Current lot qualification process
Lot selection of traditional Clostridium histolyticum collagenase (i.e., crude or enriched) products is a time consuming process. Typically, you request small samples from one or more new lots of the collagenase product you currently use. You evaluate their ability to recover sufficient numbers of functional cells, as compared to your current lot of collagenase. The goal of lot qualification is a seamless transition from existing to new lot of product. Typically, several different concentrations are used to evaluate new lots with the midpoint of this range often the collagenase concentration used for the current lot of product. If the performance of a new lot is similar to the current lot, then a larger amount of that new lot of product is purchased. In other cases, you may not find a “good” lot of product that meets your needs. Here, you often request samples of other lots of collagenase products from the same or different suppliers. This lot qualification process is repeated until a new “good” lot of product is identified. This “Groundhog Day” approach to lot qualification is re-lived whenever the current lot of collagenase product is about to be depleted.
What was learned from process?
If you routinely follow the process described above, ask yourself: “what new knowledge did I gain?” The only reasonable answer is that the new lot of collagenase appears to recover cells similar to those that were obtained with the prior lot. You have no objective data on how the new lot of collagenase compares to lots used over any from previous years. Your lack of objective data reflects the deficit in knowledge regarding the activity of key enzymes (collagenases and neutral proteases) required for releasing cells from the extracellular matrix. You also have no knowledge about how other contaminates in these products affect the function of the cells you are isolating. This process has remained the same over the last 50 years.
New knowledge enables learning from lot qualification
A proposed mechanism of how C. histolyticum collagenases degrade native collagen and the role bacterial neutral proteases play in releasing cells from the extracellular matrix has been summarized in a recent publication(1), earlier blog posts(2, 3), and in white papers(4, 5) available by download from the VitaCyte web site. Collagen degradation activity (CDA) is the critical collagenase activity required for successful cell recovery, since it measures the activity required to release cells from tissue. As native collagen is degraded, the matrix loosens, exposing sensitive sites on other matrix proteins that can be cleaved by proteases. These include proteins that hold cells to the matrix. To achieve reliable and consistent enzyme-mediated release of cells from tissue, the ideal collagenase-protease enzyme product must have 2 critical characteristics:
- Enriched or purified collagenase with consistent, specific collagen-degrading activity (CDA U/mg dry weight)
- Tight control of protease activity
Why are these two characteristics critical?
- Collagenase’s primary function is to degrade native collagen, the predominant protein in the extracellular matrix that protects other matrix proteins from proteolysis
- CDA is the critical collagenase activity for consistent recovery of cells from tissue, since degradation of native collagen initiates the tissue dissociation process
- As collagenase cuts native collagen, the extracellular matrix loosens, leading to exposure of protease-sensitive sites on other matrix proteins (including “cell anchoring” proteins) that are directly or indirectly attached to collagen fibrils or fibers
- Once a sufficient number of cell anchoring proteins are cut, cells are released from tissue
- It is essential that excess CDA is present in the collagenase-protease enzyme mixtures, to ensure effective degradation of native collagen
- Excess CDA from a highly enriched or purified collagenase product has minimal adverse effect on cell viability, because of the low percentage of contaminants and restricted substrate specificity, only cuts native or denatured (i.e., gelatin) collagen
- Neutral protease
- Neutral protease complements collagenase’s CDA by degrading unraveled (i.e., denatured) collagen and other matrix proteins
- Tight control of the neutral protease activity (NPA) is critical for reproducible cell recovery, since sub-optimal NPA is not sufficient to degrade key matrix proteins, and supra-optimal NPA will damage cells, leading to low viability and poor cell recovery.
Learning is limited by “black box” composition of traditional collagenase products
The inability to learn from lot qualification of traditional collagenase products is due to several factors (see reference 4 for more detail):
- Low concentration of collagenases and proteases that make up only 3-20% (w/w) of the product; > 80% of the biochemical components in these products are contaminants
- Each lot has variable CDA because of proteolysis of intact collagenase during the manufacturing process
- Each lot has variable amounts of two different proteases, C. histolyticum neutral protease and clostripain. This further complicates lot selection, since each protease has a different specificity and the synergy between their activities varies between different lots of product.
This is the biochemical basis for why each lot of traditional collagenase performs differently when assessed for use in cell isolation. One lot could have low specific CDA due to proteolysis of intact collagenase or secretion of low amounts of collagenase. This leads to a higher concentration of product required for release of cells from tissue. If the protease activity has a low to moderate specific activity [neutral protease activity (NPA)/mg dry weight product], then the lot may be identified as a “good” lot product. However, if the specific NPA is high, cells exposed to excess NPA are damaged, resulting in lower cell viability. These lots will likely be considered as “bad”, since cells supra-optimal amounts of NPA damage cell membranes. In contrast, if there is adequate specific CDA and NPA, then there is more flexibility in adjusting collagenase concentration to successfully isolate a specific cell population of interest. The problem is that it is difficult to solve this riddle when the biochemical composition of a traditional collagenase product is a “black box”.
DE Collagenases overcome the limitations of traditional collagenase products
The design of VitaCyte’s DE Collagenase product line overcomes the “black box” composition associated with traditional collagenase products. This new, low cost product line consists of 5 products, each containing increasing amounts of enriched collagenase added to a fixed amount of purified BP Protease (a Dispase™ equivalent enzyme). These products are identified by the total amount of collagenase activity in each bottle, as measured by a collagenase specific peptide substrate (FALGPA). These products come in two pack sizes (containing 100 mg or 1 g of product) and are labelled DE Collagenase 10/100, 20/200, 40/400, 60/600, 80/800 with the double and triple digit number reflecting the 100 mg and 1 g pack sizes, respectively.
The four primary advantages DE Collagenase products have over traditional collagenase products are:
- DE Collagenase is highly enriched, containing primarily intact collagenase enzymes with minimal contamination by other enzymes or other contaminates; products with a high percentage of intact collagenase will have the highest and most consistent specific CDA
- DE Collagenase products are manufactured to achieve consistent, specific CDA
- NPA is tightly controlled: a fixed amount of purified BP Protease is added to each DE Collagenase product so that the specific activity is consistent for every DE Collagenase product
- By using a fixed, excess amount of collagenase activity for cell isolation, it enables you to directly assess the effect of NPA on your cell isolation process
For more detail see reference 3.
How DE Collagenase contributes to learning from lot qualification
How does the design of the DE Collagenase products enable learning from lot qualification?
- These products contain minimal contaminants (>85% collagenase) and minimal contamination with clostripain
- The collagenase is primarily intact, ensuring consistent, specific CDA in the products you use.
- The NPA is contributed by purified BP Protease (> 95% pure) which has the same specific activity (NPA U/mg dry weight) in all DE Collagenase products
- Titration of NPA, in the presence of a fixed and excess amount of collagenase, enables you to learn from lot qualification because the problem is simplified to controlling only one critical variable: the NPA required to release your cell of interest from tissue.
These steps are summarized below in more detail in the prior blog post or in the white paper: “DE Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery”. The instructions below summarize the first 2 of the 4 steps used in the optimization process. By completing these steps, you will gain greater insight into the enzyme composition required to isolate your cell of interest than any of the effort you have made in the past because you are determining the effect of NPA required to isolate a sufficient number of functional cells from your isolation procedure.
- Determine the mass of collagenase you have used in the past
- Review the collagenase concentrations from your last 3 to 5 lots of traditional collagenase products used to prepare your enzyme solution for your cell isolation procedure, then determine an average optimal concentration of product that was used
- Consult the reference table on VitaCyte’s website or call VitaCyte’s technical support to translate this concentration (mg/mL) into an equivalent concentration of the appropriate DE Collagenase product.
- The concentration of DE Collagenase determined above is translated into FALGPA U/ mL of enzyme solution, and is referred to as the collagenase reference activity
- The reference FALGPA U/mL enzyme solution is used to fix the concentration of collagenase used in the cell isolation procedure
- The 5 DE Collagenase products found in the DE Collagenase Optimization kit are used in this experiment
- For the purpose of illustration, the example described below assumes you have used 2 mg/mL of a Worthington Type 1 product for you cell isolation procedures in the past
- DE Collagenase 10 or 100 can be considered to be equivalent to Worthington Type I collagenase
- Calculate the reference collagenase activity by using the formula below
Designated DE Collagenase (X FALGPA U/mg product)] x [Concentration (Y mg/mL)] = Collagenase concentration (Z FALGPA U/mL)
Applying this formula to the illustration above:
DE 20 @ 0.2 FALGPA U/mg x 2 mg/mL = 0.4 FALGPA U/mL
X x Y = Z
0.4 FALGPA U collagenase/mL is the reference collagenase activity
- To perform the calculation below, use the specific FALGPA activity per dry weight of product, as summarized in the table below
|Product||Specific FALGPA activity
(U/mg dry weight
Since all 5 DE Collagenase products contain a fixed amount of NPA (4.9 NPA U/mg), as you fix the collagenase activity used in the enzyme mixture, you reduce the NPA/mL of enzyme solution.
DE 10 (0.4 U/mL) /(0.1 FALGPA U/mg) = 4 mg/mL x 4.9 NPA U/mg = 19.6 NPA U/mL
DE 20 (0.4 U/mL) /(0.2 FALGPA U/mg) = 2 mg/mL x 4.9 NPA U/mg = 9.8 NPA U/mL
DE 40 (0.4 U/mL) /(0.4 FALGPA U/mg) = 1 mg/mL x 4.9 NPA U/mg = 4.9 NPA U/mL
DE 60 (0.4 U/mL) /(0.6 FALGPA U/mg) = 0.67 mg/mL x 4.9 NPA U/mg = 3.3 NPA U/mL
DE 80 (0.4 U/mL) /(0.8 FALGPA U/mg) = 0.50 mg/mL x 4.9 NPAU/mL = 2.4 NPA U/mL
- Plot the cell yield per g tissue (or any other parameter you choose) on the y axis vs NPA U/mL of the x axis. The plots can be defined by the shape of the curve: sigmoidal, positive slope, negative slope or no slope; hypothetical results from first 3 plots are shown below; the no slope plot is not shown since it would be a horizontal line parallel to the x axis
- The peak of a sigmoidal curve identifies the optimal dose of NPA to use in the isolation procedure.
- The other two curve types indicate that cell yield will increase (positive slope) or decrease (negative slope) as specific NPA changes in the enzyme mixture.
- In these latter two cases, further experiments will need to be performed if you want to determine the optimal NPA to use in your isolation procedure
What was learned?
Assuming a sigmoidal curve was obtained in your experiment, you can repeat the cell isolation procedure to confirm your results. Once confirmed, you can purchase the appropriate DE Collagenase product for use in your cell isolation procedures. As you use a new lot of DE Collagenase in your cell isolation procedure, you can be assured that the collagenase and neutral protease activities will be consistent, leading to minimal changes in the concentration of product used in your cell isolation procedure.
If you want to further optimize the enzyme composition used in your cell isolation procedure, you may consider performing additional experiments to assess the effect of collagenase activity on cell recovery. This is accomplished by completing steps 3 and 4 of the enzyme optimization process used described in the white paper “Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery”. The experiment outlined above is repeated, but the neutral protease activity is fixed and the collagenase activity assessed for its impact on cell recovery. Assuming a sigmoidal curve is obtained after plotting these data, then it is a simple matter of determining the optimal dose of DE Collagenase product used in the isolation procedure. The end result of this effort is new knowledge of the enzyme composition and dose of a collagenase-protease enzyme mixture required to isolate your cell of interest. If at a later time, you want to translate your research observations to a clinical application, please contact VitaCyte to discuss if it is beneficial to replace the DE Collagenase with a collagenase-protease enzyme mixture using purified enzymes.
If you have any questions on this method, call Technical Support at 317-917-3457, option 2 or send an email to firstname.lastname@example.org.