(M0930-09-57) Identification of Isomerization in IgG1 and Its Impact on Potency: Case Study of a Mab Biosimilar

Purpose: Isomerization of Asp to form IsoAsp introduces a minimal charge difference. However, isomerization can cause a conformational change because of the introduction of an additional methyl group to the peptide backbone. Isomerization was identified and quantified in the complementarity determining region (CDR) of the biosimilar candidate ADL-018 as well as in the reference product using LC-MS/MS and hydrophobic Interaction Chromatography (HIC). The isomerization in CDR impacted target binding and thereby, potency of the molecules, which was demonstrated by the potency assay.

Methods: Estimation of Isomerization was performed using HIC chromatography which employed digestion of the IgG1 into Fab and Fc. Site specificity of the isomerization in CDR was confirmed by characterization of HIC Peak as well as by using a multi–Attribute Method (MAM) on LC-MS/MS. The HIC peaks were characterized and confirmed to correspond to IsoAsp modification by collection followed by peptide mapping and LC-MS/MS analysis. The HIC method as well as the MAM methods were verified for their accuracy and precision. Potency was estimated by using IgE binding ELISA, which utilizes detection of IgG 1-IgE complex with Horseradish peroxidase (HRP) conjugated recombinant protein A/G. Tetramethyl benzidine (TMB) is used as a substrate. The color intensity is directly proportional to the amount of Omalizumab bound to the IgE. A dose-response curve is obtained with the reference standard and test samples with varying concentrations of each. 4-parameter logistic (4PL) nonlinear regression model is used for curve fitting and relative potency calculation.

Results: Isomerization was found to increase with time as estimated by HIC in the Biosimilar candidate as well as the reference product. Site of isomerization was confirmed to be an aspartic acid in the CDR which increased consistently with time. An increase in isomerization correlated with consistent drop in potency. Many other chemical modifications were detected in the MRM analysis but none had an impact on potency. The results clearly indicated that the IgG1 has tendency to show a drop in potency due to formation of Iso-Aspartate in liquid state, when stored at 2-8 deg C. The modification was arrested in frozen states. The conversion rate and its correlation with potency helps estimating the potential shelf-life of the product. Formulation and storage strategies that slow down the rate of conversion are also deemed as critical.

Conclusion: A clear correlation of the rate of isomerization and rate of decrease in potency was established for the IgG1. The isomerization in CDR alone impacted potency while other chemical modifications in Fab did not have an impact on potency or IgE binding. Site specificity of the isomerization was confirmed by MRM as well as by characterization of peaks in HIC method.

Correlation of %Isomerization levels on %potency over time