Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes.
Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Markus
M
Haberger
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Katrin
K
Bomans
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Katharina
K
Diepold
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Michaela
M
Hook
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Jana
J
Gassner
Pharma Research and Early Development; Roche Diagnostics GmbH; Penzberg, Germany.
Tilman
T
Schlothauer
Pharma Research and Early Development; Roche Diagnostics GmbH; Penzberg, Germany.
Adrian
A
Zwick
Pharma Research and Early Development; Roche Diagnostics GmbH; Penzberg, Germany.
Christian
C
Spick
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Jochen Felix
JF
Kepert
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Brigitte
B
Hienz
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Michael
M
Wiedmann
Pharma Technical Development Basel; F. Hoffmann-La Roche Ltd; Basel, Switzerland.
Hermann
H
Beck
Pharma Technical Development Basel; F. Hoffmann-La Roche Ltd; Basel, Switzerland.
Philipp
P
Metzger
Pharma Research and Early Development; Roche Diagnostics GmbH; Penzberg, Germany.
Michael
M
Mølhøj
Pharma Technical Development Basel; F. Hoffmann-La Roche Ltd; Basel, Switzerland.
Constanze
C
Knoblich
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Ulla
U
Grauschopf
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Dietmar
D
Reusch
Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.
Patrick
P
Bulau
D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D022801:Complementarity Determining Regions / Q000378:metabolism*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D013058:Mass Spectrometry; D018384:Oxidative Stress; D010449:Peptide Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D022801:Complementarity Determining Regions; D017127:Glycation End Products, Advanced; D007074:Immunoglobulin G; D011994:Recombinant Proteins
critical quality attributes; deamidation; developability; glycation; mass spectrometry; oxidation; protein degradation; quality by design; recombinant antibodies