Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

Abstract

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

Culture temperature modulates monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures

“…2d). Basic charge variants were found to be generated by a number of modifications, such as Cterminal modifications (lysine cleavage and proline amidation), methionine oxidation and aspartate isomerization (Du et al 2012). In the present study, the main cause for the higher basic variant levels obtained at sub-physiological temperatures will be further elucidated in the following section.…”

Cysteinylation of a monoclonal antibody leads to its inactivation

“…[14][15][16][17] Analytical chromatography techniques are effective tools for detecting post-translational modifications of antibodies, either through changes to the antibody charge variance (ion exchange) or hydrophobic profile (HIC). 4,14,18 Cation exchange chromatography in particular can be an effective tool for identifying antibody charge variations that result from these types of modifications, 19,20 but the antibody exhibited a complex profile that was not amenable for analysis by this method (data not shown). Modifying the gradient conditions did not lead to a significant improvement in chromatographic resolution (data not shown).…”

A new tool for monoclonal antibody analysis

“…55,56 Many of the post-translational modifications could lead to charge variants either directly by changing the net charge on the molecule or indirectly by altering protein structure, and thus pK a of charged residues. Deamidation is one of the common modifications that introduce charge heterogeneity in mAbs.…”

“…Separate charge profiles of Fab and Fc domains have been obtained using limited Lys-C or papain proteolysis in combination with CEX separation. [57][58][59][60][61] It is known that the variable regions of both LC and HC could contribute to the overall charge heterogeneity of the antibody product, 55,56 and deamidation of LC and HC CDR asparagine residues could cause loss in bioactivity. 44,62 There is, however, no previous report on domain-specific charge profiling of LC and Fd domains.…”