Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature

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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 mAbl 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.

Section snippets

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.

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