- Manufactured for superior lot-to-lot enzyme consistency for improved performance consistency
- Sufficient enzyme activity (>1,500 FALGPA Units) to release islets from average size adult human pancreata
- Purification process eliminates the majority of the contaminating protease activity including clostripain (trypsin-like activity)
- Low contaminating endotoxin levels (<25 EU/mg)
- Combine with choice of neutral protease for your established protocol
- Lot specific Certificate of Analysis provided with complete QC analysis results
(Cat. # 011-2000)
Collagenase Gold+ is an aseptically prepared, lyophilized enriched collagenase that contains both Class I and Class II Collagenase from Clostridium histolyticum.
Collagenase Gold+ is manufactured for the recovery of human islets from non-diseased donor organs. The product has negligible amounts of protease activity and must be combined with AF Thermolysin or BP Protease for the successful release of islets from the extracellular matrix.
- White lyophilized cake under vacuum in amber bottle sealed with butyl rubber stopper
- Highly purified collagenases for greater enzyme potency (>3.4 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, Collagenase Gold+ vials are sealed under vacuum. A 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”
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.