Disulfide bond characterization of endogenous IgG3 monoclonal antibodies using LC-MS: an investigation of IgG3 disulfide-mediated isoforms

Abstract

The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years. From a structural perspective, mAbs have high disulfide bond content, and the correct disulfide connectivity is required for proper folding and to maintain their biological activity. Therefore, disulfide linkage mapping is an important component of mAB characterization for ensuring drug safety and efficacy. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been well established since the late 1960s. Among these IgG subtypes, disulfide mediated isoforms have been identified for IgG2 and IgG4, and to a lesser extent in IgG1, which is the most studied IgG subclass. However, no studies have been carried out so far to investigate whether different IgG3 isoforms exist due to alternative disulfide connectivity. In an effort to investigate the presence of disulfide-mediated isoforms in IgG3, we employed a bottom-up mass spectrometry approach to accurately determine the disulfide bond linkages in endogenous human IgG3 monoclonal antibody and our results show that no such alternative disulfide bonds exist. While many antibody-based drugs are developed around IgG1, IgG3 represents a new, and in some cases, more desirable drug candidate. Our data represent the first demonstration that alternative disulfide bond arrangements are not present in endogenous IgG3; and therefore, they should not be present in recombinant forms used as antibody-based therapeutics.

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

“…The sample preparation workflow for the non-reduced approach, which is the most commonly used method, is shown in Figure 2. This approach requires alkylation of any free Cys residues, deglycosylation (for glycoproteins, and only if necessary; see Section 2.4 below for more details), proteolytic digestion of the protein without disulfide bond reduction, and the disulfide linked peptides are investigated to decipher the disulfide connectivity in the protein [52–54]. For the intact/reduced approach, two batches of enzymatically digested samples are prepared, one with the disulfide bonds intact (same as the previous approach) and the other with reduced disulfide bonds.…”

“…The widely-used separation technique is reverse phase liquid chromatography (RPLC) by means of columns packed with either C 8 or C 18 stationary phases and mobile phases consisting of polar (e.g water) and non-polar (e.g acetonitrile) solvents containing modifiers such as formic acid and trifluoroacetic acid (0.01 to 0.1%, v/v). Different types of columns can be used for peptide separation, including microbore (e.g 1.0 and 2.1 mm internal diameter, i.d) [54, 79], capillary (e.g 0.5 mm i.d) [80] and nano columns (typically 0.075 mm i.d) [65]. The typical particle size is between 3 to 5 μm.…”

“…Collision induced dissociation (CID), where a neutral reagent gas collides with the analyte ion, and electron transfer dissociation (ETD), where an electron carrier reacts the ion of interest, are the most commonly used fragmentation techniques for mass spectrometry-based disulfide bond analysis [54, 80, 81]. Figure 4 shows CID and ETD spectra of a simple interchain disulfide-bonded peptide from IgG3 monoclonal antibody that exhibits the characteristic fragment ion peaks that result from subjecting disulfide-bonded peptides to CID (Figure 4A) and ETD (Figure 4B) fragmentation.…”

“…Backbone (amide) cleavage also occurs, by generation of c and z ions, although this fragmentation pathway generally occurs at a lesser extent [83, 84]. As a result, fragment peaks representing the disulfide-linked peptides (e.g peaks at m/z 250.4 and 1179.3 in Figure 4B) are usually more intense in ETD spectra than c/z ion peaks [53, 54, 83]. Peaks resulting from backbone fragmentation of the bonded peptides may or may not contain the intact disulfide bond (fragment ions labeled in red and green in Figure 4B).…”

“…CID is preferable in such cases because small disulfide bonded peptides may not ionize at high charge states, which are required for ETD analysis, and peptide chains containing adjacent proline residues fragment efficiently when subjected to CID but not ETD fragmentation, due to the N-C α ring structure of proline [94]. For example, the hinge region of IgG3 contains a small dipeptide, CPEPK linked to CPEPK, and SCDTPPPCPR disulfide-linked to SCDTPPPCPR and a recent analysis of IgG3 disulfide bonds showed that these peptide chains did not fragment well under ETD because of the small size (for the first disulfide-linked peptide) and several adjacent proline residues on the peptide chains (for both disulfide-linked peptides), but they were unambiguously assigned using CID data [54]. Hence, in addition to the size of a disulfide-linked peptide, the amino acid sequences of the Cys-containing peptides could also be a factor in selecting an efficient fragmentation technique for disulfide analysis.…”