Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1053, Issue 1-2, Pages 299-305Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2004.08.058
Keywords
recombinant antibody; charge variants; characterization; heterogeneity; degradation
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Analytical characterization of monoclonal antibodies has been hindered by the lack of appropriate chromatographic methods to be used in conjunction with high-resolution MS. Current methodologies for standard RP-HPLC are incompatible with antibodies due to irreproducibility, low recovery, short column lifetimes, and poor resolution of degradation products. An analytical RP-HPLC-MS method was developed for monitoring and characterizing intact IgG1 antibodies. Key parameters required for improved chromatographic resolution included long alkyl chains of the stationary phase (Zorbax SB300 C-8), column temperatures elevated to 65-70 degreesC and combination of trifluoroacetic acid and heptafluorobutyric acid ion-pairing agents. RP chromatographic separation of degradation species and C-terminal lysine variants along with the characterization of glycosylation profile by mass spectrometry demonstrates the capability of this method for whole antibody analysis. (C) 2004 Elsevier B.V. All rights reserved.
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