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Evaluation of various purified components in the development of an application-specific enzyme formulation for the isolation of porcine islets

Mike Green, Kelley Nonweiler, Andrew Breite, Francis Dwulet, Robert McCarthy

VitaCyte, Indianapolis, IN, United States

Our efforts to develop a robust, reproducible enzyme formulation for isolating porcine islets led us to assess the efficacy of several potential purified components on selected outcome parameters. Pancreata obtained from 18-36 month old retired Landrace breeders were processed using a standardized Ricordi islet isolation procedure. The enzyme candidates tested fell within one of three broad categories, including Class 1 collagenase (C1), Class 2 collagenase (C2), and neutral protease. Enzymatic activities of the components were normalized per gram of tissue using internally developed assays for collagen degradation, neutral protease, and BAEE activity. Quantified outcome parameters included percent undigested tissue, switch times, packed cell volume (PCV) and islet equivalents per gram (IEQ/g). The molecular form of recombinant C1 [ i.e. intact (rC1) vs truncated C1 (rC1c)] had no effect on PCV when the collagen degrading activity was targeted at 25,000 U/g. However, achieving such target activities required over 14-fold higher (P < .0001) enzyme masses with rC1c. Similarly, IEQ/g did not differ between isolations performed with rC1 and rC1c, but percent undigested tissue was lower (P < .05) in isolations performed with C1c (23.5% vs 31.8%). Recombinant C2 (rC2) targeted at 7.5 Wunsch units/g caused a 36% reduction (P < .001) in undigested tissue, an approximate 5 minute quicker (P < .001) switch time and a 55% increase (P < .01) in PCV relative to naturally-derived C2 at the same target levels. Clostripain, purified from C. histolyticum, reduced (P < .01) the amount of undigested tissue by over 20% when targeted at 25 activated BAEE units/g. No differences were observed between isolations that utilized a neutral protease sourced from B. polymyxa or C. histolyticum for any parameter measured. Within the context of porcine islet isolation, these data 1) indicate that total enzyme activity is more important than protein mass itself; 2) suggest that recombinant C2 has some yet to be identified competitive advantage over naturally-sourced C2; 3) stress the value of targeting multiple enzyme activities in the development of an optimal application-specific enzyme formulation which can only be accomplished by the use of purified sources of the various components. Currently, we are utilizing the above results in a DOE where multiple levels of each factor (rC1, rC2, clostripain, a neutral protease source) are being evaluated to identify the formulation which maximizes islet yield.