Evidence for Trisulfide Bonds in a Recombinant ... - ACS Publications

Jul 10, 2009 - The hinge region of human IgG2 contains four cysteine residues involved in disulfide linkages between the heavy chains, as well as the ...
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Anal. Chem. 2009, 81, 6148–6155

Evidence for Trisulfide Bonds in a Recombinant Variant of a Human IgG2 Monoclonal Antibody Pavlo Pristatsky, Steven L. Cohen,† Debra Krantz, Jillian Acevedo, Roxana Ionescu, and Josef Vlasak* Merck Research Laboratories, West Point, Pennsylvania 19486 The hinge region of human IgG2 contains four cysteine residues involved in disulfide linkages between the heavy chains, as well as the heavy and light chains. These linkages provide the fundamental framework of three distinct IgG2 disulfide isoforms recently described. Here, we detail another, disulfide-related post-translational modification in a recombinant variant of human IgG2. Heterogeneity associated with this antibody was separated into several fractions by anion-exchange chromatography (AEX), which is an important initial step that highlights the resolving power of surface charge-based HPLC techniques. Mass spectrometry of the intact antibody revealed weakly resolved discrete covalent additions of 25-35 Da in one of the two main AEX fractions. Digestion by endoproteinase Lys-C performed under nonreducing conditions, as well as tandem MS experiments, narrowed the modification to the peptide-containing disulfide-bridged hinge structure. High mass resolution and accuracy measurements of the peptide strongly suggested an addition of one or two S atoms. The modification could be eliminated by a mild reducing treatment of the intact antibody. Overall, these findings are consistent with the replacement of up to two disulfide bridges (S-S) with a like number of trisulfides (S-S-S) in the antibody hinge. The trisulfide modification is rather uncommon for proteins and its possible origins in the IgG2 variant are discussed. Immunoglobulin G (IgG) monoclonal antibodies (Mabs) represent a rapidly growing class of protein biopharmaceuticals for the treatment and prevention of disease.1-3 Advances in recombinant bioprocess technologies and purification schemes, combined with the application of robust analytical methods, have permitted and facilitated high levels of production of consistent protein products.4-11 IgG Mabs are large (150 kDa) multidomain disulfide-bridged glycoproteins that display various forms of * To whom correspondence should be addressed. Fax: 215-652-5299. E-mail: [email protected]. † Current address: The Rockefeller University, 1230 York Ave., New York, NY 10065. (1) Brekke, O. H.; Sandlie, I. Nat. Rev. Drug. Discovery 2003, 2, 52–62. (2) Bebbington, C.; Yarranton, G. Curr. Opin. Biotechnol. 2008, 19, 613– 619. (3) da Silva, F. A.; Corte-Real, S.; Goncalves, J. Biodrugs 2008, 22, 301– 314. (4) Birch, J. R.; Racher, A. J. Adv. Drug Delivery Rev. 2006, 58, 671–685. (5) Aldington, S.; Bonnerjea, J. J. Chromatogr. B: Biomed. Sci. Appl. 2007, 848, 64–78.

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structural and molecular heterogeneity.12,13 A particularly common form of heterogeneity involves cysteine residues (e.g., oxidation status, disulfide linkage variation, or decomposition via β-elimination),14,15 whose understanding can present notable analytical challenges. Of the three common IgG subclasses currently in clinical investigations and commercial production (IgG1, IgG2, and IgG4), deciphering the disulfide heterogeneity of the IgG2 subclass has eluded a clear understanding. Recently, however, several outstanding publications have appeared that reconcile and describe, in detail, the disulfide-mediated structural isoforms of both native and recombinant IgG2 antibodies.16-19 The studies revealed two novel disulfide isoforms, in addition to the traditionally expected one. The isoforms were termed IgG2A, IgG2-B, and IgG2-A/B. The A isoform is the known classic IgG2 structure with all four hinge cysteines engaged in disulfide bonding with cysteine counterparts from the second heavy chain. In the B form, two of the four hinge cysteines are bridged to the second heavy-chain hinge cysteines, the third is bridged to the light chain, and the fourth is bridged to a cysteine in the CH1 domain (this CH1 cysteine connects with the light chain cysteine in the A isoform). The A/B isoform is an intermediate containing both A- and B-isoform disulfide linkages.19 (6) Flatman, S.; Alam, I.; Gerard, J.; Mussa, N. J. Chromatogr. B: Biomed. Sci. Appl. 2007, 848, 79–87. (7) Kelley, B. Biotechnol. Prog. 2007, 23, 995–1008. (8) Filpula, D. Biomol. Eng. 2007, 24, 201–215. (9) Gottschalk, U. Biotechnol. Prog. 2008, 24, 496–503. (10) Zhou, J. X.; Tressel, T.; Yang, X.; Seewoester, T. Biotechnol. J. 2008, 3, 1185–1200. (11) Zhang, Z. Q.; Pan, H.; Chen, X. Y. Mass Spectrom. Rev. 2009, 28, 147–176. (12) Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. J. Pharm. Sci. 2008, 97, 2426–2447. (13) Vlasak, J.; Ionescu, R. Curr. Pharm. Biotechnol. 2008, 9, 468–481. (14) Tous, G. I.; Wei, Z.; Feng, J.; Bilbulian, S.; Bowen, S.; Smith, J.; McGeehan, P.; Casas-Finet, J.; Schenerman, M. A. Anal. Chem. 2005, 77, 2675–2682. (15) Cohen, S. L.; Price, C.; Vlasak, J. J. Am. Chem. Soc. 2007, 129, 6976– 6977. (16) Dillon, T. M.; Ricci, M. S.; Vezina, C.; Flynn, G. C.; Liu, Y. D.; Rehder, D. S.; Plant, M.; Henkle, B.; Li, Y.; Deechongkit, S.; Varnum, B.; Wypych, J.; Balland, A.; Bondarenko, P. V. J. Biol. Chem. 2008, 283, 16206–16215. (17) Liu, Y. D.; Chen, X.; Zhang-van Enk, J.; Plant, M.; Dillon, T. M.; Flynn, G. C. J. Biol. Chem. 2008, 283, 29266–29272. (18) Martinez, T.; Guo, A.; Allen, M. J.; Han, M.; Pace, D.; Jones, J.; Gillespie, R.; Ketchem, R. R.; Zhang, Y.; Balland, A. Biochemistry 2008, 47, 7496– 7508. (19) Wypych, J.; Li, M.; Guo, A.; Zhang, Z. Q.; Martinez, T.; Allen, M. J.; Fodor, S.; Kelner, D. N.; Flynn, G. C.; Liu, Y. Q. D.; Bondarenko, P. V.; Ricci, M. S.; Dillon, T. M.; Balland, A. J. Biol. Chem. 2008, 283, 16194– 16205. 10.1021/ac9006254 CCC: $40.75  2009 American Chemical Society Published on Web 07/10/2009

The principal methods used to analyze Mab heterogeneity, such as the disulfide isoforms, typically include various combinations of chromatography, electrophoresis, and mass spectrometry. Heterogeneity generally results in changes in the surface charge of the antibody13 enabling charge-sensitive methods (e.g., ionexchange chromatography) as the optimal primary analytical approaches. For example, cation-exchange chromatography (CEX) proved useful for resolving the IgG2 disulfide heterogeneity.16,18,19 In this report, we describe, for the first time, another form of Mab cysteine-related heterogeneity, namely, the detection of trisulfide bonds in the antibody hinge region of an IgG2 variant. Anionexchange chromatography (AEX), followed by enzymatic digestion and mass spectrometry, were critical for resolving the heterogeneity and establishing the presence of up to two trisulfide bridges in lieu of a like number of canonical disulfides in the Mab hinge region. EXPERIMENTAL SECTION Materials. The IgG2 monoclonal antibody variant was manufactured by Merck & Co., Inc., and purified according to standard manufacturing procedures. The antibody has a lambda light chain and several mutations in the CH2 domain. Human myeloma IgG2, both kappa and lambda, purified from human plasma were purchased from Sigma. Guanidine-HCl, N-ethylmaleimide (NEM), and hydroxylamine hydrochloride were purchased from Acros; acetonitrile, formic acid, urea, sodium acetate, and TFA were purchased from Pierce; Tris-HCl and MOPS buffer were purchased from Sigma; imidazole and piperazine were purchased from Fluka; HPLC-grade water was obtained from Fisher; and lysyl endoproteinase (Lys-C) was obtained from Wako Pure Chemicals. Anion-Exchange Chromatography (AEX). AEX was performed on a ProStar HPLC system (Varian, Walnut Creek, CA) using a ProPac WAX-10 4 × 250 mm column (Dionex, Sunnyvale, CA) with pH-gradient elution (mobile phase A and B contained 2.4 mM MOPS, 1.4 mM imidazole, 11.6 mM piperazine at pH 7.0 and 5.5, respectively; gradient: 0-100% B in 40 min at a flow rate of 1 mL/min). For fraction purification, a larger, 9 × 250 mm column was used with a flow rate of 4 mL/min. Mass Spectrometry of AEX FractionssIntact Protein. The AEX fractions were stored (2-8 °C) in polypropylene microtubes until needed. The fractions were diluted with aqueous 0.1% formic acid (v/v) to ∼0.1 mg/mL Mab concentration and placed into a refrigerated (2-8 °C) autosampler for injection (1-3 µL) onto an liquid chromatography-electrospray ionization-time of flight (LCESI-TOF) mass spectrometer (Model 6210, Agilent, Santa Clara, CA). Protein was resolved on a reversed-phase 0.5 mm × 50 mm PS-DVB (polystyrene-divinylbenzene) PepSwift Monolithic capillary column (LC Packings/Dionex, Sunnyvale, CA). The column was held at 80 °C. Mobile phase A was 0.1% formic acid (v/v) in water and phase B was 0.1% formic acid (v/v) in acetonitrile. The proteins were eluted by a gradient from 10% B to 40% B in 15 min at a flow rate of 20 µL/min. The ESI-TOF was calibrated with continuous co-injection of Agilent’s proprietary perfluoro compound, providing a mass accuracy of better than 10 ppm. Spectral viewing and data analysis (deconvolution) was performed with Protein Confirmation software (Agilent).

Mass Spectrometry of AEX FractionssLys-C Digestion. The Lys-C digestion and HPLC procedures were based on methods reported by Wypych et al.19 with some modifications. Mab samples (100 µg) were desalted on YM10 spin filters (Millipore, Billerica, MA) and evaporated to dryness on a SpeedVac (Savant/Thermo). The proteins were denatured by adding 10 µL of denaturing buffer (8 M guanidine-HCl, 10 mM NEM, 0.1 M NaOAc, pH 5.0) and incubating for 3 h at 37 °C. Then, 290 µL of digestion buffer (4 M urea, 20 mM NH4OH, 0.1 M Tris, pH 7.0) and 5 µL Lys-C enzyme (2 mg/mL or 0.74 au/mL, final enzyme-to-mAb ratio ) 1:10 (w/w)) was added to each sample and incubated overnight at 37 °C. Urea was included in the digestion buffer to keep the nonreduced protein in the denatured state during the digestion. In the absence of urea, the digestion was incomplete. The digestion was quenched by the addition of TFA (0.1% v/v final) and analyzed by LC/MS using an Agilent 1100 HPLC system coupled to a Q-TOF-II mass spectrometer (Waters, Milford, MA). An aliquot of the digest (100 µL) was loaded onto a C4 column (Grace Vydac 214TP52, 5 µm, 300 A, 2.1 mm × 250 mm) held at 60 °C. Mobile phase A was 0.1% TFA (v/v) in water and mobile phase B was 0.1% TFA (v/v) in 90% acetonitrile. The peptides were eluted by a gradient from 2% B to 50% B during 100 min at a flow rate of 0.2 mL/min. The online mass spectra were obtained by spraying the eluent into the mass spectrometer using an ESI source. The eluent was also monitored by UV detection at 214 nm. The capillary needle voltage was set at 3500 V, the cone voltage was 35 V, the relative collision energy was set to 10, the source temperature was maintained at 110 °C, and the desolvation temperature was 250 °C. The time-of-flight (TOF) analyzer acquired spectra using an m/z range of 300 to 2000. Data acquisition and analysis was performed with Micromass MassLynx 4.1 software. For the tandem MS experiments, 500 µL of the LysC digest was loaded onto the column and the gradient was applied as described previously. The triply charged ions corresponding to the hinge dipeptide (m/z ) 1784.5) and its modified variant with one trisulfide (m/z ) 1795.2) were selected for collision-induced fragmentation (CID). Argon was used as the collision gas, and the relative collision energy was set at 70. All other parameters were as previously described. Mild Reduction. First, a screening of different redox conditions was performed. The antibody (5 mg/mL) was formulated in 20 mM Tris-HCl, pH 7.5 with varying ratios of one of the two constituents of a redox pair. Cysteine/cystamine or glutathione/ glutathione disulfide was used at molar excess ranging from 0 to 100. The solutions were incubated overnight at 4 °C or room temperature. The reaction was stopped by reducing the pH to 5.0 with 3 M sodium acetate (control samples were quenched immediately after the addition of the redox pair). The results were evaluated by AEX. After this initial screen, incubation with 20 molar excess of cysteine (over Mab molecules) was deemed to be the optimal treatment and selected for experiments with AEX fractions. The antibody, as well as its AEX-II and AEX-III fractions formulated at 1 mg/mL (6.7 µM) in 20 mM sodium acetate, were dialyzed in three different buffers: (1) 20 mM Tris-HCl, pH 7.5, 134 µM cysteine (20× molar excess); (2) 20 mM Tris-HCl, pH 7.5 (no cysteine); and (3) 20 mM sodium acetate, pH 5.0, 134 µM Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 1. AEX profile of the human IgG2 variant. Individual fractions and their approximate percentage in the sample are indicated.

cysteine (20× molar excess). The dialysis was conducted for 24 h at 4 °C. The reaction was quenched by transferring dialysis cassettes into 20 mM sodium acetate, pH 5.0, followed by a thorough dialysis. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) experiments were performed on a Capillary DSC platform (MicroCal, LLC, Northampton, MA) at a protein concentration of 1 mg/mL and a scan rate of 1 °C/min (as described earlier20). The excess heat capacity was obtained using the Origin software for buffer subtraction, normalization to protein concentration, and interpolation of a cubic baseline in the transition region. RESULTS AND DISCUSSION Heterogeneity of Human IgG2 Variant Resolved by AnionExchange HPLC (AEX). In this report, we describe the identification of a novel AEX heterogeneity of a recombinant variant of human IgG2 monoclonal antibody. When analyzed by AEX, the antibody exhibits significant heterogeneity consisting of four fractions, designated as AEX-I, AEX-II, AEX-III, and AEX-IV (see Figure 1). The majority of the sample appears in fractions AEX-II and AEX-III, which have approximately equal contributions. Because this monoclonal antibody only differs from human IgG2 in four amino acid substitutions and it contains the same cysteine residues, we reasoned that the observed heterogeneity is likely due to the disulfide isoforms of human IgG2.16,19 Both AEX Fractions II and III Contain IgG2-A Disulfides. To determine disulfide connectivity, individual AEX fractions (see Figure 1) were subjected to nonreducing Lys-C peptide mapping, as described by Wypych et al.19 The portion of the chromatogram that contains disulfide-connected peptides characteristic for individual isoforms (IgG2-A, IgG2-A/B, and IgG2-B) were carefully examined. No evidence of IgG2-B isoform was found in any of the four fractions. Fraction AEX-I (i.e., the early eluting (basic) variant) was predominantly IgG2-A/B. Surprisingly, all of the remaining three fractions were identified as IgG2-A. Similar conclusions were obtained by analyzing the intact antibody from individual fractions by reversed-phase HPLC (see Figure S-1 in the Supporting Information). This method was shown to resolve IgG2 disulfide isoforms (the isoforms elute in the following order: (20) Ionescu, R. M.; Vlasak, J.; Price, C.; Kirchmeier, M. J. Pharm. Sci. 2008, 97, 1414–1426.

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Figure 2. Mass spectrometry of IgG2 variant Mab samples. LCESI-TOF-MS of (A) the entire sample and AEX fractions ((B) AEX-II, (C) AEX-IIIA, and (D) AEX-IIIB). Depicted is the portion of the deconvoluted mass spectra showing the intact monoclonal antibody. See text for full description.

IgG2-B, IgG2-A/B, and IgG2-A) reliably and was used as a surrogate method for the more-direct, but more-involved, peptide mapping approach.16,19 By reversed-phase HPLC, the retention times of the main peak of AEX-II, AEX-III, and AEX-IV fractions were comparable, supporting the fact that they, indeed, represent the same disulfide isoform (see Figure S-1 in the Supporting Information). In contrast, the AEX-I fraction eluted earlier, which would be consistent with it being IgG2-A/B, compared to the remaining fractions being IgG2-A. The lack of IgG2-B and the low abundance of IgG2-A/B is likely related to the fact that this antibody contains a lambda light chain, which was shown to bias the distribution of disulfide isoforms to IgG2-A.16,19 Importantly, this data demonstrated that the two main AEX fractions, AEX-II and AEX-III, do not represent disulfide isoforms and therefore are present for different reasons. AEX Fraction III Contains One to Two Low-Mass Covalent Additions. Mass spectrometry of the entire Mab sample, as well as the AEX fractions, provided initial evidence that the heterogeneity observed by AEX could arise from covalent modifications (see Figure 2). Electrospray ionization (ESI), coupled with timeof-flight (TOF) mass spectrometry, yields one of the better ways to obtain reasonably accurate mass (below 30 ppm) and resolution of proteins, especially large proteins such as intact Mabs.21 LCESI-TOF-MS analysis of the entire unfractionated antibody (see

Figure 2A) shows two main peaks, with measured masses of 145,223 and 145,384. These correspond to the intact Mab glycosylated with two “G0F”{(GlcNAc)2(Man)3(GlcNAc)2Fuc}21 Nlinked glycans (calculated mass of 145,222 Da) and the intact glycosylated Mab with an additional hexose (162 Da) attached to one of the G0F oligosaccharides, respectively. Also notable is a poorly resolved and unexpected split of the two Mab peaks. The mass separation caused by the split of the main peak (at 145,223) is ∼25 ± 10 Da and the exact identity of a single modification with this mass could not be readily assigned. Repeat analyses eliminated the possibility that the peak splitting is artifactual, and the modification was expected to be covalent, because the mass spectrometry analysis was performed under strongly denaturing conditions. ESI-TOF analysis of the AEX fractions shed additional light on the heterogeneity. Fraction AEX-II showed intact Mab with the expected mass (145,222 Da) and much less evidence for the split-peak heterogeneity (see Figure 2B). In contrast, fraction AEXIII contained the split-peak modification (not shown), suggesting that this modification is causing the separation of AEX fractions AEX-II and AEX-III. To better understand the modification, AEXIII was further fractionated into A and B, representing the left and right side of the AEX-III peak, respectively. The ESI-TOF mass spectra of AEX-IIIA and AEX-IIIB appear to be similar: dominated by Mab with at least one or two modifications (each modification with a mass of 25 ± 10 Da) (see Figures 2C and 2D). The principal difference between fractions AEX-IIIA and AEX-IIIB is the presence of a greater level of unmodified Mab (left shoulder in the mass spectra) in AEX-IIIB, compared to that for AEX-IIIA. Assuming that this covalent modification causes AEX separation, it is surprising that the later-eluting fraction IIIB contains a smaller amount of this modification than the earlier-eluting AEX-IIIA fraction. We believe that this apparent elevated level of unmodified Mab in the AEX-IIIB fraction corresponds to acidic variants of the AEX-II fraction that coincidently coelutes with the AEX-IIIB fraction (see below for further discussion). The Hinge Region of Fraction AEX-III Contains One or Two Trisulfides. The entire Mab sample and each AEX fraction were further analyzed by peptide mapping under nonreducing conditions to localize and determine the identity of the suspected modification. Prior to Lys-C digestion, the proteins were denatured in guanidine-HCl in the presence of NEM, to tag any free cysteines and prevent disulfide shuffling. The most notable differences between AEX fractions II and III were observed between retention times of 75-80 min, as seen in Figure 3. The measured mass of the 76.5 min peak corresponded to the hinge structure of IgG2A, which is a homodimer dipeptide connected by four pairs of disulfides spanning residues Cys214-Lys239 (see Figure 5A, presented later in this work). Eluting immediately after the hinge peptide were two other peaks at 77.3 and 78.3 min, the intensities of which differed between the AEX fractions. Treatment of the digests with a reducing agent resulted in the disappearance of all three of the HPLC peaks, suggesting an involvement of disulfide bridges in the two later eluting peaks (data not shown). Figure 4A depicts a portion of the raw mass spectra of the HPLC peaks. The spectra show three isotopic clusters with a charge state of (21) Gadgil, H. S.; Pipes, G. D.; Dillon, T. M.; Treuheit, M. J.; Bondarenko, P. V. J. Am. Soc. Mass Spectrom. 2006, 17, 867–872.

Figure 3. Section of HPLC traces of Lys-C digest depicting the differences among the entire Mab sample and AEX fractions II and III.

Figure 4. Mass spectra of the disulfide-linked hinge dipeptide from AEX fraction III. (A) A portion of the mass spectra from a Lys-C digest (performed under nonreducing conditions) of the three HPLC peaks at 76.5, 77.3, and 78.3 min (Figure 3), showing the corresponding isotopic clusters with 3+ charge state of the disulfide-linked hinge dipeptide. (See Figure 5a for the peptide sequence and the text for full description of the figure.) (B) Summary of the charge-reduced mass differences measured between the isotope clusters; small vertical bars represent mass differences between corresponding isotope peaks within a pair of isotopic clusters (for example, ∆ and ∆′ represent mass differences between the first isotope peaks from one cluster the next). The overall average of the measured mass differences is noted and is compared to the theoretical monoisotopic masses of a S atom and two O atoms.

3+ and m/z values (rounded to one decimal place) of 1785.5, 1796.2, and 1806.8. These values denote the most intense isotope peak in each cluster, and the three clusters themselves correspond to the disulfide-linked hinge dipeptide and the dipeptide modified by the successive addition of +32 and +64 Da, respectively. Each isotopic cluster consists of ∼10 resolved isotope peaks (whose mass accuracies are in the third decimal place). To minimize systematic errors and obtain the most accurate determination of the masses of the modifications, we used the mass differences between corresponding pairs of isotope peaks from one cluster Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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to the next. For example, ∆ and ∆′ in Figure 4A represent the masses that separate the first isotope peaks among the three clusters. In this fashion, >50 individual accurate mass differences were calculated, and these are plotted in Figure 4B (as small vertical bars), along with the average value of the differences (31.9713 Da) and the theoretical values (based on monoisotopic masses)22 for a single S atom (31.9721 Da) and two O atoms (31.9898 Da). From these measurements, we concluded that the modification is likely to be an insertion of up to two S atoms into the hinge peptide. Because reduction eliminated the hinge disulfide peptide entirely (leaving no indication of the additional S atoms by MS (data not shown)), we further suspected that insertion of the additional S atoms was associated with the disulfide bonds, resulting in one or two trisulfide bonds within the hinge of IgG2. The extent of the modification was estimated by calculating area percent of extracted ion chromatograms of each variant. This method of quantification is based on the reasonable assumption that the ionization efficiency is approximately the same for each variant. Thus, in the entire sample, the relative amounts of unmodified hinge, modified with one trisulfide, and modified with two trisulfides were 69%, 22%, and 9%, respectively. The HPLC traces in Figure 3 show that AEX fraction III is greatly enriched in trisulfides, whereas fraction II is almost devoid of them, which is in agreement with the intact mass data shown in Figure 2 and discussed previously. Efforts were made to further localize the trisulfide bonds within the hinge dipeptide. In the first approach, we performed tandem MS experiments on the hinge dipeptide. The triply charged ions corresponding to two forms of the hinge dipeptide (namely, the unmodified form and the proposed trisulfide variant containing the 32 Da addition) were selected for CID fragmentation. Most of the y ions (ranging from y2 to y18) were detected up to the C-terminal-most cysteine of the genetic hinge (Cys221) (summarized in Figure 5A) and determined to be identical and without any modifications for both forms of the dipeptide. Only two fragment ions (b8*-NH3 and b10*-NH3) that contain the genetic hinge were detected; the short peptide segment was comprised of one heavy chain peptide with all four cysteine pairs bridged to the opposing heavy chain (see Figure 5B and the associated caption for further description). It can be seen that the two b ions of the proposed single trisulfide-containing variant were indeed modified by an additional mass of 32 Da (see Figure 5C). In summary, the tandem MS data indicates that the +32 Da modification is localized to the genetic hinge. There were no cleavages found that were associated with the genetic hinge itself, despite the presence of the intervening proline residues. This finding is in accordance with what has been typically observed with CID of the cysteine-bridged hinge peptide of IgG1.23 Application of a new generation of MS gas-phase fragmentation technologies (e.g., electron-capture24 and electron-transfer dissociation) has recently permitted mapping of the disulfide-bridged hinge dipeptide of an IgG1 monoclonal antibody (with two disulfides).23 Although we believe that extending this method to the more complex IgG2 hinge dipeptide (with four disulfides) (22) McCloskey, J. A. Methods Enzymol. 1990, 193, 869–870. (23) Wu, S. L.; Jiang, H.; Lu, Q.; Dai, S.; Hancock, W. S.; Karger, B. L. Anal. Chem. 2009, 81, 112–122. (24) Zubarev, R. A. Curr. Opin. Biotechnol. 2004, 15, 12–16.

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Figure 5. (A) Schematic drawing of the hinge homodimer dipeptide. This peptide spans Cys214 to Lys239 and contains the genetic hinge (coded by a separate exon44), the lower hinge, and the N-terminal part of the antibody CH2 domain. Four disulfide bridges within the genetic hinge between the two heavy chains denoting the IgG2-A isoform19 are shown. The detected y and b ion fragments from tandem MS experiments performed on the unmodified dipeptide and the dipeptide with one proposed trisulfide are indicated (see text). The asterisk after the b ions indicates that the fragment also contain the entire, 26residue long, opposite heavy-chain peptide, connected by four disulfides (or three disulfides and one trisulfide). (B) Detail of the CID spectrum of unmodified hinge dipeptide displaying ions for fragments y16, y18, b8*, and b10*. (C) CID spectrum of the hinge dipeptide modified with one proposed trisulfide showing the increase in the mass of cysteine-containing b fragments (by ∼32 amu).

offers a more promising route to mapping the proposed trisulfide bonds, it is beyond the scope of this current study and will be considered for future experiments. A second approach to localize the proposed trisulfide bonds in the Lys-C hinge dipeptide involved subenzymatic digestion of either the unmodified or modified forms of the dipeptide. These attempts proved to be unsuccessful at cleaving between any of the hinge disulfides. It is not surprising that such a structure is impervious to protease cleavages, given the rigidity that four closely spaced disulfide bridges likely impart to the hinge dipeptide. Incubation with a Reducing Agent Leads to Elimination of the Trisulfides. Heterogeneity related to disulfide chemistry (e.g., cysteinylation, disulfide linkage variation) can be often reduced after incubation under mild reducing conditions.16,19,25 How would such treatment affect the trisulfide heterogeneity? We treated the IgG2 variant samples using a panel of conditions with different reducing strengths. The effect of the treatment was evaluated by monitoring changes in the AEX profile. An overnight

incubation at 4 °C or at room temperature in the presence of a reducing agent (cysteine or glutathione) at pH 7.5 resulted in conversion of a significant portion of AEX-III fraction to AEX-II. The presence of the oxidized counterpart (e.g., cystamine or glutathione disulfide) was not needed. The pH of the solution was an important factor; when the incubation was performed at pH 5.0, no change in the AEX pattern was observed. This is in agreement with the general mechanism of disulfide/thiol exchange reaction where the reactive groupsthe thiolate anion (RS-)sbecomes more abundant as the pH approaches the pKa of the thiol group (typical pKa 8-9).26 Figure 6 shows an example of one experimental set. In addition to the conversion of AEX-III to AEX-II, an increase of AEX-I (i.e., the IgG2-A/B isoform) is also apparent, which is expected based on the reported results for IgG2.16,19 These samples were further analyzed by peptide mapping to confirm that the changes in the AEX chromatogram upon the reduction treatment are indeed due to the elimination of trisulfides. As seen in Figure 7, incubation of AEX fraction III at pH 7.5 in the presence of cysteine resulted in complete elimination of any hinge peptide containing the trisulfides and only the disulfide form of the hinge dipeptide remained. The reduction performed at pH 5 or in the absence of cysteine at pH 7.5 showed no change in the level of trisulfides. These data are in agreement with the AEX results. We must note that, although the treatment with cysteine at pH 7.5 led to complete elimination of the trisulfides (determined by the peptide mapping analysis; see Figure 7), not all material in fraction AEX-III was converted to AEX-II (see Figure 6). This is in agreement with the peptide mapping (Figure 3) and intactmass data (Figure 2), which show that not all antibody molecules in fraction AEX-III contain trisulfides, suggesting that a portion of AEX-III contains modifications other than the trisulfides. When individual AEX fractions were analyzed by isoelectric focusing (IEF) (see Figure S-2 in the Supporting Information), the presence of trisulfides was not resolved. Instead, IEF revealed a presence of acidic and basic variants in the AEX fractions. The distribution of these charge variants was significantly different among the four AEX fractions and followed the fundamentals of AEX separation. The most basic fraction, AEX-I, contained a significant amount of basic IEF variants but no acidic IEF variants. In contrast, more-acidic fractions, such as AEX-III, did not contain any basic variants and were enriched in acidic variants. Based on these results, we conclude that a portion of the AEX-III fraction contains IEF-resolved acidic variants of fraction AEX-II and, therefore, it cannot be converted to AEX-II by the mild reducing treatment. Differential Scanning Calorimetry of AEX-II and AEX-III Fractions. The thermal stability of the AEX-separated fractions was evaluated by differential scanning calorimetry (DSC). The thermograms of fractions AEX-II and AEX-III are shown in Figure 8 and they overlap very well. The temperature-induced unfolding profile for each of these fractions presents two transitions. Based on the enthalpies of reaction for each transition and previous observation for monoclonal antibodies,20 one may infer that the (25) Banks, D. D.; Gadgil, H. S.; Pipes, G. D.; Bondarenko, P. V.; Hobbs, V.; Scavezze, J. L.; Kim, J.; Jiang, X. R.; Mukku, V.; Dillon, T. M. J. Pharm. Sci. 2007, 97, 775–790. (26) Creighton, T. E. Proteins: Structures and Molecular Properties, 2nd Edition; W. H. Freeman and Co.: New York, 1993; pp 17-20.

Figure 6. Reducing treatment analyzed by AEX. The traces represent AEX chromatograms. Antibody samples at 1 mg/mL (6.7 µM) were incubated overnight at 4 °C in four different formulations, with or without 20× molar excess of cysteine (134 µM), at pH 7.5 or 5.0.

first transition represents the unfolding of the Fab fragment and CH2 domain, and the second transition represents the unfolding of the CH3 domain. The DSC results on AEX-II and AEX-III fractions suggest that the presence of trisulfides does not significantly impact the thermal stability of a monoclonal antibody and/or the independent unfolding of Fab and Fc fragments. These findings are in contrast with the reports on IgG2-A and IgG2-B isoforms,16 which were found to have significantly different thermal stabilities. Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 7. Reducing treatment of fraction AEX-III analyzed by peptide mapping. The fraction was treated with different mild reducing conditions overnight at 4 °C (as noted) and then subjected to Lys-C digest under nonreducing conditions. Depicted are mass spectra of the portion of the chromatogram containing the three peaks eluting at 76.5, 77.3, and 78.3 min (e.g., the forms of the hinge dipeptide with zero, one, or two trisulfides). Isotopic clusters of the 3+ charge state are shown (described in Figure 4). AEX traces of the same samples are in Figure 6.

Figure 8. Differential scanning calorimetry (DSC) thermograms of fractions AEX-II (solid line) and AEX-III (symbols). The proteins were analyzed at 1 mg/mL.

Trisulfides and Their Origin. Trisulfides in nature are commonly associated with small organic molecules with biotic and abiotic origins ranging from diallyl polysulfides derived from garlic27 to polysulfanes found in sulfur-rich fuels, natural rubber, and soils.28 In contrast, the presence of trisulfides associated with peptides and proteins seems to be rare. One of the early reported detections of protein trisulfide was in superoxide dismutase extracted from bovine erythrocytes,29 a finding which was later determined to be an artifact caused by the protein extraction and purification process.30 Another example is trisulfide detected in a truncated interleukin-6 mutein expressed recombinantly in E. coli.31 The most commonly reported trisulfide-containing protein is the recombinant human growth hormone (rhGH) that is expressed from E. coli,32-35 whereas no trisulfide has been detected in native hGH extracted from pituitary gland.33 Although possible routes of trisulfide formation in rhGH have been postulated,34 the exact mechanism remains unknown. Other polypeptide trisulfides have been derived from accelerated degradation studies (27) Kim, Y. H. In Organic Sulfur Chemistry: Biochemical Aspects; Oae, S., Okuyama, T., Eds.; CRC Press: Boca Raton, FL, 1992; pp 137-194. (28) Steudel, R. Chem. Rev. 2002, 102, 3905–3945. (29) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049–6055. (30) Briggs, R. G.; Fee, J. A. Biochem. Biophys. Acta 1978, 537, 100–109.

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conducted on wool keratin proteins36 and on aqueous solutions of the salmon calcitonin peptide.37 One possible mechanism of trisulfide formation in recombinant protein involves reaction of dissolved hydrogen sulfide (H2S)38 generated during the fermentation process.39,40 Recently, a treatment of rhGH with solutions that contained dissolved H2S has been shown to lead to the formation of elevated levels of trisulfide forms of the protein.41 For monoclonal antibodies, the hinge region is more solvent accessible than other disulfide-containing regions of the Mab and thus a possible target for reaction with H2S and the formation of trisulfide. We speculate that this mechanism is also involved in the case of the recombinant IgG2 Mab described here. In addition, we found no evidence for trisulfides in commercially available IgG2 isolated from immunoglobulin or in any number of recombinantly expressed IgG1 Mabs (data not shown). Various approaches have been proposed to minimize trisulfide formation in recombinant proteins while maintaining biological activity including treatment with mercapto compounds (e.g., cysteine, DTT),42 sulfite,39 inclusion of metal salts and lowering of the pH.41 More recently, a “stripping” method that uses an inert gas (e.g., nitrogen, argon, air) bubbled through the fermentation solution to displace and remove dissolved H2S has been proposed.40 CONCLUSIONS It has been known for some time that antibodies under longterm storage in solution can form a nonreducible entity that appears on SDS-PAGE. This has recently been confirmed to arise from the expulsion of sulfur from a disulfide, resulting in a thioether bridge (-CH2-S-CH2-).14,15 Here, for the first time, we present evidence of the insertion of sulfur into the hinge to form a trisulfide bridge (-CH2-S-S-S-CH2-). The finding expands our understanding of the sphere of influence of cysteine residues in Mabs and highlights the critical role that (31) Breton, J.; Avanzi, N.; Valsasina, B.; Sgarella, L.; Lafiura, A.; Breme, U.; Orsini, G.; Wenisch, E.; Righetti, P. G. J. Chromatogr. A 1995, 709, 135– 146. (32) Jespersen, A. M.; Christensen, T.; Klausen, N. K.; Nielsen, P. F.; Sorensen, H. H. Eur. J. Biochem. 1994, 219, 365–373. (33) Canova-Davis, E.; Baldonado, I. P.; Chloupek, R. C.; Ling, V. T.; Gehant, R.; Olson, K.; Gillece-Castro, B. L. Anal. Chem. 1996, 68, 4044–4051. (34) Andersson, C.; Edlund, P. O.; Gellerfors, P.; Hansson, Y.; Holmberg, E.; Hult, C.; Johansson, S.; Kordel, J.; Lundin, R.; Mendel-Hartvig, I.; Noren, B.; Wehler, T.; Widmalm, G.; Ohman, J. Int. J. Peptide Protein Res. 1996, 47, 311–321. (35) Ribela, M. T. C. P.; Gout, P. W.; Oliveira, J. E.; Bartolini, P. Curr. Pharmaceut. Anal. 2006, 2, 103–126. (36) Ghadimi, M.; Hill, R. R. J. Chem. Soc. Chem. Commun. 1991, 903–904. (37) Windisch, V.; DeLuccia, F.; Duhau, L.; Herman, F.; Mencel, J. J.; Tang, S. Y.; Vuilhorgne, M. J. Pharm. Sci. 1997, 86, 359–364. (38) Purdie, J. W.; Gravelle, R. A.; Hanafi, D. E. J. Chromatogr. 1968, 38, 346– 350. (39) Christensen, T. A method of converting a hydrophobic derivative of a polypeptide into the native form, World Patent WO96/02570, February 1, 1996. (40) Becker, P.; Christensen, T. Method for preventing formation of trisulfide derivatives of polypeptides, World Patent WO2006/069940, July 6, 2006. (41) Hemmendorrf, B.; Castan, A.; Persson, A. Method for the production of recombinant peptides with a low amount of trisulfides, U.S. Patent 7,232,894, June 19, 2007. (42) Sørensen, H. H.; Christensen, T. A method of detecting the presence of and converting of a polypeptide, World Patent WO94/24157, October 27, 1994.

robust analytical methods play in the overall characterization of therapeutic proteins.43

ACKNOWLEDGMENT We would like to thank Katie Lancaster for developing the AEX method and Van Hoang, Lorenzo Chen, and Marc Kirchmeier for their insightful discussions and support. (43) Datola, A.; Richert, S.; Bierau, H.; Agugiaro, D.; Izzo, A.; Rossi, M.; Cregut, D.; Diemer, H.; Schaeffer, C.; Van Dorsselaer, A.; Giartosio, C. E.; Jone, C. ChemMedChem 2007, 2, 1181–1189. (44) Brekke, O. H.; Michaelsen, T. E.; Sandlie, I. Immunol. Today 1995, 16, 85–90.

NOTE ADDED AFTER ASAP PUBLICATION This paper posted to the Web on July 10, 2009 with the corrections not implemented. The correct version was posted on July 10, 2009. SUPPORTING INFORMATION AVAILABLE Reversed-phase HPLC and IEF traces of AEX fractions. (PDF) This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 26, 2009. Accepted June 24, 2009. AC9006254

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