A study in glycation of a therapeutic recombinant humanized monoclonal antibody: Where it is, how it got there, and how it affects charge-based behavior
Autor: | Irena Petkovska, Domenic Matthews, Ron Taticek, Cynthia P. Quan, Emily W. Alcala, Stacey Ma, Eleanor Canova-Davis |
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Rok vydání: | 2008 |
Předmět: |
Models
Molecular Glycosylation medicine.drug_class Lysine Ion chromatography Biophysics CHO Cells Monoclonal antibody Tandem mass spectrometry Biochemistry Chromatography Affinity law.invention chemistry.chemical_compound Cricetulus Tandem Mass Spectrometry Glycation law Cricetinae medicine Animals Humans Molecular Biology Chromatography Chemistry Isoelectric focusing Antibodies Monoclonal Galactose Cell Biology Chromatography Ion Exchange Boronic Acids Recombinant Proteins Glucose Immunoglobulin G Recombinant DNA Chromatography Liquid |
Zdroj: | Analytical Biochemistry. 373:179-191 |
ISSN: | 0003-2697 |
DOI: | 10.1016/j.ab.2007.09.027 |
Popis: | The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product. |
Databáze: | OpenAIRE |
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