Setting a new paradigm for low cost collagenase

Setting a new paradigm for low cost collagenase

The current paradigm for collagenase:

  • Crude collagenase is impure, as reflected by the brown to black color of the product.
  • Biochemical heterogeneity between different lots of crude collagenase is due to impurities and is reflected by inconsistent results after use in cell isolation procedures.
  • Collagenase products are often sold as different “Types” that reflect their enzymatic characteristics or by supplier’s internal assays that confirm the ability of the lot to isolate specific cell types.
  • Collagenase “Type” bears no relationship to its ability to degrade the different collagen types found in mammalian tissue.
  • Many users control problems associated with lot to lot variability by pre-qualifying specific lots of collagenase for their application.

The current paradigm reflects the collagenase manufacturing process. Crude collagenase is derived from a Clostridium histolyticum cell supernatant recovered after anaerobic fermentation. The supernatant is concentrated and dialyzed, then poured into trays, frozen, and lyophilized. The resulting protein cake is mechanically treated to create a granular powder that can be dispensed into bottles for final packaging.

Lot to lot variability of crude collagenase led manufacturers to categorize individual lots of product into different “Types” and to offer lot sampling or qualification programs. This enabled customers to assess product performance prior to purchase. Some customers pre-qualified lots by determining that a sufficient number of viable cells were recovered for their purpose. Other customers pre-qualified lots based upon cell yield, cell viability, and functional performance . As expected, this was a time consuming process that often led customers to purchase large amounts of product that would last for years, to avoid repeating the time-consuming and non-productive process of lot qualification.

The core point to remember is that crude collagenase lots are inherently variable since they reflect the unique biochemical composition of their source, C. histolyticum culture supernatants. The active enzymes responsible for cell release from tissue, collagenase and neutral protease, comprise only 3-8% of the dry weight of crude collagenase. The remainder of dry weight is biopigment, other enzymatic activities, undefined contaminants, and endotoxin.

The biochemical variability between different lots of the same product makes it very difficult to apply crude collagenase to clinical applications. Cell isolation methods, used directly or indirectly for clinical application, will likely require the use of defined or purified enzyme mixtures to ensure development of a reproducible protocol that preserves cell function.

The new paradigm for low cost collagenase

The current paradigm is overcome by basing the improvements on our current understanding of tissue dissociation. The essential facts relevant to this discussion are listed below.

  • Collagenases and neutral protease enzymes work synergistically to degrade the extracellular matrix and release cells from tissue.
  • Collagenase’s sole function is to degrade native and denatured collagen (i.e., gelatin) that is measured by collagen degradation activity (CDA) and gelatinase activity, respectively.
  • Collagenase’s CDA and gelatinase activities degrades native collagen, loosening up the extracellular matrix.
  • Relaxing the matrix leads to exposure of new sites on extracellular matrix proteins that are now susceptible to proteolytic degradation; this includes the anchoring proteins that hold cells to the tissue.
  • CDA is a critical analytical determination that is not often measured, or is assessed by imprecise and/or inaccurate methods
  • Proteolytic degradation of intact collagenase during manufacturing can decrease CDA, which leads to variability of CDA between lots.
  • Neutral protease activity found in crude collagenase is variable, and comprises two enzymes: histolyticum neutral protease and clostripain.

VitaCyte’s approach to develop a defined, low cost collagenase product line started by improving the process used to generate the raw material, thereby increasing the recovery of intact collagenase with a high CDA. The DE Collagenase product line is manufactured by adding increasing amounts of defined, enriched collagenase to a fixed amount of purified BP Protease (a Dispaseä equivalent enzyme). The DE Collagenase product line is summarized in the table below. The five DE Collagenase products are labelled by the targeted amount of total collagenase activity per bottle (total FALGPA U), each with two different pack sizes: 100 mg and 1 g of crystalline powder. Each product pair has the same specific FALGPA activity (FALGPA U/mg dry weight). To simplify the manipulation of the FALGPA U numbers in the table below, they are expressed as milli-FALGPA U (multiply the FALGPA U/mg by 1000; for example DE 100 has 0.1 FALGPA U/mg x 1000 = 100 milli-FALGPA U per mg). The amount of purified BP Protease in each product pair is fixed; the neutral protease specific activity remains constant across all DE products. By increasing the amount of collagenase added to a fixed amount of protease, the collagenase:protease activity ratios increase incrementally, enabling users to reduce the amount of protease used in their cell isolation procedures.

DE Collagenase* Product Line
Product Name Amount per bottle Milli-FALGPA U/mg NPA U/mg Collagenase: protease ratio
DE 10

DE 100

100 mg

1 g

100 mU/mg ≈ 4.9 U/mg 20
DE 20

DE 200

100 mg

1 g

200 mU/mg ≈ 4.9 U/mg 41
DE 40

DE 400

100 mg

1 g

400 mU/mg ≈ 4.9 U/mg 82
DE 60

DE 600

100 mg

1g

600 mU/mg ≈ 4.9 U/mg 122
DE 80

DE 800

100 mg

1g

800 mU/mg ≈ 4.9 U/mg 163
*DE Collagenase contains an inert peptide excipient that protects enzymes from degradation and increases convenience by enabling product to be weighed out prior to use


Summary

All DE Collagenase products contain a low hygroscopic peptide excipient that preserves enzyme activity during storage and enables the required amount of product to be weighed out at time of use; the white crystalline powder readily dissolves and passes easily through a sterilization filter

The design of DE Collagenase Products overcomes many of the problems associated with the current, crude collagenase paradigm. These differences are summarized in the Figure below, comparing the key features of the current “black box” paradigm to the new “transparent box” paradigm. This shift to a transparent box paradigm is driven by improvements to collagenase raw material manufacturing, enabling increased recovery of intact collagenase, which translates into higher CDA per mg of product. A subsequent processing step removes the majority of contaminants that include the C. histolyticum neutral protease. The simplicity of this design, adding increasing amount of collagenase to fixed amount of protease, allows use of a broader range of collagenase:protease ratios in cell isolation than can be found when using crude collagenase products. If you can reduce the protease activity required to recover cells, it will likely lead to higher viabilities, less cell damage and improved recovery of functional cellular activity. Tight control of protease activity is key to minimizing differences between lots of collagenase product, and key to achieving a consistent cell product. This statement assumes there is excess collagenase activity in the collagenase-protease enzyme mixture. Excess collagenase has minimal impact on isolated cells because of its narrow specificity, degrading only native and denatured collagen (i.e., gelatin) found in the extracellular matrix.

Additional information on the DE Collagenase product line can be found on VitaCyte’s website. Three white papers can be downloaded that review the following issues

Additional information

If you cannot find the information you need for application of the DE Collagenase products, review the content under the Applied Use menu on VitaCyte’s web site or call VitaCyte’s technical support number at 317-917-3457, option #2, between 8:30 am to 5:30 pm Monday through Friday.

A webinar on this topic has been scheduled for 12:00 noon EDT (-5 UTC) on Thursday September 14th, 2017. To sign up for this presentation, complete the registration form found at

https://register.gotowebinar.com/register/4599699527690572035

 

 

 

 

 

 

 

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