Andrew S. Friberg

The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation

Background. The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. Methods. Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. Results. Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. Conclusions. 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.

Human Islet Separation Utilizing a Closed Automated Purification System

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.

National Institutes of Health–Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities

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.

Large-Scale Comparison of Liberase HI and Collagenase NB1 Utilized for Human Islet Isolation

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.

Large-scale bioreactor expansion of tumor-infiltrating lymphocytes.

BACKGROUND The aim of this study was to evaluate an improved technique for expansion of tumor-infiltrating lymphocytes (TILs) based on the WAVE Bioreactor system with perfusion and tube-welding techniques. Our hypothesis was that the bioreactor would allow for optimized provision of nutrients and removal of spent media while minimizing culture volumes. These refinements might lead to a better quality of expanded cells with lower amounts of exhausted cells compared to static expansions in culture bags. PROCEDURES Tumor-infiltrating lymphocytes from 4 melanoma patients were expanded and compared in parallel using either the WAVE Bioreactor 2/10 System or traditional static culture methods. The parameters viability, final cell number, phenotype and effector function were measured. RESULTS Our results show that the bioreactor system with perfusion is suitable for large-scale expansion of tumor-infiltrating lymphocytes and allows for higher cell densities and absolute cell numbers as compared to static culture conditions. Phenotypic characteristics of TILs were compared pre and post expansion and showed no consistent difference between the two expansion methods. TILs harvested had the phenotype and function corresponding to intermediate to late effector cells. The system allows one technician to operate several bioreactors simultaneously, thereby reducing the labor for one expansion to approximately 1/3 compared to static expansion. DISCUSSION The WAVE Bioreactor system is suitable for large-scale expansion of TILs. Due to constant perfusion of fresh media and removal of spent media much higher cell densities were achieved while the culture volume and the glucose and glutamine levels were kept constant. Expansion of TILs in the bioreactor system represents a labor- and cost-effective method to reach large numbers of T cells for adoptive cell transfer therapy in the clinic. CONCLUSION The system presented herein offers an effective alternative to large-scale production of cell products for clinical use while meeting requirements of therapeutic cell quantities and qualities of current protocols for treatment of malignant melanoma.

Quantification of the Islet Product : Presentation of a Standardized Current Good Manufacturing Practices Compliant System With Minimal Variability

Background. Accurate islet quantification has proven difficult to standardize in a good manufacturing practices (GMP) approved manner. Methods. The influence of assessment variables from both manual and computer-assisted digital image analysis (DIA) methods were compared using calibrated, standardized microspheres or islets alone. Additionally, a mixture of microspheres and exocrine tissue was used to evaluate the variability of both the current, internationally recognized, manual method and a novel GMP-friendly purity- and volume-based method (PV) evaluated by DIA in a semiclosed, culture bag system. Results. Computer-assisted DIA recorded known microsphere size distribution and quantities accurately. By using DIA to evaluate islets, the interindividual manually evaluated percent coefficients of variation (CV%; n=14) were reduced by almost half for both islet equivalents (IEs; 31% vs. 17%, P=0.002) and purity (20% vs. 13%, P=0.033). The microsphere pool mixed with exocrine tissue did not differ from expected IE with either method. However, manual IE resulted in a total CV% of 44.3% and a range spanning 258 k IE, whereas PV resulted in CV% of 10.7% and range of 60 k IE. Purity CV% for each method were similar approximating 10.5% and differed from expected by +7% for the manual method and +3% for PV. Conclusion. The variability of standard counting methods for islet samples and clinical quantities of microspheres mixed with exocrine tissue were reduced with DIA. They were reduced even further by use of a semiclosed bag system compared with standard manual counting, thereby facilitating the standardization of islet evaluation according to GMP standards.

Vitacyte collagenase HA: a novel enzyme blend for efficient human islet isolation.

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

Transplanted Functional Islet Mass: Donor, Islet Preparation, and Recipient Factors Influence Early Graft Function in Islet-After-Kidney Patients

Background. The ability to predict clinical function of a specific islet batch released for clinical transplantation using standardized variables remains an elusive goal. Methods. Analysis of 10 donor, 7 islet isolation, 3 quality control, and 6 recipient variables was undertaken in 110 islet-after-kidney transplants and correlated to the pre- to 28-day posttransplant change in C-peptide to glucose and creatinine ratio (&Dgr;CP/GCr). Results. Univariate analysis yielded islet volume transplanted (Spearman r=0.360, P<0.001) and increment of insulin secretion (r=0.377, P<0.001) as variables positively associated to &Dgr;CP/GCr. A negative association to &Dgr;CP/GCr was cold ischemia time (r=−0.330, P<0.001). A linear, backward-selection multiple regression was used to obtain a model for the transplanted functional islet mass (TFIM). The TFIM model, composed of islet volume transplanted, increment of insulin secretion, cold ischemia time, and exocrine tissue volume transplanted, accounted for 43% of the variance of the clinical outcome in the islet-after-kidney data set. Conclusion. The TFIM provides a straightforward and potent tool to guide the decision to use a specific islet preparation for clinical transplantation.

Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation

Background Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure. Methods Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age. Results There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases. Conclusions The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Human islet isolation processing times shortened by one hour : minimized incubation time between tissue harvest and islet purification

Human Islet Isolation Processing Times Shortened By One Hour : Minimized Incubation Time Between Tissue Harvest and Islet Purification