Protein drugs are the most active and fastest-growing part of the current pharmaceutical research and development field. Recombinant protein therapeutics is different from small molecule drugs due to its structural specificity and protein molecule instability, so more strict control is needed in bio-synthesis, purification and storage steps. The instability of the protein is mainly concentrated at its C- and N-terminal, and the analysis of the variations at the C-terminus and N-terminus is particularly important.
What Is C-terminal Lysine Variant?
The C-terminal lysine variant is a common phenomenon in monoclonal antibodies and recombinant protein, and this modification was first reported more than a decade ago. It mainly manifests as variation in the presence or absence of lysine on the C-terminal of the heavy chain. Even though the C-terminal lysine residue may not contribute to the bioactivity, the degree of heterogeneity of C-terminal lysine variants reveals the manufacturing consistency and should be monitored for product consistency. Recently, several different analytical methods were used to analyze C-terminal lysine variant, for example, isoelectric focusing (IEF), capillary isoelectric focusing (cIEF) and reversed-phase HPLC. Now, matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatograph mass spectrometer (LC-MS) are more popular because they are used to directly determine the relative percentage of C-terminal lysine contained in the original antibody.
Figure 1. Schematic diagram illustrating C-terminal lysine-variants
Analysis of C-Terminal Lysine Variants at Creative Proteomics
At Creative Proteomics, we have developed a professional platform for analysis of C-terminal lysine variants that contains various techniques, including but not limited to:
These methods will generate data to show that charge differences exist between different lots of proteins, so that we can qualitatively verdict if the C-terminus of the protein has changed. In addition, by MALDI-TOF MS and LC-MS, the protein is fragmented regularly in the mass spectrum to obtain fragments of the protein peptide, and the spectrum is analyzed to obtain the C-terminal sequence of the protein. These methods can directly determine the relative percentage of C-terminal lysine.
Creative Proteomics has spent decades of efforts to develop and explore protein analysis methods in protein structure as well as its kinds of variants. As a global company, we place a high value on effectiveness and innovation to deliver comprehensive solutions with the highest levels of quality. If you have any questions or specific needs, please don’t hesitate to contact us.
Dick Jr, et al. C‐terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes. Biotechnol Bioeng. 2008, 100(6): 1132-1143.