Qualification of a Rapid In-house PCR-based Mycoplasma Assay for Screening of Cell Therapy Products

Recent journal publication demonstrates qualification of a commercial Roche CustomBiotech MycoTOOL PCR-based kit for a rapid, in-house mycoplasma test to speed mycoplasma final product testing and release of cell therapy products.

Cell therapy products require microbial testing including, mycoplasma, sterility, and endotoxin analysis as part of the final product release criteria for patient safety starting in Phase I. This is a regulatory requirement as product contamination can create real risk for patients, particularly if patients are pediatrics, geriatrics or immunocompromised. However, cell therapy products have unique properties, which make using traditional compendial microbial methods difficult or even impractical. This is especially true for mycoplasma testing where the conventional compendial culture (broth and agar) method requires a large testing volume, takes 28 days for results, and is not able to be easily automated or scaled. These are especially challenging in cell therapy products, where products do not have a long shelf life and production volume is often limited. In addition, in the case of CAR T products, patients are often being treated with these therapeutics as a last option and as a result, timing is crucial. Thus a short manufacturing length, including quick quality control testing turnaround times for these therapeutics can be critical.

A quicker turnaround time from sampling to results also supports final release testing closer to actual final product release, thus enhancing the product’s safety profile and distribution speed. Faster turnaround times for testing also enables real time in-process testing, thereby enabling more process analytics at additional testing points and quality by design initiatives. Another important benefit is that after initial qualification is complete, in-house testing is often more cost effective than sending samples out for testing providing the therapeutic manufacturer increased control over investigations if contamination incidents arise, and to adjust testing strategies, if needed. However, the main drawback to outsourcing samples for testing is that the turnaround time is too long to be relevant for final product release criteria.

Nucleic Acid Amplification Technique for Rapid Mycoplasma Screening

Since the traditional compendial culture (broth and agar) method is too slow to be suitable for most cell therapy applications, Nucleic Acid Amplification Technique (NAT)-based assays are a great alternative. NAT-based mycoplasma assays detect mycoplasma based on the presence of a nucleic acid sequence and have been shown to be highly sensitive often only requiring a small test volume and because no lengthy culture is required, can be conducted very rapidly.

One NAT-based technique, polymerase chain reaction (PCR)-based assays have demonstrated both rapid and specific detection of mycoplasma. However, these rapid assays must pass qualification that show they can meet or exceed the sensitivity of the compendial method and there is not matrix interference from the test components in order to be approved by regulatory bodies. While there has been a bit of a misconception about the difficulties around qualification, many companies have successfully qualified rapid testing methods with regulatory agencies.

In order to achieve the highest benefit from using a rapid mycoplasma testing strategy, the test should be qualified in-house using the sponsors’ facilities, equipment, consumables, cells, media and matrices to meet regulatory testing requirements. If companies opt instead to send samples out for testing at independent labs, even if the lab uses rapid methods, the shipping, testing of samples, and report creation, negates time-savings that could have been gained from the rapid testing. Benefits of in-house rapid microbial testing include: significantly faster results, reduced product release times, reduced sample volume required, improved process control and cost savings, and results representative of final therapy product.

Qualification of a Rapid and Sensitive NAT Technique for Mycoplasma Screening

In a recently published paper titled, “A Rapid and Sensitive Nucleic Acid Amplification Technique for Mycoplasma Screening of Cell Therapy Products” (Molecular Therapy: Methods & Clinical Development, June 2020), authors qualify a mycoplasma detection assay that utilizes “the commercially available MycoTOOL PCR Mycoplasma detection kit (Roche) with a modified protocol in order to obtain the required 10 CFU/mL sensitivity level. The protocol involves DNA extraction from samples of cell therapy products, amplification of mycoplasma species nucleic acid via highly sensitive touchdown PCR, and visualization by gel electrophoresis. The use of live mycoplasma species to demonstrate assay sensitivity is avoided through the addition of defined quantities of mycoplasma species genomic DNA (gDNA) that are converted to CFU/mL values using empirically derived genome copy to CFU (GC/CFU) ratios.” By using this method, authors demonstrated a lower limit of detection that exceeded regulatory requirements set by Health Canada and the assay is currently being used to screen clinical cell therapy products.

I was fortunate to be able to interview Lisa Dreolini, Research Assistant in the Holt Lab at Canada’s Michael Smith Genome Sciences Centre (GSC) in Vancouver and lead author of the study. We discussed the qualification process and she provided information for others who are interested in following a similar approach.

Faster Turnaround Time Required for Mycoplasma Testing on Cell Therapy Products – Solution In-house rapid Mycoplasma testing

I began by asking Lisa about the 28-day turnaround time for conventional mycoplasma testing discussed in her paper. She explained why a 28-day timeline was such a problem and why authors were looking for a faster solution. She explained that many cell therapies, including theirs, are administered fresh and have a limited shelf life. Health Canada requires demonstration that the cell product is mycoplasma-free prior to patient administration. The 28-day turnaround time for compendial methods makes it impossible to deliver a fresh product before its expiry. As a result the BC Cancer Immunotherapy Program, identified a commercial provider of a PCR-based testing protocol that delivered results in less than 28-days, however, due to the shipping process plus testing time they were unable to meet the strict timing requirements of a fresh product. To solve these delays, they developed an in-house rapid testing method by leveraging a commercial mycoplasma kit that had been previously accepted by Health Canada for testing other clinical cell therapy products that could generate results in as little as 8 hours.

Value and Benefit of In-house Rapid Mycoplasma Test

Lisa then explained the importance and benefit of developing and implementing a rapid mycoplasma method for their CAR T clinical trial. She said that Health Canada requires that cell therapy products be extensively tested for safety prior to administration to a patient, including screening for mycoplasma species contamination. Early on they realized that existing methods were unable to meet this need and that they would need to develop an in-house method to be able to proceed with their trial. The primary benefit of running the tests in-house was flexibility and control over when the results would be available. Another critical value was that once all the development and qualification steps were complete, the in-house method was more cost effective than sending samples out for testing. Going through the qualification process also gave them in-house expertise, which allows them to be more confident in the interpretation of results.

Commercial Rapid Mycoplasma Testing Kit Selection

Next I asked Lisa why her team selected the commercial Roche CustomBiotech MycoTOOL Endpoint PCR method (MycoTOOL Mycoplasma Sample Prep Kit + MycoTOOL Mycoplasma Detection Amplification Kit) to leverage for their rapid mycoplasma method?

She explained that the Roche CustomBiotech MycoTOOL Endpoint PCR method was chosen because it was approved for use by the FDA, EMA, as well as Health Canada for mycoplasma species screening of other clinical cell therapy products and select commercial biopharmaceutical products. In order to be accepted as a release test by Health Canada, they needed to demonstrate that the method was comparable to the compendial methods. They were able to leverage the full kit qualification performed by Roche and execute a shortened product-specific qualification, which saved them a substantial amount of time and money.

Since Roche CustomBiotech offers two PCR-based kits, I followed up by asking why Lisa’s lab chose the MycoTOOL Endpoint PCR kit (i.e. MycoTOOL Mycoplasma Detection Amplification Kit) vs. the MycoTOOL Mycoplasma Real-Time PCR Kit. Lisa said that the Endpoint PCR-based kit was chosen from a resource standpoint as most institutions have at least one thermocycler, whereas the real-time PCR machines are in shorter supply. They wanted to develop a protocol that could easily be transferred to additional manufacturing and clinical sites.

Regulatory Qualification of MycoTOOL

Next, we discussed the regulatory steps that Lisa and her team took to implement the MycoTOOL Endpoint PCR method with regulators. She said that to receive regulatory qualification, they prepared a validation report demonstrating that multiple operators were able to detect mycoplasma species at the required sensitivity without matrix interference using the MycoTOOL Endpoint PCR method with the modified protocol. This report was included in the Clinical Trial Application (CTA) submitted to Health Canada. Their method has been approved for use with in-process and final cell products manufactured at their Center.

Implementation of the MycoTOOL for Rapid Mycoplasma Screening

I asked Lisa about any modifications that they had to make to the Roche MycoTOOL Mycoplasma Detection Amplification Kit manual protocol when implementing the testing process. She described two major modifications to the protocol provided with the MycoTOOL Mycoplasma Detection Amplification Kit: the first was increasing the PCR reaction volumes, and the second was the substitution of the GAPDH control primers with human β-actin specific primers. They determined during the validation process that an increased PCR reaction volume was required to be able to reliably detect the positive control mycoplasma species genomic DNA. The control primer substitution was required because the kit includes primers to be used with the Chinese Hamster Ovary (CHO) cell line that we found were unable to amplify GAPDH from human samples.

Additionally, they chose to spike in genomic mycoplasma species genomic DNA to demonstrate assay sensitivity instead of using live mycoplasma. This was an important aspect of the method that allowed them to perform the method within their labs, in close proximity to cell therapy production that otherwise would have been jeopardized by the introduction of live mycoplasma.

To follow up, I asked why they chose β-actin primers to confirm cell lysis and DNA recovery in control PCR reactions. She said it was important to demonstrate that a negative test result is due to the sample being truly negative for mycoplasma, and not because of the absence of genetic material in the reactions to detect. The β-actin primer reactions confirm that the cells were properly lysed and that DNA was not lost in the processing steps. β-actin was chosen as a target because it is a standard housekeeping gene that has been regularly used as a PCR control in their lab and within the community at large.

I then asked at what points in their process they are testing for mycoplasma and why they selected these points. Lisa said that they are testing an in-process sample taken at Day 5 of the culture process, as well as the final infusion product. The results of both in-process and final time points are a requirement set forth by Health Canada. In addition, they like to have the in-process results prior to lymphodepletion of the patient to guide their decision making in the clinic.

Advice for Others Interested in Implementing In-house Rapid Mycoplasma Testing

I mentioned to Lisa that it seems their lab had no problem developing and qualifying the rapid mycoplasma assay leveraging the Roche MycoTOOL Endpoint PCR method for their CAR T clinical products.  She said that the process development and validation of the modified protocol took over a year of concerted effort to accomplish, and was not a simple undertaking. The requirement to detect 10 CFU/mL requires an exquisitely sensitive assay, and thus proper spatial segregation (DNA preparation and PCR), unidirectional workflow, and PCR trained expertise are important safeguards that must be put in place. The method ideally requires multiple workspaces and dedicated processing hoods, which may not be available to all groups.

She went on to say that once they developed a robust and reliable method, they found the assay to be straightforward to execute. They recommend others consult the journal publication for clear instructions on how to perform the testing themselves.

BC Cancer Immunotherapy Program is also offering mycoplasma testing as a non-profit service to the cell therapy community. Interested parties can contact the Project Manager, Dr. Mhairi Sigrist at msigrist@bcgsc.ca.

For more information and study details, please see A Rapid and Sensitive Nucleic Acid Amplification Technique for Mycoplasma Screening of Cell Therapy Products


About Lisa Dreolini

Lisa Dreolini is a Research Assistant in the Holt Lab at Canada’s Michael Smith Genome Sciences Centre (GSC) in Vancouver. Her Bachelor of Science in Microbiology & Immunology and Master’s degree in Genetics/Immunology were received from the University of British Columbia. She has been at the GSC for 15 years, working on a wide range of exciting projects involving genomics, neurobiology and immunology. More recently, Lisa has been a key team member of the BC Cancer Immunotherapy Program where she was responsible for establishing a large scale plasmid production pipeline following GMP guidelines as well as developing a Mycoplasma detection assay for screening cell therapy products.

 The BC Cancer Immunotherapy Program is an investigator-lead group, embedded in the Canadian health care system.  Their vision is to create and clinically translate the next generation of cancer immunotherapies. Their goal is to develop safe, potent and cost-effective immunotherapies that can be deployed against a broad range of cancers. https://www.bccrc.ca/programs/immunotherapy

For more information on Roche CustomBiotech mycoplasma testing offerings, please see the MycoTOOL Mycoplasma Detection page.

MycoTOOL Mycoplasma Sample Preparation Kit, QC Sample Preparation Kit, MycoTOOL Mycoplasma Detection Amplification Kit, and MycoTOOL Mycoplasma
Real-Time PCR Kit are for use in quality control / manufacturing process only.
MycoTOOL is a trademark of Roche. Other brands or product names are trademarks
of their respective holders.

Lisa Dreolini has been paid a fee by Roche Diagnostics for her participation in this interview.

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