Charge variants are considered as a Critical Quality Attribute of therapeutic monoclonal antibodies, as they can have a significant impact on the efficacy of the drug. State-of-the-art analytical techniques are required to both quantify and identify the variants. In this application note, we present the portfolio proposed by Quality Assistance, including capillary isoelectric focusing (cIEF) and ion-exchange chromatography (IEX), with applications to commercial monoclonal antibodies. Approaches based on (2D-)LC/MS are presented for the identification of the variants.
Monoclonal antibodies (mAbs) constitute a major and fast-growing biotherapeutic class, thanks notably to their outstanding selectivity for specific targets. Their physico-chemical characterisation is complex due to their size (around 150 kDa), numerous variants caused by post-translational modifications, or their tendency to aggregate. Besides, biotherapeutics are subject to various degradation pathways, including – but not limited to – oxidation, deamidation, aggregation and fragmentation.
So-called “charge variants” are variants that have an impact on the net charge of the protein, and therefore on its isoelectric point. For monoclonal antibodies, acidic variants are commonly due to the presence of sialic acids on the glycans or to deamidation of asparagine residues converted to aspartic and isoaspartic acids, while basic variants may come from the presence of lysine residues at the C-terminus of heavy chains or from succinimide formation (intermediate in the deamidation reaction).
Some of these modifications have limited impact on the safety and/or efficacy of the product but others, such as deamidation, especially when occurring in the CDR (Complementary Determining Region) of the antibody, may cause a significant loss in activity. Therefore, it is necessary to characterise and quantify charge variants at release and during stability studies.
Together with a strong expertise in the field, Quality Assistance offers a wide range of techniques for charge variant analysis of monoclonal antibodies. Three types of cIEF systems are available: PA800+ (Sciex), iCE3 and MaurICE (Protein Simple). Ion-exchange chromatography can be performed either in salt gradient or pH gradient. Identification of the variants can be tailored to the project, using either 2D-LC/MS or IEX/MS. This allows to meet regulatory requirements for the analysis of charge variants which are considered as Critical Quality Attributes of therapeutic monoclonal antibodies.