Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins

Abstract

Modern size exclusion chromatography (SEC) can be defined by the use of relatively small columns (e.g., 150 × 4.6 mm) packed with sub-3 μm particles, allowing a 3- to 5-fold increase in method throughput compared to that of conventional SEC. The quick success of the first sub-2 μm SEC column introduced in 2010 led to the development of numerous ultra-high performance (UHP)-SEC columns for the analysis of therapeutic monoclonal antibody (mAb)-based products. Aggregates also known as high-molecular-weight species (HMWS) are indeed one of the most important critical quality attributes (CQAs) of mAbs, as HMWS may decrease the product efficacy or cause immunogenicity effects. Therefore, the confident characterization of mAbs requires strong knowledge of not only modern SEC performance (i.e., selectivity and efficiency) but also the inherent limitations caused by non-specific interactions more likely to occur with complex antibody drug conjugates (ADCs) and some commercial mAb products. This review discusses the importance of liquid chromatographic (LC) instrumentation in order to exploit the full potential of modern SEC columns and current trends to hyphenate SEC to mass spectrometry (MS). Recent applications for antibody-based products (i.e., mAbs, ADCs, Fc-Fusion proteins and bispecific antibodies) are presented. Finally, tips and tricks are provided to further optimize SEC separations and maintaining their performance over time with better understanding of unexpected SEC results.

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

“…These future studies would benefit from the expansion of analysis approaches to include both disulfide bond mapping, and a suitable charge variant analysis approach in lieu of the RP-LC-MS approach used here. Additional considerations for experimental methods that provide aggregate level information, including size-exclusion chromatography 74 and subvisible particle analysis approaches, 75 would provide valuable data on the downstream effects of these changes in HOS and stability.…”

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

“…Because of these safety risks, the industry and regulatory authorities consider aggregation as a CQA [28]. SEC separates proteins into three major species: (i) high molecular weight species (HMWS), (ii) main peak (predominantly the monomeric form), and (iii) low molecular weight species (LMWS) [29,30]. Compounds are separated based on the difference between their hydrodynamic volumes, which results in different residence times that solutes spend in the internal pores of the stationary phase.…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…HIC, RP, SCX, and SEC can employ water‐rich mobile phases that prevent denaturing of proteins. Several reviews are available on these separation modes . A total of 32 IgGs were separated on a HIC column, and 10 to 12 mAbs eluted in 22 to 24 min .…”