Eight manufacturing facilities participating in the National Institutes of Health–sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.
We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.
A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 +/- 1.1% vs. 98.2 +/- 2.0%, p < 0.05). Islet yield (120,468 +/- 15,970 vs. 114,570 +/- 15,313 IE, NS) and purity (51.7 +/- 4.8% vs. 54.4 +/- 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.
For more than a decade Liberase HI was commonly used as the standard enzyme blend for clinical human islet isolation until enforced replacement by collagenase NB1 (NB1). This change resulted initially in a reduction in islet isolation outcome and transplant activities worldwide. This retrospective study was initiated to compare the efficiency of NB1 premium grade with Liberase in 197 human islet isolations. All pancreata were processed between January 2006 and June 2008 utilizing the same procedures for isolation and quality assessment except the administration of preselected lots of either Liberase (n = 101) or NB1 (n = 96). Utilizing Liberase, significantly more digested tissue and purified islet yield was produced compared to NB1. In contrast, the use of NB1 was associated with significantly higher purity and glucose stimulation index during dynamic perifusion. The expression of proinflammatory markers was almost identical except tissue factor expression, which was higher after utilization of Liberase. No difference was found in the percentage of pancreata fulfilling the criteria for clinical islet transplantation. The results suggest that Liberase is more efficient for pancreas dissociation than collagenase NB1 but seems to be more harmful to exocrine cells and islet tissue.
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