Asparagine deamidation dependence on buffer type, pH, and temperature

被引:123
作者
Pace, Amanda L. [1 ]
Wong, Rita L. [1 ]
Zhang, Yonghua Taylor [2 ]
Kao, Yung-Hsiang [2 ]
Wang, Y. John [1 ]
机构
[1] Genentech Inc, Late Stage Pharmaceut Dev, San Francisco, CA 94080 USA
[2] Genentech Inc, Prot Analyt Chem, San Francisco, CA 94080 USA
关键词
deamidation; formulation; kinetics; protein formulation; stability; buffer; pH; temperature; MONOCLONAL-ANTIBODY; CHARGE HETEROGENEITY; STABILITY; RESIDUES; RATES; IDENTIFICATION; DEGRADATION; PEPTIDE; ISOMERIZATION; FRAGMENT;
D O I
10.1002/jps.23529
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
The deamidation of asparagine into aspartate and isoaspartate moieties is a major pathway for the chemical degradation of monoclonal antibodies (mAbs). It can affect the shelf life of a therapeutic antibody that is not formulated or stored appropriately. A new approach to detect deamidation using ion exchange chromatography was developed that separates papain-digested mAbs into Fc and Fab fragments. From this, deamidation rates of each fragment can be calculated. To generate kinetic parameters useful in setting shelf life, buffers prepared at room temperature and then placed at the appropriate stability temperatures. Solution pH was not adjusted to the same at different temperatures. Deamidation rate at 40 degrees C was faster in acidic buffers than in basic buffers. However, this trend is reversed at 5 degrees C, attributed to the change in hydroxide ion concentration influenced by buffer and temperature. The apparent activation energy was higher for rates generated in an acidic buffer than in a basic buffer. The ratepH profile for mAb1 can be deconvoluted to Fc and Fab. The Fc deamidation showed a V-shaped profile: deamidation of PENNY peptide is responsible for the rate at high-pH, whereas deamidation of a new site, Asn323, may be responsible for the rate at low-pH. The profile for Fab is a straight line without curvature. (c) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:17121723, 2013
引用
收藏
页码:1712 / 1723
页数:12
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