title: Asparagine deamidation dependence on buffer type, pH, and temperature.
abstract: 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°C was faster in acidic buffers than in basic buffers. However, this trend is reversed at 5°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 rate-pH 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.
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California 94080.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Pace
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California 94080.
authors.forename: Rita L
authors.initials: RL
authors.lastname: Wong
authors.affiliation: Protein Analytical Chemistry, Genentech Inc., South San Francisco, California 94080.
authors.forename: Yonghua Taylor
authors.initials: YT
authors.lastname: Zhang
authors.affiliation: Protein Analytical Chemistry, Genentech Inc., South San Francisco, California 94080.
authors.forename: Yung-Hsiang
authors.initials: YH
authors.lastname: Kao
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California 94080. Electronic address: wang.john@gene.com.
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D000577:Amides / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001216:Asparagine / Q000032:analysis* / Q000378:metabolism; D002021:Buffers; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D055550:Protein Stability; D013696:Temperature
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D002021:Buffers; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D001216:Asparagine; D010206:Papain