Robert McCarthy
Author’s note: I will make a similar presentation to the one cited below as part of VitaCyte’s webinar series at 11 AM EST on Thursday, December 11, 2025
Importance of Controlling Critical Ancillary Materials
Ancillary materials (AMs) come in contact with the cellular therapeutic products during cell processing but are not intended to be part of the final product formulation. For human islet isolation, the Clostridium histolyticum collagenase and neutral protease enzymes are AMs. Applying a Quality by Design approach to islet manufacturing treats tissue dissociation as a critical process parameter, with the critical quality attributes of the collagenase and protease mixtures crucial to the success of islet isolation. The enzyme composition and dose impact
- speed of tissue dissociation,
- pre-purification islet recovery,
- post-purification islet yield,
- cell viability and function,
- recovery after short-term cell culture.
I recently gave a presentation at a VitaCyte-sponsored industry symposium at the Cell Transplantation and Regenerative Medicine meeting held in Tokyo last month on the “ISO-20399 Standard for Ancillary Material: Relevance to Tissue Dissociation Enzymes.” The presentation focused on Section 11 of ISO 20399, which addresses change management.
Case Study: New Insights Gained From the Clinical Islet Transplantation Consortium Clinical Trial
The case study reviewed the impact of switching from Liberase™ HI to the Nordmark purified collagenase and protease enzymes in 2007 for use in the Clinical Islet Transplantation Consortium (CITC) clinical trial. Three of the most experienced islet transplant centers that successfully replicated the Edmonton Protocol as part of the Immune Tolerance Network clinical trial were unable to isolate enough islets for transplantation when using the Nordmark enzyme products.
A collaboration between VitaCyte and Bernhard Hering’s lab at the University of Minnesota (UMN) led to a series of human islet isolations demonstrating the importance of using intact class I (C1) collagenase in the C1 & class II (C2) collagenase-protease mixtures used for human islet isolation.(1) Further studies summarized results from 94 islet isolations performed at UMN, confirming the importance of using intact C1 in C1-C2-neutral protease mixtures and highlighting the need to select an appropriate neutral protease. In these experiments, Nordmark’s C. histolyticum neutral protease (NB Protease) was superior to Thermolysin in terms of islet yields and morphology when a split pancreas was used to compare the effectiveness of each enzyme mixture. These results led UMN to recommend the use of a “new enzyme mixture” (NEM) that contains VitaCyte’s Collagenase HA and Nordmark’s NB Protease.(2)
These observations led AM Suppliers to improve the collagenase enzymes used for islet isolation. All the products sold today contain predominantly intact class I collagenase.(3) VitaCyte’s BP Protease has higher purity than Nordmark’s NB protease and can substitute for NB Protease in the NEM.(4)
Need for Continuous Improvement of Enzymes Used for Human Islet Isolation
The case study above emphasizes the need for testing new enzyme mixtures to continuously improve human islet isolation. Camillo Ricordi was the catalyst for funding an R&D project at Boehringer Mannheim in 1991 after meeting with senior management and urging them to help realize the promise of islet transplantation to treat adult diabetic patients at Boehringer Mannheim to fund an R&D project.(5) Today, VitaCyte is poised to collaborate with any interested islet isolators to test new enzyme formulations that will improve islet yield and function (If you want to collaborate on a project improving enzyme formulations for human islet isolation, please contact me directly at rcmccarthy@vitacyte.com).
The two challenges islet manufacturing groups face today are increasing islet recoveries from younger organ donors and from pancreata recovered from patients with chronic pancreatitis as part of the total pancreatectomy-islet auto-transplant (TP-IAT) procedure. In the former application, younger individuals now represent a predominant category of organ donors because of the opioid crisis. Younger donors have higher percentages of embedded islets, which translates into failed islet isolations, as these islets cannot be purified.(6) In patients undergoing TP-IAT, insulin independence directly correlates with the mass of islets infused.(7)
Allo-islet and auto-islet transplantation continues to grow. The last Collaborative Islet Transplant Registry Reports showed that allo islet transplants grew at ≈ 10% cumulative average growth rate from 2005 to 2017, whereas auto-islet transplants grew at ≈ 17.5% rate from 2009 to 2019.
Three factors impact the success of human islet isolation:
- Quality of the pancreata
- Technical expertise of the islet isolator
- Optimization of the collagenase-protease enzyme mixtures
To apply a total quality management approach using QbD principles to improve a manufacturing process, you need to start with a controllable factor. Adopting purified, well-characterized collagenase and protease enzymes is the best place to start. This action also aligns with statement in Section 11 of the ISO 20399 Standard to protect the security of supply of the collagenase-protease enzyme products used for tissue dissociation.
References
- Balamurugan AN, Breite AG, Anazawa T, Loganathan G, Wilhelm JJ, Papas KK, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplant. 2010;89:954-61.
- Balamurugan AN, Loganathan G, Bellin MD, Wilhelm JJ, Harmon J, Anazawa T, et al. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplant. 2012;93:693-702.
- Loganathan G, Hughes MG, Szot GL, Smith KE, Hussain A, Collins DR, et al. Low cost, enriched collagenase-purified protease enzyme mixtrues successfully used for human islet isolation. OBM Transplantation. 2019;3(2). Available from https://www.lidsen.com/journals/transplantation/transplantation-03-02-064
- Balamurugan AN, Green ML, Breite AG, Loganathan G, Wilhelm JJ, Tweed B, et al. Identifying effective enzyme activity targets for recombinant class I and class II collagenase for successful human islet isolation. Transplant Direct. 2016;2:e54.
- McCarthy RC, Green ML, Dwulet FE. Evolution of enzyme requirements for human islet isolation. OBM Transplantation. 2018; 2: Available from: http://www.lidsen.com/journals/transplantation/transplantation-02-04-024.
- Loganathan G, Subhashree V, Narayanan S, Tweed B, Andrew Goedde M, Gunaratnam B, et al. Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix. Am J Transplant. 2019; 19:831-43.
- Bellin MD, Beilman GJ, Sutherland DE, Ali H, Petersen A, Mongin S, et al. How durable Is total pancreatectomy and intraportal islet cell transplantation for treatment of chronic pancreatitis? J Am CollSurg 2019;228:329-39.