Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms.
Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other proteins, therapeutic mAbs can under go various enzymatic and non-enzymatic reactions that can affect their structural integrity and stability. Among the degradation reactions, isoaspartate (isoAsp) formation is one of the major sources of charge heterogeneity of mAbs. This paper reports the detection and quantification of isoAsp in a recombinant mAb and its charge isoforms resolved by cation exchange high performance liquid chromatography. The assay utilizes the enzyme protein isoaspartyl methyltransferase in conjunction with strong cation exchange separation and UV detection (at 260 nm) of S-adenosyl-L-homocysteine, which is produced stoichiometrically in the enzymatic reaction. The mAb is found to contain an average 0.2 mol of isoAsp per mol of protein, however, various charge isoforms were found to contain different levels of isoAsp. The most acidic isoforms contain approximately 0.7 mol of isoAsp per mol of protein, and no isoAsp is detected in the most basic isoform. It appears that the majority of isoAsp in the mAb is formed as a result of asparagine deamidation.
Zhang
Wei
W
Wyeth BioPharma, Genetics Institute Campus, One Burtt Road, Andover, MA 01810, USA. wei.zhang@immunogen.com
Czupryn
Marta J
MJ
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D026581:Isoaspartic Acid / Q000032:analysis* / Q000737:chemistry; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
D000911:Antibodies, Monoclonal; D026581:Isoaspartic Acid; D020033:Protein Isoforms; D011994:Recombinant Proteins