Determination of Fab− Hinge Disulfide Connectivity in Structural

Dec 29, 2009 - sequential hinge cysteines. Furthermore, the connectivity of a novel subvariant of IgG2-B containing an intrachain disulfide linkage in...
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Anal. Chem. 2010, 82, 1090–1099

Determination of Fab-Hinge Disulfide Connectivity in Structural Isoforms of a Recombinant Human Immunoglobulin G2 Antibody Bing Zhang, Adam G. Harder, Heather M. Connelly, Lorna L. Maheu, and Steven L. Cockrill* Analytical Sciences, Amgen, Inc., 4000 Nelson Road, Longmont, Colorado 80503 The detection and characterization of unexpected disulfide-mediated structural variants of human immunoglobulin G2 (IgG2) antibodies was recently the subject of two copublications.1,2 In this paper, we present data to confirm the previously reported structures and elucidate the complete disulfide connectivity of each variant through the application of a novel analytical methodology. In this manner, the data illustrate the presence of at least five structural variants, including the classical structure with independent Fab domains and a hinge region. Multiple subvariants of the IgG2-A/B and IgG2-B structures are identified; these subvariants of each structure differ through the order of attachment of Fab peptides to the sequential hinge cysteines. Furthermore, the connectivity of a novel subvariant of IgG2-B containing an intrachain disulfide linkage in the lower hinge region is elucidated. The results presented in this paper reveal that the population of IgG2 disulfide structural variants is yet more complex than recently reported. Structural models of immunoglobulin G (IgG) subclasses were first proposed in the 1960s and 1970s.3-9 The greatest differences between the IgG subtypes in these models were related to the interchain linkages, whereas the intrachain disulfide bonding patterns were determined to be conserved across the subclasses. Of particular note is the fact that the various subclasses are typified by differences in the amino acid composition and linkage structure in the hinge region.10 Unexpected disulfide-related structures have been identified recently in the IgG4 subclass, in which stable intrachain bonds between the two hinge region cysteines were identified and * Corresponding author. E-mail: [email protected]. (1) Wypych, J.; Li, M.; Guo, A.; Zhang, Z.; Martinez, T.; Allen, M. J.; Fodor, S.; Kelner, D. N.; Flynn, G. C.; Liu, Y. D.; Bondarenko, P. V.; Ricci, M. S.; Dillon, T. M.; Balland, A. J. Biol. Chem. 2008, 283, 16194–16205. (2) 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. (3) Pink, J. R. L.; Milstein, C. Nature 1967, 214, 92–94. (4) Pink, J. R. L.; Milstein, C. Nature 1967, 216, 941–942. (5) Frangione, B.; Milstein, C.; Franklin, E. C. Biochem. J. 1968, 106, 15–21. (6) Frangione, B.; Milstein, C. J. Mol. Biol. 1968, 33, 893–906. (7) Frangione, B.; Milstein, C.; Pink, J. R. Nature 1969, 221, 145–148. (8) Edelman, G. M.; Cunningham, B. A.; Gall, W. E.; Gottlieb, P. D.; Rutishauser, U.; Waxdal, M. J. Proc. Natl. Acad. Sci. U.S.A. 1969, 63, 78–85. (9) Milstein, C.; Frangione, B. Biochem. J. 1971, 121, 217–225. (10) Brekke, O. H.; Michaelson, T. E.; Sandlie, I. Immunol. Today 1995, 16, 85–90.

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characterized by the presence of “half-molecule” species.11-13 Similar linkages have been suggested, albeit with much lower prevalence, in antibodies of the IgG1 subclass.14 A pair of landmark publications revealed the detection and characterization of two new disulfide-mediated structural variants of human IgG2 antibodies, termed IgG2-B and IgG2-A/B.1,2 Unlike the classical structure (IgG2-A) first published in the 1960s and 1970s in which the hinge region and Fab domains were separated, the two new structures identified the presence of linkages between cysteine residues in the Fab domain and hinge region. Variant IgG2-B contained linkages connecting two copies of the Fab peptides with two copies of the hinge peptide, whereas variant IgG2-A/B represented a hybrid structure incorporating partial features of both IgG2-A and IgG2-B, such that a single Fab domain is linked to two copies of the hinge peptide. Determination of these structures was accomplished through characterization of species present in the nonreduced peptide maps of IgG2 antibodies, but absent when separated under identical conditions after treatment with reducing agent. For species containing a single disulfide linkage between two peptides the connectivity is established directly from identification of these constituent peptides. However, for species containing more than two cysteine residues, the precise connectivity cannot be determined in this manner. Species containing multiple Cys residues have been characterized either by automated Edman N-terminal sequencing or via simplification of the nonreduced peptide structure by secondary digestion. The utility of Edman sequencing has been reported previously for identification of parallel linkages between dimeric hinge peptides in IgG4.15 The observation of diPTH-cystine in cycles in which a cysteine residue is expected confirms the parallel “ladder”-type structure consistent with the classical model.15 However, as noted in prior communications, this result does not preclude the presence of alternative bonding arrangements between cysteine residues. The classical Fab disulfide-linked tripeptide species (H78(s-s)H6(s-s)L12) has been characterized by orthogonal methods, including secondary digestion with endoprotease Glu-C on (11) Schuurman, J.; Van Ree, R.; Perdok, G. J.; Van Doorn, H. R.; Tan, K. Y.; Aalberse, C. Immunology 1999, 97, 693–698. (12) Schuurman, J.; Perdok, G. J.; Gorter, A. D.; Allberse, R. C. Mol. Immunol. 2001, 38, 1–8. (13) Aalberse, R. C.; Schuurman, J. Immunology 2002, 105, 9–19. (14) Bloom, J. W.; Madanat, M. S.; Marriott, D.; Wong, T.; Chan, S. Y. Protein Sci. 1997, 6, 407–415. (15) Zhang, W.; Marzilli, L. A.; Rouse, J. C.; Czupryn, M. J. Anal. Biochem. 2002, 311, 1–9. 10.1021/ac902466z  2010 American Chemical Society Published on Web 12/29/2009

account of the glutamic acid residue fortuitously located between the two cysteines in peptide H6.1 Alternatively, the corresponding tripeptide in an IgG4 antibody was characterized by tandem-MS analysis and detection of fragmentation products containing neither, one, or both disulfides.15 While the tandem-MS approach is appropriate for simpler species, given the complex nature (number of disulfide linkages) and large size of nonreduced peptides from the newly reported IgG2 variants (∼15 and ∼25 kDa for IgG2-A/B and IgG2-B, respectively1), the application of tandem-MS methodologies is severely limited through inability for efficient fragmentation and the vastly complex resulting spectra due to the presence of multiple disulfide linkages between multiple peptides and/or multiple copies of the same peptide. A recognized limitation in the analytical characterization of recently reported IgG2 disulfide variants was the paucity of data regarding the specific linkages between the Fab arm and hinge cysteines. Given the closely spaced cysteine residues of the IgG2 hinge peptide, determination of the precise connectivity poses a significant analytical challenge. Several methods exist for the elucidation of bonding between closely spaced residues, including limited reduction/alkylation16 and cyanylation-induced cleavage.17 We recently reported a novel methodology that couples manual Edman chemistry with LC-MS analysis to provide comprehensive linkage analysis of peptides containing closely spaced cysteine residues.18 However, until recently no reports existed of successful application to such complex systems as the human IgG2 disulfide variants. One paper describing a study employing limited reduction and differential alkylation of IgG2 structural variants was recently published, in which three unexpected structural variants of a therapeutic recombinant IgG2 in addition to the classical structure were isolated from cation exchange chromatography.19 In this paper we present data that describe the application of a novel analytical approach to afford elucidation of the connectivity of the unique linkages for the recently reported disulfide variants (IgG2-A/B and IgG2-B). Our data show partial agreement with those of the previous paper on IgG2 variant connectivity determined by limited reduction and alkylation;19 however, we identify different linkages for IgG2-A/B and multiple previously unreported linkages for IgG2-B. MATERIALS AND METHODS Materials. The recombinant human IgG2 monoclonal antibody was expressed with κ-type light chains and γ2-type heavy chains. The antibody was produced from Chinese hamster ovary (CHO) cell culture and purified using established techniques.20 Endoprotease Lys-C was sourced from Wako Chemicals (Richmond, VA), and endoproteinase Glu-C (sequencing grade) was purchased from Roche (Indianapolis, IN). Reagents for manual execution of Edman sequencing including trifluoroacetic acid (TFA), phenyl isothiocyanate (PITC), and N-methylpiperidine/water/methanol solution were sequencing grade materials obtained from Applied Biosystems (Foster City, Yen, T.-Y.; Yan, H.; Macher, B. A. J. Mass Spectrom. 2002, 37, 15–30. Wu, J.; Watson, J. T. Methods Mol. Biol. 2002, 194, 1–22. Zhang, B.; Cockrill, S. L. Anal. Chem. 2009, 81, 7314–7320. 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. (20) Shukla, A. A.; Hubbard, B.; Tressel, T.; Guhan, S.; Low, D. J. Chromatogr., B 2007, 848, 28–39. (16) (17) (18) (19)

CA). Pyridine was ACS reagent grade from Fluka (Buchs, Switzerland). Preprepared mobile phases (0.1% TFA in water and 0.1% TFA in acetonitrile), as well as HPLC-grade water and 1-propanol, were from J.T. Baker (Phillipsburg, NJ). Tris was from Calbiochem (La Jolla, CA); tris(2-carboxyethyl)phosphine (TCEP) and trifluoroacetic acid (TFA) for mobile-phase preparation were purchased from Pierce (Rockford, IL). Sodium acetate, urea, hydroxylamine hydrochloride, guanidine hydrochloride solution (8 M), and N-ethylmaleimide (NEM) were obtained from SigmaAldrich (St. Louis, MO). Separation of Disulfide Variants. Disulfide variants were isolated using a nonreduced reversed-phase high-performance liquid chromatography (RP-HPLC) method using an Agilent 1100 system on the basis of previous reports.1,2,21 Mobile phases consisted of (A) 0.11% TFA in 0.39% 1-propanol, 0.11% acetonitrile and (B) 0.10% TFA in 70% 1-propanol, 20% acetonitrile. Maximal loading of an analytical scale RP-HPLC column (Agilent Zorbax SB300 C18, 4.6 × 250 mm, 5 µm particle size) without resolution loss was employed and elution accomplished with a linear gradient between 24% and 29% mobile phase B in 25 min after a 5 min equilibration at 15% mobile phase B. The flow rate was 0.5 mL/ min, and detection was monitored at 215 nm. Each isoform was enriched from multiple rounds of fraction collection and pooling. Fractions were dried by vacuum centrifugation and stored at -20 °C until analysis. Mass analysis was also performed by coupling an ESI-TOF instrument (Applied Biosystems Q-STAR Pulsar I, Foster City, CA) to the chromatographic instrument. Deconvolution of the charge envelope across the isoform profile was accomplished using Analyst QS software (version 2.0, Applied Biosystems). Nonreduced Peptide Mapping of Unfractionated and Enriched Disulfide Variants. A 150 µL volume containing 900 µg of rIgG2 was denatured using 350 µL of denaturation buffer (8 M guanidine hydrochloride, 10 mM NEM, 0.1 M sodium acetate, pH 5.2) and incubated for 3 h at 37 °C. NEM was present to alkylate free sulfhydryl and mitigate the potential for disulfide scrambling.22 The entire 500 µL volume was added to 20 mL of Lys-C digestion buffer (4 M urea, 20 mM hydroxylamine, 0.1 M Tris, pH 7.0) and treated with 450 µL of endoprotease Lys-C (2 mg/mL in water). Proteolysis was accomplished by incubation at 37 °C overnight, after which the digestion was quenched by addition of 250 µL of 5% TFA. Samples for assessment of the reduced peptide map were then subjected to reduction by incubation with 5 µL of tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 30 min. Enriched disulfide variants were processed with different volumes of reagent, depending upon their proportion in the nonreduced RP-HPLC separation (RP-1 and RP-3 were treated with higher volumes; RP-2, RP-4, and RP-5 were treated with lower volumes). Each fraction was reconstituted in 10 or 20 µL of denaturation buffer, denatured by incubation at 37 °C for 3 h, and then treated with 400 or 800 µL of Lys-C digestion buffer. Each fraction was treated with 9 or 18 µL of Lys-C solution and digested (21) Dillon, T. M.; Bondarenko, P. V.; Rehder, D. S.; Pipes, G. D.; Kleemann, G. R.; Ricci, M. S. J. Chromatogr., A 2006, 1120, 112–120. (22) Bures, E. J.; Hui, J. O.; Chow, D. T.; Katta, V.; Rohde, M. F.; Zeni, L.; Rosenfeld, R. D.; Stark, K. L.; Haniu, M. Biochemistry 1998, 37, 12172– 12177.

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overnight at 37 °C, after which the digest was quenched by addition of 5 or 10 µL of 5% TFA. Separation was accomplished using an Agilent 1200 HPLC instrument using a reversed-phase column (Vydac 214TP52 C4, 2.1 × 250 mm) thermostated to 60 °C. Mobile phases were (A) 0.1% TFA in water and (B) 0.1% TFA in 90% acetonitrile. Digested samples (∼150 µg) were injected and separated using a linear gradient of 0-45% mobile phase B in 90 min after a 10 min equilibration step. A flow rate of 200 µL/min was employed and peptide elution monitored by absorbance at 215 nm and online mass spectrometry using an ESI-TOF instrument (Agilent MSDTOF, Santa Clara, CA) in positive ion mode. Fraction collection of disulfide-linked peptides for further characterization was accomplished via bypassing the mass spectrometer inlet and using an automated fraction collector (Agilent). Isolated peptides were dried by vacuum centrifugation and stored at -20 °C until analysis. Secondary Digestion of Disulfide-Linked Peptides. Isolated peptide G was reconstituted in 100 µL of Glu-C digestion buffer (2 M guanidine hydrochloride, 100 mM Tris, pH 6.5) and incubated with 5 µL of endoprotease Glu-C (1 mg/mL in digestion buffer) overnight at room temperature. The digest products were then separated by HPLC (Agilent 1200, Santa Clara, CA) using a reversed-phase column (Agilent Zorbax 300SB C8, 2.1 × 150 mm, 5 µm particle size) thermostated to 50 °C. Mobile phases were (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and separation was accomplished using a linear gradient of 2-50% mobile phase B in 45 min after a 5 min equilibration step. A flow rate of 200 µL/min was employed and peptide elution monitored at 215 nm and by online mass spectrometry using an ESI-TOF instrument (Agilent MSD-TOF) in positive ion mode. Manual fraction collection was performed under identical conditions, except that the mass spectrometer inlet was bypassed. Disulfide-linked peptides b, c, and f were collected from nonreduced Lys-C digests of 10 mg of rIgG2. Each peptide pool was dried by vacuum centrifugation and reconstituted in 400 µL of Glu-C digestion buffer. Digestion was performed by addition of 20 µL of endoprotease Glu-C (1 mg/mL in digestion buffer) and carried out overnight at room temperature. The digest products were then analyzed by LC-MS with separation and detection parameters as summarized above. Manual N-Terminal Sequencing. Manual coupling and cleavage of amino acid residues from disulfide-linked peptides was performed according to an optimized protocol using traditional Edman chemistry.18 Briefly, each disulfide-linked peptide after secondary digestion was collected and dried by vacuum centrifugation. Samples were resuspended in 10 µL of 10 mM NEM and incubated in the dark at room temperature for 30 min. Samples were then treated with 40 µL of anhydrous pyridine, 5 µL of PITC, and 5 µL of N-methylpiperidine/water/methanol solution and incubated at 50 °C for 3 min under dry nitrogen. Samples were dried by vacuum centrifugation and cleavage of N-terminal residues performed by resuspension in 5 µL of TFA and incubation at 50 °C for 2 min under dry nitrogen, followed by addition of 40 µL of 4 M guanidine hydrochloride, 100 mM Tris, pH 6.5. The leaving and residual groups were identified by loading the complete volume and separation by capillary scale RP-HPLC (Agilent 1200 HPLC instrument) coupled to a linear ion trap ESI instrument (Thermo LTQ XL, Waltham, MA). Separation was 1092

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performed using a reversed-phase column (Agilent Zorbax 300SB C8, 1.0 × 150 mm, 3 µm particle size) thermostated to 50 °C. Mobile phases were as noted above. Samples were separated using a linear gradient of 2-71% mobile phase B in 65 min after a 5 min equilibration step. A flow rate of 50 µL/min was employed and peptide elution monitored at 215 nm and by online mass spectrometry. Data from the LC-MS analysis was parsed for specific mass values corresponding to potential residual and leaving groups, depending on the known inventory of peptides associated with each unique peptide. A second identical preparation of each sample was employed to collect the residual group(s) for additional processing cycles. Iterative coupling and cleavage cycles were executed by repeating the steps noted above. RESULTS AND DISCUSSION Identification of Disulfide-Linked Peptides by Peptide Mapping. Comparison of peptide maps of unfractionated IgG2 following digestion with endoprotease Lys-C under nonreduced and reduced conditions provided determination of peptides bound through disulfide linkages by their appearance in the nonreduced map and absence following reduction. The nonreduced map (Figure S1, Supporting Information) shows peaks A-G which are absent upon reduction. Disappearance of these peptides after reduction confirms that they are involved in disulfide linkages in the native protein. The highlighted region of Figure S1 indicates additional species that also disappear upon reduction; these are disulfide-linked peptides corresponding to variants of the IgG2 disulfide structure, and their characterization is discussed later. Determination of Connectivity for the Classical IgG2-A Variant. The disulfide connectivity of the classical IgG2-A variant was accomplished using an analytical approach analogous to that previously reported.1 Comparison of theoretical to measured mass values confirmed the identities of the constituent species from the disulfide-bound peptides and for peptides containing a single disulfide bond (peptides A-E) allowed direct determination of the disulfide connectivity. Peptide F was identified as the hinge-hinge peptide (H11(s-s)H11-12) through its observed mass. Analysis of peptide F by automated Edman sequencing resulted in the detection of di-PTH-Cys moieties in each of cycles 1, 2, 5, and 8 (data not shown), confirming the presence of four parallel disulfides.1,15 However, the presence of alternate connectivity cannot be ruled out, due to the limitations of the N-terminal sequencing method. Reduction of peptide G showed the generation of three product peaks with identities confirmed by mass analysis as H6, H7-8, and L12. Digestion of the nonreduced peptide with endoprotease Glu-C resulted in cleavage at position 142 of peptide H6, forming H6a (N-terminal portion) and H6b (C-terminal portion). This confirmed linkages as H6a (Cys-136) bound to L12 (Cys-215) and H6b (Cys-149) coupled to H7-8 (Cys-205), consistent with the classical IgG2-A structure.1 Note that digest products of the alternative structure were not observed, indicating that a single homogeneous bonding pattern exists for the Fab arm intrachain linkage in the IgG2 molecule and that disulfide exchange is not observed during sample processing. Taken together, characterization of the disulfide-linked peptides A-G elucidated the linkages between specific Cys

residues, summarized in Table S1, Supporting Information, which confirm the presence of the expected classical disulfide structure (IgG2-A). Characterization of Nonreduced Peptides Unique to Disulfide Variants. In addition to characterization of nonreduced peptides A-G which confirmed the classical IgG2-A disulfide structure, the nonreduced Lys-C peptide map also showed the presence of other species eluting between 90 and 100 min (peaks a-i) that disappeared following treatment with reducing agent (Figure S1, Supporting Information). The observed masses and putative constituent species of these nonreduced peptides are summarized in Table S2, Supporting Information. Three distinct groupings of characteristic masses (∼12, ∼15, and ∼25 kDa) are observed in the mass spectrometric data for peaks a-i (Table S2, Supporting Information). Multiple nonreduced peptides corresponding to each grouping are observed; for example, peaks b, c, and f contain peptides with masses of 15450.88, 15225.49, and 15000.19 Da, respectively, all of which are related to the ∼15 kDa grouping. The mass difference between these peptides is consistent with the presence of zero, one, or two copies of the H12 peptide through variable cleavage at the Lys-Pro sequence in H11-12. Given that the observed mass distribution for each grouping is the result of partial cleavage, it was postulated that the related species of each grouping share a common disulfide structure, and therefore, characterizations of peaks containing representative species for each grouping was performed. The three groupings of nonreduced Lys-C peptides correspond to disulfide variants, determined on the basis of the constituent masses of each grouping. The ∼15 kDa variant contains two copies of the hinge peptide connected to a single copy of each of the Fab arm peptides and corresponds to the IgG2-A/B subclass.1 The ∼25 kDa variant contains two copies of the hinge peptide connected to two copies of each of the Fab arm peptides and corresponds to the IgG2-B subclass.1 The ∼12 kDa variant contains a single copy of the hinge peptide (H11) connected to a single copy of each of the Fab arm peptides (H6, H7, and L12); the presence of this ∼12 kDa peptide was noted in the prior paper, but was not discussed.1 Isolation and Enrichment of Disulfide Variants. Disulfide variants were isolated by nonreduced RP-HPLC. Five fractions were isolated (Figure S2, Supporting Information) and assessed for observed molecular weight, purity,and proportional distribution of each fraction (Table S2, Supporting Information). The masses are essentially identical within the capability of the MS instrument, confirming that these species are differentiated by conformation or structural features and not biochemical modification. The purified fractions were employed for further evaluation of nonreduced peptide maps and subsequent analyses. Characterization of Disulfide-Linked Peptides in Nonreduced RP-HPLC Fractions. To ascertain which disulfide variant eluted at what position in the nonreduced RP-HPLC distribution, enriched fractions (RP-1 through RP-5) were subjected to nonreduced peptide mapping with endoprotease Lys-C. Through this approach the presence of peptides specific to a particular disulfide variant were assessed. Similar profiles throughout most of the chromatograms were obtained from the peptide maps of all RP fractions, including the presence of a subset of the disulfide-

containing peptide peaks corresponding to the classical IgG2 structure, peptides A-E (data not shown). This observation indicates that the expected intrachain disulfide linkages were uniformly present across all fractions. The major differences between the peptide maps were observed in the peak distribution between 90 and 100 min (Figure S3, Supporting Information). Observed masses for the peptides from each fraction (Table S4, Supporting Information) indicate that each fraction corresponded to a specific group of peptides characteristic of a given variant. The presence of multiple peaks within a grouping is related to the presence of partial cleavage at Lys-Pro sequences. Accordingly, fraction RP-1 is characterized by the ∼25 kDa variant (IgG2-B) and RP-3 by the ∼15 kDa variant (IgG2-A/B), and RP-4 and RP-5 show only the peptides corresponding to the classical structure (IgG2-A). Notably, RP-2 is characterized by a ∼12 kDa variant, which was not addressed in previous reports. Determination of Disulfide Connectivity for Each Variant. As previously described, the combination of manual Edman sequencing steps and analysis of the resulting products by LC-MS after each coupling and cleavage cycle affords specific and sequential identification of the connectivity of each disulfide present in nonreduced peptides.18 Fortuitously, the conserved hinge peptide sequence of the IgG2 subclass contains a lysine residue immediately prior to the first hinge cysteine, and hence, the N-terminal residue of the H11 hinge peptide is Cys-224. Therefore, the connectivity of disulfide variants can be readily determined by the specific monitoring of leaving and/or residual groups. This approach is illustrated in the following section for the IgG2-A/B (∼15 kDa) variant. The fundamental concept for this novel methodology for connectivity assignment is not dissimilar to the protein ladder sequencing method, in which matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis is used to differentiate cleavage products following sequential reactions.23,24 This approach is elegant in that single amino acid residues are sequentially removed without separation, resulting in the presence of a set of nested peptides, each differentiated by the loss of one N-terminal amino acid residue compared to its predecessor. The observed mass difference affords identification of the amino acid residue lost in successive cycles. In theory, this methodology could also be applied to the disulfide-bearing species, as the loss of different leaving groups yields residual groups of specific and characteristic mass. However, the approach is confounded by the inherent nature of MALDI-MS to induce prompt dissociation of disulfide bonds, such that the constituent disulfide-linked peptides are observed in a reduced state.25-27 Principle of the Method To Differentiate Structural Variants. Figure 1 illustrates three possible structures of the ∼15 kDa (IgG2-A/B) variant nonreduced Lys-C peptide following secondary digestion with endoprotease Glu-C. These structures are employed to demonstrate the principal of the methodology for determination (23) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89–92. (24) Bartlet-Jones, M.; Jeffery, W. A.; Hansen, H. F.; Pappin, D. J. C. Rapid Commun. Mass Spectrom. 1994, 8, 737–742. (25) Patterson, S. D.; Katta, V. Anal. Chem. 1994, 66, 3727–3732. (26) Crimmins, D. L.; Saylor, M.; Rush, J.; Thoma, R. S. Anal. Biochem. 1995, 226, 355–361. (27) Qiu, Z.; Cui, M.; Li, H.; Liu, Z.; Liu, S. Rapid Commun. Mass Spectrom. 2007, 21, 3520–3525.

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Figure 1. Representations of three putative structures of IgG2-A/B disulfide variants.

of disulfide linkage connectivity, but do not represent an exclusive list of possible structures. Each of the three structures shown in Figure 1 contains differing connectivity between the Fab cysteines of H6a and L12 with the first two cysteine residues of the hinge peptides. In each case, the PITC coupling and cleavage cycle results in the generation of “leaving” and “residual” groups that confer elucidation of the disulfide connectivity of the initial nonreduced peptide. Indeed, the technique has the power to identify the presence of mixed populations in spite of the fact that the leaving groups are consistent between variants. For example, the L12 species is common to the leaving groups of both structures 2 and 3; however, the corresponding residual groups for each structure possess unique masses, affording detection and discrimination of multiple species simultaneously. Nonreduced peptides characteristic of each disulfide variant were analyzed through repetitive cycles of Edman MS to ascertain the specific connectivity associated with each, as described in the following sections. Characterization of the ∼15 kDa (IgG2-A/B) Variant. The IgG2-A/B variant, characterized by the ∼15 kDa nonreduced peptide following digestion with endoprotease Lys-C, contains two copies of the hinge (H11) peptide and a single copy of the Fab peptides (L12, H6, H7-8). The IgG2-A/B variant was analyzed by repetitive cycles of PITC coupling, cleavage, and LC-MS following secondary digestion with endoprotease Glu-C, and the LC-MS data were parsed for masses corresponding to potential leaving and residual groups, noted in Figure 1. Resulting EICs for leaving and residual groups are presented (panels A and B, respectively, of Figure 2). 1094

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Of particular note is the fact that signals corresponding to both the L12* and H6a* leaving groups are observed (Figure 2A). [Note that the number of asterisks in the peptide nomenclature corresponds to the number of cycles and therefore also to the number of amino acids truncated from the corresponding N-terminus of the peptide. Therefore, L12* is the L12 peptide following a single cycle, with concomitant removal of the native N-terminal serine residue.] This suggests that either (1) both L12 and H6a peptides are connected to Cys-224 of the two copies of H11 or (2) a mixture of structures is present or (3) both (1) and (2) are true. Examination of the EICs for the residual groups (Figure 2B) revealed a signal for two different speciessIgG2-A/B3 [H11-12*(s-s)H11*] and IgG2-A/B4 [H6a*(s-s)H11-12*(s-s)H11*(s-s)L12*]. This indicates the presence of a mixture of disulfide variants related to the ∼15 kDa species, the first where L12 and H6a are connected to Cys-224 of both H11 peptides (consistent with structure 3 in Figure 1) and the second where L12 and H6a are connected to Cys-225 of both H11 peptides (consistent with structure 1 in Figure 1). Although not a quantitative analysis, the relative proportions of H6a* and L12*, as well as the various characteristic residual groups, are indicative that structure 3 corresponds to IgG2-A/B3, i.e., the predominant variant for IgG2-A/B. Note that the labeling nomenclature used here (IgG2-A/B3 and IgG2-A/B4) is consistent with the fact that IgG2-A/B1 and IgG2-A/B2 have been used in the contemporary literature to describe other disulfide connectivities,19 and the structures determined in this study demonstrate distinct linkage patterns. Interestingly, leaving groups are sometimes characterized by the presence of a doublet of PTH-labeled species eluting in the

Figure 2. EICs of leaving and residual groups after the first cycle for the ∼15 kDa (IgG2-A/B) variant.

chromatogram following execution of the conversion step analogous to traditional Edman sequencing, as evidenced for the L12* leaving group (Figure 2A). Both species in the doublet presented identical masses and fragmentation patterns (data not shown). It is considered that these two species may be the result of racemization during the conversion step to the final PTH derivative.28 Analysis of the second cycle revealed the presence of a single residual group H11-12**(s-s)H11** (data not shown), confirming that the two structures elucidated from the first cycle data were differentiated by linkage patterns among L12, H6a, and the first two cysteine residues of the hinge peptide (H11). Given that structure 3 is the predominant variant for IgG2-A/ B, the major leaving group for the second cycle would be the labeled cystine group. This species is considered too small and hydrophilic to be retained by the RP-HPLC conditions used and therefore was not detected. However, analysis of the second cycle data for the L12** and H6a** leaving group of the minor species (IgG2-A/B4, structure 1) did not reveal the presence of these species. This is considered an issue related to the low relative proportion of structure 1 and the cumulative losses associated with repetitive cycling.18 For similar reasons the connectivity between the Cys-228 and Cys-231 residues in the remaining portion of the hinge-hinge dipeptides could not be determined; however, it is known that there are interchain linkages in the residual group from the second cycle as this species contained both hinge peptides, obviating intrachain disulfide bonds. The elucidated disulfide structures for IgG2-A/B are depicted in Figure 3. Note that the dashed lines reflect the expected linkage consistent with the classical hinge structure, and although it is known from these data that two interchain linkages exist, these connectivities have not been explicitly confirmed. Characterization of the ∼25 kDa (IgG2-B) Variant. The IgG2-B variant, characterized by the ∼25 kDa nonreduced peptide following digestion with endoprotease Lys-C, contains two copies of the hinge peptide (H11) and two copies of each of the Fab (28) Iida, T.; Matsunaga, H.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. J. Chromatogr., A 1998, 813, 267–275.

peptides (L12, H6, H7-8). After secondary digestion of peptide f with endoprotease Glu-C, the resulting nonreduced peptide was analyzed by multiple cycles of PITC coupling, cleavage, and LC-MS. The LC-MS data were parsed for masses corresponding to potential leaving and residual groups. Reconstructed EICs of the observed leaving and residual groups from the first cycle are shown (panels A and B, respectively, of Figure 4). Similar to the IgG2-A/B variant, data analysis confirmed the presence of two leaving groups (L12* and H6a*) as well as two residual groups for the IgG2-B variant. The observed residual groups (IgG2-B1*, mass 8483.6 Da, and IgG2-B2*, mass 7630.6 Da) correspond to subvariants of IgG2-B; the masses are consistent with species containing either two copies of the hinge peptide (H11* and H11-12*) bound to two copies of H6a* for IgG2-B1 or two copies of the hinge peptide (H11* and H11-12*) bound to a single copy each of L12* and H6a*. This suggests that the two subvariant species are differentiated by the connectivity between the Cys-224 of each hinge peptide. For IgG2-B1 the Cys-224 residue on both hinge peptides is connected to Cys-215 of L12 in a symmetrical arrangement, whereas for IgG2-B2 the residual group is consistent with the loss of single copies of both L12* and H6a*, suggesting that connectivity exists between Cys-224 of one hinge peptide and Cys-205 of L12 and between Cys-224 of the other hinge peptide and Cys-136 of H6a. No other residual groups were observed, indicating the absence of other subvariant species (data not shown). Upon analysis of the two coeluting residual groups following a second round of coupling and cleavage, it was found that the H6a** group was detected (Figure 5A). Although no signal was confirmed for the other leaving group (L12**), this was not unexpected due to L12** being characteristic of the minor variant (IgG2-B2) and the cumulative losses from multiple rounds of analysis. Furthermore, the relatively small size of the L12** species may compromise retention and detection using the RPHPLC separation. A single residual group was observed (Figure 5B). Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

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Figure 3. Elucidated Fab-Hinge disulfide connectivity for IgG2 structural variants.

Figure 4. EICs of leaving and residual groups after the first cycle for the ∼25 kDa (IgG2-B) variant.

The mass of the residual group (5125.7 Da) corresponds to H11**(s-s)H11-12**, i.e., the dimeric hinge-hinge peptide after loss of Cys-224 and Cys-225 from both peptides. Importantly, this illustrates the linkage between Fab and hinge peptides is limited to the first two cysteine residues only (Cys-224 and Cys-225) on both hinge peptides and therefore that interchain disulfide bonds exist between the two hinge peptides. Given the previously identified limitations of this technique for the cycling efficiency, data could not be obtained to ascertain the geometry of the 1096

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linkages between Cys-228 and Cys-231 of each hinge peptide; however, it is known that there are interchain linkages in the residual group from the second cycle as this species contained both hinge peptides, obviating intrachain disulfide bonds. Hence, overall the IgG2-B variant contains two distinct disulfide substructures characterized by the ∼25 kDa nonreduced Lys-C peptides, denoted IgG2-B1 and IgG-2B2, as illustrated in Figure 3. Note that the dashed lines reflect the expected linkage consistent with the classical hinge structure, and although it is known from these

Figure 5. EICs of leaving and residual groups after the second cycle for the ∼25 kDa (IgG2-B) variant.

Figure 6. EICs of leaving and residual groups after the first cycle for the ∼12 kDa variant.

data that two interchain linkages exist, these connectivities have not been explicitly confirmed. The connectivity of IgG2-B1 is also consistent with that elucidated for another rIgG2 molecule.19 Characterization of the ∼12 kDa Variant. This variant, characterized by the ∼12 kDa nonreduced peptide following digestion with endoprotease Lys-C, contains a single copy of the hinge (H11) and Fab peptides (L12, H6, H7-8). The smaller number of structural elements limits the number of possible arrangements; therefore, only two cycles were needed to afford complete elucidation of disulfide connectivity. The EICs for potential leaving and residual groups are shown (panels A and B, respectively, of Figure 6). Examination of the data indicated that only the L12* leaving group was observed, along with the associated residual group consisting of H11*(s-s)H6a* (m/z value 1354.9 for the 3+ ion). There was no evidence for the H6a* leaving group or the corresponding H11*(s-s)L12* residual group (Figure 6B). The data indicate that issues of disulfide scrambling are not apparent in this methodology, as previously noted.18

In the second cycle, EICs showed the detection of the H6a** leaving group and the H11** residual group (panels A and B, respectively, of Figure 7). No signal was observed for the L12** leaving group (the peak at ∼45 min in Figure 7A is an unrelated species, confirmed by MS/MS analysissdata not shown). Importantly, the H11** residual group corroborates the precept that the ∼12 kDa variant contains an intrachain disulfide linkage in the hinge peptide between Cys-228 and Cys-231. Therefore, the connectivity of the ∼12 kDa peptide characteristic of this disulfide variant is as depicted in Figure 3. It is noteworthy that this structure potentially constitutes only half of the antibody homodimer. A forthcoming paper will describe in more detail the structural nuances of this subvariant. Categorization of this species within the IgG2 subclasses is most appropriate as a subvariant of the IgG2-B variant; each intact monoclonal antibody of this variant structure contains two independent copies of the ∼12 kDa species. As each copy requires Fab to hinge linkage, this is most closely related to the IgG2-B structure, characterized by both light chains Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

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Figure 7. EICs of leaving and residual groups after the second cycle for the ∼12 kDa variant.

connected to the hinge. Therefore, the ∼12 kDa subvariant is termed IgG2-B3. CONCLUSIONS In this paper we present data using a novel methodology to conclusively elucidate the previously uncharacterized disulfide connectivities between Fab and hinge peptides among recently reported disulfide variants of recombinant IgG2 molecules.1,2 The analytical methodology utilized was a newly developed technique involving the coupling of traditional Edman N-terminal sequencing chemistry and LC-MS analysis of sequential cycling products.18 Prior analysis of recombinant insulin as a well-characterized test protein demonstrated that this powerful technique affords facile analysis and data interpretation and obviates the occurrence of disulfide scrambling and therefore affords a comprehensive assessment of the architecture of complex disulfide-linked peptides. Of the three IgG2 disulfide variants categorized (IgG2-A, IgG2A/B, and IgG2-B), two of the populations demonstrated the presence of multiple linkage geometries between the hinge and Fab regions (i.e., IgG2-A/B3 and IgG2-A/B4, and IgG2-B1, IgG2B2, and IgG2-B3). The proportions of each variant (Table S3, Supporting Information) demonstrated that the classical IgG2-A structure was not the predominant species for the studied rIgG2 antibody, consistent with earlier reports.1,19 For IgG2-B, two of the variants differ through the symmetry of the disulfide linkages. In IgG2-B1, the predominant species, the two copies of the L12 peptides are connected through their cysteines (Cys-215) to the first cysteine (Cys-224) of each hinge peptide, H11. The H6 cysteine (Cys-136) on both “sides” of the molecule is connected to the second hinge cysteine (Cys225), resulting in a symmetrical arrangement. In the minor subvariant, IgG2-B2, this symmetry is absent; one L12 peptide is bound through its cysteine residue (Cys-215) to the first cysteine (Cys-224) on one side of the hinge-hinge dipeptide, whereas the H6 cysteine (Cys-136) is connected to the first cysteine (Cys-224) on the other hinge peptide, H11. Similarly, the second hinge cysteine residues (Cys-225) are linked to both remaining L12 and H6. These linkage patterns are illustrated in Figure S4, Supporting Information. 1098

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IgG2-B3 is a newly reported species that is categorized as a subvariant of IgG2-B due to the fact that the proportions of its constituent peptides (Fab and hinge) are present in a 1:1 ratio and that the two light chains are connected to the hinge in an intact monoclonal antibody monomer. In IgG2-B3 the L12 cysteine (Cys-215) is coupled to the first hinge cysteine (Cys224) and the H6 cysteine (Cys-136) is linked to the second hinge cysteine (Cys-225). The absence of dimeric hinge-hinge structures in the IgG2-B3 subvariant requires the presence of intrachain linkage between the remaining hinge cysteines (Cys228 and Cys-231). The bonding structure for IgG2-B3 is presented in Figure S5, Supporting Information. In the case of IgG2-A/B3, the predominant species of the IgG2-A/B variant for this molecule, linkages were elucidated between the light chain L12 peptide (Cys-215) and the first cysteine residue (Cys-224) of one heavy chain hinge peptide, H11. Concomitantly linkage between heavy chain H6 peptide (Cys-136) and the first cysteine residue (Cys-224) of the other heavy chain hinge peptide was determined. For the minor subvariant, IgG2-A/B4, the linkage between L12 and H6 Fab peptides was determined to be to the second cysteine residue (Cys-225) of the H11 hinge peptides. These structures are depicted in Figure S6, Supporting Information. Detailed evaluation of the classical IgG2-A structure was not undertaken beyond confirmation of the connectivity of peptides A-G in the nonreduced Lys-C peptide map of unfractionated material; the presence of multiple nonreduced RP-HPLC fractions bearing the same apparent disulfide linkages (RP-4 and RP-5) suggests possible subtle differences between the two. Whether this is related to differential bonding in the hinge-hinge dipeptide (i.e., parallel “ladder” vs “crossed” linkages) or some other conformational disparity remains the subject of future investigation. As noted in the contemporary literature, the immunoglobulin G subclasses possess demonstrable differences in their physicochemical2,29 and biological30 properties. Furthermore, it has been reported that the IgG2 structural variants may potentially have (29) Roux, K. H.; Stretlets, L.; Michaelsen, T. E. J. Immunol. 1997, 159, 3372– 3382. (30) Jeffries, R.; Lund, J. Immunol. Lett. 2002, 82, 57–65.

discernible differences in their characteristic properties.2 In a regulatory environment, that is, engineered biotherapeutic molecules for human use, complete understanding of the molecular properties and their implication to potential synergistic interactions between physicochemical characteristics and biological function is a growing expectation for licensure and is fundamental to demonstrating appropriate knowledge of a therapeutic product. The data and methodologies presented in this paper provide a prospective approach for detailed characterization of the disulfide structures of rIgG2 molecules. The observation of multiple subvariants for particular IgG2 disulfide isoforms demonstrates the degree of characterization required for full characterization of a biotherapeutic product. Of interest for future evaluation will be the influence of cell line and/ or culture conditions on the distribution of the subvariant population or potentially even the presence of alternative disulfide architecture in rIgG2 molecules. Certainly ongoing investigations have demonstrated the ability to exert a degree of control over the heterogeneity of IgG2 disulfide variants through site-directed mutagenesis involving Cys f Ser mutations.31 At present the (31) Allen, M. J.; Guo, A.; Martinez, T.; Han, M.; Flynn, G. C.; Wypych, J.; Liu, Y. D.; Shen, W. D.; Dillon, T. M.; Vezina, C.; Balland, A. Biochemistry 2009, 48, 3755–3766.

implication of the presence of multiple subvariants with respect to biological properties remains to be fully understood. Additionally future considerations include improvements in the resolution of the nonreduced RP-HPLC separation to afford more accurate quantitation of the disulfide variants, as well as the confirmation of linkage specificity in the lower hinge region (Cys228 and Cys-231) for all disulfide variants. This may resolve the apparent differences between the two RP fractions (RP-4 and RP5) that demonstrate the classical IgG2-A disulfide structure. ACKNOWLEDGMENT We are particularly grateful to Edward Towers and Brent Kendrick for their insightful input into the early scientific development and thank Jette Wypych, Greg Flynn, Pavel Bondarenko, and Alain Balland, among others, for stimulating technical discussion. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 10, 2009.

October

29,

2009.

Accepted

AC902466Z

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