Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

In the accompanying report (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., and Balland, A. (2008) J. Biol. Chem. 283, 16194–16205), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1β ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 ≫ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2λ composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a “blood-like” environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms. In the accompanying report (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., and Balland, A. (2008) J. Biol. Chem. 283, 16194–16205), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1β ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 ≫ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2λ composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a “blood-like” environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms. Recombinant monoclonal antibodies, typically human or humanized, are used as protein-based therapeutic agents because of their high degree of specificity and the ability to alter their functional properties when desired. In vivo, therapeutic antibodies can function via several mechanisms, including as antagonists that compete with ligand binding and interfere with receptor response, as signaling molecules that elicit a response in the target cells (e.g. apoptosis), or as agents that target specific cells populations (1Brekke O.H. Sandlie I. Nat. Rev. Drug Discov. 2003; 2: 52-62Crossref PubMed Scopus (445) Google Scholar). The latter mechanism may involve attaching an effector moiety (e.g. enzymes, toxins, and radionuclides) to the antibody or using the antibody's natural effector functions, which are mediated through the immunoglobulin Fc domain. These natural functions include antibody-dependent cellular cytotoxicity and activation of the complement cascade, leading to complement-dependent cytotoxicity. Effector functions have been shown to be dependent on the immunoglobulin γ (IgG) subclass affinity for Fc receptors (IgG1 > IgG3 > IgG4 > IgG2) (2Burton D. Mol. Immunol. 1985; 22: 161-206Crossref PubMed Scopus (366) Google Scholar, 3Canfield S.M. Morrison S.L. J. Exp. Med. 1991; 173: 1483-1491Crossref PubMed Scopus (252) Google Scholar), and this feature serves as a common determinant for therapeutic use. The human IgG2 subclass in particular has emerged as an attractive framework for therapeutic antibodies in clinical applications for which effector functions are undesirable or unnecessary for therapeutic activity (4Beenhouwer D.O. Yoo E.M. Lai C.W. Rocha M.A. Morrison S.L. Infect. Immun. 2007; 75: 1424-1435Crossref PubMed Scopus (65) Google Scholar, 5Jefferis R. Expert Opin. Biol. Ther. 2007; 7: 1401-1413Crossref PubMed Scopus (250) Google Scholar). The increased prevalence of therapeutic IgGs has led to a renewed interest in understanding antibody structure and its relationship to biological function. Structural heterogeneity in proteins can result from genetic differences or from many common post-translational modifications, such as glycosylation, protein folding, disulfide bond formation, and chemical modifications to amino acid side chains or the peptide backbone (6Liu, H., Gaza-Bulseco, G., Faldu, D., Chumsae, C., and Sun, J. (2008) J. Pharm. Sci., in pressGoogle Scholar). For example, structural changes caused by glycan variants have been shown to impact antigen binding and antibody effector functions (7Matsumiya S. Yamaguchi Y. Saito J. Nagano M. Sasakawa H. Otaki S. Satoh M. Shitara K. Kato K. J. Mol. Biol. 2007; 368: 767-779Crossref PubMed Scopus (235) Google Scholar, 8Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. Annu. Rev. Immunol. 2007; 25: 21-50Crossref PubMed Scopus (1008) Google Scholar, 9Walsh G. Jefferis R. Nat. Biotechnol. 2006; 24: 1241-1252Crossref PubMed Scopus (748) Google Scholar, 10Harris R.J. Dev. Biol. (Basel). 2005; 122: 117-127PubMed Google Scholar). Other examples demonstrate how cysteinylation of cysteines and incomplete disulfide bond formation in antibodies can interfere with antigen recognition and ultimately lead to reduced binding or inactivity (11Banks 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. 2008; 97: 775-790Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Chaderjian W.B. Chin E.T. Harris R.J. Etcheverry T.M. Biotechnol. Prog. 2005; 21: 550-553Crossref PubMed Scopus (100) Google Scholar). Disulfide heterogeneity of human IgG4 molecules represents a clear example of how unstable disulfide bonds can disrupt the structural integrity of an antibody, generating half-molecule forms. In this case, the half-molecule IgG4 is still capable of specific binding, although in a diminished capacity due to the loss of multivalent binding. Disulfide bond formation is a post-translational process that can affect the structure and function of proteins. Incomplete or incorrect disulfide bonds have the potential to generate improperly folded proteins. Although disulfide heterogeneity is less common in mammalian expression systems possessing the proper intra-cellular redox environment and post-translational machinery for protein folding, incomplete or improper disulfide bond formation of bacterially expressed mammalian proteins is commonly observed. Restoring native disulfide bonds in these proteins (often produced as inclusion bodies or soluble aggregates) has typically been accomplished by solubilization in the presence of a high concentration chaotropic agent, typically 6 m guanidine hydrochloride (GuHCl), followed by exposure to redox agents while slowly decreasing the concentration of the chaotrope (13Lilie H. Schwarz E. Rudolph R. Curr. Opin. Biotechnol. 1998; 9: 497-501Crossref PubMed Scopus (396) Google Scholar). Additionally, redox procedures without chaotropic agents have been used for Fc fusion proteins and antibodies produced in mammalian cells (12Chaderjian W.B. Chin E.T. Harris R.J. Etcheverry T.M. Biotechnol. Prog. 2005; 21: 550-553Crossref PubMed Scopus (100) Google Scholar, 14Sassenfeld, H. M., Remmele, R. L., and McCoy, R. E. (Sep. 6, 2002) World Patent Cooperation Treaty, Patent Application WO 02/068455A3Google Scholar), primarily to modify the disulfide structure without denaturing the proteins and improve binding to their targets. In the companion report (15Wypych 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: 16194-16205Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), we describe the existence of multiple disulfide isoforms of human IgG2 antibodies that can be partially resolved by cation exchange, capillary electrophoresis, and reversed-phase chromatography. Three discrete isoforms were identified, each having different disulfide linkages between the light chain (LC) 3The abbreviations used are: LC, light chain; HC, heavy chain; mAb, monoclonal antibody; RP-HPLC, reversed-phase high performance liquid chromatography; ESI, electrospray ionization; IL-1RI, interleukin-1 cell surface receptor type 1; IL-1β, interleukin-1β; GuHCl, guanidine hydrochloride; CEX, cation exchange; CH1, constant region one of heavy chain; SEC, size-exclusion chromatography; Fc, fragment crystallizable; PBS, phosphate-buffered saline. and heavy chain (HC), as detailed by nonreduced peptide mapping. In the first part, thorough covalent characterization of the IgG2 disulfide isoforms has been presented. In this study, we describe a redox procedure for enrichment of the IgG2 disulfide isoforms utilizing a relatively low concentration of GuHCl. We investigated the individual biophysical properties of the human IgG2 isoforms, their potency, and the mechanism of disulfide conversion. Materials—Recombinant human monoclonal IgG antibodies were produced from Chinese hamster ovary cells and purified using well established purification protocols (16Shukla A.A. Hubbard B. Tressel T. Guhan S. Low D.J. J. Chromatogr. B. 2007; 848: 28-39Crossref PubMed Scopus (699) Google Scholar) at Amgen. The purified proteins were stored in a formulation buffer at 4 °C. Unless otherwise noted, the same representative mAb expressed as an IgG1κ versus IgG2κ construct was used throughout this study. Antibodies purified from human plasma were purchased from Sigma and ICN biochemicals. The antibodies were purified from human plasma using well established purification protocols, which generally include acid precipitation of non-IgG proteins, ammonium sulfate precipitation, and affinity chromatography. Reversed-phase Chromatography—An Agilent 1100 HPLC system with a binary pump was equipped with a UV detector and an autosampler. The proteins were injected onto a Zorbax 300SB C8 column (150 × 2.1 mm, 5 μm, 300 Å) operated at 75 °C. The flow rate was 0.5 ml/min. Mobile-phase A was water containing 0.1% trifluoroacetic acid. Mobile-phase B was 70% isopropyl alcohol, 20% acetonitrile, and aqueous 0.1% trifluoroacetic acid. Samples were injected at a loading condition of 10% B and increased to 19% B over 2 min. A linear elution gradient of 1.1% B/min started at 2 min and ended at 24 min. The column was then flushed for 5 min with 95% B. The column was reequilibrated with the loading condition for 5 min. Size-exclusion Chromatography—An Agilent 1100 HPLC system with a binary pump was equipped with a UV detector and an autosampler. The proteins were injected onto two Tosoh Bioscience TSK-Gel G3000 SWxl columns in series (7.8 mm × 300 mm, 5-μm particles) operated at 25 °C. The flow rate was 0.5 ml/min. The mobile phase contained 100 mm sodium phosphate, 500 mm sodium chloride, 5% ethanol, pH 7.0. Sedimentation Velocity Analytical Ultracentrifugation—The solution properties of the IgG2 isoforms were evaluated by sedimentation velocity measurements conducted with a temperature-controlled Beckman XL-I analytical ultracentrifuge equipped with an An-60 Ti rotor and a photoelectric scanner (Beckman Instruments, Palo Alto, CA). Samples were diluted to 0.5 mg/ml in the desired buffer (see Fig. 6). Protein samples were loaded in a double sector cell equipped with a 12-mm Epon centerpiece and a sapphire optical window. The reference compartment was loaded with the matching buffer. The samples were monitored at 280 nm at a rotor speed of 40,000 rpm at 20 °C. Analyses of the raw data were completed using Sedfit v. 8.9 to obtain concentration distribution (c(s)) as a function of sedimentation coefficient. A confidence level of 0.7 (1 standard deviation) was used for the analysis. Meniscus and frictional ratio (f/f0) were fitted. Other fitting parameters used were default parameters suggested by Sedfit software. Replicate runs showed standard deviation of average sedimentation coefficients of <0.01 s. Partial specific volume for each antibody, buffer density, and buffer viscosity were calculated using Sednterp (v. 1.08) and used for hydrodynamic radii calculations. Differential Scanning Calorimetry—Samples were analyzed using a MicroCal VP-Capillary differential scanning calorimeter system. All samples were diluted to 1 mg/ml in acetate buffer at pH 5. A scan rate of 1 °C/min was used. Redox Treatment—To enrich for IgG2-B and IgG2-A, the antibodies were incubated at 3 mg/ml in two buffers: 1) 200 mm Tris buffer at pH 8.0; 2) 200 mm Tris buffer at pH 8.0 with 0.9 m GuHCl. A combination of cysteine and cystamine was added at concentrations of 6 mm to 1 mm. The ratios of redox reagents were determined by testing multiple conditions and monitoring for optimal IgG2 isoform conversion by reversed-phase chromatography. Several redox reagents listed in a previous study (14Sassenfeld, H. M., Remmele, R. L., and McCoy, R. E. (Sep. 6, 2002) World Patent Cooperation Treaty, Patent Application WO 02/068455A3Google Scholar) and their ratios were evaluated before choosing the above conditions. The samples were protected from light and placed at 2-8 °C for 24-48 h. Chondrocyte Bioassay—The anti-IL-1RI IgG samples were serially diluted from 400 nm to 1.5 pm in assay media. The diluted test antibodies (50 μl) were added to the wells of 96-well plates seeded with human chondrocytes at a density of 10,000 cells/well in a 100-μl volume. The final antibody concentration ranged from 100 nm to 0.38 pm. After a 30-min incubation, 50 μl of recombinant human IL-1β was added to a final concentration of 10 pm. After incubation overnight, the antibody activities were analyzed using an IL-6 immunoassay with electrochemiluminescence detection (Meso Scale Discovery, Gaithersburg, MD). The inhibition of IL-6 production was calculated as a percentage of maximum IL-1β activity. The inhibition-response curve for each test antibody was established, and the corresponding IC50 values (the concentration of antibody which reduces the signal by 50%) were derived using GraphPad Prism software. IL-1β-induced IL-6 Human Whole Blood Bioassay—The anti-IL-1RI IgG samples were evaluated in 50% human whole blood (final concentration) from 10 nm to 0.30 pm in half-log increments for a 10-point IC50 curve. After a 45-min preincubation with anti-IL-1RI IgG samples, the blood was stimulated with recombinant IL-1β for a final concentration of 30 pm (∼IC50). After incubation overnight, the antibody activities were analyzed using an IL-6 immunoassay with electrochemiluminescence detection (Meso Scale Discovery). The inhibition of IL-6 production was calculated as a percentage of maximum IL-1β activity. IC50 values were calculated using six separate donors (three donors on two different days). Statistical Analysis—Bioassay data were analyzed using GraphPad Prism. IC50 values were derived by nonlinear regression (variable slope). Where dose-response curves were incomplete, the bottom was constrained to zero. Significance was calculated using a one-way analysis of variance with a Tukey multiple comparison post-test. A p value <0.05 was considered significant. Crystal Structure Analysis—Display and measurements were made using the DeepView/Swiss-Pdb-Viewer version 3.7. Structural Heterogeneity of Human IgG2 and Homogeneity of IgG1 by RP-HPLC—RP-HPLC coupled with mass spectrometry has emerged as an effective analytical tool for characterizing and monitoring structural heterogeneity of antibodies (17Dillon T.M. Bondarenko P.V. Ricci M.S. J. Chromatogr. A. 2004; 1053: 299-305Crossref PubMed Google Scholar, 18Dillon T.M. Bondarenko P.V. Rehder D.S. Pipes G.D. Kleemann G.R. Ricci M.S. J. Chromatogr. A. 2006; 1120: 112-120Crossref PubMed Scopus (156) Google Scholar). In this study, several human IgG1 (Fig. 1, k-q) and IgG2 (Fig. 1, a-j) antibodies were analyzed generating the RP-HPLC profiles shown in Fig. 1. The IgG2 molecules consistently produced heterogeneous profiles for both monoclonal (Fig. 1, a-f) and endogenous antibodies purified from human serum (Fig. 1, g-j), whereas those of IgG1 were homogeneous. Furthermore, IgG2 antibodies containing kappa (κ) and lambda (λ) LC differed in their RP-HPLC profile. Both antibodies displayed a characteristic four-peak profile but displayed a different abundance of the earliest eluting peak. These results clearly show the inherent differences between the IgG1 and IgG2 subclass in addition to subtle variations among the LC type of IgG2. To further study the effects of this structural phenomenon, monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1 (IL-1RI) (supplemental Fig. S1). The most significant differences between the human IgG1 and IgG2 subclasses are the primary structure of the hinge and two serine to cysteine substitutions (Ser → Cys) in IgG2 (Table 1). The IgG2 upper hinge sequence is three amino acids shorter than that of IgG1 and contains a cysteine substitution at position 219. Additionally, the CH1 loop of the IgG2 HC has a cysteine substitution at position 131 that is available for disulfide bonding to the LC. Although the overall IgG1 and IgG2 amino acid sequences are 95% identical, the proteins exhibited significant structural differences, as determined by the RP-HPLC analysis (Fig. 2). As shown in the accompanying report, the heterogeneity in the RP-HPLC profile was a result of disulfide linkage differences in the IgG2s (15Wypych 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: 16194-16205Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Therefore, as expected, high resolution mass spectral analysis in-line with RP-HPLC found the calculated molecular weight values of the four chromatographic peaks to be identical (data not shown) (18Dillon T.M. Bondarenko P.V. Rehder D.S. Pipes G.D. Kleemann G.R. Ricci M.S. J. Chromatogr. A. 2006; 1120: 112-120Crossref PubMed Scopus (156) Google Scholar). Interestingly, the individual RP-HPLC peaks displayed unique mass spectral features, suggesting underlying structural differences. We measured the number of protons on the surface of each of the RP-HPLC peaks to be different (supplemental Fig. S2), according to the method initially proposed by Chait and coworkers (19Chowdhury S.K. Katta V. Chait B.T. J. Am. Chem. Soc. 1990; 112: 9012-9013Crossref Scopus (603) Google Scholar). The results indicated that peak 1 had decreased solvent exposure and a more compact structure while peaks 2-4 showed an increased solvent exposure with a larger number of protons on the antibody surface and a more open structure.TABLE 1Human IgG1 and IgG2 sequence alignments of the CH1 loop and upper hinge of the HCEu #128129130131132133134135136137138IgG1LeuAlaProSerSerLysSerThrSerGlyGlyIgG2LeuAlaProCysSerArgSerThrSerGluSerEu #219220221222223224225226227228229IgG1SerCysAspLysThrHisThrCysProProCysIgG2CysCysValGluCysProProCys Open table in a new tab Biological Activity of IgG1 and IgG2 Anti-IL1-R1—IgG1 and IgG2 constructs were designed to compete with the IL-1β ligand, thereby inhibiting IL-1-mediated cellular events, including the production of IL-6 (20Guerne P.A. Carson D.A. Lotz M. J. Immunol. 1990; 144: 499-505PubMed Google Scholar). IL-1RI is present on the surface of many cell types, including endothelial cells, fibroblasts, T cells, and chondrocytes. In response to IL-1, chondrocytes switch from synthesizing cartilage matrix molecules such as collagens and proteoglycans to releasing matrix degrading molecules like matrix metalloproteinases, leading to cartilage degradation (21McInnes I.B. Schett G. Nat. Rev. Immunol. 2007; 7: 429-442Crossref PubMed Scopus (1912) Google Scholar). Biological activity of the anti-IL-1RI IgG1 and IgG2 mAbs were assessed by monitoring the inhibition of IL-1β-induced IL-6 production by primary human chondrocytes. The results of the bioassays showed statistically significant differences in IL-6 inhibition (Fig. 3) with the IgG2 having approximately one-third of the IgG1 potency. This raised the question of whether the lower IgG2 mAb potency was due to the disulfide heterogeneity, as observed by RP-HPLC. Ideally, the RP-HPLC peaks would be collected and tested for potency, but because the RP-HPLC assay utilizes denaturing conditions (high temperature, organic solvent, and low pH), the collected fractions would not retain native structure and function. Therefore, a different assay using nondenaturing conditions was needed. Fractionation of the disulfide heterogeneity was attempted by cation exchange (CEX) chromatography, which has been shown to partially resolve IgG2 disulfide isoforms (15Wypych 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: 16194-16205Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). When the collected CEX fractions were analyzed by RP-HPLC, they showed low purity (<50%) of the isoforms. However, differences in activity were observed that correlated with differences in the disulfide isoforms. The normalized potencies (IC50) of CEX fractions ranged from 70 ± 13% to 125 ± 12% (data not shown). Although not definitive, these results indicated that this naturally occurring structural heterogeneity affects the bioactivity of the antibody. To confirm these results, a more efficient enrichment technique was sought. Reduction-oxidation (Redox) Treatment of IgG2—Redox treatment of proteins from the inclusion body state is a common practice in the microbial production of recombinant proteins but is not typically implemented in mammalian cell production. However, because the IgG2 isoforms were discovered to be disulfide-mediated, the reactivity of the isoforms to redox treatment was tested. In the example shown, the anti-IL-1RI IgG2 mAb was subjected to a variety of redox conditions in the presence and absence of GuHCl. By changing the redox treatment conditions, either RP-HPLC peak 1 or peak 3 could preferentially be enriched (Fig. 4). Peak 1 was redox-enriched without using GuHCl, and peak 3 was enriched using ∼1 m GuHCl, a concentration well below that known to affect overall secondary or tertiary structure of the antibody (11Banks 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. 2008; 97: 775-790Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Teerinen T. Valjakka J. Rouvinen J. Takkinen K. J. Mol. Biol. 2006; 361: 687-697Crossref PubMed Scopus (25) Google Scholar). The major RP-HPLC peaks in the redox-treated materials eluted at approximately the same retention times as peaks in the untreated (no redox) IgG2 control material. Full characterization of IgG2 heterogeneity by RP-HPLC has been previously completed by performing nonreduced peptide mapping on collected RP-HPLC fractions (15Wypych 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: 16194-16205Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). That characterization showed that the RP-HPLC method was able to resolve three unique disulfide structures in the elution order of IgG2-B (peak 1), IgG2-A/B (peak 2), and IgG2-A (peaks 3-4). The RP-HPLC elution profiles suggested that the two redox procedures preferentially enrich for the newly discovered IgG2 isoforms (IgG2-A and IgG2-B). Additionally, several other IgG2 mAbs with alternative antigen targets were redox-treated using similar conditions (supplemental Fig. S3). These results showed that all IgG2 mAbs tested were reactive to redox treatment. The redox-enriched isoforms showed no further conversion after being removed from the redox solution and stored in a PBS solution. To confirm the identity of the redox-enriched IgG2 isoforms, nonreduced peptide mapping was performed on the redoxtreated material and IgG2 control material (supplemental Fig. S4) using the earlier described protocols for nonreduced peptide mapping and mass spectrometry identification. The analysis confirmed that the redox procedures were able to enrich for two of the IgG2 isoforms (IgG2-A and IgG2-B) that were previously shown to exist in the IgG2 control material (15Wypych 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: 16194-16205Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Therefore, redox treatment of the IgG2 antibodies did not introduce new disulfide forms but effectively populated structural isoforms already present within the control material. Based on the above results, the IgG2 disulfide isoforms were classified into three predominant structures, IgG2-A, -B, and -A/B (Fig. 5), defined by their disulfide connectivity. The structures differed by their interchain disulfide attachment of LC to the CH1 region or to the hinge. Two of the isoforms (IgG2-A and IgG2-B) contained symmetrical interchain disulfide connectivity in that the same light-to-heavy chain (LC-HC) attachment was present on both sides of the molecule (Fig. 5). In the third isoform (IgG2-A/B), one light chain was connected to CH1, whereas the other was connected to the hinge, which made it an asymmetrical disulfide structure. The IgG2-A/B disulfide form was an intermediate of the A and B forms. Biophysical Characterization of Redox-enriched Isoforms—The structural and thermodynamic properties of the redox-enriched isoforms (IgG2-A and IgG2-B) were compared using a series of biophysical assays. These tests included far UV CD, size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation, and differential scanning calorimetry. Far-UV CD analysis was used to assess the overall secondary structure of the antibody. Comparison of the individual CD spectra for each of the enriched IgG2 isoforms and IgG2 control material showed no significant difference in secondary structure (data not shown). SEC analysis, which separates based on hydrodynamic radius, was used to assess the apparent molecular sizes of the IgG2 isoforms. The enriched isoforms and the control material generated similar peak shapes but eluted at slightly different retention times (Fig. 6a). The enriched IgG2-A eluted earlier than IgG2-B, with the untreated sample, containing a mixture of the isoforms, eluting at an intermediate time. These results suggested that IgG2-A had a larger apparent molecular size than IgG2-B. An alternate explanation of the retention time shift is that the isoforms were differentially interacting with the column matrix due to differences in solvent exposed surface area or hydrophobicity. Sedimentation velocity analytical ultracentrifugation was therefore used as an orthogonal technique to investigate the hydrodynamic properties of the disulfide isoforms. Similar to the SEC data, the sedimentation coefficient distribution was unique for each of the IgG2 isoforms analyzed in various buffers (Fig. 6b). The enriched IgG2-B isoform consistently showed a greater sedimentation coefficient (s) value than IgG2-A, supporting the SEC assessment of a more compact conformation for IgG2-B. In addition, the thermal stability of IgG2-A and IgG2-B was tested using differential scanning calor