Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping.

Deamidation is one of the most common degradation pathways for proteins and frequently occurs at "hot spots" with Asn-Gly, Asn-Ser or Asn-Thr sequences. Occasionally, deamidation may occur at other motifs if the local protein structure can participate or assist in the formation of the succinimide intermediate. Here we report the use of a chymotryptic peptide mapping method to identify and characterize a deamidated form of an IgG1 which was observed as an acidic peak in the cation exchange chromatography (CEX). The antibody was formulated in sodium acetate buffer, pH 5.3 and this deamidated form was observed mainly under thermal stress conditions. It was found that the IgG1 molecule with deamidation in the Fc region at asparagine residue 330 (in a Val-Ser-Asn-Lys motif) is the predominant form in this CEX peak, and was missed by tryptic mapping because the peptides are hydrophilic and elute near the void volume. In addition, a domain-based CEX method using papain digestion was developed to monitor the Asn 330 deamidation. These methods revealed that the Fc deamidation occurs mainly at Asn 330 in the VSNK motif at pH 5.3, whereas at pH 7.5, deamidation occurs predominantly at Asn 389 and Asn 394 in the NGQPENNYK motif.

Asparagine deamidation dependence on buffer type, pH, and temperature.

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.

Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations.

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Γ23 ) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein-solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1182-1193, 2013.