Why the Adoption of Ancillary Material Standards is Important for Cell Therapy

Bob McCarthy

Windermere’s “Buying Hierarchy” model categorizes the life cycle of a product into four categories: functionality, reliability, convenience, and price.(1) At the product launch, the customer asks, “Is it doing what it is supposed to do?” (functionality/performance) and whether it is reliable (i.e., does it work all the time?). As the customer becomes familiar with the product, convenience becomes essential (i.e., is it easy to use?). As similar products enter the market, the customer evaluates products primarily on convenience and price, making them commodities.

The biggest challenge for developing cell therapeutic products or any new medical procedure is proving it works.

Importance of Collagenase-Protease Enzyme Mixtures for Human Islet Isolation

For allo-islet transplantation, the first report describing the treatment of an adult type 1 diabetic patient was in 1977.(2) Twenty-three years later, the University of Alberta reported that all seven patients who received allo-islet transplantation to treat refractory type 1 diabetes remained insulin-independent a year after transplant.(3) The “Edmonton Protocol” was hailed as a breakthrough in managing these patients since, before this report, only 8.2% of 267 patients remained insulin-independent for more than one year.

What were the keys to Edmonton’s success? First, the immunosuppressive regimen used to treat these patients was changed, avoiding glucocorticoids and combining tacrolimus, sirolimus, and daclizumab, which inhibited T lymphocyte activation. And second, an important but unrecognized improvement was using a specifically designed, purified collagenase-protease enzyme mixture for human islet isolation. Development of the Liberase™ HI Purified Enzyme Blend at Boehringer Mannheim Biochemicals in 1994 enabled islet isolators to use a consistent collagenase-protease enzyme mixture to recover sufficient islets for transplantation. Before 1994, these scientists spent significant time screening different lots of collagenase products to “qualify” a lot that gave acceptable islet yields from human pancreas.(4)

A change in the manufacturing procedure in the early 2000s led to suboptimal performance and instability of Liberase HI. This change significantly impacted the NIH-funded Clinical Islet Transplantation Consortium (CITC) clinical trial to assess human islet transplantation’s utility in treating adult type 1 diabetic patients. The managers of this trial dropped the use of Liberase HI because of a potential threat of transmission of spongiform encephalitis, as bovine proteins were used in the culture media to generate collagenase from Clostridium histolyticum. However, the switch to a different enzyme supplier did not overcome the problem of suboptimal islet yields. (4) Analysis of the purified collagenase products used to recover human islets for transplantation showed that Liberase HI and these new enzymes contained degraded C. histolyticum class I collagenase. The problem was overcome when collagenase-protease mixtures containing intact class I collagenase achieved results equivalent or superior to those obtained with the original Liberase HI product.(5, 6)

The key lesson from this illustration was that the characterization of the collagenase-protease enzymes is essential to improve the outcome of human islet isolation. Recovery of islets after enzyme-mediated tissue dissociation is the critical go-no-go step for islet transplantation, and more importantly, increasing the mass of islets transplanted into the patient directly affects the likelihood of insulin independence.

The CITC clinical trial aimed to establish a standardized isolation process that all islet isolation manufacturing sites could adopt. Once adopted, participants in the trial could elect to file a biological license application for this treatment with the FDA.

However, 52.5% of the islet isolations performed as part of the CITC 07 clinical trial were used for subsequent transplantation.(7) This percentage is similar to those achieved at experienced islet isolation labs before implementing the CITC isolation protocols.(8) Human islet isolation is an expensive procedure. Estimates from the GRAGIL Consortium in 2004 reported the cost of performing 5.6 islet isolations to obtain sufficient islet yields for one transplantation was € 23,775.(9) Assuming that the percentage of islet isolations has not changed since 2004, the costs today are estimated at € 40,507, assuming an average inflation rate of 2.7%. This cost does not include organ procurement and supply, which cost $25,000 to $35,000 per pancreas in the US.

Three variables can account for the percentage success rate:

1. Variability of the collagenase-protease enzyme mixtures used for islet isolation
2. The unique characteristics of each donor organ
3. The skill of the islet isolation team since these procedures rely on the expertise of an experienced islet isolator

One Step to Improve the Reliability of Human Islet Isolation

How do you systematically improve human islet isolation or any other cell isolation manufacturing process that uses collagenase enzymes? You begin with the simplest element you can control, the collagenase-protease enzyme mixtures used in cell isolation.

Human islet isolators can improve the reliability of their isolation processes by adopting and following voluntary consensus standards (VCS) published by Standards Development Organizations. Section 3036 of the 21^st Century Cures Act charged the FDA to adopt the appropriate standards to accelerate the approval of regenerative medicine therapies. The FDA also funded a Standards Coordinating Body to support community efforts to develop regenerative medicine standards and to maintain a web portal that lists standards applicable to regenerative medicine. In October 2023, the FDA published a Guidance for Industry on this topic.

The International Standards Organization recently published an International Standard on Ancillary Materials (ISO 20399). ISO defines ancillary materials (AM) as “materials that come in contact with the cellular therapeutic product during cell processing but are not intended to be part of the final product formulation.”

Adopting and applying the ISO 20399 Standard for human islet isolation requires understanding the characteristics of the enzymes used in the islet isolation process. These enzymes impact islet yield, viability, and in vitro function. The enzyme supplier should provide the appropriate physicochemical and functional characteristics of the collagenase and protease enzymes (i.e., AM Supplier). The islet isolator is responsible for qualifying these products for their intended use. Here, the islet isolator and AM supplier must have a “cooperative and transparent” relationship to set product specifications that reflect the product’s intended application. If this degree of relationship does not exist, additional risk to the success of the islet isolation process can occur, as illustrated by the problems described above in the CITC clinical trial.

Additionally, the US Pharmacopeia published USP <1043> on Ancillary Materials. The accompanying blog post compares and contrasts the content of the USP <1043> and ISO 20399 Ancillary Materials Standards.

Adopting Standards for ancillary material for human islet isolation will minimize the effect of variability of collagenase-protease enzymes on the outcome of the isolation process. Additional work on the qualification of donor pancreata before performing an islet isolation and the development of more rigorous training of islet isolators should improve the reliability and cost-effectiveness of this cell therapy. These improvements should increase the reliability of human islet isolation performed to generate islets for transplantation, improving the cost-effectiveness of these procedures and enhancing the quality of life for these patients.

References

1. Christensen CM. Innovator’s Dilemma, When New Technologies Cause Great Firms to Fail. Boston MA: Harvard Business School Press; 1997.
2. Najarian JS, Sutherland DE, Matas AJ, Steffes MW, Simmons RL, Goetz FC. Human islet transplantation: a preliminary report. Transplant Proc. 1977;9:233-6.
3. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343:230-8.
4. 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.
5. 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.
6. 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.
7. Ricordi C, Goldstein JS, Balamurugan AN, Szot GL, Kin T, Liu C, et al. National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities. Diabetes. 2016;65:3418-28.
8. Sakuma Y, Ricordi C, Miki A, Yamamoto T, Pileggi A, Khan A, et al. Factors that affect human islet isolation. Transplant Proc. 2008;40:343-5.
9. Guignard AP, Oberholzer J, Benhamou PY, Touzet S, Bucher P, Penfornis A, et al. Cost analysis of human islet transplantation for the treatment of type 1 diabetes in the Swiss-French Consortium GRAGIL. Diabetes Care. 2004;27:895-900.