Joshua J. Wilhelm

A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products

Background. The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). Methods. We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. Results. The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510±2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681±147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893±181,329 total IEQ and 5979±1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. Conclusions. A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay.

Background. Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. Methods. Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. Results. These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100±150,900 islet equivalent number (IEQ) (4147±1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500±152,400 IEQ (2134±1524 IEQ/g pancreas). Conclusions. These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.

Regulation of hepatocyte cell cycle progression and differentiation by type I collagen structure.

Cell behavior is strongly influenced by the extracellular matrix (ECM) to which cells adhere. Both chemical determinants within ECM molecules and mechanical properties of the ECM network regulate cellular response, including proliferation, differentiation, and apoptosis. Type I collagen is the most abundant ECM protein in the body with a complex structure that can be altered in vivo by proteolysis, cross-linking, and other processes. Because of collagens complex and dynamic nature, it is important to define the changes in cell response to different collagen structures and its underlying mechanisms. This chapter reviews current knowledge of potential mechanisms by which type I collagen affects cell behavior, and it presents data that elucidate specific intracellular signaling pathways by which changes in type I collagen structure differentially regulate hepatocyte cell cycle progression and differentiation. A network of polymerized fibrillar type I collagen (collagen gel) induces a highly differentiated but growth-arrested phenotype in primary hepatocytes, whereas a film of monomeric collagen adsorbed to a rigid dish promotes cell cycle progression and dedifferentiation. Studies presented here demonstrate that protein kinase A (PKA) activity is significantly elevated in hepatocytes on type I collagen gel relative to collagen film, and inhibition of this elevated PKA activity can promote hepatocyte cell cycle progression on collagen gel. Additional studies are presented that examine changes in hepatocyte cell cycle progression and differentiation in response to increased rigidity of polymerized collagen gel by fiber cross-linking. Potential mechanisms underlying these cellular responses and their implications are discussed.

Islet autotransplantation to preserve beta cell mass in selected patients with chronic pancreatitis and diabetes mellitus undergoing total pancreatectomy.

Objectives Islet autotransplantation (IAT) is performed in nondiabetic patients with chronic pancreatitis at the time of total pancreatectomy (TP) to minimize risk of postoperative diabetes. The role of TP-IAT in patients with chronic pancreatitis and C-peptide–positive diabetes is not established. We postulate that IAT can preserve beta cell mass and thereby benefit patients with preexisting diabetes undergoing TP. Methods Preoperative metabolic testing, islet isolation outcomes, and subsequent islet graft function were reviewed for 27 patients with diabetes mellitus and chronic pancreatitis undergoing TP-IAT. The relationships between the results of preoperative metabolic testing and islet isolation outcomes were explored using regression analysis. Results Mean islet yield was 2060 (SD, 2408) islet equivalents/kg. Peak C-peptide (from mixed meal tolerance testing) was the strongest predictor of islet yield, with higher stimulated C-peptide levels associated with greater islet mass. Half of the patients who had C-peptide levels measured after transplantation demonstrated C-peptide production at a level that conveys protective benefit in type 1 diabetes (≥0.6 ng/mL). Conclusions These findings provide proof of concept that significant islet mass can be isolated in patients with chronic pancreatitis and C-peptide–positive diabetes mellitus undergoing TP-IAT. Stimulated C-peptide may be a useful marker of islet mass before transplantation in these patients.

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.

Proposed thresholds for pancreatic tissue volume for safe intraportal islet autotransplantation after total pancreatectomy.

The simple question of how much tissue volume (TV) is really safe to infuse in total pancreatectomy–islet autotransplantation (TP–IAT) for chronic pancreatitis (CP) precipitated this analysis. We examined a large cohort of CP patients (n = 233) to determine major risk factors for elevated portal pressure (PP) during islet infusion, using bivariate and multivariate regression modeling. Rates of bleeding requiring operative intervention and portal venous thrombosis (PVT) were evaluated. The total TV per kilogram body weight infused intraportally was the best independent predictor of change in PP (ΔPP) (p < 0.0001; R2 = 0.566). Rates of bleeding and PVT were 7.73% and 3.43%, respectively. Both TV/kg and ΔPP are associated with increased complication rates, although ΔPP appears to be more directly relevant. Receiver operating characteristic analysis identified an increased risk of PVT above a suggested cut‐point of 26 cmH2O (area under the curve = 0.759), which was also dependent on age. This ΔPP threshold was more likely to be exceeded in cases where the total TV was >0.25 cm3/kg. Based on this analysis, we have recommended targeting a TV of <0.25 cm3/kg during islet manufacturing and to halt intraportal infusion, at least temporarily, if the ΔPP exceeds 25 cmH2O. These models can be used to guide islet manufacturing and clinical decision making to minimize risks in TP–IAT recipients.

Prediction of pancreatic tissue densities by an analytical test gradient system before purification maximizes human islet recovery for islet autotransplantation/allotransplantation.

Background. Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. Methods. Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. Results. Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. Conclusions. Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.

Metabolic assessment prior to total pancreatectomy and islet autotransplant: Utility, limitations and potential

Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass ≥2500 IEQ/kg. The acute C‐peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values −0.33 to −0.40, p ≤ 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C‐peptide on MMTT ≥4 ng/mL were associated with greater odds of receiving an islet mass ≥2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87–1.0; OR 7.9 for C‐peptide, CI 1.75–35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C‐peptide ≥4 ng/mL on MMTT conveyed eight times the odds of receiving ≥2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.

Islet size index as a predictor of outcomes in clinical islet autotransplantation.

Background The islet size distribution in a preparation may contribute to islet transplant outcomes. At the same islet equivalent (IE) dose, larger islets may exhibit poorer therapeutic value and this may be because of oxygen diffusion limitations that worsen in proportion to islet size. Methods To test this hypothesis, we studied the impact of islet size index (ISI) and other islet product characteristics on outcomes after islet autotransplant (IAT) in recipients receiving a marginal islet dose (2000–4999 IEs per kg body weight) from January 1, 2009 to June 11, 2012, at the University of Minnesota (n=58). ISI was defined as the number of IE divided by the number of islet particles (IPs) in a preparation; an ISI less than 1 indicates a mean islet diameter that is less than 150 &mgr;m. The primary post-IAT outcome was 6-month insulin use status. Results Logistic regression analysis indicate that IPs/kg (P=0.001), IEs/kg (P=0.019), total IPs transplanted (P=0.040), and ISI (P=0.074) were most strongly correlated with the primary outcome. The ISI (mean±standard error) was lower for recipients achieving insulin independence at 6 months (0.71±0.05) versus those partially (0.83±0.05) or completely (1.00±0.07) insulin dependent. The combination of islet dose (expressed as units IPs/kg) and ISI exhibited a sensitivity of 75% and specificity of 74% in predicting insulin independence in this population of patients. Conclusion Islet autotransplant recipients of a marginal islet doses were more likely to achieve insulin independence when transplanted with a greater number of smaller islets.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

Background Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. Methods We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Results Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. Conclusions A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.