Rational Method for Optimizing Collagenase-Protease Mixtures for Cell IsolationCurrent State

Traditional crude and enriched collagenase enzymes are ill-defined biochemical products where enzyme activities reflect the composition of the Clostridium histolyticum fermentation supernatant used to manufacture these products. The manufacture of Liberase HI Purified Enzyme Blend, the first purified collagenase-protease enzyme mixture designed for human islet isolation, showed that at least three enzymes were needed for cell isolation: class I (C1) and class II (C2) collagenases from C. histolyticum and one or more neutral proteases. The enzyme composition for this product was derived from performing biochemical analyses on “good” lots of Collagenase P. The advantages of this approach are that only the required enzymes are used in the isolation procedure and lot pre-qualification is eliminated as long as the product is consistently manufactured.

VitaCyte developed 3 other “reverse engineered,” purified collagenase-protease mixtures for isolation of mouse islets, human hepatocytes, and human adipose stromal vascular fraction from lipoaspirate. However, the limitations for using “reverse engineering” to optimize collagenase-protese enzyme formulation are:

• Assumption that the “good” lot traditional collagenase was the best formulation for cell isolation
• It requires collaboration with an enzyme supplier who has the skills required to perform the required biochemical analyses
• The supplier has the capabilities to formulate a purified collagenase-protease enzyme mixture that can replace the good collagenase lot

The new approach is based on feedback from customers who further optimized the cell specific  purified enzyme mixtures described above by reducing neutral protease activity and by applying principles from a hypothetical model for enzyme mediated cell isolation. This approach does not require any biochemical analysis of the your current lot of collagenase product. However, you must estimate the mass of collagenase in the enzyme solution you use for cell isolation.

Four key assumptions of this model are:

• Excess purified collagenase (with minimal protease contamination) will not have an adverse effect on cell viability or function
• Collagenase’s collagen degradation activity (CDA) must be in excess to ensure loosening of the extracellular matrix
• If CDA in excess, then neutral protease activity is critical to the speed of tissue digestion, and yield of functional viable cells
• Neutral protease activity required for successful cell isolation is equivalent or less than the neutral protease activity found in Worthington Type 1 collagenase

Step 1: Find the collagenase mass in the product you plan or currently use for isolating cells. Roche Liberase research products are sold in 5 or 50 mg bottles of collagenase. To determine the mg of collagenase in Worthington Types 1, 2, 3, or 4 collagenase products, divide the dry weight of collagenase to prepare your enzyme solution by 16.67 (these product contain about 6% collagenase). For other products, contact us.

Step 2: Determine the amount of collagenase required for your isolation procedure. This amount is the mass you use to prepare the enzyme solution in the cell isolation process. Make the appropriate calculations to determine the mass of collagenase in the enzyme solution you use to isolate cells.

Step 3: Prepare three enzyme solutions for lot testing using the PD Collagenase 100, PD 800 products and a 50:50 mixture of PD 100:PD 800. The amount of collagenase, protease and excipient for these 3 products are listed below.

*mg of each component in 1 g product

 

For illustrative purposes, assume you want to make an enzyme solution with 10 mg of collagenase (note, collagenase mass ≠ dry weight of traditional collagenase product) in 100 mL. You can assume the collagenase is in excess if you successfully isolate your cells of interest most of the time.

Three enzyme solutions are prepared, each containing 10 mg of collagenase. The amount is determined by dividing 10 by the percentage of collagenase in each product.

• For PD 100: 10 mg/0.055 = 181.8 mg
• For 50:50 mixture of PD100:PD800: 10 mg/0.248 = 40.3 mg
• For PD 800: 10 mg/0.44 = 22.7 mg

The table below shows that each enzyme solution contains the same amount of collagenase (10 mg in 100 mL or 100 ug/mL of enzyme solution) but differing amounts of neutral protease.

*Specific activity of purified BP Protease 140,000 NPA U/mg

 

Step 4: Determine if the VitaCyte’s products provides comparable results. If the results are comparable to those obtained with your current lot of product, congratulations! You know know more about the enzyme formulation required for isolating your cell of interest.

If you do not obtain equivalent results with any of the enzyme mixtures above when compared to your current lot of collagenase, the enzyme formulation may need to adjusted or supplemented with another protease, Clostripain, as described in the package insert. Clostripain may be needed because it has a complementary enzyme activity when compared to BP Protease. Clostripain is a trypsin-like enzyme that cuts at different regions of the protein than BP Protease.

The effort required to assess the performance of the three PD Collagenase enzyme solutions above will not change the effort you presently take to qualify new lots of traditional crude or enriched collagenase products. The benefits of adopting the PD Collagenase enzyme in place of traditional collagenase are summarized in the table below.

Sigma and Nordmark collagenase are very hererogenous and may be more difficult to replicate.

If you have any questions on the information or if you want to use another VitaCyte purified collagenase or protease enzymes for this application, contact us.