C-Terminal Lysine Variant Analysis

Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents for a wide range of human diseases, including many types of cancer. Process changes in production scale, media, and cell culture process parameters can result in product quality inconsistencies among different antibody production batches. Heterogeneity analysis in levels of post-translational modifications (PTMs) of mAbs is an indispensable part of their characterization as well as quality control. Creative Biolabs applies a strategic approach to conduct PTM analysis for clients globally as part of our mAb structural characterization services. Here, we focus on the analysis of C-terminal lysine variants of mAb products.

Introduction to C-Terminal Lysine

Large-scale production of mAb products often involves the use of cell cultures that are known to produce varying levels of heterogeneity. One of the potential sources of heterogeneity involves C-terminal lysine residues, which is typically found on the heavy chains of antibody molecules. More specifically, C-terminal lysines on antibody heavy chains can be lost, so that individual antibodies in a production batch can vary at their C-terminus as to whether a lysine residue is present. Therefore, C-terminal lysine can be potentially present on both the heavy chains of an antibody (Lys 2), on either one of the heavy chains (Lys), or neither of them (Lys 0). C-terminal Lys variants are frequently observed in mammalian cell-derived recombinant antibodies, usually produced by proteolysis of endogenous carboxypeptidases during the manufacturing process.

Fig.1 The C-terminal heterogeneities of mAbs. (Beck, 2013)

Impact of C-Terminal Deletion

It has been reported that C-terminal Lys deletion has no impact on antibody structure, thermal stability, antigen binding and potency, FcRn binding, and pharmacokinetics in rats. Thus, it is not considered as a critical quality attribute (CQA). However, since lysine contains a positive charge, antibodies lacking the basic C-terminal lysine(s) differ in the charge state from the ones with lysine, contributing to mAb charge heterogeneity. Therefore, it is still an important factor to include in the characterization parameters.

C-Terminal Lysine Variant Analysis Services Provided by Creative Biolabs

Our laboratories are equipped with a broad range of techniques to satisfy various requirements. Because Lys is readily charged, its loss results in a decrease in positive charge that allows for resolution of the modified and unmodified structures by charge-based separation techniques such as ion exchange chromatography (IEX) and capillary isoelectric focusing (cIEF). What’s more, loss of the terminal Lys residues gives a mass shift, which can be detected and quantified through the application of mass spectrometry (MS)-based techniques.

Based on our advanced techniques and abundant experience, Creative Biolabs is proud to offer the most comprehensive PTM analysis services, covering a wide range of modifications, such as glycosylation, C-terminal Lys variant, N-terminal cyclization, deamidation and isomerization, oxidation, glycation, etc. We design customized strategic analytic programs to meet the requirements of regulatory standards and industry guidance such as ICH Q6B.

If you are interested in our service, please do not hesitate to contact us for more details.

Reference

1. Beck, A.; et al. Characterization of Therapeutic Antibodies and Related Products. Analytical Chemistry. 2013, 85(2): 715-736.

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