Thermolysin & human islet isolation: Truth vs. Fiction

More human islet isolations have been performed using purified collagenase and thermolysin than any other purified enzyme mixture (1). Several reports have shown the adverse effects when using thermolysin for human islet isolation (2, 3). This blog post reviews the truth and fiction when using thermolysin for human islet isolation. It will also briefly discuss the recent change in thermolysin made by the supplier to an animal origin-free product.

Choice of thermolysin in Liberase HI formulation based on cost, not performance: Cost was the primary factor for use of thermolysin in Liberase™ HI Purified Enzyme Blend. Purified collagenase and neutral protease are required to release islets from pancreatic tissue. Initially, the goal of the Liberase project team was to purify Clostridium histolyticum neutral protease (CHNP) from the same C. histolyticum raw material used as a source of the collagenase. However, this enzyme was difficult to purify and was unstable. The assumption was other neutral proteases would be as effective as CHNP for human islet isolation. Purified thermolysin and Dispase™ were found to have acceptable purity and stability with thermolysin chosen based on cost. Dose-response experiments correlating Liberase HI mass to islet yields showed that the use of 0.75, 1.5, or 3.0 mg of Liberase HI per mL of solution did not affect human islet yields (4).

Importance of the extinction coefficient in translation mass of enzyme to its activity: The Liberase HI enzyme formulation used since 2001 contained about 11.5 mg of thermolysin and  500 mg of purified collagenase. Participants of Immune Tolerance Network islet transplantation trial evaluated results from many human islet isolations before arriving at a consensus to use this dose. The protein concentration of thermolysin was determined using a 0.1% extinction coefficient at A280 nm = 1.1 (e0.1% 280 nm = 1.1). Here, 1 mg/mL neutral pH solution from multiple lots of thermolysin was prepared, the A280 nm for each solution determined, with the mean A280 nm  = 1.1. The extinction coefficient is a critical parameter for characterizing enzymes since the mass of protein (i.e., enzyme) is directly related to total enzyme activity. The specific activity of an enzyme is the enzyme units per mg of material (U/mg).

VitaCyte also used e0.1% 280 nm = 1.1 to determine the mass of thermolysin product to be consistent with the thermolysin concentration used for Liberase HI. In contrast, Roche now uses a different extinction coefficient for thermolysin: e0.1% 280 nm = 1.76 (reported on their Thermolysin Certificate of Analysis). This change occurred when they introduced the Liberase MTF kit. It translates to a 60% increase in the mass of protein per mg when compared to the Liberase HI or VitaCyte Thermolysin products. VitaCyte verified this fact after analysis of the enzyme activity of Roche MTF or VitaCyte Thermolysin and calculation of their specific activities using e0.1% 280 nm = 1.1. Both enzymes gave equivalent specific activities. Based on these data, a 15 mg bottle of the MTF Thermolysin product contained neutral protease activity equivalent to about 24 mg of Liberase HI or VitaCyte Thermolysin activity.

Consequences of this difference in thermolysin activity between suppliers: The change in the concentration of thermolysin led many laboratories to unknowingly add excessive amounts of thermolysin activity when using the Roche MTF kit. For those who view a kit as a “ready to use product,” some isolators likely used the entire 15 mg bottle for human islet isolation. However, others realizing thermolysin’s strong protease activity, either reduced the dose of thermolysin(5, 6) used for islet isolation or replaced it with another neutral protease enzyme (2, 7-9).

Other neutral protease enzymes for human islet isolation: A.N. Balamurugan (i.e., Bala) and colleagues achieved respectful results when using VitaCyte’s thermolysin with purified collagenase for human islet isolation (10). However, Bala believed that CHNP was superior to thermolysin for human islet isolation based on his earlier work with Rita Bottino at the University of Pittsburgh where they showed the best quality islets were those isolated using Roche’s Collagenase P product. Bala and his University of Minnesota (UMN) colleagues prepared a “new enzyme mixture” (NEM), composed of  VitaCyte’s Collagenase HA with Serva/Nordmark’s NB Protease (CHNP) (2). The enzyme dose used for isolation was 18-20 Wunsch U of collagenase and 1.25-1.5 DMC U of protease per g of trimmed pancreas. Analysis of results from 90 islet isolations using eight different enzyme mixtures showed that islets isolated with NEM gave shorter digestion times, higher recovery of purified islets expressed as total IEQ/pancreas and per g pancreas than the majority of enzyme mixtures compared in this report. Further experiments compared the effectiveness of CHNP or thermolysin mixed with a fixed dose of VitaCyte’s Collagenase HA to recover islets from a split human pancreas (2). These results showed that NEM gave significantly higher total purified islet yields and percentage of islets with mean diameters > 200 μm. Analysis of these data, expressed as IEQ per g pancreas, was not significantly different likely because of the small number of experiments performed to compare the performance of these enzymes.

Further collaborative studies with Bala and his UMN colleagues with VitaCyte led to the evaluation of VitaCyte’s purified BP Protease (a Dispase™ equivalent enzyme) for human islet isolation. The BP Protease activity used per g of tissue (23,400 neutral protease U) was equivalent to the activity used in an updated NEM formulation (1.75 DMC U/g tissue). The initial studies used a fixed amount of BP Protease with one of four different doses of recombinant C. histolyticum class I and class II. These results showed that collagenase dose did not affect the recovery of functional islets from the body-tail of human pancreas (8). Further studies by Bala and colleagues at the University of Louisville using intact pancreas showed that this dose of BP Protease was very effective in recovering islets when excess purified recombinant (12 or 20 WU/g pancreas) or natural collagenase (20 WU/g pancreas) was used in the isolation procedure (9). The mean islet yield per g tissue was 5,309 IEQ when either purified recombinant or natural collagenase was used in excess (11).

Change in thermolysin product, now labeled animal origin-free: In 2017, the supplier of purified thermolysin discontinued manufacture of the thermolysin product used since 1994 for human islet isolation and replaced it with an animal origin-free product. VitaCyte’s discussion with the supplier indicated that the only change in the manufacturing process was the omission of using bovine casein in the fermentation media so that the product could be labeled animal origin-free. Internal evaluation of the new product at VitaCyte led the scientific staff to conclude that there could be changes in the biochemical characteristics of this enzyme. These changes led VitaCyte to notify customers who currently purchase thermolysin of this product change. The CIzyme Thermolysin (catalog #002-1000 and #002-2000) were replaced with a new Animal Free Thermolysin product (#002-3000).

VitaCyte continues to use the same extinction coefficient to determine the thermolysin protein concentration. The few lots of the new product that VitaCyte evaluated appear to have the same specific activity as the discontinued thermolysin product. However, we do not know how this product change will impact its effectiveness to isolate human islets.

If you are interested in learning more about the change in thermolysin or if you want to evaluate BP Protease as a replacement for thermolysin in human islet isolation, contact technical support at VitaCyte by phone (317-917-3457) or email (feedback@vitacyte.com).

References

  1. Balamurugan AN, Naziruddin B, Lockridge A, Tiwari M, Loganathan G, Takita M, et al. Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010. American Journal Transplantation. 2014;14(11):2595-606.
  2. 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. Transplantation. 2012;93(7):693-702.
  3. Brandhorst H, Friberg A, Nilsson B, Andersson HH, Felldin M, Foss A, et al. Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation. Cell Transplantation. 2010;19(1):3-8.
  4. Fetterhoff TJ, Cavanagh TJ, Wile KJ, Wright MJ, Dwulet FE, Gill J, et al. Human pancreatic dissociation using a purified enzyme blend. Transplantation Proceedings. 1995;27(6):3282-3.
  5. O’Gorman D, Kin T, Imes S, Pawlick R, Senior P, Shapiro AM. Comparison of Human Islet Isolation Outcomes Using a New Mammalian Tissue-Free Enzyme Versus Collagenase NB-1. Transplantation. 2010;90:255-59.
  6. Stahle M, Foss A, Gustafsson B, Lempinen M, Lundgren T, Rafael E, et al. Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation. Transplantation Direct. 2015;1(5):e19.
  7. Bucher P, Mathe Z, Morel P, Bosco D, Andres A, Kurfuest M, et al. Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation. Transplantation. 2005;79(1):91-7.
  8. 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. Transplantation Direct. 2016;2(1):e54.
  9. Loganathan G, Subhashree V, Breite AG, Tucker WW, Narayanan S, Dhanasekaran M, et al. Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield. American Journal Transplantation. 2018;18(2):478-85.
  10. 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. Transplantation. 2010;89(8):954-61.
  11. McCarthy RC. New insights for using purified enzymes for human islet isolation 2018 [Available from: https://www.vitacyte.com/news/new-insights-for-using-purified-enzymes-in-human-islet-isolation/. Accessed 23 May 2018.