Comparative study on enzymatic activity and molecules stability of some commercial proteolytic enzymes used in pancreatic islet isolation.

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (COLL G and COLL H). They are used in a defined tissue dissociation enzyme (TDE) mixtures together neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). The TDE mixtures were in part responsible for the success of the Edmonton protocol; however, just to now, people working in islets purification found discrepancy in an application to another one. This variability in application see in the enzymatic blend composition the higher accused, such as the contamination from endotoxines due to extractive production methods to obtain them. Using electrophoresis and gelatin zymography approaches; together densitometry evaluation assays we compared in composition, stability and auto-digestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have investigated about the stability of analyzed enzymes when they are in solution in relation to their storage processes at different temperatures (-20°C; 0°C and r.t.); such as, concerning their digestive activity when applied at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results shown an heterogeneous composition of the different blend of collagenases from Roches and Serva, furthermore, heterogeneity is observed by lot to lot; on the other hand, we have found several more proteins and/or fragments respect the HPLC profiles publish by vendors. In gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, in neutral protease from Serva contaminants with gelatinolytic activities were detected; not in thermolysin. In stability experiments we investigated an inactivation/auto-digestive processes of analyzed enzymes; nevertheless, at different working temperatures, a more stable activity of analyzed molecules at 25°C were observed; activity that can be compared to that detectable at 37°C at the beginning of digestive process, at this temperature in the times inactivation enzymes were observed. These data take together strongly imply a not controlled digestive processes due to several contaminants present in the blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities mean the impossibility to control the islets digestion process due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.

In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.