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J Pharm Sci. 2018 Oct;107(10):2570-2580. doi: 10.1016/j.xphs.2018.06.015. Epub 2018 Jun 21.

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay.

Author information

1
Protein Analytical Chemistry, Genentech Inc., South San Francisco, California 94080; Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047. Electronic address: yyang@gene.com.
2
Protein Analytical Chemistry, Genentech Inc., South San Francisco, California 94080.
3
Bioprocess Development, Genentech Inc., South San Francisco, California 94080.
4
Lancaster Laboratories, Inc., Lancaster, Pennsylvania 17605.
5
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047. Electronic address: schoneic@ku.edu.

Abstract

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.

KEYWORDS:

Lucifer yellow carbohydrazide (LY CH); acidic charge variants; aggregation; charge heterogeneity; metal-catalyzed oxidation; protein carbonylation; quality attributes; recombinant mAb

PMID:
29935298
DOI:
10.1016/j.xphs.2018.06.015

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