Large Scale Purification and Characterization of A21 Deamidated Variant-Most Prominent Post Translational Modification (PTM) for Insulins Which Is Widely Observed in Insulins Pharmaceutical Manufacturing
Authors: Shukla Vibhava , Sriva Koduru , S Madhu Kumar M , Vajpai Navratna , Agarwal Neha , S Nethra , P Somesh B , Kulshrestha Abhishek , Hazra Partha 
Published: December 2020

Abstract
BackgroundThe rapid growth of global diabetic population attracts the large scale manufacturing of insulin and analogues to meet the unmet demand. Biopharmaceutical development demands appropriate understanding of product variant, which are formed due to post-translational modification. Deamidation is the most common post‐translational modification occurring in insulins and insulin analogues. Same time, the same variant is most prominent product variant occur during biopharmaceutical manufacturing process or during the storage. Often, one or the other deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of highly purified deamidated material is always being challenging due to very similar mass, similar ionic properties, and high structural similarity of the variant compared to the parent productResultsIn the present work authors tried to come up with a novel, easy and large scale process of purification of deamidated impurity with high purity from insulin. Anion exchange resin PolyQuat offers a high ionic capacity for both strong anion exchange and weak anion exchange species. Due to this unique functionality, PolyQuat offers enhanced performance and better selectivity than conventional ion exchange media, which enables to purify closely related A21 desamido variant from insulin. This single chromatography step can enrich the purity of desamido insulin to >96%. The PolyQuat elution pool was concentrated through the preparative RP-HPLC column in the bind elute mode. The RP-HPLC elution pool was precipitated and lyophilized in powder form. Detailed characterization of human insulin variant (+1 Da desamido) was carried out for its primary, secondary and tertiary structure through state-of-the-art mass spectrometry methods, circular dichroism and intrinsic fluorescence and was compared with human insulin. Intact, reduced and Peptide Mass Fingerprinting analysis identified an increase of 1Da mass in the peptide bearing A21 amino acid. We have also compared the extent of IR-B phosphorylation by A21 desamido insulin against human insulin reference standard. As biological functions are governed by protein structure, IR-B phosphorylation assay was performed to compare functional activity of AspA21 human insulin variant and human insulin. The comparative phosphorylation profiles and relative potency clearly suggested no impact of A21 deamidation on the functional activity of recombinant human insulin. ConclusionsThe present work reveals efficient scalable process to manufacture insulin desamido variant in the powder form with more that 60% recovery with 97% purity. This simplified process for manufacturing deamidated variant presented in this study can be handy for the insulin manufacturers to make large quantity variant standard and use for their process development as well as routine manufacturing process. Purified insulin desamido variant allowed detailed characterization of the species as well helps in using as an impurity standard for process monitoring in QC, which aids in robust control for insulin manufacturing.