Verifying Removal of Residual Production Enzymes from Your Final Product

Enzymes such as Trypsin and Collagenase are often critical components for product processing, modification of proteins and cell preparation for stem cell therapies. As these final cell-based products are intended for administration into patients, it is important that any residual enzyme material from the production process be assessed and its final concentration minimized to reduce risk. Potential negative impacts of residual enzymes could include immunogenic activity, reduced efficacy of the final product, and toxicity in patients. Although there are no specific regulatory guidelines at this time to specify allowable amounts of residual Trypsin or Collagenase, these are still considered process residuals and potential product contaminants, and thus should be assessed as part of an overall risk mitigation strategy.

While activity assays are often used to verify removal of residual enzymes, these have limited sensitivity and may only provide partial information as they will only detect active enzyme, while missing inactive fragments that might still be immunogenic. ELISA technology that is independent from activity is a favorable alternative for detecting residual enzymes such as Trypsin and Collagenase.

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Question 1

Is using your kits easier than preparing homebrew ELISA assays?

The Roche ELISA assays can be performed in any laboratory without specialized training. Additionally, they are available in an all-inclusive kit format with validated reagents, so all components necessary are accessible and ready to use. This creates a simpler, more reliable approach than with many homebrew assays, which may be subject to reagent lot-to-lot variability, potentially impacting accuracy of the final result.

Question 2

What type of enzymes will be detectable with these assays?

The Residual Protein Trypsin assay detects Roche Recombinant Trypsin from porcine pancreas, expressed in Pichia Pastoris. The Residual Protein Liberase assay detects constituents of the Roche Liberase Blends containing Collagenase I, Collagenase II, and Thermolysin. Enzymes from other suppliers may show a different reactivity with regard to sensitivity and accuracy; therefore compatibility with individual products must be verified.

Question 3

We are interested in conducting in process testing for residual enzymes to check our process efficiency at a couple stages. Can you explain what kind of information we will gain by running your kits particularly with respect to in process tests? Also do you have any recommendations for using you kits in this manner?

These ELISA kits are intended for quantitative detection of the respective enzyme proteins in manufacturing processes of biopharmaceutical and cell therapy products. They can be used with sample material from aqueous buffer solutions from biotechnological processes, as well as cell culture supernatant. The wide measuring ranges for each assay make them suitable for different matrices and a broad range of sample types from both in-process samples to bulk drug substances. Thus, they can be used at different stages of your product development process.

The kits include the necessary standards and controls. Instructions for Use for these kits provide an example of typical results for a calibration curve. The appropriate calibration values need to be generated for each run, with the standards placed on the same plate as the samples within a run. With the calibration curve you generate you will be able to derive a quantitative result for your samples. Individual results may vary depending on the sample matrix and the test run.

In conclusion, the kits can help to determine the efficiency of your purification techniques at multiple stages of the development process. This information can help you make adjustments to existing procedures and optimize your overall time to release.

Question 4

What if you are not using Roche enzymes, can you still use these kits?

The Residual Protein Liberase Kit was designed to detect the constituents of Liberase Blends containing Collagenase I, Collagenase II, and Thermolysin, specifically the formulations found in the following Liberase Blends available from Roche: Liberase MNP-S, Liberase MNP-S GMP Grade, and Liberase MTF C/T GMP Grade.

The Residual Protein Trypsin Kit was designed to detect Trypsin, recombinant from porcine pancreas, expressed in Pichia pastoris, available from Roche.

The polyclonal antibodies in these ELISA kits detect a multitude of epitopes, so they may detect similar enzymes from other suppliers, but may show a different reactivity with regard to sensitivity and accuracy. Therefore, the compatibility of the kit calibration with other enzyme products must be verified. Roche can provide samples for evaluation in these situations. Also, please note that the Residual Protein Trypsin Kit is not suitable for measurement of non-porcine trypsin.

Question 5

What is the time to results for these assays?

Total assay time is approximately 2 hours and 30 minutes. Incubation time is 1 hour and 55 minutes. Therefore, minimal hand-on time is required to run these assays.

Question 6

What are the sensitivity and measuring ranges of your assays?

The Residual Protein Trypsin assay has a sensitivity of ≤0.5 ng/ml and measuring range of 0.5 ng/ml up to approximately 50 ng/ml. The Residual Protein Liberase kit has a sensitivity of 0.1 ng/ml and measuring range of 0.1 ng/ml up to approximately 5 ng/ml.

Question 7

At what stage(s) of the production cycle should you test for residual enzymes?

This is very much up to the discretion of the individual company and your risk mitigation strategy. Testing can be done as an in-process control to monitor efficiency of the enzyme removal procedure, as well as for a final release testing criteria. The Roche assays offer a broad measuring range suitable for in-process samples to bulk drug substances.

Question 8

What type of samples are compatible with your assays? Is there any sample preparation required?

The assays are intended for use with aqueous buffer solutions from biotechnology processes and cell culture supernatant. If cell-free aqueous solutions are used, no sample preparation is required. For testing of cell culture samples, the samples must be centrifuged and the supernatants can be used for enzyme determination.

Question 9

Do you have a clinical grade trypsin?

Roche's Recombinant Trypsin is manufactured under ISO 13485 and in accordance to good manufacturing practice (GMP) guidelines. Additionally, no animal derived products are used in the fermentation, purification, or final formulation. Roche’s Recombinant Trypsin is currently used as a critical raw material for the production of active pharmaceutical ingredients (API), i.e., insulin, vaccines and for cell dissociation. Click here to access more information from the Roche Custom Biotech website.

Question 10

We use BAPNA to test for residual trypsin activity. How does your kit compare?

The Residual Protein Trypsin kit is an antibody-based immunoassay which provides data indicating the quantity of Trypsin protein present in a sample. The BAPNA test detects the rate of enzyme activity in the sample, i.e. enzymatic cleavage of the BAPNA molecule. In other words, the BAPNA test indicates how much enzyme activity is present, whereas our Roche kit will tell you how much Trypsin protein is present (whether active or not). This is important as these inactive pieces may still be immunogenic and have potential consequences for patients and end product performance.

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