Antibody Mixtures Digested using GingisREX

The formulation of antibodies in mixtures has revealed significant clinical advantages but causes increased analytical challenges. Long-term studies of formulated antibody mixtures over time are both difficult and time consuming. An example of a post-translational modification that could occur during storage is the oxidation of tyrosine that may induce conformational changes of an antibody.


In a recent article by scientist at Merck in New Jersey, USA, new analytical workflows were explored to study a formulated mixture of antibodies of subclass IgG1, IgG2 and IgG4. To study conformational changes in single antibodies and mixtures, the scientists utilized fluorescent labeling of oxidized tyrosine to 3,4-dihydroxyphenylalanine for analysis using LC-MS.


In a complex sample containing antibody mixtures with similar primary sequences, the authors found that by using the arginine specific protease GingisREX®, longer peptides could be generated which resulted in the formation of unique peptides. The GingisREX protease is highly specific for c-terminal digestion after arginine residues and has showed improved performance over Arg-C in a direct comparison (GingisREX Applications).


Using the GingisREX based LC-MS workflow, the scientists could study a particular oxidation hotspot in peptide 20 of the IgG4 antibody that only occurred when the antibodies were studied in a mixture. The authors conclude that orthogonal methods for study of antibody mixtures are needed and that the combination of fluorescent labeling of oxidized residues in combination with mass spectrometry is a promising approach.



Mozziconacci, O. et al., 2020. Probing Protein Conformation Destabilization in Sterile Liquid Formulations through the Formation of 3,4-Dihydroxyphenylalanine. Molecular Pharmaceutics, 17(10), pp.3783–3793.


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Protease that digests C-terminally of Arginines