Method of downstream processing for conversion of human insulin precursor molecule into a functional human insulin protein molecule

A method of downstream processing for in-situ conversion of a protein precursor molecule having the amino acid sequence set forth in SEQ ID NO:

• 1 into a functional protein molecule, said method comprising:
• a) concentrating the protein precursor molecule through a tangential flow filtration cassette to obtain a concentrate of the protein precursor molecule and mixing the concentrate with an organic solution to obtain a retentate reaction mixture,
• b) incubating the retentate reaction mixture in a tosyl phenylalanyl chloromethyl ketone trypsin immobilized column for a period of time, and then passing the retentate reaction mixture through the tosyl phenylalanyl chloromethyl ketone trypsin immobilized column to obtain a protein ester, and
• c) mixing the protein ester with a deblocking buffer and purifying the protein ester using a hydrophobic interaction chromatographic column and a size exclusion chromatographic column to obtain the functional protein molecule having purity of at least 98.5%.