio between iso-Asp and Asp is approximately 3 to 1 based on studies using synthetic peptides, although it can be more variable when the hydrolysis occurs as part of a native protein.6,7 We performed hydrolysis experiments at pH 8.4 using a thermal stressed MAb1 sample which initially contained ∼34% succinimide at Asp30. The hydrolysis reaction was carried out at 37 °C for periods of 1 and 2 days. The resulting samples were then subjected to high fidelity trypsin digestion peptide map analysis. 5917

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Table 2. Determination of Succinimide Hydrolysis Rate by High Fidelity Trypsin Digestion Peptide Map Analysisa high fidelity peptide map pH 8.4 treatment

% iso-Asp

% Asp

% Asu

T=0

8

58

34

T = 24 h

4

78

18

2 32

89 68

9 ND

T = 48 h T = 2 h denatured a

Relative quantification is estimated to have a % RSD of e20%.

As shown in Table 2, the succinimide level was found at 18% after 1 day and 9% after 2 days of hydrolysis, with a half-life of approximately 1 day. Interestingly, the hydrolysis product appeared to be Asp only, even as a low level of the iso-Asp form present in the starting stressed material was seen to reduce over increasing hydrolysis time. Since the Asp30 residue is part of the original primary sequence of MAb1, we expected the hydrolyzed samples to show recovered activity as compared with the starting stressed material. This was confirmed with measured biological activities of 76% ((4%, n = 3) in the original sample before treatment, as compared with 82% ((3%, n = 3) after 1 day and 89% ((9%, n = 3) after 2 days of alkaline treatment. Since there was no substantial amount of iso-Asp present, we were not able to determine the relative activity of the protein containing iso-Asp at the Asp30 position. The long half-life of succinimide and the observation of Asp as the only hydrolysis product at the Asp30 position were intriguing. Studies using synthetic peptides suggest expected halflives of less than 20 min for succinimide at pH 8.5 and a distribution ratio of approximately 3:1 for the hydrolysis products of iso-Asp and Asp.18 20 We hypothesized that our observation may be relatively unique because the hydrolysis was allowed to occur with the protein in a properly folded conformation. If we remove these structural constraints by subjecting the protein samples to denaturing conditions, the hydrolysis would be expected to proceed at a much faster rate and result in a more typical distribution of iso-Asp and Asp residues. An experiment was performed in which stressed MAb1 sample was incubated at pH 8.4 in the presence of 4.5 M guanidine hydrochloride. In distinct contrast to alkaline incubation under native conditions, the hydrolysis of succinimide at Asp30 was complete after 2 h of incubation with no succinimide detected by the high fidelity trypsin digestion peptide map analysis. Since the starting material had approximately 34% succinimide at the Asp30 position, we estimated that a distribution ratio of 3:1 would yield equivalents of 25% iso-Asp and 9% Asp with complete hydrolysis of succinimide. With addition to the original 8% iso-Asp and 58% Asp seen in the starting material, the final relative amount of isoAsp and Asp were expected to be 33% and 67%, respectively. This estimate agrees well with the apparent 32% iso-Asp and 68% Asp as determined by the high fidelity trypsin digestion peptide map analysis (Table 2). These results, together with the observation of Asp as the only hydrolysis product after native condition alkaline treatment, support our conclusion that protein conformation plays a key role in deciding the fate of succinimide hydrolysis. Similar observations of Asp and not iso-Asp as the predominant hydrolysis product of succinimide have been reported7,8 as well as isomerization of degradation product isoAsp to Asp in IgG1 light chain CDR1.21

High Fidelity Trypsin Digestion for Minimization of Assay Induced Deamidation. As one would expect, the minimal

exposure to alkaline pH during reduction and trypsin digestion also resulted in a significantly lower level of artificial deamidation. We examined the extent of deamidation in a tryptic peptide in two IgG1 molecules (MAb1 and Mab2) under both conventional and high fidelity trypsin digestion conditions. The peptide is located in the heavy chain Fc region and is commonly present in many IgG1 mAb molecules. The amino acid sequence of this peptide is GFYPSDIAVEWESN14GQPEN19NYK, with Asn residues N14 and N19 reported to have significant levels of deamidation.24 Using conventional trypsin digestion with 5 h of digestion at pH 8.2, we found significant amounts of deamidation species at the N14 position in both MAb1 and MAb2 samples. With an estimated % RSD of e20%, the levels of isoAsp species at N14 relative to that of the nondeamidated species was estimated to be 11% and 12%, respectively. An earlier study using a third IgG1 molecule (MAb3) revealed ∼11% iso-Asp at N14 (by UV absorbance peak area) after 4 h of digestion at pH 8.0 and 37 °C and a linear increase of 1.9% per hour between 4 and 7 h of incubation. In sharp contrast, the same iso-Asp species was observed to be no more than 0.3% in both MAb1 and MAb2 using high fidelity trypsin digestion conditions. It is clear that the use of conventional trypsin digestion conditions would have grossly overestimated the amount of deamidation in this peptide and likely other peptides that contain labile Asn residues. This result further demonstrates that high fidelity trypsin digestion peptide map analysis is superior in offering fast, simple, and accurate results in the detection and characterization of deamidation and succinimide degradation products. Trypsin is widely used in many life science applications, including proteomic research in discovery and detailed product characterization in biopharmaceutical manufacturing such as we describe in this work. A number of studies have been published in recent years that provided further insight into the optimal digestion conditions for these various applications.25 31 There is evidence that chemical modification can result in reduced autolysis and improved thermal stability.25,26 Improved digestion efficiency was reported using trypsin that was chemically modified, immobilized, or pressurized.27 29 It was also demonstrated that detergent-assisted as well as microwave-assisted trypsin digestion conditions produced faster and more efficient digestion.30,31 Consistent with our observations in this study, it has been reported that porcine trypsin is more robust and active at relatively low pH when compared with bovine trypsin.32 35 These characteristics make porcine trypsin particularly well suited for high fidelity peptide map analysis of succinimide, deamidation, and isomerization degradation products. We also believe that careful studies in the future could yield even faster and more efficient trypsin digestion conditions that would result in further improvement in the fidelity and throughput of peptide map analysis.

’ CONCLUSIONS When succinimide formation is studied as a potential degradation pathway for protein therapeutic products, peptide map analysis is an essential analytical technique for the identification of the location and nature of degradation products. Hydrolysis of succinimide occurs inadvertently during sample handling and with prolonged exposure to alkaline pH during proteolytic digestion, resulting in increased amounts of Asp and iso-Asp products. The presence of succinimide can be significantly under5918

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Analytical Chemistry represented or missed entirely with the use of conventional peptide map conditions. We demonstrate here in this report the relevance and importance of having a high fidelity peptide map procedure for the study of succinimide in proteins. The experimental approach described here not only allows correct identification of degradation products, it provides an essential tool to quantitatively describe the degradation event with regard to the relative amounts of succinimide, iso-Asp, and Asp forms and, consequently, the degradation kinetics. This method also effectively eliminates artificial deamidation that is observed under conventional trypsin digestion conditions, allowing accurate determination of deamidation sites and levels in protein therapeutic products and their stability characteristics.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (650) 225-1138. Fax: (650) 225-3554. E-mail: yu.c@ gene.com.

’ ACKNOWLEDGMENT We thank Yi Shen for her work in stressed panel sample preparation; Leslie Welch, Louie Basa, Melissa Alvarez, and Oleg Borisov for mass spectrometry instrument support; Armando Cordoba for Edman sequencing and Yongmei Chen for binding kinetics experiments; and Yongjian Wu and Margaret Lin for their assistance with biological activity measurements. We are grateful for the analysis and discussions of protein crystal structure with Christian Wiesmann. We greatly appreciate discussions with Jun Ouyang, James Andya, Bruce Kabakoff, Sue Skieresz, Judy Shimoni, Jing Qing, Yan Wu, Jun Liu, Aditya Wakankar, and Reed Harris and for the thoughtful review of the manuscript by John Stults. ’ REFERENCES (1) Clarke, S. Int. J. Pept. Protein Res. 1987, 30, 808–821. (2) Radkiewicz, J. L.; Zipse, H.; Clarke, S.; Houk, K. N. J. Am. Chem. Soc. 2001, 123, 3499–3506. (3) Chu, G. C.; Chelius, D.; Xiao, G.; Khor, H. K.; Coulibaly, S.; Bondarenko, P. V. Pharm. Res. 2007, 24, 1145–1156. (4) Huang, H. Z.; Nichols, A.; Liu, D. Anal. Chem. 2009, 81, 1686–1692. (5) Yan, B.; Steen, S.; Hambly, D.; Valliere-Douglass, J.; Vanden Bos, T.; Smallwood, S.; Yates, Z.; Arroll, T.; Han, Y.; Gadgil, H.; Latypov, Y.; Wallace, A.; Lim, A.; Kleemann, G. R.; Wang, W. Balland, A. J. Pharm. Sci. 2009, 98, 3509 3521. (6) Athmer, L.; Kindrachuk, J.; Georges, F.; Napper, S. J. Biol. Chem. 2002, 277, 30502–30507. (7) Xiao, G.; Bondarenko, P. V.; Jacob, J.; Chu, G. C.; Chelius, D. Anal. Chem. 2007, 79, 2714–2721. (8) Napper, S.; Prasad, L.; Delbaere, L. T. J. Biochemistry 2008, 47, 9486–9496. (9) Harris, R. J.; Kabakoff, B.; Macchi, F. D.; Shen, F. J.; Kwong, M.; Andya, J. D.; Shire, S. J.; Bjork, N.; Totpal, K.; Chen, A. B. J. Chromatogr., B 2001, 752, 233–245. (10) Teshima, G.; Stults, J. T.; Ling, V.; Canova-Davis, E. J. Biol. Chem. 1991, 266, 13544–13547. (11) Kwong, M. Y.; Harris, R. J. Protein Sci. 1994, 3, 147–149. (12) Cacia, J.; Keck, R.; Presta, L. G.; Frenz, J. Biochemistry 1996, 35, 1897–1903. (13) Valliere-Douglass, J.; Jones, L.; Shpektor, D.; Kodama, P.; Wallace, A.; Balland, A.; Bailey, R.; Zhang, Y. Anal. Chem. 2008, 80, 3168–3174.

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