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C-terminal Lysine Cleavage Assay

Lysine is a basic amino acid with a very unstable side-chain e-NH2. It has strong nucleophilic activity and can undergo nucleophilic reactions with various groups. Lysine residues may undergo various modifications, including acetylation, methylation, ubiquitination, ubiquitination, propionylation, and butyrylation recently discovered by scientists. Pilot studies have shown that these modifications can affect the chemical stability of your drug candidates and finally cause a reduced drug activity in clinical use. With a wealth of background knowledge and a strong manufacturability assessment technical team, Creative Biolabs provides a number of high-quality C-terminal lysine cleavage services to assess the chemical stability and the efficacy of your testing drugs.

Effect of Lysine Modification on Antibody Drugs

Structure of an IgG antibody with possible modifications. Fig.1 Structure of an IgG antibody with possible modifications. (Beyer, 2017)

Our C-terminal Lysine Cleavage Assay

C-terminal lysine cleavage, that is, the post-translational modification (PTM) of the lysine which has occurred in the antibody-drug is removed by a certain cleavage method. In Creative Biolabs, the C-terminal lysine cleavage is often treated by protein digestion. Here are a few common methods for enzyme digestion:

Trypsin specifically recognizes and cleaves the lysine or arginine carboxy terminus of the antibody/polypeptide chain and has extremely high site-specificity. The peptides produced by digestion are often terminated with lysine and arginine. These peptides are easily charged with a bivalent or higher valence positive charge under acidic conditions, are easily detected by mass spectrometry, and are widely used in research.

Lys-C is a protease that specifically recognizes and cleaves the carboxy terminus of lysine and still has enzymatic activity under strongly denaturing conditions. Often used in combination with trypsin, it can be used to analyze macromolecular complexes to make enzyme digestion more complete.

It is the most commonly used enzyme digestion system in proteomics research because of its advantages of simple and convenient operation and low sample loss.

The antibody will be digested with Lys-C and further digested with trypsin. In this way, the length of the peptide cut out is suitable, and is easier to extract from the gel in order to distinguish by mass spectrometry. Therefore, sequential digestion is suitable for complex proteomics research.

Highlights

As a global contract research organization, Creative Biolabs worked on a wide range of technical services based on the C-terminal lysine cleavage assay for assessing the manufacturability of various drugs. We utilize unrivaled, proprietary drug design, study data, product candidate, advanced project lifecycle management, as well as real-time data to ensure the ideal outcomes. If you want to know more information, please contact us or send us an inquiry.

Reference

  1. Beyer, B.; et al. Microheterogeneity of recombinant antibodies: analytics and functional impact. Biotechnology Journal. 2017, 13(1): 1700476.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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