Process for preparation of insulin compounds

摘要

The present invention relates to the preparation of insulin compounds including their analogs or derivatives thereof from their corresponding precursor forms by a one step enzymatic reaction involving the combinatorial and concurrent use of optimal quantities of trypsin and carboxypeptidase B that work synergistically directing the reaction in a controlled manner to avoid production of random undesired byproducts. Particularly, the enzymatic conversion reactions of the instant invention offer advantages of reduction in the number operational steps, higher yield and purity of the desired end products.

主权项

1. A process for converting insulin aspart, insulin lispro, and insulin glulisine from their corresponding insulin precursors represented by the formula:where,
R is hydrogen or a chemically or enzymatically cleavable amino acid residue or a chemically or enzymatically cleavable peptide comprising at least two amino acid residues; R1 is OH or an amino acid residue, or Y1-Y2 in which Y is an amino acid residue; the moieties A1 to A21 corresponds to insulin A chain and the moieties B1 to B27 corresponds to insulin B chain including amino acid substitution, deletion and/or additions thereof; R2 is Z1-Z2 wherein Z1 is selected from Pro, Lys, Asp and Z2 is selected from Lys or Pro or Glu or Z1-Z2-Z3 wherein Z1 is selected from Pro, Lys, Asp and Z2 is selected from Lys or Pro or Glu and Z3 is threonine or a peptide moiety of at least three amino acid residues with the provision that the amino acid corresponding to B30 is threonine; X is a polypeptide that connects the A chain to the B chain which can be cleaved enzymatically without disrupting either the A chain or the B chain bearing at least two amino acids wherein the first and the last amino acid is Lysine or Arginine, said precursor defined by an amino acid sequence which is at least 95% homologous to the amino acid sequence as set forth in SEQ IDs 1, 2, or 3, wherein A and B chains of such precursor are identical to A and B chains of aspart; or said precursor defined by an amino acid sequence which is at least 95% homologous to the amino acid sequence as set forth in SEQ IDs 4, 5 or 6, wherein A and B chains of such precursor are identical to A and B chains of lispro; or said precursor defined by an amino acid sequence which is at least 95% homologous to the amino acid sequence as set forth in SEQ IDs 7, 8 or 9, wherein A and B chains of such precursor are identical to A and B chains of glulisine; and wherein the process consists of treating said precursor at pH of about 7.0 to 9.0 with trypsin and carboxypeptidase used combinatorially and concurrently provided that the relative concentration ratio of trypsin to carboxypeptidase is from 5:6 to 50:1 to yield at least 75% of aspart, lispro or glulisine respectively devoid of impurities des-octapeptide and des-threonine.