Fragmentation of a recombinant monoclonal antibody at various pH.
OBJECTIVE
To determine the relative importance of direct hydrolysis and beta-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH.


METHODS
A recombinant monoclonal antibody was incubated in buffers of various pH at 40 degrees C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS).


RESULTS
Direct hydrolysis was accelerated by acidic and basic pH, while beta-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5-9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain.


CONCLUSIONS
Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.
Gaza-Bulseco
Georgeen
G
Process Sciences Department, Abbott Bioresearch Center, Worcester, Massachusettes 01605, USA.
Liu
Hongcheng
H
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D009844:Oligosaccharides; D010455:Peptides; D011994:Recombinant Proteins; D001216:Asparagine