Process for the production and purification of the collagenase enzyme from vibrio alginolyticus
The present invention claims a novel process for the production and purification of microbial collagenase (Microbial Collagenase EC 3.4.24.3) produced by the non-pathogenic aerobic bacterium Vibrio alginolyticus chemovar. iophagus (NCIMB Number: 1 1038, synonym LMG 3418, hereinafter called Vibrio alginolyticus ), which said process provides high production levels of collagenase with a stable, reproducible, cheap fermentation process. The collagenase produced from Vibrio alginolyticus according to the process described herein also presents a specific activity superior to that of other microbial collagenases, is stable in aqueous solution, and can be frozen without significant damage. A further subject of the present invention is pharmaceutical compositions containing collagenase obtained according to the production and purification process described, for the purpose of therapeutic treatment of disorders characterized by collagen accumulation or for the treatment of blemishes/imperfections that benefit from reducing local collagen accumulations.
1. A process for the production and purification of collagenase from Vibrio alginolyticus chemovar. lophagus , comprising the following stages:
Stage A: Inoculation of Vibrio Alginolyticus chemovar. lophagus into an Erlenmeyer flask and fermentation with culture broth of non-bovine animal origin;
Stage B: Clarification of the fermented broth thus obtained by tangential flow ultrafiltration with 100-500 kD Molecular Weight Cut-Off (MWCO) cassettes;
Stage C: Dialysis and concentration of the clarified medium obtained in stage B, by tangential flow ultrafiltration with 5-30 kD MWCO cassettes;
Stage D: Purification of the solution containing collagenase obtained in Stage C, by anion-exchange resin carrying weak basic groups, at a pH of between 6.9 and 7.4;
Stage E: Dialysis and concentration of the fractions with collagenolytic activity collected in Stage D, by tangential flow ultrafiltration with 10-50 kD MWCO cassettes;
Stage F: Purification of the solution thus obtained, by anion-exchange resin carrying strong basic groups, at a pH of between 6.9 and 7.4;
Stage G: Diafiltration and concentration of the fractions with collagenolytic activity ≧95% originating from stage F, by tangential flow ultrafiltration with 10-50 kD MWCO cassettes;
Stage H: Filtration of the solution containing collagenase thus obtained, through an 0.2 μm absolute filter, and storage at a temperature of between −20° and −80° C.;
wherein the thus obtained collagenase from Vibrio alginolyticus chemovar. lophagus being characterized by:
molecular weight 82 Kda;
specific activity between 1000 and 1800 nkat/mg;
purity between 98.0 and 100%;
no microbial or protein contaminants;
stability at a pH of between 5.5 and 11;
stability in aqueous solution at a temperature ranging between 4° and 40° C.;
stability in aqueous solution at 4° C. for 30 days;
stability in aqueous solution at a temperature ranging between −20° C. and −80° C. for 24-48 months;
lyophilisability to obtain a stable lyophilic powder; and
caseinase activity is less than 1.0 U/ml.
2. The process according to claim 1 , wherein the culture broth is of porcine animal origin or is a mixture of porcine and plant origin.
3. The process according to claim 1 or 2 , wherein the anion-exchange resin carrying the weak basic groups carries diethylaminoalkyl groups and the anion-exchange resin carrying strong basic groups carries quaternary ammonium groups.