- Manufactured for superior lot-to-lot enzyme consistency for improved performance consistency
- Sufficient enzyme activity (>1,900 Wunsch Units) to release islets from average size adult human pancreata
- Purification process eliminates the majority contaminating protease activity including clostripain (trypsin-like activity)
- Low contaminating endotoxin levels (<10 EU/mg)
- Combine with choice of neutral protease for your established protocol
- Lot specific Certificate of Analysis provided with complete QC analysis results
Cat. # 001-1000
Collagenase HA is an aseptically prepared, lyophilized mixture of 60% purified Class I (C1) Collagenase and 40% purified Class II (C2) Collagenase from Clostridium histolyticum.
Collagenase HA is manufactured to provide the optimal collagenase activity for isolating cells from tissue or recovering adherent cells after in vitro culture. The product is also used in the following cell isolation application, when combined with the appropriate neutral protease for your protocol (suggestions follow in parentheses, below):
- White lyophilized cake under vacuum in amber bottle sealed with butyl rubber stopper
- Highly purified collagenases for greater enzyme potency (>2.8 U/mg)
- Stable for at least 2 years when stored at -20°C
- Lyophilized in a buffer containing calcium to ensure enzymes stability during initial reconstitution
This product is purified from culture supernatants of C. histolyticum fermentation that contain porcine gelatin and pancreatic enzymes derived from US and Canadian sources. No bovine derived animal products are used in any step of the fermentation or purification process. Standard chromatography techniques are used to purify the collagenase. After assessing enzyme potency, the collagenase is aseptically dispensed into depyrogenated vials and lyophilized. When dry, the Collagenase HA vials are sealed under vacuum. I representative sample from the batch is reconstituted and analyzed by a variety of quality control tests. Results are compared to established specifications before release into inventory.
The Pz-peptide substrate (Wünsch Assay) has historically been used to characterize collagenase activity. While this assay has advantages in terms of reproducibility and historical precedence, it also has several limitations. The Wünsch Assay is strongly biased towards C2 and is not sensitive to the different molecular forms of C1. In addition, this activity assesses the catalytic activity of the enzyme and not its ability to degrade native collagen. Degraded C2 without a collagen binding domain is as active as intact C2, potentially providing misleading information about the quality of the enzyme. The limitations of the Wünsch assay led us to develop a fluorescent microplate CDA using fluorecein isothiocyanate (FITC) labeled calfskin collagen fibrils as substrate.
The intact molecular form of purified C1 with two collagen binding domains (C1 116kDa) has approximately 10-fold higher CDA when compared the CDA found with same amount of purified C1 containing only one collagen binding domain (C1 100kDa) or intact C2 (C2 114kDa). A recent report in the literature describes the importance of intact C1 in successful human islet isolations.
Breite AG, Dwulet FE, McCarthy RC. 10th Annual Rachmiel Levine Diabetes and Obesity Symposium 2010 – Las Vegas, NV P#8 “Purification and Characterization of Clostridium histolyticum Neutral Protease used for Human Islet Isolation”
Collagenase HA FAQ
This product is stable for at least two years from date of manufacture if stored unopened between -15 to – 25°C. This product is not manufactured under U.S. Good Manufacturing Procedures nor are individual lots tested for sterility.
Internal studies have shown the reconstituted enzyme is stable as a frozen solution between -15 to – 25°C for at least 1 year as long as no other protease enzymes had been added to the solution. Additional studies have shown the reconstituted collagenase was successfully frozen and thawed three times as a concentrated or dilute solution without apparent loss of potency as assessed by the CDA assay. The product is shipped on dry ice to provide the most stable conditions during shipment.
“HA” refers to “High Activity,” designating the benefit of the HA product’s particular properties.
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Collagenase HA and MA differ only in the amount of truncated class 1 collagenase present. Both are 60%:40% blends of highly purified class 1 (C1) and class 2 collagenase (C2), respectively. The Collagenase MA will have a lower CDA specific activity relative to the HA product, because a portion of the C1 protein in the MA product has lost a collagen binding domain whereas HA is comprised of only intact C1. The two products have comparable Wunsch specific activities.
General Product FAQ
Thermolysin and BP Protease are entirely animal origin free.
A porcine gelatin peptone is used during the fermentation step of natural collagenase production. While this animal sourced material is required to stimulate collagenase production from the biosynthesis from the organism Clostridium histolyticum during anaerobic fermentation, it represents a very low adventitious agent risk as the media is sterilized at 121°C for 30 minutes prior to inoculation, which has been shown to inactivate many viruses.
The gelatin is removed during the purification process and no animal sourced materials are used in the remainder of the process.
Additional manufacturing details are available in product insert literature for each product.
There are two components to inactivation of the digestive enzymes commonly used for tissue dissociation. To inactivate protease activity, a serum-containing media or buffer is most effective. Buffers or media containing human serum albumin (HSA) are ineffective in suppressing protease activity, even at very high concentrations of protein (>10%). However, serum-containing buffers do not inhibit collagenase activity. The most effective method for inactivating collagenase activity is reducing temperature. Collagenases will lose about 80% their maximal activity at 26°C. Thus, introduction of an ice-cold, serum- containing media should virtually eliminate all collagenolytic and proteolytic activity of an enzyme mixture.
General Collagenase FAQ
VitaCyte’s technical service team has a significant amount of experience in transitioning from crude enzyme products to the more defined purified ones and is glad to provide you with assistance. If you can provide us details of the cell of interest; the enzyme product that is currently being used for the isolation; the concentrations (or activities) being targeted; volumes utilized; and, a certificate of analysis for the particular lot you would like to mimic; we can provide some reasonable guidance to which product(s) and what target activities best fit your application. Our defined enriched (DE) collagenase products are specifically designed to encompass the broad range of activities found in the crude enzyme products and to make the transition to a highly purified product seamless.
In developing an Application Specific Formulation, we generally rely on individuals considered “expert in the art” to ß-test potential formulations and guide us in the refinement of the final composition. These formulations are usually engineered from existing protocols and crude enzyme targets. Once a formulation has been identified and deemed acceptable by at least a couple independent labs, it may be included in our product portfolio. We are always eager to assist you with virtually any request for a custom blend so do not hesitate to contact us to discuss your application.