Elucidation of Mg2+ induced size and charge heterogeneity in monoclonal antibody therapeutics.

Abstract

Changes in charge variant profile are known to affect mAb stability and vice versa. This report elucidates the effects of magnesium metal (0.5 mM Mg²⁺) on trastuzumab (IgG1 antibody). Mg²⁺ is often used as an excipient (50-100 mM) and lubricant (5-10 % w/w) in biopharmaceutical formulations. Analytical size-exclusion chromatography (SEC) and cation-exchange chromatography (CEX) coupled with mass spectrometry (MS) were used to evaluate the size and charge heterogeneity in the thermal and metal stressed samples and compared to the control sample (room temperature). The present study unveils that presence of Mg²⁺ significantly increases the rate of aggregation with 9 % aggregation observed in Mg²⁺ stressed samples as compared to that from thermal stress (~2 %) or control sample (<1 %). Similarly, a 2-fold elevation in acidic variants was observed both in presence of Mg²⁺ and thermal stress, when contrasted with the control sample. Application of stress also led to the formation of 17 additional chemical modifications (7 due to thermal stress and 10 due to Mg²⁺ stress) which were not identified in control, predominantly involving deamidation, isomerization of aspartic acid, oxidation, and succinimide modifications. The results indicate the need for a detailed analysis of the impact of presence of metals in biotherapeutic formulations.