Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature
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INTRODUCTION
Monoclonal antibodies (mAbs) have emerged as an important class of biopharmaceuticals to treat a variety of diseases, with a majority of those treatments in oncology and immunology. Although mAbs are relatively stable proteins, they can be vulnerable to physical and chemical degradations during the manufacturing of drug substance and drug product, and storage. To maintain the stability of a therapeutic antibody, it must be protected from the various forms of degradation that can decrease its
Antibody
The IgG 1 full-length mAb, mAb1, used in these studies was produced from Chinese hamster ovary cells at Genentech (South San Francisco, California).
Sample Preparation
To generate the various formulation conditions, buffer exchange was performed using a dialysis cassette (Slide-a-Lyzer Cassette from Thermo Fisher Scientific Inc., Logan, Utah) according to the manufacturer’s protocol with a 100:1 buffer to sample ratio and three buffer exchanges at 5°C. The protein formulation with the acidic or basic buffer was
Comparison of Fc Deamidation Rate as Determined by Papain-IEC and Other Methods
Deamidation of Fc Asn has been extensively characterized using LC-MS by Chelius et al.2 and Sinha et al.15 In those studies, either the mAb2 or the Fc fragment15 was stored in 100 mM Tris buffer, pH 7.4, at 37°C. In our study to assess the effectiveness of the papain-IEC assay in analyzing Asn deamidation, the same reaction conditions were used. The only difference was the protein concentration; a concentration of 30 mg/mL was used, as most mAb products are currently formulated at concentrations
CONCLUSIONS
Deamidation is a source of nonenzymatic protein degradation, and it is important to monitor it during the course of formulation development. Being directly proportional to the [OH-] in a solution, the rate of Asn deamidation is sensitive to pH, temperature, and buffer type. At different temperatures, the buffering species will affect deamidation rate trends because of the changing [OH-] in the solution. The kinetic parameters calculated herein indicate that a buffer species that may not seem
ACKNOWLEDGMENTS
The authors would like to thank Dr. Thomas Patapoff for the use of and references on Hbf, Benjamin Tran and Jennifer Hu for assistance in developing the papain-IEC assay, and Dr. Mary Cromwell for her support of this project and critiques of this manuscript. Editorial assistance by Eileen Y. Ivasauskas is greatly appreciated.
REFERENCES (28)
- et al.
Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization
J Pharm Sci
(2006) - et al.
Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides
J Biol Chem
(1987) - et al.
Identification of multiple sources of charge heterogeneity in a recombinant antibody
J Chromatogr B
(2001) - et al.
Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgGl heavy chain
J Pharm Sci
(2009) - et al.
Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody
Structure
(2001) - et al.
Characterization of the stability of a fully human monoclonal IgG after prolonged incubation and elevated temperature
J Chromatogr B
(2006) - et al.
Isolation and characterization of a succinimide variant of methionyl human growth hormone
J Biol Chem
(1991) - et al.
Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion
J Pharm BiomedAnal
(1997) - et al.
Self-buffering antibody formulations
J Pharm Sci
(2008) - et al.
Controlling deamidation rates in a model peptide: Effects of temperature, peptide concentration, and additives
J Pharm Sci
(2001)
Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies
Anal Chem
Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides
Pharm Res
Kinetics and mechanism of succinimide ring formation in the deamidation process of asparagine residues
J Chem Soc Perkin Trans
Asparagine deamidation: pH- dependent mechanism from density functional theory
Biochemistry
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