Detection and Quantitation of IgG 1 Hinge Aspartate Isomerization: A Rapid Degradation in Stressed Stability Studies

Abstract

In biopharmaceutical process development, it is desirable to identify sites of covalent degradations to ensure product consistency. One characterization method used for therapeutic immunoglobulin gamma (IgG) 1 antibodies is limited LysC proteolysis followed by reversed-phase LC/MS. Limited LysC proteolysis leads to high efficiency cleavage at the C-terminal side of the hinge lysine 222 residue, generating Fab and Fc fragments. In this report, we show that IgG 1 samples incubated under mildly acidic conditions at elevated temperatures were partially resistant to LysC cleavage at the hinge and resulted in a species where one of the Fab arms remained connected to the Fc region (Fab-Fc). The growth of the Fab-Fc species was proportional to the duration and storage temperature of the incubation period and correlated with the amount of isomerization of the aspartic acid residue preceding lysine 222, determined by peptide mapping. The isomerization rates of samples stored for up to one year at 4 degrees C, 6 months at 29 or 37 degrees C, or 3 months at 45 degrees C were determined, and the activation energy for this conversion was calculated to be approximately 33 kJ mol(-1). The apparent isomerization rate constant was only 0.02 week(-1) for samples stored at 4 degrees C, which resulted in a modest increase from 5.1 to 6.0% isoD after twenty four weeks of storage and, hence, is not a significant concern under normal storage conditions typically used for monoclonal antibodies. However, when stored at 29 degrees C, the apparent rate constant of this reaction was found to be 0.06 week(-1) and resulted in an increase from 5.1 to 21.1% isoD after twenty four weeks of storage and is a major degradant in stressed IgG 1 antibodies.

Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

“…To this end we first obtained a uniform data set that contains residue-specific quantitative data on antibody degradation products. Where available, these detected modifications are in accordance with known hotspot information from published data [11], [12], [54], [91]. The pH was kept constant at 6.0 during forced degradation and sample preparation to detect the succinimide intermediate that quickly hydrolyzes at alkaline pH, Asp isomerization, which occurs mainly at slightly acidic pH, and Asn deamidation without method-induced deamidation events.…”

Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

“…Although isomerization of aspartic or glutamic acid residues does not directly result in generation of charged species, it could indirectly affect surface charge distribution by inducing local conformational perturbation resulting in solvent exposure of neighboring residues Hambly et al have shown isomerization of Asp223 in the hinge region to be the fastest degradation in another IgG1 (40). They pointed that hinge tryptic peptide SCD 223 K is a polar peptide that elutes early in the void volume in reduced-alkylated reversed phase chromatogram in alkylated form (iodoacetic acid alkylation, +58 Da), and therefore difficult to detect.…”

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

“…Protein therapeutics are inherently structurally complex biological drugs whose active components are not a single, well-defined molecule, but a mixture of similar molecules which can differ by type and extent of post-translational modification, 1–5 chemical modifications, 6–11 and three dimensional conformations. 12,13 In addition, batch to batch variation of active components and production impurities can further complicate defining of the analytical characteristics of a protein drug.…”