Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.
The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.
Late Stage Pharmaceutical and Processing Development, Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Caroline
C
Loew
Constanze
C
Knoblich
Jürgen
J
Fichtl
Nadja
N
Alt
Katharina
K
Diepold
Patrick
P
Bulau
Pierre
P
Goldbach
Michael
M
Adler
Hanns-Christian
HC
Mahler
Ulla
U
Grauschopf
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002853:Chromatography, Liquid; D057230:Limit of Detection; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013205:Staphylococcal Protein A / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D013205:Staphylococcal Protein A; D008715:Methionine