Controlling the enzymatic digestion during the production of biologics is one of the key issues associated with proteolytic enzymes such as trypsin. Uncontrolled enzymatic activity results in generation of impurities which puts tremendous pressure on the purification tools to obtain therapeutic level of purity. Even though the inhibitors such as phenylmethyl sulfonyl fluoride, Soyabean trypsin inhibitor, etc. are commonly used in production strategies, complete inhibition is not achieved, thus laying a platform for the use of immobilized trypsin. The use of immobilized enzyme provides better control on enzymatic reactions and also on the traces of residual trypsin in the finished product. In the current work, recTryp has been immobilized using NHydroxysuccinimide (NHS) activated Biotin and Streptavidin coated magnetic (SCM beads). The coupling between recTryp and NHS activated biotin was performed at a ratio of 1:15 (w/w) in the presence of sodium carbonate buffer, pH 9.5. The excess biotin was removed by dialysis against PBS, followed by coupling between SCM beads and Biotinylated recTryp. The non-specifically adsorbed enzyme on the beads was removed by extensive washing against PBS. The activity of the same was identified by performing BSA digestion assay. The immobilized system can also be used in the insulin production.
