Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

Abstract

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

“…Native MS and ion mobility (IM) MS are emerging as important tools for the characterization of antibody-based products 20 . For example, native MS coupled to size-exclusion chromatography 21 and native IM MS 22 have been used to analyze BsIgG obtained from the CrossMab technology and antibody-drug conjugates, respectively, under more physiologically representative conditions.…”

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

“…In these cases, the mobile phases are composed of largely volatile components, facilitating ionization and allowing for direct coupling to MS [54]. IEX and SEC may also be coupled directly to ESI-MS under conditions where ESI-compatible mobile phases with reduced ionic strengths are used [55,56]. Other modes of LC, such as HIC and affinity LC, for example, must be performed in an offline fashion, under which separations are performed separately and individual fractions are collected, which are then analyzed independently by MS. With HIC and affinity LC, mobile phases typically contain non-volatile components that must be removed prior to ESI-MS [46].…”

Maintaining consistent quality and clinical performance of biopharmaceuticals

“…Further, progress in the ability of mass spectrometers to analyze large intact proteins is increasing the utility of this technology for identifying and monitoring functionally relevant product variants. Example include strategies for top down elucidation of site-specific variants as well as additional capabilities for characterizing variants and their impact on protein interactions in intact molecules under native conditions [59][60][61][62][63][64].…”