(M0930-01-03) The Tale of Two Methods: Understanding the Roles of IEX and icIEF for Monitoring Charge Heterogeneity of Stressed Biotherapeutics

Purpose: Forced degradation of biologics is the intentional degradation of a therapeutic molecule to various degrees through conditions such as heat, light, pH, and other chemical stresses to estimate possible degradation that may occur through manufacturing, storage, shelf life, and distribution. Samples subjected to various stress conditions are then analyzed for aggregation, fragmentation, charge variants, post-translational modifications, and potency to assess what liabilities may be present and to help inform the analytical control strategy. Charge variant assays in particular highlight the charge heterogeneity in the sample due to post translational modifications on the protein and play an important aspect to the analytical control strategy. The purpose of this study is to highlight the differences between two different methods for analyzing charge variants in biotherapeutic molecules: Ion-Exchange Chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The comparison of these two methods help inform on the differences that can lead to an offset in some forced degradation conditions and can establish a plan of analysis for future studies.

Methods: A therapeutic antibody (mAb1) was subjected to various stress conditions analyzing the impact of heat stress, UV and Visible light stress, low pH, high pH, oxidative stress, and glucose stress over various time periods. These samples were then frozen after each time point and tested using IEX and icIEF to compare both methods for charge variant assessment. Each method grouped samples into total acidic, total basic, and main peak, and the percent areas of each group were compared between the methods.

Results: The charge variant profile of mAb1 from each method is comparable, showing similar profiles with comparable area percentages (differing by 2-10% area) of total acidic, total basic, and main peaks (See Figure 2).  During stresses such as heat or pH, the two assays show comparable data, and while the IEX trace does show more resolution compared with icIEF, the trends of total acidic percent area, total basic percent area, and main peak percent area correspond well between the two methods (Figure 1 and Figure 2). However, light stress and oxidative stress conditions show a deviation where the total basic percent area increases significantly in IEX, whereas this is not detected in icIEF. A similar phenomenon occurs with glucose stress, where a significant increase in total acidic percent area is seen in icIEF but not detected to the same degree in IEX. (Figure 2). These differences highlight that different methods can result in different charged variant readouts, depending on what type of modification is being observed. Light and oxidation stress, in particular,   results in methionine oxidation within mAb1 and gives a significantincrease in basic species in IEX but not in icIEF. Meanwhile, glycation due to glucose exposure gives an increase in acidic species that is not as readily detected in IEX.

Conclusion: While certain stress conditions give similar results regardless of charge variant assay, significant differences between icIEF and IEX results for mAb1 have occurred with oxidative and glucose stresses. These differences could be a result of the different techniques, particularly how icIEF relies on denaturation to separate by charge, whereas IEX occurs in its folded state but with higher salt presence. IEX measuring only the surface charge of the protein could play a critical role in result differences, particularly for glycation and oxidation.  Follow-up studies using mass spectrometry techniques will be utilized to further understand what these differences could be.