Restoring the biological activity of crizanlizumab at physiological conditions through a pH-dependent aspartic acid isomerization reaction

Abstract

In this study, we report the isomerization of an aspartic acid residue in the complementarity-determining region (CDR) of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping. The biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The influence of pH on this isomerization reaction was investigated using capillary zone electrophoresis. Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long-term storage conditions of 2 to 8°C at pH values below 6.3, a complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C), resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions.

Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

“…Therefore, protein therapeutics are generally stored under mildly acidic pH (4–6) conditions which in turn favor isomerization . Studies aimed at deciphering the factors that modulate Asp/Asn isomerization have shown that peptides and proteins containing nonbulky amino acids, primarily Gly, at the carboxyl side of Asn/Asp are more susceptible to isomerization and deamidation reactions. ,− CDRs and other regions of mAbs that are particularly flexible and/or have higher solvent accessibilities are also more prone to deamidation and isomerization. , …”

“…Considering the impact of Asn/Asp isomerization, identification of the resulting isomerized residues is vital for unraveling disease mechanisms and for the comprehensive characterization of biotherapeutics prior to an investigational new drug application filing at the Food and Drug Administration or other foreign regulatory agency. , While deamidation results in a mass shift of 0.984 Da and can be easily detected using mass spectrometry (MS), isomerization is difficult to detect because there is no mass change associated with it. Several tandem MS (MS/MS)-based methods have been developed to address the challenging differentiation of Asp/ iso Asp in proteins. ,− In one of the earlier reports, low-energy CID was used to identify iso Asp peptides based on the presence of diagnostic b ( N – 1) + H 2 O ( N is the position of iso Asp) and y ( L – N + 1) – 46 ions ( L is the length of the peptide), depending on the location of the basic residue .…”

“…14,17 Considering the impact of Asn/Asp isomerization, identification of the resulting isomerized residues is vital for unraveling disease mechanisms and for the comprehensive characterization of biotherapeutics prior to an investigational new drug application filing at the Food and Drug Administration or other foreign regulatory agency. 8,13 While deamidation results in a mass shift of 0.984 Da and can be easily detected using mass spectrometry (MS), isomerization is difficult to detect because there is no mass change associated with it. been developed to address the challenging differentiation of Asp/isoAsp in proteins.…”

“…Physical and chemical stress during manufacturing and storage leads to the degradation of protein therapeutics or loss of activity. The isomerization of Asp and deamidation of Asn in the complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs) are the major degradation pathways that lead to the loss of target binding and ultimately their potency. ,− It is well known that exposure to high temperatures and high pH accelerates deamidation, oxidation, enzymatic cleavages, dimer formation, and aggregation of proteins . Therefore, protein therapeutics are generally stored under mildly acidic pH (4–6) conditions which in turn favor isomerization .…”