Method of obtaining a purified, biologically active heterologous protein

A process of obtaining a recombinant insulin glargine having purity of at least 96% and containing less than 1% glycosylated impurity by a yeast expression system, said process comprising steps of:

• a) culturing Pichia pastoris transformed by a vector containing a DNA sequence defined by formula X—B—Y-A encoding insulin glargine precursor wherein,
• X is a leader peptide sequence comprising at least one amino acid,
• B is the amino acid sequence of the B chain of the insulin molecule, its derivatives or analogs,
• Y is a linker peptide of amino acid sequence set forth as SEQ ID NO. 3,
• A is the amino acid sequence of the A chain of the insulin molecule, its derivatives or analogs,
• and the A and B chain can be modified by amino acid substitution, deletion and/or addition;
• b) recovering the yeast expressed insulin glargine precursor, said recovering comprises separating the insulin glargine precursor from the yeast to obtain a recovered insulin glargine precursor preparation;
• c) optionally subjecting the recovered preparation of step (b) to a step of crystallization,
• d) subjecting the recovered preparation of step (b) or crystals of step (c) to enzymatic conversion at pH ranging from about 6 to about 10, in presence of trypsin or trypsin like enzyme and water miscible organic solvent in the concentration ranging from about 40% to about 60%, to obtain insulin glargine containing at least one related impurity;
• e) purifying said insulin glargine containing at least one related impurity, said purifying comprises contacting the said insulin glargine with a RP-HPLC chromatographic matrix wherein multiple purification steps are carried out by employing:
• i) a first step of equilibrating the matrix with acetonitrile at a concentration of about 10%, in acetic acid at a concentration of about 250 mM;
• followed by eluting the said insulin glargine with said acetonitrile;
• ii) a second step of re-equilibrating the matrix with acetonitrile at a concentration of about 10%, in an organic acid buffer A at concentration ranging from about 20 mM to about 200 mM at pH ranging from about 3 to about 8.5, and re-eluting the said insulin glargine with said acetonitrile;
• and
• iii) a third step of re-equilibrating the matrix with ethanol at a concentration of about 6%, in an organic acid buffer A at concentration ranging from about 10 mM to about 50 mM;
• and re-eluting the said insulin glargine with said ethanol;
• and
• f) precipitating the purified insulin glargine of step (e) by adding a combination of citric acid buffer and zinc chloride at pH ranging from about 6 to about 8 to obtain the insulin glargine having purity of at least 96% and containing less than 1% glycosylated impurity.