Tri uoroacetic acid

Trypsin and formic acid were obtained from Fluka (Buches, Germany) and Promega (Fitchburg, WI), respectively. Iodoacetamide, dithiothreitol, urea, EDTA, NAM and TSA were purchased from Sigma (St. Louis, MO). A TMT kit was obtained from Thermo (Waltham, MA). Pure water and acetonitrile were obtained from Fisher Chemical. Tri uoroacetic acid was purchased from Sigma-Aldrich. Protease inhibitors were obtained from Calbiochem. A BCA kit was obtained from Beyotime.

Recombinant insulin drug substance of analytical purity of >99% pure containing deamidation variant of 0.3% was procured from Biocon Limited, Bangalore, India. Puri ed insulin drug substance was solubilized in 0.01N HCl targeting concentration of 4-10 mg/ml. Liquid solution was incubated at 45°C for minimum 24.0 hours. The sample was analysed in analytical HPLC to measure the level of enrichment of A21 deamidated variant in forced degradation condition. Processing of deamidated enriched material is pH adjusted to 7±0.1 using 1N NaOH. Conductivity of this solution was maintained at ≤ 3mS/cm and nal load was ltered through 1.2 and 0.45µm lters After attaining this level, the material was used for feed for 1st step of puri cation through Anion Exchange Chromatography. The other regular laboratory chemicals and chemicals required for chromatography were procured from quali ed vendors. The raw materials used for characterization studies were Hydrochloric acid (12N 37% Sigma Aldrich), Acetonitrile (J.T Baker (ACN)), Tri uoroacetic acid (Sigma Aldrich (TFA), C18 column (ACE), C8-column (Waters), Dithiothreitol (DTT), Glu-C (Endoproteinase, Roche), tris(hydroxymethyl) aminomethane (Sigma Aldrich (TRIS). Insulin phosphorylation in-vitro assay was performed using engineered CHO-K1 cells overexpressing insulin receptor B (IR-B).