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J Chromatogr A. 2006 Jan 13;1102(1-2):164-75. Epub 2005 Nov 17.

Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics.

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  • 1Amgen, Department of Pharmaceutics, One Amgen Center Drive, MS 8-1-C, Thousand Oaks, CA 91320, USA.


A reversed-phase LC/MS method was developed for reduced antibodies that provides efficient separation of light chain and two variants of heavy chain containing N-terminal glutamine and pyroglutamic acid. The best separation was achieved on Zorbax CN and Varian Pursuit DiPhenyl columns eluted with increasing percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid. Although glutamine was genetically coded for the N-terminal residue of heavy chain of a monoclonal antibody used in this study, we found that most of it (70%) was converted to pyroglutamate during production. The conversion process continued in vitro and was monitored by the method. Deconvoluted electrospray ionization mass spectrum of the heavy chain revealed the glycosylation profile of a single N-linked sugar including a-, mono-, and di-galactosylated biantennary glycans and a 5-mannose sugar form.

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