ARTICLE pubs.acs.org/ac
Revealing a Positive Charge Patch on a Recombinant Monoclonal Antibody by Chemical Labeling and Mass Spectrometry Liangyi Zhang,† Wayne Lilyestrom,‡ Charlene Li,† Thomas Scherer,‡ Robert van Reis,§ and Boyan Zhang*,† †
Protein Analytical Chemistry, ‡Pharmaceutical Development, §Purification Development, Genentech Inc., South San Francisco, California 94080, United States
bS Supporting Information ABSTRACT: During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometrybased footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.
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ecombinant monoclonal antibodies (rmAbs) have large and complex three-dimensional (3D) structures that contain multiple weak acidic and basic groups on their surfaces. The ionization of these groups results in heterogeneous charge structures. Taking advantage of their complex but unique ionexchange equilibria, highly selective ion-exchange chromatography is commonly used for the purification of rmAbs in biotechnology industries.1,2 Evolved from the development in the past two decades, standardized platform purification approaches that are applicable to multiple rmAbs have been established to streamline the development efforts.35 A platform purification process typically involves Protein A affinity chromatography for capturing rmAbs and two ion-exchange chromatographic polishing steps to complete the purification.4 Most rmAbs fit well using a platform process; however, rmAbs that do not fit well using the platform approach have been observed. For those outliers, extra effort and time are often required to develop a robust purification process to produce rmAb products with acceptable yield, purity, and quality. Ion-exchange chromatography (IEC) and other charge-sensitive assays, such as capillary isoelectric focusing (cIEF), are commonly employed in the analytical characterization and quality control systems to assess the batch-to-batch production consistency and to monitor degradation of rmAbs.6,7 The separation mechanism is different between IEC and cIEF.7 In cIEF, a rmAb and its variants, which normally result from various post-translational r 2011 American Chemical Society
modifications in the manufacturing process, are separated on the basis of their difference in the isoelectric point (pI) values. In IEC, rmAb variants are resolved according to their surface electrostatic interactions with the oppositely charged stationary resin in a column. The electrostatic interaction is not only affected by the net charge of the intact rmAb but also influenced by the rmAb tertiary structure, in particular the distribution of the charges on the surface.8 For most rmAbs, the two chargesensitive assays give results with consistent charge heterogeneity profiles.9 Inconsistency between the results from these two methods may indicate that the rmAb has unique tertiary surface charge features. To understand why a rmAb does not fit the platform purification process and/or why its charge profiles are not consistent between IEC and cIEF assays, assessment of rmAb surface charge distribution is necessary. On the basis of a theoretical simulation study, Freitag and co-workers have recently reported that positive charges on a basic rmAb (pI = 9.3) are concentrated in the variable domains.10 They further proposed that the variable domains of the heavy and light chains of the rmAb form a positive charge patch, which dominate the electrostatic interaction with the apatite resin. Unfortunately, the amino acid Received: July 13, 2011 Accepted: October 17, 2011 Published: October 17, 2011 8501
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Analytical Chemistry composition of the charge patch could not be specified by structure modeling. In 1996, Zhu and Karlin defined that a “3D-charge cluster”, which could be considered the same as the definition of “charge patch”, is formed when three or more charge residues cluster in a region with a radius of 8 Å of a protein.11 Among the protein 3D structures they surveyed, about 10.2% contain at least one 3D-charge cluster and most of them are of mixed type involving about equally anionic and cationic residues. However, there was only one positive charged cluster found in the photosynthetic reaction center structure (PDB code: 1prc).11 This indicates that there is a scarcity of positive charge patches in endogenous proteins. To date, there are still no experimental identification reports on the amino acid composition of a surface charge patch on any recombinant monoclonal antibody. Mass spectrometry (MS) has been used to study protein surface topology and tertiary structure.1214 In these approaches, protein surface residues are chemically labeled and the percentage of labeling is then determined by peptide mapping. Since the percentage of residue labeling reflects solvent accessibility, protein conformation and conformational dynamics can be implied. 12 Recently, hydrogendeuterium exchange mass spectrometry (HDX-MS) has been applied to study the conformation of rmAbs, and conformational changes resulting from post-translational modifications such as methionine oxidation can be revealed.15,16 However, HDX-MS is not an ideal method to study protein surface charge features because the deuterium labels at the side chains are not stable and are lost when the sample reaches the mass analyzer.17 Complementary to deuterium labeling, stable chemical labeling methods in conjunction with MS have been also used to study protein conformation.12,13,18,19 Because the labeling tags are stable during sample preparation and characterization, the dynamics of amino acid side chains can be obtained. Furthermore, most labeling chemistries are selective to amino acids.13,20 As a result, amino acids of particular interest to protein conformation or interaction can be individually probed. One recombinant human antibody, which is referred to as rmAb1 in this work, showed atypical behavior in cation-exchange chromatography (CIEC) and did not fit into an established platform purification process.3,5 Chemical labeling and MS-based footprinting methods were developed to map the positive charge distribution on the surface of rmAb1. The interfaces between rmAb1 and cation-exchange resin were further characterized by labeling rmAb1 while bound to the resin. Multiple peptide mapping methods and homology modeling approaches were used to specifically identify residues involved in the interaction and understand the positive charge patch phenomenon.
’ EXPERIMENTAL SECTION Materials. Seven rmAbs were manufactured in-house at Genentech (South San Francisco, CA). The stressed rmAb1 samples were prepared by exchanging its buffer to 50 mM sodium acetate at pH 5.5 followed by incubation at 40 °C for 2 and 4 weeks, respectively. Sulfo-succinimidyl acetate (also called sulfo-NHS acetate, SNA), p-hydroxyphenylglyoxal (HPG), and tris(2-carboxyethyl) phosphine HCl (TCEP) were purchased from ThermoFisher (Rockford, IL). Dithiothreitol (DTT) was acquired from Sigma Aldrich (St. Louis, MO). Iodoacetic acid (IAA) was purchased from Fluka (Steinheim, Switzerland). Trypsin, Asp-N, and Lys-C were bought from Roche (Indianapolis, IN). ProPac WCX10 columns were bought from Dionex (Sunnyvale, CA). Fractogel
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SE Hicap cation-exchange resin was obtained from EMD (San Diego, CA). Cation-Exchange Chromatography (CIEC) and Imaged Capillary Isoelectric Focusing (icIEF). CIEC of rmAbs was performed on a Waters 2796 BioAlliance HPLC instrument (Milford, MA) controlled by Dionex Chromeleon software (Sunnyvale, CA). Solvent A was 50 mM HEPES at pH 7.5 (pH was adjusted by 50% sodium hydroxide). Solvent B was 0.1 M sodium sulfate in solvent A. rmAb stock solutions were diluted to 2 mg/mL with solvent A, and 25 μL of the diluted sample was injected onto a ProPac WCX-10 column (4 250 mm). rmAbs were eluted with a linear salt gradient of 280% B over 30 min at a flow rate of 1.0 mL/min; they were subsequently detected by ultraviolet (UV) absorbance at 280 nm. icIEF of rmAbs was carried out following the reported protocol,21 and the method details can be found in the Supporting Information section. In-Solution Chemical Labeling. Lysine and arginine residues on rmAb1 were labeled by sulfo-NHS acetate (SNA)22 and p-hydroxyphenylglyoxal (HPG),23 respectively (Scheme S-1, Supporting Information). The rmAb1 stock solution was diluted to 1 mg/mL with solvent A (50 mM HEPES at pH 7.5). SNA and HPG were freshly prepared as 140 mM stock solutions in solvent A. Both labeling reactions were performed at a series of reagentto-protein molar ratios of 100, 200, 400, 800, 1600, and 2000. Reaction mixtures were incubated at 37 °C for 2 h. The reactions were then quenched by addition of 10 μL of 10% TFA. The chemically labeled rmAbs were stored at 80 °C prior to analysis. On-Resin Chemical Labeling. rmAb1 was loaded onto Fractogel SE Hicap cation-exchange resin and then labeled with SNA and HPG, respectively. The resin (100 μL) was washed twice with 1 mL of solvent A and was then added to 1 mL of rmAb1 at 1 mg/mL in solvent A. The mixture was gently agitated for 1 min at room temperature. After centrifugation, the supernatant was discarded. The resin bound with rmAb1 was washed twice with 1 mL of solvent A and subsequently suspended into 1 mL of solvent A. It is noted that, in a separate experiment, the binding capacity of the resin was tested for rmAb1. The bound rmAb1 was eluted with the solvent B, and its concentration was measured by UV absorbance. It was found that the yield was nearly 100%, indicating that all of 1 mg of rmAb1 stayed bound to the 100 μL resin after the wash, and suspension in the solvent A SNA or HPG was added at a series of reagent-to-protein molar ratios of 100, 200, 400, 800, 1600, and 2000, respectively. After incubation at 37 °C for 2 h, the reaction mixtures were centrifuged and the supernatant was discarded. The resin was washed with 1 mL of the solvent A twice. The labeled rmAb1 was eluted using 1 mL of solvent B. After the salt elution, any rmAb1 that remained bound to the resin was eluted by 6 M guanidine hydrochloride treatment. Mass Spectrometry. The progress of the chemical labeling reaction and the final extent of global labeling of rmAb1 were monitored and determined by measuring the increase of the molecular masses of rmAb1 light and heavy chains using an Agilent Micro-TOF mass spectrometer (Santa Clara, CA). The rmAb1 samples were reduced by TCEP prior to injection onto a chip-based C8 column (Agilent, Santa Clara, CA) followed by ESI-TOF MS detection. The percentage of labeling of lysine residues was determined by a combined Asp-N and trypsin peptide mapping approach. The SNA-modified rmAb1 samples were digested into peptides 8502
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with Asp-N and trypsin, respectively. Because Asp-N digestion yielded the same peptide sequences for both unmodified and modified rmAb1, the extracted ion chromatogram of Asp-N peptide mapping was used as the primary method to quantify the percentage of labeling of lysine residues. However, when the Asp-N peptides contain multiple lysine residues, tryptic peptide mapping was used to confirm the labeling site and estimate the percentage of labeling at the individual lysine sites. For the same reason, Lys-C peptide mapping as the primary quantification method was combined with tryptic peptide mapping to quantify the percentage of HPG-modified arginine residues of rmAb1 samples. Peptide mapping was carried out on an Agilent 1200 HPLC online coupled with an LTQ Orbitrap XL mass spectrometer (ThermoFisher Scientific, San Jose, CA). HPLC conditions used were the same as previously reported.24 Details can be found in the Supporting Information section. Homology Modeling and Electrostatic Potential Calculations. A homology model of rmAb1 was generated using a Modeller9v725 software and the crystal structural coordinates of anti-Her2 (PDBs 3D6G and 3N85). The electrostatic surface of the rmAb1 model was calculated using the Adaptive PoissonBoltzmann Solver version 1.3 (APBS).26 The electrostatic surface charge distribution of rmAb1 was calculated with a Linearized PoissonBoltzmann (PB) equation and cubic B-spline discretization of the charge distributions. The resulting electrostatic surfaces were visualized by PyMoL (Schr€odinger LLC, San Diego, CA).
’ RESULTS AND DISCUSSION Anomalous Charge Heterogeneity Characteristics of rmAb1. The cation-exchange chromatograms for seven rmAbs with pI values ranging from 8.2 to 9.3 are shown in Figure 1A. For rmAbs 27, the retention time increases gradually as the pI value increases. In contrast, rmAb1, which has a same apparent pI value as rmAb7, elutes much later than rmAb7. The unusually high retentive power of rmAb1 implies that it has a unique distribution of surface positive charges that favors strong binding to the negatively charged surface of the resin. The charge heterogeneity profiles of native and thermally stressed rmAb1 were also assessed by CIEC. Different from the other six rmAbs examined, charge variants of rmAb1 are not well separated by CIEC even though the method was optimized to achieve the best resolution by adjusting mobile phase conditions (buffer components, pH, salt types) and HPLC instrumental parameters (column chemistry, temperature, gradient slope etc.) (Figure 1B). The rmAb1 main peak is dominant, which accounts for 96% of the total charge variants for the time zero control sample. Although a shoulder peak appeared to the left of the main peak for the thermally stressed samples, it is difficult to integrate as a separate peak. As a result, the main peak including the left shoulder still accounts for 96% of the total charge variants for the stressed sample. Furthermore, the basic charge variants of rmAb1 are not observed for either the control or the stressed samples by CIEC. In contrast, the charge variants of rmAb1 are well separated by icIEF for both control and thermally stressed samples (Figure 1C). The relative abundance of the main peak is 62% for the control sample; the main peak decreased to 49% and 39% for the rmAb1 samples that were thermally stressed for 2 and 4 weeks, respectively. The reduction in main peak was accompanied by an increase in the relative percentage of acidic peaks from 34%
Figure 1. (A) Cation-exchange chromatograms (CIEC) of 7 rmAbs with pI values ranging from 8.2 to 9.3. pI values of rmAbs are shown following their names. (B) CIEC and (C) icIEF profiles of native and thermally stressed rmAb1. Acidic, main, and basic represent the acidic charge variants, the main peak, and the basic charge variants for rmAb1.
for control sample to 46% and 54% for the two thermal stressed samples. These results indicate that the icIEF method is able to monitor the degradation of rmAb1 while this CIEC method is not. To explain the atypical CIEC profile of rmAb1, we hypothesized that several positively charged residues cluster together on the protein surface to form a “positive surface charge patch” that exhibits extremely strong binding to the negatively charged chromatographic resin. If the charge patch dominates the surface electrostatic interaction in CIEC, degradation on residues outside the patch may not change the retention of the protein such that the corresponding charge variants cannot be separated from the native isoform. The surface charge patch should not affect the separation of charge variants by icIEF because the net charge of the intact protein, not the surface charge distribution pattern, determines the migration of the protein in the liquid pH gradient that is driven by an electric field in icIEF. Hence, this hypothesis can be used to explain the inconsistent charge heterogeneity profiles of rmAb1 that were detected by CIEC and icIEF. To verify if a positive charge patch is present on the surface of rmAb1, selective chemical labeling protocols for basic lysine and arginine residues were developed. By combining the lysine and arginine labeling chemistry with mass spectrometry analysis, the positive charge distribution on rmAb1 surface was mapped as discussed below. Distribution of the Positive Charges on rmAb1 Surface. The distribution of positive charges on rmAb1 surface was mapped on the basis of the percentage of labeling of lysine and arginine residues in-solution. Development and optimization of 8503
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Table 1. Percentage of Labeling of Arginine and Lysine Residues on rmAb1 Fab
Fc
light chain residue
heavy chain
percent labeled (%) b
residue
R17
35.8
R19 and R38
a
99.8
K43
49.4
a
K31
K46 R55 and R62 K67
b,c
3.6
d
42.8
b
heavy chain
percent labeled (%) b,c
n/ae
62.7
R259
43.1b
b
K278
68.1d
d
K292 and K294
71.8c
b,c
R296 and R304
n/ae
K321, K323, K325
n/ae
89.4
a
K65
80.5
R67 and R72
0.4
d
R80
31.8
K106
32.0
R87
6.0
K330, K338, K342, K344
K114 K133
81.1 1.9
R98a R100a
n/ae 77.5
R348 and R359 K364
K153
0d
K125
26.1
K374
16.5
K160
89.5
K137
60.6
K396
64.7d
K170
28.2
K151
0
K413
0d
R420
13.8b
K175
46.4
K76
percent labeled (%)
K226, K250, K252
59.7
R57
residue
87.8
K209 and K214
n/a
e
44.2b,c 0.3
K190
46.9
K217
100.0
K418
90.3
R193
24.6b
K218
73.8
K443
12.7
K208
47.8
K222
a
n/ae b
The residues that are shown in italic are located in the CDRs of rmAb1. The percentages of labeling of arginine residues were determined on the basis of Lys-C peptide map analysis. Unless specified, the percentages of labeling of lysine and arginine residues were determined on the basis of tryptic peptide map analysis. c The number represents the percentage of single labeling of the two amino acid residues that reside in a same peptide after Asp-N or Lys-C digestion. d The percentages of labeling of lysine residues were determined on the basis of Asp-N peptide map analysis. e The percentages of labeling are not available for those residues because (1) most of those peptides contain multiple labeled lysine or arginine residues, and the percentage of labeling of the specific residues cannot be differentiated from each other; (2) a few of those peptides are short (di-, tri- or tetra-) peptides that were unable to be detected as they eluted in the solvent front in the LC-MS/MS peptide mapping method.
the in-solution labeling methods are described in detail in the Supporting Information section. The labeling reagent to protein molar ratio (R/P) was 200:1 for lysine labeling and 400:1 for arginine labeling. The percentage of labeling of each lysine and arginine residue was determined by calculating the ratio of the peak area of the modified peptide to the sum of the modified and unmodified peptides measured in the peptide maps. Results from this determination are displayed in Table 1 in three columns, grouped by their location in primary structure: light chain, Fab heavy chain, and Fc heavy chain. As can be seen in Table 1, the percentages of labeling have been obtained for the 32 out of total 37 lysine and arginine residues in the Fab region (light chain and Fab heavy chain); their average percentage of labeling is calculated to be 43.3%. The average percentage of labeling of those 13 lysine and arginine residues in the Fc heavy chain domains is 32.7%. This suggests that the lysine and arginine residues in the Fab region are more accessible to react with the labeling reagents. This conclusion is supported by the observation that the (Fab)’2 of rmAb1 eluted much later than the Fc species on the CIEC (data not shown). It is noteworthy that the percentages of labeling have not been obtained for 13 lysine and 3 arginine residues, most of which are located in the Fc region. This is because the corresponding proteolytic peptides contained multiple labeled lysine or arginine residues and the percentage of labeling of specific residues cannot be differentiated from each other. The percentage of labeling of lysine residues spans a wide range, from 0% for HC-K151 and HC-K413 to 99.8% for LCK31 and 100% for HC-K217. For arginine residues, the percent
Figure 2. Solvent accessibility of arginine (A) R57, (B) R67, and (C) R72 and D73 on the heavy chain of rmAb1. The 3D model for rmAb1 was generated by homology modeling. The guanidino groups in arginine residues are highlighted in blue while the carboxylic groups in aspartic or glutamic acids are in red. (D and E) The electrostatic surface of the 3D model of rmAb1 at pH 7.5. Positive potentials are shown in blue and negative are in red. (F) The composition of a positive charge patch in the variable domains of rmAb1. “H-” and “L-” in the labels represent the heavy and light chain of rmAb1, respectively.
labeled also varies over a wide range, from 0.4% for HC-R67 and HC-R72 to 89.4% for HC-R57. These percentages of labeling reflect a charge residue’s solvent accessibility and reactivity. To explore if the percentage of labeling of one residue correlates with 8504
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Figure 4. Change in the percentage of labeling of (AD) lysine and (E, F) arginine residues as a function of the reagent-to-protein ratio (R/P). The percentage of in-solution labeling is plotted as open circles, while the percentage of on-resin labeling is shown as solid circles. Figure 3. (A) Base peak ion chromatogram of Asp-N peptides derived from in-solution (upper) and on-resin (lower) labeled rmAb1. Collisioninduced dissociation (CID) spectra of (B) native and (C) acetylated peptides HC 6272 (DSVKGRSTISR). rmAb1 was labeled with SNA at a reagent-to-protein ratio of 200. The bold letters displayed in the sequence inset are those b- and y-ions that are observed in the CID spectra. Asterisk (/) denotes loss of ammonia.
its location and microchemical environment in rmAb1’s tertiary structure, a 3D model of rmAb1 generated by homology modeling (Figure 2) is used. As shown in Figure 2A, HC-R57 is fully exposed and does not interact with any surrounding residues on the 3D model. This correlates well with its high percentage of labeling of 89.4%. In contrast, HC-R67 is buried inside on the 3D model (Figure 2B), which is consistent with the extremely low reactivity observed in the labeling experiment. As shown in Figure 2C, HC-R72 seems partially solvent-accessible on the model. Its low percentage of labeling could be explained by its spatial vicinity toward HC-D73 (labeled red color), as a salt bridge could occur between the two oppositely charged residues, preventing the arginine from being labeled. Therefore, the 3D model generated by homology modeling correlates well with the surface features of rmAb1 revealed by our chemical labeling experiments. To understand the experimental charge distribution results, the electrostatic surface of rmAb1 at pH 7.5 was simulated on the basis of the 3D model generated by homology modeling. Figure 2D,E displays the side views of the 3D model, and although the positive charges in the constant domains seem evenly distributed, they seem more concentrated in the variable domains, which is consistent to some extent with the chemical labeling experimental data that lysine and arginine residues in the variable domains are relatively highly labeled. This finding is
consistent with the recent report by Freitag and co-workers that positive charges on a basic rmAb (pI = 9.3) were concentrated in the variable domains.10 However, without pinpointing the specific basic residues involved, they proposed that the collective variable domains form a positive charge patch.10 We think that a small, strong charge patch dominates the surface electrostatic interaction for this rmAb1; this in-solution labeling and 3D modeling approach allows us to view the overall charge distribution on rmAb1 surface, but an on-resin labeling approach is required to identify specific charge residues that may be strongly involved in the proteinresin interaction. Identifying the Charge Residues that Interact with Resin. In order to identify the lysine and arginine residues that interact with resin, rmAb1 was labeled while bound to the cation-exchange resin. On the basis of protein mass measurement by ESI-TOF mass spectrometry, an average of 2.3 lysine and 0.25 arginine residues on the light chain and 3.6 lysine and 0.65 arginine on the heavy chain were shielded from labeling upon resin-binding (Figures S-1 and S-2, Supporting Information). To pinpoint the shielded residues, peptide maps of the in-solution and onresin labeled rmAb1 samples were compared (Figure 3A), Five pairs of peptides show obvious differences in their abundance: P1P10 , P2P20 , P3P30 , P4P40 , and P5P50 . P1P5 are unmodified peptides while their corresponding P10 -P50 are acetylated peptides, which elute slightly later than the unmodified peptides. They correspond to the differential labeling on HC-K65, LC-K67, HC-K76, LC-K31, and HC-N-terminus, respectively. To demonstrate how these labeling sites were identified, the MS/MS spectra of peptides P1 and P10 are shown in Figure 3B,C, respectively. Both spectra are composed of a series of b- and y-type ions that correspond to the peptide sequence. Although the y7 ions in the two spectra have the same m/z values, the y8 ion derived from the acetylated peptide 8505
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Analytical Chemistry (Figure 3C) is 42 Da heavier than that from the native peptide (Figure 3B), suggesting that the K65 residue is acetylated. It is noteworthy that the acetylated peptide eluted later than the native form, which is consistent with the fact that acetylation increases the hydrophobicity of lysine residues. Likewise, in the arginine labeling experiments, two arginine residues (HC-R57 and -R100) were evidently less labeled when rmAb1 was bound to the resin. In total, seven charge residues were found to be shielded and they likely interact with the cation-exchange resin. These residues are all located in or close to the CDRs of rmAb1. To further assess the binding dynamics of the seven charge residues to the resin, we labeled rmAb1 at a series of labeling reagent to protein ratios (R/P) for both in-solution and on-resin labeling experiments. The justification for selecting the range of R/P ratios for binding dynamic studies for respective lysine and arginine residues are described in the Supporting Information section. The change in the percentage of residue labeling is plotted as a function of the R/P in Figure 4, referred to as doseresponse curves. First, for LC-K67 (Figure 4A), the percentage of insolution labeling increases significantly with an increase in the reagent concentration, but the percentage of on-resin labeling does not evidently increase. This suggests that LC-K67 strongly binds to the resin and is thus not affected by the labeling reaction. Second, for HC-K65 (Figure 4B), the curve shows a similar pattern to that of LC-K67 except that the percentage of on-resin labeling increases at a slightly higher rate than the former, suggesting that HC-K65 is also strongly bound to the resin but with a slightly lower binding strength than LC-K67. Third, for HC-K76 (Figure 4C), its percentages of labeling from in-solution and on-resin experiments increase with the R/P at a similar rate. This suggests that the binding of this residue to the resin is weak and can be disrupted by the labeling reaction. Fourth, as shown in Figure 4D, LC-K31’s percentage of in-solution labeling is high at all three R/P ratios, suggesting the residue is highly flexible in solution. In contrast, the percentage of on-resin labeling is low at the R/P of 200 and then significantly increases with the reagent concentration, indicating that the residue weakly binds to the resin and can be readily disrupted by the labeling reaction. Likewise, for HC-R57 (Figure 4E), the percentage of in-solution labeling is high, but its percentages of on-resin labeling remains low until the R/P reaches 2000, suggesting that the residue binds to the resin strongly. On the other hand, the percentage of onresin labeling of HC-R100 (Figure 4F) increases with the reagent concentration and thus HC-R100 binds weakly to the resin. On the basis of the doseresponse curves, three charge residues were found to strongly bind to the resin: HC-R57, HC-K65, and LC-K67. They are all located on rmAb1 surface on the basis of their relatively high percentages of labeling in solution (Table 1) and thus likely form a surface charge patch that strongly binds to the cation-exchange resin. Revealing the Charge Patch on the Surface of rmAb1. When the seven resin-binding charge residues identified from the comparison of the in-solution and on-resin labeling results are highlighted on the 3D model of rmAb1, four of these residues, including the three strongly binding residues (HC-R57, HCK65, and LC-K67) and a weakly binding residue LC-K31, appear to be close to each other, leading to a highly basic surface region (Figure 2F). We thus consider that these four residues form a positive surface charge patch that binds strongly to the cation-exchange resin. The other three charge residues
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Figure 5. Cation-exchange chromatograms for (top) native rmAb7, (middle) on-resin and in-solution labeled rmAb1, and (bottom) native rmAb1. rmAb1 was labeled with SNA at the reagent-to-protein ratio (R/P) of 100.
(HC N-terminus, HC-K76, and HC-R100) also appear to be close to each other on other side of the structure (Figure S-3, Supporting Information). However, these residues are not located on the same plane, and thus, they may not be able to bind to the resin simultaneously. This surface topology is once again consistent with the labeling experiment because the three charge residues bind to the cation-exchange resin to a lower extent and can be easily disrupted by the chemical labeling reaction. To confirm that the presence of the surface charge patch is responsible for the anomalous CIEC profile of rmAb1, the lysine residues were neutralized in solution by SNA-labeling at the R/P of 100. About 5.5 lysine residues were labeled for the entire antibody on the basis of the molecular mass determination of TCEP-reduced SNA-labeled rmAb1 sample, which is described in Supporting Information section. According to peptide mapping, 1.8 of the charge patch residues (HC-K65, LC-K31, and LC-67) were labeled. For comparison, rmAb1 was also labeled on-resin. Only 2.4 lysine residues were labeled, and they were primarily located outside the charge patch according to peptide mapping. The charge patch residues were not labeled because they were shielded from labeling by the resin. The in-solution and on-resin labeled rmAb1 were reanalyzed using the CIEC method, and the chromatograms are shown in Figure 5. The retention time of the main peak of the on-resin labeled rmAb1 is very close to the main peak of the native rmAb1. In contrast, the in-solution labeled rmAb1 elutes much earlier and in a much broader peak distribution than the on-resin labeled rmAb1. Both the in-solution and on-resin labeled rmAb1 showed broader distribution than the native form does. This is likely because the labeling reactions yield a protein mixture that varies in the site and extent of labeling. The in-solution labeled rmAb1 has similar modifications as the on-resin labeled protein except that an average of 1.8 lysine residues in the charge patch are additionally neutralized. The slight changes in the charge patch appear to have significant impact on the CIEC retention time of rmAb1, suggesting that the charge patch residues play a critical role in rmAb1’s retention. Furthermore, the retention time of the in-solution labeled rmAb1 becomes comparable to that of native 8506
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Analytical Chemistry rmAb7, which has the same pI as the unlabeled rmAb1 and exhibits a typical CIEC profile. This implies that the binding of rmAb1 to cation-exchange resin becomes normal when charge patch residues are neutralized. All this evidence supports that charge patch residues are responsible for the unusually high binding strength of rmAb1 to the cation-exchange resin. The positive surface charge patch that we have identified on rmAb1 can be used to explain its anomalous CIEC profile observed in the purification process and analytical characterization. In the purification process, because of the high binding strength of the positive charge patch, rmAb1 requires high salt for elution from cation-exchange columns. In the analytical CIEC characterization, because the charge patch dominates the surface electrostatic interaction between rmAb1 and resin, charge variants, resulting from chemical modifications on the surface regions away from the positive charge patch, coelute with the native isoform of rmAb1. Recently, rmAbs with excessive positive charges are shown to have fast clearance rate in vivo.27 It is believed that these molecules favorably bind to negatively charged cell surfaces, facilitating the pinocytosis-related antibody clearance pathway.27,28 Likewise, rmAb1 was also found to have a slightly high clearance rate in humans. A study is underway to generate charge patch mutants of rmAb1 and to determine if they have an improved pharmacokinetic profile compared to the native rmAb1.
’ CONCLUSIONS In this work, an atypical charge heterogeneity profile was observed for rmAb1 and the surface charge distribution of rmAb1 was investigated. Methods for selective chemical labeling of arginine and lysine residues in conjunction with MSbased footprinting were developed and used to map the distribution of positive charges on rmAb1 surface. The positive charges in rmAb1 were found to be more distributed in the Fab region. When bound to cation-exchange resin, seven surface charge residues on rmAb1 were found to be shielded from labeling. Three lysine and one arginine residue that strongly interacted with resin clustered on the surface to form a positive charge patch, which was responsible for the atypical CIEC profile of rmAb1. Although a surface charge patch concept has previously been proposed to explain the ion-exchange chromatography of a monoclonal antibody,10 we believe that this work is the first report to reveal the residue composition of a charge patch on a rmAb. With the known composition residues of the charge patch, site-directed mutagenesis can be conducted to produce charge patch mutants for further investigation of how the charge patch influences the biological function and pharmacokinetics of the antibody. Furthermore, with slight changes in the labeling chemistries, the developed MS-based footprinting methods can also be applied to investigate proteinprotein interactions such as antibody aggregation and antibody antigen binding. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in the text; experiment details, scheme of chemical labeling reactions (Scheme S-1), development of in-solution and on-resin labeling chemistries, global extent of labeling of lysine (Figure S-1) and
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arginine (Figure S-2), and reproducibility of the in-solution and onresin labeling. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Address: Protein Analytical Chemistry, MS 96B, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.
’ ACKNOWLEDGMENT We would like to thank Pat Rancatore and Trevor Swartz for critical review of the manuscript, Chris Teske for the discussion about antibody binding to cation-exchange resins, and Charles Eigenbrot for his help on homology modeling of the variable fragment of rmAb1. ’ REFERENCES (1) Wang, N. W. Bioprocess Technol. 1990, 9, 359–400. (2) Jungbauer, A.; Hahn, R. Methods Enzymol. 2009, 463, 349–371. (3) Low, D.; O’Leary, R.; Pujar, N. S. J. Chromatogr., B 2007, 848, 48–63. (4) Shukla, A. A.; Hubbard, B.; Tressel, T.; Guhan, S.; Low, D. J. Chromatogr., B 2007, 848, 28–39. (5) Kelley, B. mAbs 2009, 1, 443–452. (6) Ahrer, K.; Jungbauer, A. J. Chromatogr., B 2006, 841, 110–122. (7) Vlasak, J.; Ionescu, R. Curr. Pharm. Biotechnol. 2008, 9, 468–481. (8) Regnier, F. E. Science 1987, 238, 319–323. (9) Sosic, Z.; Houde, D.; Blum, A.; Carlage, T.; Lyubarskaya, Y. Electrophoresis 2008, 29, 4368–4376. (10) Schubert, S.; Freitag, R. J. Chromatogr., A 2009, 1216, 3831–3840. (11) Zhu, Z. Y.; Karlin, S. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8350–8355. (12) Kaltashov, I. A.; Eyles, S. J. Mass Spectrom. Rev. 2002, 21, 37–71. (13) Mendoza, V. L.; Vachet, R. W. Mass Spectrom. Rev. 2009, 28, 785–815. (14) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158–170. (15) Houde, D.; Arndt, J.; Domeier, W.; Berkowitz, S.; Engen, J. R. Anal. Chem. 2009, 81, 2644–2651. (16) Houde, D.; Peng, Y.; Berkowitz, S. A.; Engen, J. R. Mol. Cell. Proteomics 2010, 9, 1716–1728. (17) Zhang, Z. Q.; Smith, D. L. Protein Sci. 1993, 2, 522–531. (18) Xu, G. Z.; Chance, M. R. Anal. Chem. 2005, 77, 4549–4555. (19) Jumper, C. C.; Schriemer, D. C. Anal. Chem. 2011, 83, 2913–2920. (20) Hager-Braun, C.; Hochleitner, E. O.; Gorny, M. K.; ZollaPazner, S.; Bienstock, R. J.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2010, 21, 1687–1698. (21) Wu, J.; Huang, T. Electrophoresis 2006, 27, 3584–3590. (22) Brier, S.; Pflieger, D.; Le Mignon, M.; Bally, I.; Gaboriaud, C.; Arlaud, G. J.; Daniel, R. J. Biol. Chem. 2010, 285, 32251–32263. (23) Liu, Y. Y.; Kvaratskhelia, M.; Hess, S.; Qu, Y. X.; Zou, Y. J. Biol. Chem. 2005, 280, 32775–32783. (24) Yang, Y.; Strahan, A.; Li, C.; Shen, A.; Liu, H.; Ouyang, J.; Katta, V.; Francissen, K.; Zhang, B. mAbs 2010, 2, 285–298. (25) Eswar, N.; Marti-Renom, M. A.; Webb, B.; Madhusudhan, M. S.; Eramian, D.; Shen, M.; Pieper, U.; Sali, A. Comparative Protein Structure Modeling With MODELLER. In Current Protocols in Bioinformatics, Supplement 15, 5.6.15.6.30, John Wiley & Sons, Inc.: Hoboken, NJ, 2006. 8507
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