title: Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures.
abstract: The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.
authors.affiliation: Pharma Research and Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Diepold
authors.forename: Katrin
authors.initials: K
authors.lastname: Bomans
authors.forename: Michael
authors.initials: M
authors.lastname: Wiedmann
authors.forename: Boris
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Andreas
authors.initials: A
authors.lastname: Petzold
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Robert
authors.initials: R
authors.lastname: Mueller
authors.forename: Bernd
authors.initials: B
authors.lastname: Moritz
authors.forename: Jan Olaf
authors.initials: JO
authors.lastname: Stracke
authors.forename: Michael
authors.initials: M
authors.lastname: Mølhøj
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D000577:Amides; D000906:Antibodies; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine