Article pubs.acs.org/ac
Characterization of the N‑Terminal Heterogeneities of Monoclonal Antibodies Using In-Gel Charge Derivatization of α-Amines and LCMS/MS Daniel Ayoub,†,§ Diego Bertaccini,‡,∥,§ Hélène Diemer,‡,∥ Elsa Wagner-Rousset,† Olivier Colas,† Sarah Cianférani,‡,∥ Alain Van Dorsselaer,‡,∥ Alain Beck,*,† and Christine Schaeffer-Reiss*,‡,∥ †
Centre d’Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France ∥ IPHC, CNRS, UMR7178, 67087 Strasbourg, France ‡
S Supporting Information *
ABSTRACT: The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein Nterminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized.
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direct effect on the safety, potency, and other pharmaceutical properties of the antibody, must be validated for submission to regulatory agencies like the European Medicines Agency (EMA) or the American Food and Drug Administration. The sequence variants described in the literature include fragmentation (i.e., backbone cleavage of the polypeptide chain),16 truncation,17 and incomplete processing of the signal peptide.8,18,19 Fragmentation and truncation are forms of antibody degradation that both involve cleavage of the polypeptide sequence, the latter though referring specifically to deletions of one or a few residues from the N- or C-terminus of the protein. For instance, the truncation of the C-terminal lysine of the heavy chain of antibodies is very common,17 while cleavage of C-terminal glycines followed by proline amidation has also been reported.20 N-Terminal truncation due to degradation has seldom been reported in the literature; however, N-terminal heterogeneity has repeatedly been shown to arise from incomplete processing of the signal peptide resulting in N-terminal elongated heavy or
ith advances in protein engineering technologies, monoclonal antibodies (mAbs) and derivatives have emerged as one of the largest drug classes in human therapeutics.1 More than 50 molecules have already been approved to treat various conditions including autoimmune diseases and cancer, while more than 30 others are being investigated in advanced clinical trials.2 The success of mAbs is mainly due to their high specificity and long circulating halflives. Furthermore, their ability to induce immune cell effector response and the possibility of conjugation with cytotoxic drugs increase their potency against targeted cells. Like all bioproducts, monoclonal antibodies present different levels of heterogeneity.3 Indeed, different glycosylation patterns,4 cysteine linkage,5 and sequence alterations6−8 can arise from the production system while glycation,9 oxidation,10 deamidation,11 and aggregation,12 among others, can occur during purification and storage. To be considered for marketing approval by regulatory agencies, the molecules must be characterized extensively with a battery of methods specific to each structural modification or alteration (see the review by Beck et al.13). In particular, with numerous generic versions of off-patent mAbs (biosimilars) being filed for approval,14,15 the primary amino-acid sequence, whose alteration may have a © 2015 American Chemical Society
Received: November 27, 2014 Accepted: March 13, 2015 Published: March 13, 2015 3784
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Analytical Chemistry light chains.8,18,21,22 The most common extension of the Nterminus of light and heavy chains is due to the valine− histidine−serine (VHS) alternate signal peptidase cleavage site.22 C-Terminal truncations result in a more basic protein and can therefore be detected and isolated using charge-based techniques such as cation exchange chromatography and isoelectric focusing.21 Similarly, N-terminal elongation and truncation generally alter the charge and thereby the isoelectric point of mAbs.18,22 Therefore, their presence can also be detected with charge-based separation techniques (ion exchange chromatography,23 isoelectric focusing, capillaryisoelectric focusing (IEF), off gel-IEF,21 and capillary electrophoresis-sodium dodecyl sulfate (SDS)8). However, while these techniques are useful for the detection of unexpected modifications, the characterization of these variants requires more direct and specific methods such as mass spectrometry (MS).20 Here, we describe a MS-based method to characterize mAb N-terminal sequence variants arising from truncation, degradation, or incomplete processing of signal peptides. For this purpose, a specific in-gel N-terminal protein labeling method was developed using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP), followed by enzymatic digestion and liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis. In an approach named N-TOP (N-terminal oriented proteomics), Gallien et al.24 have shown how TMPP can be used as a chemical tag for the identification of protein N-terminal positions in proteomic studies, achieving significantly higher (up to 20 times) electrospray ionization (ESI) efficiency due to the permanent positive charge of the modified peptides. Moreover, in reversed-phase LC-MS/MS, the hydrophobic nature of the reagent delays the elution of the modified Nterminal peptides and groups them away from other peptides thereby favoring their sampling in data dependent acquisition MS/MS experiments. At pH 8.2, the TMPP reaction is specific to N-terminal amines such that ε-amino groups of lysines are not modified. However, mAbs are often formulated in acidic buffers that are not compatible with the derivatization reaction, and they usually contain high amounts of histidine that would compete for reaction with TMPP. We have therefore adapted the N-TOP protocol for specific in-gel TMPP protein α-amine derivatization. An et al. have described a method based on TMPP in-gel labeling of protein and peptides to enhance matrix-assisted laser desorption/ionization-MS detection. However, their method is not N-terminal oriented and leads to the labeling of all free amines (N-terminal and lysine residues).25 In this study, we applied our N-terminal oriented in-gel TMPP labeling strategy to characterize N-terminal heterogeneities in Hz6F4-2, an antibody for which LC-MS middle-up analysis and cation exchange chromatography experiments suggested an incomplete processing of its signal peptide. We also qualitatively evaluated the sensitivity of our method for the identification and characterization of low abundance polypeptide chain breakdown products by spiking a solution of Hz6F42 with a known amount of protease cleaved Hz6F4-2. This methodology was then successfully applied to assess, in a single LC-MS/MS analysis, the N-terminal heterogeneity of a mixture of five therapeutic mAbs, a mimic for the mAb cocktails already in current clinical trials. Altogether, our results demonstrate the efficiency of the in-gel N-TOP approach for the character-
ization of unexpected mAb N-terminal sequence variants present in complex mixtures at contaminant levels.
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EXPERIMENTAL SECTION Reagents and Monoclonal Antibodies Used in the Study. Hz6F4-2 was expressed in eukaryotic cell lines and purified using standard manufacturing procedures at the Centre d’Immunologie Pierre Fabre (Saint Julien-en-Genevois, France) as previously described.26 Trastuzumab (Herceptine, Genentech), Natalizumab (Tysabri, Biogen Idec), Ofatumumab (Arzerra, Genmab), Palivizumab (Synagis, MedImmune), Cetuximab (Erbitux, Merck KGaA), and Bevacizumab (Avastin, Roche) are EMA approved versions and formulations, available commercially. Hemoglobin and all the reagents and solvents used in this study were purchased from Sigma-Aldrich unless otherwise stated. Middle-Up LC-MS Analysis of Hz6F4-2. Hz6F4-2 was cleaved in the hinge region by limited proteolysis using the immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) (Fabricator, Genovis, Lund, Sweden). The disulfide bonds were subsequently reduced to produce ∼25 kDa subunits as described previously.14,27 The LC-MS system used was an Acquity UPLC system (Waters, Manchester, UK) coupled to an ESI-time of flight (TOF) mass spectrometer (LCT premier, Waters). Solvent A consisted of 0.1% formic acid in water while solvent B consisted of 0.1% formic acid in 60% acetonitrile and 40% isopropanol. Hz6F4-2 subunits (0.5 μg) were loaded on a 1 mm × 150 mm BEH300 C4 1.7 μm column (Waters) with 5% solvent B under 0.1 mL/min flow. Elution was performed with the percentage of solvent B increased from 5 to 15% over 3 min, 15−45% over 30 min, and then 45−80% in 1 min followed by 5 min at 80%. The column was finally reconditioned at 5% solvent B. Mass spectra were acquired in positive ion mode for m/z ranging from 500 to 5000. Sample Preparation for N-Terminal TMPP Derivatization. An equimolar solution mixture of the antibodies Trastuzumab, Ofatumumab, Palivizumab, Cetuximab, and Hz6F4-2 was prepared. For analysis of N-terminal processing, Hz6F4-2 was cleaved using IdeS without reducing the disulfide bonds. The cleaved antibody was then spiked at 1% into an intact Hz6F4-2 solution. In-Gel N-Terminal Protein Derivatization. Samples (hemoglobin (2 μg)), individual antibodies (2 μg), and antibody mixtures (5 μg) were loaded into a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel. After migration, the gel bands were excised and placed in a 96well plate. Using an automated robot platform (Massprep station, Waters), the gel slices containing protein samples were washed twice in 25 mM NH4HCO3 and CH3CN. The cysteine residues where subsequently reduced in 10 mM (tris(2carboxyethyl)phosphine) at room temperature and then alkylated with 30 mM iodoacetamide. After dehydration with acetonitrile, 20 μg of (N-succinimidyloxycarbonyl-methyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) was added to each well in the reaction buffer (50 mM Tris-HCl, 8 M urea, 2 M thiourea, pH 8.2). After 1 h, 2 μL of a 50% solution of hydroxylamine was added to each well. The gel slices were then washed three times in 25 mM NH4HCO3 and CH3CN before dehydration with CH3CN. Separate enzymatic digestions were performed in-gel overnight at 37 °C using porcine trypsin (Promega, Madison WI, USA), chymotrypsin (Roche, Mannheim, Germany), or endoproteinase AspN 3785
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Analytical Chemistry (Roche). The peptides were then extracted using 60% CH3CN in 1% formic acid in water. The volumes were reduced in a vacuum centrifuge and adjusted to 10 μL using 0.1% formic acid in water before nanoLC-MS/MS analysis. LC-MS/MS and Data Analysis. NanoLC-MS/MS analyses were performed on a nanoACQUITY ultraperformance-LC system coupled to a Q-TOF SYNAPT HDMS mass spectrometer (Waters, Manchester, UK). The peptides were trapped on a 0.18 mm × 20 mm, 5 μm Symmetry C18 precolumn (Waters) and then separated on an ACQUITY UPLC BEH130 C18 column (Waters), 75 μm × 200 mm with 1.7 μm particle size. The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Trapping was performed for 1 min at 15 μL/min with 99% of solvent A and 1% of solvent B. Elution was performed at a flow rate of 300 nL/min, using a 1−50% gradient (solvent B) over 38 min at 45 °C, after which 90% solvent B was maintained for 1 min before the column was reconditioned at 99% of solvent A over 6 min. The Q-TOF system was operated in positive ion mode, with the capillary voltage and cone voltage set to 3.5 kV and 35 V, respectively. The mass calibration and online correction of the TOF were performed by lock-mass on product ions derived from the Glu-fibrino-peptide B. For the tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides, preferably doubly and triply charged ions, were selected on each MS spectrum for further isolation and collision-induced dissociation (CID) at two energies set using the collision energy profile. Tandem MS fragmentation was performed using argon as the collision gas. The complete system was controlled using MassLynx 4.1 (SCN 639, Waters). Mass data collected during analysis were processed and converted into “.pkl” using ProteinLynx Global Server 2.4 (Waters) which were then submitted to the Mascot search engine (version 2.4.1, Matrix Science) installed on a local server. The searches were performed against an in-house generated database containing the sequences of the antibodies with their signal peptides and common contaminants (trypsin, human keratins). Searches were performed with a 15 ppm tolerance on the precursor ion mass and a 0.05 Da tolerance for the fragment ions. Carbamidomethylation of cysteine residues was defined as a fixed modification while methionine oxidation, N-terminal cyclization of glutamine and glutamic acid to pyroglutamic acid, and N-terminal TMPP derivatization were searched as variable modifications. Spectra yielding an identification of an N-terminal peptide (TMPP or pyroglutamic acid) were reinspected manually.
Figure 1. Middle-up LC-MS analysis of Hz6F4-2. (a) Chromatogram showing the three subunits of Hz6F4-2. (b, c) Deconvoluted mass spectra of the Fd subunit with (b) the main peak at a molecular mass consistent with N-terminal cyclization of glutamine (major form) and (c) low intensity peaks at molecular masses higher than those of the Fd subunits, from fragments that partially coelute with the light chain. These peaks may correspond to N-terminal elongated heavy chains.
charge variants were also observed by cation exchange chromatography (data not shown). These observations point toward an incomplete processing of the signal peptides for the Hz6F4-2 heavy chain. This hypothesis can only be confirmed using extensive LC-MS/MS sequencing to unambiguously characterize these minor forms by determining the N-terminal positions of the polypeptide chains present in the mAb sample. Classical peptide mapping does not generally allow the identification of alternative N-terminal peptides for several reasons. First, their low abundance renders them uncompetitive both during the ionization process and during sampling for further MS/MS sequencing in data dependent experiments. Second, database searches are usually performed with fully specific enzymatic rules that tend to miss unexpected peptides. Finally, the MS/MS spectra obtained are typically not informative enough, even when selected and fragmented, due to the low intensity of the precursor ions. Furthermore, in the case of low abundance truncation or degradation (shorter protein sequences), the alternative N-terminus is within the sequence of the protein. In this case, the assignment of semitryptic peptides is ambiguous, since they can arise both from unspecific enzymatic cleavage29 and from a degradation product. Specific N-terminal labeling, before digestion, of αamines at the protein level allows alternative N-termini to be assigned unambiguously. The in-gel N-TOP strategy described here is based on in-gel derivatization of the free N-terminal αamines. Performing the derivatization in-gel circumvents buffer compatibility problems. Peptide Mapping of Hz6F4-2 and Characterization of Its N-Terminal Sequence Variants. A key requirement for the in-gel N-TOP strategy is that the reaction should remain specific to the α-terminal amines, leaving the ε-amines of the lysine side chains intact. Unspecific derivatization of the latter would prevent trypsin cleavage at these sites and complicate the analysis. At pH 8.2, the ε-amino groups of lysines are protected by protonation and are therefore significantly less reactive to TMPP. Maintaining the pH at 8.2 therefore ensures highly selective TMPP derivatization while minimizing unspecific lysine derivatization (Supporting Information, Figure S1). Another important requirement for this method is the proper elimination of excess TMPP and its hydrolysis products, which may coelute in LC with TMPP-labeled peptides due to their similar hydrophobicities. Futhermore, they may also compete with the TMPP-labeled peptides during ESI, potentially leading to ion suppression and reduced sensitivity. The reaction side
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RESULTS AND DISCUSSION Middle-Up Analysis of Hz6F4-2. Middle-up LC-MS analysis is a technique used routinely for the characterization of major variants of monoclonal antibodies. IdeS cleavage followed by the reduction of disulfide bonds results in three subunits (light chain, Fc/2 domain, and Fd domain), each with a molecular mass of approximately 25 kDa. It allows for the rapid and accurate profiling of N-glycans site by site and the assessment of the presence of various charge and size variants such as C-terminal lysine clipping, N-terminal cyclization of glutamine,28 and oxidation.27 The low abundance peaks obtained for the middle-up LC-MS analysis of Hz6F4-2 (Figure 1) could be the result of the larger Fd subunits coeluting partially with the light chain. Unexplained low abundance 3786
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Analytical Chemistry products were successfully eliminated here by repeated washing and dehydration with ammonium bicarbonate and acetonitrile. These steps were automated using the Massprep robot (cf. Experimental Section). Sequence coverage of 96% and 73% was achieved, respectively, for the light and heavy chains by trypsin peptide mapping of TMPP-labeled Hz6F4-2 (97% for both chains if we account for low abundance semitryptic peptides29). Chymotrypsin and Asp-N peptide mapping brought the sequence coverage up to 100% for both chains. Figure 2 highlights the
Table 1. List of the N-Terminal Sequences Identified for Hz6F4-2 Heavy and Light Chainsa
a
The expected N-terminal position is shown in red.
Figure 2. (A) Sequence coverage (73%) of the Hz6F4-2 heavy chain achieved by tryptic peptide mapping after TMPP labeling. Residues in light blue represent the N-terminal positions due to misprocessed signal peptide; in green, the expected N-terminal position with and without cyclization of the glutamine; in dark blue, an N-terminal position corresponding to the truncation of the glutamine residue. (B) Distribution of peptide retention times for the N-terminal and internal tryptic peptides identified.
main advantage of the N-TOP strategy, namely, the identification of internal sequences and nonfree N-terminal sequences (pyroglutamylated, acetylated, etc.) in combination with the unambiguous characterization of unexpected Ntermini with the TMPP label. Furthermore, the hydrophobicity of TMPP ensures that these peptides are grouped at the end of the elution gradient, away from most internal sequencing, rendering them more competitive for MS/MS selection during data dependent acquisition. Table 1 lists the N-terminal sequences identified in this experiment. Seven amino-terminal peptides are identified for the heavy chain of Hz6F4-2, six of which are TMPP labeled. The non-TMPP labeled form is the correctly processed form with N-terminal cyclization of glutamine (Gln) to pyroglutamic acid (pGlu) and corresponds to the major form identified in the middle-up experiment. The in-gel N-TOP approach does not therefore affect the identification of blocked amino-terminal peptides with cyclized N-terminal glutamine. The minor form, without N-terminal cyclization of Gln, is also identified, among the TMPP-labeled peptides. The other five correspond to unexpected N-terminal positions. Four of these are the result of incomplete processing of the secretion (signal) peptide and one arises from the clipping of the N-terminal glutamine. Figure 3 presents a selection of annotated MS/MS spectra that identify
Figure 3. A selection of MS/MS spectra of N-terminal peptides obtained by the in-gel N-TOP analysis of Hz6F4-2, specifically (A, B) spectra of TMPP-derivatized peptides allowing the identification of two misprocessed forms and (C, D) spectra of the expected N-termini identified (C) with TMPP derivatization and (D) with a cyclized glutamine.
the N-terminal positions of different proteoforms of the heavy chain. Generally, due to their permanent positive charge, TMPP-derivatized peptides produce N-terminal bn product ion signals with intensities that depend on the peptide sequence. For example, the CID spectrum of a quadruply charged TMPPlabeled peptide (Figure 3A) displays a series of singly charged bn ions plus y-type ions. Despite the 22 amino acids length, the peptide exhibits a good MS/MS fragmentation quality that allowed unambiguous localization of the TMPP tag. No alternative N-termini were identified for the light chain whose TMPP labeled N-terminal peptide was identified by chymo3787
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Analytical Chemistry trypsin and Asp-N but was too short to be identified with trypsin due to arginine in position 3. Characterization of N-Terminal Sequence Variants of Approved Single Therapeutic mAbs. The in-gel N-TOP method was then applied to a selection of approved therapeutic antibodies (Natalizumab, Trastuzumab, and Bevacizumab) to search for eventual N-terminal sequence proteoforms. The expected N-terminal positions for all these antibodies were identified with either a cyclized N-terminal glutamine or TMPP tags. No alternative N-terminal positions were found for Trastuzumab and Bevacizumab. For Natalizumab, however, an isoform of the heavy chain with incomplete processing of its signal peptide was identified in two different experiments (trypsin and Asp-N digestions). Figure 4 shows the MS/MS
Figure 4. MS/MS spectrum of a TMPP labeled N-terminal peptide of the heavy chain N-terminal variant of Natalizumab. This minor Nterminal elongated proteoform results from an incomplete processing of the heavy chain’s signal peptide.
spectrum of the Asp-N TMPP-labeled peptide LAVAPGAHSQVQLVQAGA. This peptide corresponds to the N-terminal position of this proteoform variant. The expected N-terminal position (major form) is the first glutamine. Application of the In-Gel N-TOP Approach for the Characterization of Fragmentation/Degradation Sites in mAbs. Monoclonal antibodies are prone to peptide backbone cleavage under acidic conditions such as those used during processing. The peptidyl bond between aspartic acid and glycine or proline is particularly sensitive to acid-induced proteolysis. Proteases present in the cell culture medium and early processing steps can also induce antibody clipping, most often in the hinge region.30 To mimic this situation, we cleaved Hz6F4-2 artificially by limited IdeS proteolysis. To evaluate the sensitivity of this approach and to simulate low levels of degradations, we spiked this sample at 1% concentration in an uncleaved Hz6F4-2 sample (Figure 5). All the N-terminal peptides also detected for unprocessed Hz6F4-2 were identified. One additional TMPP-labeled peptide, GPSVFLFPPKPK, corresponding to the expected hinge region cleavage site of IdeS, is clearly identified in the MS/MS spectrum in spite of its low concentration (Figure 5). This demonstrates the potential of this approach for the identification of minor degraded forms in mAb samples. Characterization of the N-Terminal Heterogeneity of mAb Mixtures. To benchmark our in-gel TMPP methodology, the complexity of a polyclonal antibody cocktail was mimicked using a mixture of five mAbs (viz., Hz6F4-2 and four approved therapeutic mAbs). As some of the mixed mAbs present tryptic cleavage sites in the first few N-terminal residues, trypsin, chymotrypsin, and AspN were all three used for N-terminal peptide identification (as described above for the Hz6F4-2 light chain). As shown in Table 2, the expected N-
Figure 5. Flowchart outlining the procedure used to characterize the polypeptide chain breakdown site, with the resulting MS/MS spectrum of a TMPP-derivatized peptide generated by spiking 1% IdeS-cleaved Hz6F4-2 into a sample of intact Hz6F4-2.
terminal positions of the heavy chains of all five antibodies were identified with cyclized N-terminal glutamine or glutamic acid. TMPP-derivatized peptides were also identified for all five mAbs due to the presence of minor or major forms with unmodified N-terminal glutamines (for Palivizumab, Cetuximab, and Hz6F4-2) or TMPP-labeled glutamic acid (for Trastuzumab and Ofatumumab). The elongated forms of Hz6F4-2 with misprocessed signal peptides were also identified with the TMPP tag. The light chains of all five mAbs were identified with the expected TMPP-derivatized N-terminal peptides, and no form with a modified α-amine was detected (Table 2). Note that the light chains of Palivizumab and Trastuzumab have the same primary N-terminal sequence. Altogether, these results demonstrate that the in-gel N-TOP derivatization approach proposed here achieves the sensitivity necessary to characterize N-terminal heterogeneities in complex mAb mixtures.
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CONCLUSIONS The approach presented here based on in-gel α-amino-terminal derivatization with TMPP is effective in the characterization of N-terminal sequence heterogeneities in mAbs, also providing extensive peptide mapping in the same experiment. Unexpected sequence variants due to misprocessed signal peptides or truncation are systematically and unambiguously identified and characterized. The increased ionization efficiency afforded by the TMPP tag enhances the detection of low abundance peptide variants. Low-level polypeptide chain breakdown sites 3788
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Analytical Chemistry Table 2. Results of the In-Gel N-TOP Analysis of an Equimolar Mixture of Five Monoclonal Antibodiesa peptide sequence 4
EVQLVESGGGL14 EVQLVESGGGLVQPGGSLR2
4
4
DIQMTQ9
4
EVQLVESGGGLVQPGR19 EVQLVESGGGLVQPGRSLRLSCAASGFTFN33
4
4
EIVLTQSPATLSLSPGER21
4
QVTLRESGPALVKPTQTL21
4
DIQMTQ9
4
QVQLKQSGPGLVQPSQSL21
4
DILLTQSPVILSVSPGER21
11
LSGTTGVHSQVQLVQSGAEVK31 QVQLVQSGAEVK31 20 QVQLVQSGAEVK31 17 VHSQVQLVQSGA28 20
21
AIRMTQ26
enzyme TrastuzumAb Heavy Chain chymotrypsin trypsin TrastuzumAb Light Chain chymotrypsin OfatumumAb Heavy Chain trypsin Asp-N OfatumumAb Light Chain trypsin PalivizumAb Heavy Chain chymotrypsin PalivizumAb Light Chain chymotrypsin CetuximAb Heavy Chain chymotrypsin CetuximAb Light Chain trypsin Hz6F4-2 Heavy Chain trypsin trypsin trypsin chymotrypsin Hz6F4-2 Light Chain chymotrypsin
modification (Δ mass)
m/z measured (charge)
Pyro-Glu (−18) TMPP (+572)
535.280 (2+) 818.727 (2+)
TMPP (+572)
662.26 (2+)
TMPP (+572) Pyro-Glu (−18)
733.019 (3+) 1040.196 (3+)
TMPP (+572)
824.07(3+)
Pyro-Glu (−17)
961.043 (2+)
TMPP (+572)
662.26 (2+)
Pyro-Glu (−17)
939.013 (2+)
TMPP (+572)
832.75 (3+)
TMPP (+572) TMPP (+572) Pyro-Glu (−17) TMPP (+572)
899.779 619.968 634.838 912.938
TMPP (+572)
654.28 (2+)
(3+) (3+) (2+) (3+)
The expected N-terminal positions of all five antibodies were identified using one or more of the following enzymes in parallel: trypsin, chymotrypsin, or AspN. a
Strasbourg, Région Rhône-Alpes, CNRS, and Université de Strasbourg. We also thank the Fondation pour la Recherche Médicale and the Proteomic French Infrastructure (ProFI; ANR-10-INSB-08-03) for financial support.
can also be characterized, as shown in spiking experiments with a 1% IdeS cleaved mAb. This approach was also successfully used to detect and characterize a misprocessed signal peptide in the approved therapeutic antibody Natalizumab and variants in a mixture of antibodies highlighting its potential for the study of polyclonal IgG mixtures. As this method adds only one step to the classical in-gel peptide mapping protocol, it can be easily implemented for the characterization of biosimilars and most recombinant or purified bioproducts.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1, the in-gel TMPP derivatization of hemoglobin performed at two different pHs. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail: christine.schaeff
[email protected]. Phone: +33 (0)3 68 85 27 79. *E-mail:
[email protected]. Phone: +33 (0)4 50 35 35 22 Author Contributions §
D.A. and D.B. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the OptimAbs network biocluster (LyonBiopole and Alsace Biovalley) and sponsored by DGCIS, Oséo, Feder, Région Alsace, Communauté Urbaine de 3789
DOI: 10.1021/ac504427k Anal. Chem. 2015, 87, 3784−3790
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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on March 26, 2015. The title was modified and the revised version was reposted on March 27, 2015.
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DOI: 10.1021/ac504427k Anal. Chem. 2015, 87, 3784−3790
