Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry

Abstract

The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

“…Native MS utilizes 100% aqueous solutions of low-to-moderate concentrations of volatile salt (e.g., 50 mM ammonium acetate) buffered at neutral pH. 3,4 This type of analysis proceeds commonly after removal of non-volatile salts by buffer exchange followed by nanospray infusion MS. 5–7 Native MS-based intact mass analysis is required for certain classes of biotherapeutics that require preservation of non-covalent associations of protein-protein or protein-ligand complexes. 8 Native MS also affords a fundamental benefit to intact mass analysis of all types of heterogeneous samples where proteins acquire fewer charges and yield spectra at high m/z relative to denatured conditions.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…A mAb may possess glycosylation at both CH2 regions, at one of two CH2 regions, or be fully aglycosylated. Figure reprinted from [19] with permission.…”

“…Immunoglobulin G (IgG) antibodies have been the predominant type of mAbs employed as therapeutic proteins [13]. IgGs are composed of aggregated sets of two identical light and heavy chains linked by combinations of disulfide bonds and/or other non-covalent interactions, resulting in a ~150 kDa protein.…”

“…A common mAb PTM is Nglycosylation at Asn 297 of the CH2 region, resulting in considerable proteoform heterogeneity from the combination of glycans at the two glycosylation sites [14]. Other PTMs, further increasing proteoform heterogeneity, include Met 1 -loss, formation of pyroglutamic acid, Cterminal lysine clipping, carboxymethylation of lysine, oxidation, deamidation, disulfide bond formation, glycation, single site amino acid replacement, and sequence truncation, amongst others [13,[15][16][17][18] . .…”

“…However, the bottom-up approach may lead to sample preparation modifications and, importantly, precludes the ability to identify individual proteoforms, often consisting of combinations of multiple modifications [44,264]. Thus, methods for intact and middle-down protein analysis are being developed [19,265].…”