Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE

SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and β-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by β-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Free sulfhydryl in recombinant monoclonal antibodies

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other recombinant protein drugs, therapeutic mAb's need to be well characterized to ensure their structural and functional integrity. IgG mAb's are composed of two heavy and two light chains covalently linked by interchain disulfide bonds. Each domain of the heavy or light chain contains one additional disulfide bond. Native IgG mAb's, with completely formed disulfide bonds, should not bear any free sulfhydryl. This report describes detection and quantification of free sulfhydryl in recombinant mAb's produced in Chinese hamster ovary (CHO) cells using a fluorescent technique. The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide (NPM). The purified mAb's appear to be homogeneous under native conditions with approximately 0.02 mol of free sulfhydryl per mole of protein. Upon denaturation, minor species related to the mAb's are observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the free sulfhydryl level is determined to be approximately 0.1 mol/mol of protein. These results suggest that a small portion of these recombinant mAb's lack in intermolecular disulfide bonds but remain noncovalently associated under native conditions. The formation of the free sulfhydryl containing mAb species is likely to occur during the culture process and/or protein folding process in the endoplasmic reticulum (ER).     

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography–mass spectrometry

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography–mass spectrometry (LC–MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.     

Chip-based gel electrophoresis method for the quantification of half-antibody species in IgG4 and their by- and degradation products

The inter-heavy-chain disulfide bonds of the IgG4 subclass can be described as being at equilibrium with the intra-chain disulfide bonds. This means that a fraction of IgG4 has noncovalently linked heavy chains (half-antibody). The percentage of half-antibodies produced depends upon the expression system used. Nondenaturing assays fail to separate the half-antibodies from the native form because two half-molecules are held together by noncovalent forces. The pharmaceutical industry needs a reliable denaturing assay for checking batch-to-batch consistency. Until now sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the standard method used to detect the presence of half-antibodies. However, this technique is laborious and cannot be automated. Furthermore, cumbersome densitometric measurements are necessary for quantification. To overcome these disadvantages a chip-based gel electrophoresis method was investigated. In the nonreduced format the separation profile is compared with that from SDS-PAGE. The limit of quantification as a percentage of the amount applied, repeatability, reproducibility, and linearity are compared with those of SDS-PAGE. The amounts of half-antibody and of by- and degradation products were determined for several batches by using area percentage and by external calibration with IgG4 as a reference standard. Both methods allow avoidance of error introduction for the quantification as is the case by application of myosin as reference concentration. Both sets of results are compared with each other and with the results from SDS-PAGE. In the reduced format it is noted that the reduction of the inter-heavy-chain disulfide bridges proceeds faster than the reduction of the heavy-light-chain bonds. Therefore optimized conditions are necessary to obtain a complete reduction.     

Evidence of disulfide bond scrambling during production of an antibody-drug conjugate

ABSTRACT

Antibody-drug conjugates (ADCs) that are formed using thiol-maleimide chemistry are commonly produced by reactions that occur at or above neutral pHs. Alkaline environments can promote disulfide bond scrambling, and may result in the reconfiguration of interchain disulfide bonds in IgG antibodies, particularly in the IgG2 and IgG4 subclasses. IgG2-A and IgG2-B antibodies generated under basic conditions yielded ADCs with comparable average drug-to-antibody ratios and conjugate distributions. In contrast, the antibody disulfide configuration affected the distribution of ADCs generated under acidic conditions. The similarities of the ADCs derived from alkaline reactions were attributed to the scrambling of interchain disulfide bonds during the partial reduction step, where conversion of the IgG2-A isoform to the IgG2-B isoform was favored.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis

ABSTRACT

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.     

Human IgG2 antibodies display disulfide-mediated structural isoforms

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.     

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.     

Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V_H domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V_H domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Effect of the light chain C-terminal serine residue on disulfide bond susceptibility of human immunoglobulin G1λ

The light chain cysteine residue that forms an interchain disulfide bond with the cysteine residue in the heavy chain in IgG1κ is the last amino acid. The cysteine residue is followed by a serine residue in IgG1λ. Effect of the serine residue on the susceptibility of disulfide bonds to reduction was investigated in the current study using a method including reduction, differential alkylation using iodoacetic acid with either natural isotopes or enriched with carbon-13, and mass spectrometry analysis. This newly developed method allowed an accurate determination of the susceptibility of disulfide bonds in IgG antibodies. The effect of the serine residue on disulfide bond susceptibility was compared using three antibodies with differences only in the light chain last amino acid, which was either a serine residue, an alanine residue or deleted. The results demonstrated that the presence of the amino acid (serine or alanine) increased the susceptibility of the inter light and heavy chain disulfide bonds to reduction. On the other hand, susceptibility of the two inter heavy chain disulfide bonds and intrachain disulfide bonds was not changed significantly.     

The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region: IMPLICATIONS FOR FUNCTIONAL ACTIVITY*

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser²²²) and a hinge mutant IgG4(Pro²²²) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s_20,w⁰ of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration R_g was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron R_g values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser²²²) with extended hinge structures. The IgG4(Pro²²²) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.     

Disulfide bond structures of IgG molecules: Structural variations,chemical modifications and possible impacts to stability and biological function

The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG₄ and later in IgG₂ antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through β-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.Key words: recombinant monoclonal antibody, disulfide bond, trisulfide bond, free sulfhydryl, dehydroalanine, thioether, aggregation     

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post‐translational insertion of a sulfur atom into disulfide bonds at the heavy–heavy and heavy–light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run‐to‐run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench‐scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. Biotechnol. Bioeng. 2012; 109: 2523–2532. © 2012 Wiley Periodicals, Inc.     

Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H₂O₂ for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.     

Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.     

A new tool for monoclonal antibody analysis

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')₂ and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.