The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

Abstract

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

“…We denote these two species by the symbols pEpE MAb and QpE MAb respectively. These two species can be differentiated by CEX HPLC (5,17). We have measured (Table S1 in SI Appendix) molar ratios of QpE MAb to pEpE MAb, x, in both coexisting phases as well as in the original solutions.…”

“…Recently, LLPS of several pharmaceutical antibodies have been reported (5)(6)(7)(8)(9). There are five isotypes of mammalian antibodies with distinct Fc regions, including IgA, IgD, IgE, IgG, and IgM.…”

“…Here we report the study of the LLPS of a monoclonal human antibody, which is denoted by IgG2-A as in ref. 5, under solution conditions mimicking those in a blood serum. Specifically, we investigated LLPS at physiological pH (pH ¼ 7.4) in the presence of human serum albumin (HSA), which is the major protein component in blood serum.…”

Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

“…For example, it has been proposed that the C H 2 domain can trigger IgG aggregation at low pH [48], which is likely caused by reduced conformational stability of the Fc C H 2 region as compared with other domains in IgG1 molecules under stress conditions [45]. Recently, Arora et al [28] used HDX-MS to characterize the protein interfaces involved in the reversible self-association of an IgG1 mAb.…”

Pathological crystallization of human immunoglobulins

“…W ith the rapid growth in the development of antibody drugs, it has become apparent that some Igs can lose solubility at sufficiently high concentration (1)(2)(3)(4)(5)(6)(7). The resulting condensation into crystals, concentrated liquid phases, or aggregates is caused by net attractive interprotein interactions.…”