Fragmentation of a Recombinant Monoclonal Antibody at Various pH
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To determine the relative importance of direct hydrolysis and β-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.
A recombinant monoclonal antibody was incubated in buffers of various pH at 40°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).
Direct hydrolysis was accelerated by acidic and basic pH, while β-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.
Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.
KEY WORDSfragmentation hinge region mass spectrometry recombinant monoclonal antibody
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