We recently finished our Ask the Expert discussion on “Verifying Removal of Residual Production Enzymes from Your Final Product.” During this Ask the Expert session, we discussed strategies for verifying the removal of residual enzymes used in biomanufacturing, like trypsin and collagenase from the final product. Specific questions included questions regarding sample prep, timing of testing, benefits of using a kit, sensitivity and clinical grade material.

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. This information was also covered in a Cool Tool Feature on the Cell Culture Dish, “Residual Enzyme Testing – All in one Assay Kits for Liberase and Trypsin.”

In this Ask the Expert session, Dr. Noble This week, Dr. Roger Noble, Ph.D., Lead Technical Support Consultant at Roche Custom Biotech, answered questions regarding strategies for verifying removal of residual enzymes from your final product, including how to use the ELISA-based Roche Residual Protein Trypsin and Liberase kits.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Verifying Removal of Residual Production Enzymes from Your Final Product.

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?

The Answer:

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.

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

The Answer:

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.

Is using your kits easier than preparing homebrew ELISA assays?

The Answer:

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.

For more information, please see custombiotech.roche.com/home/contact.html