The strategy of qualifying a commercially available, in-house, rapid mycoplasma test enables cost-effective and timely in-process and final release testing solution.

Introduction

Cell Therapy products have unique manufacturing requirements that necessitate the development of new processes and technologies to support manufacturing. Like other biologics, most cell therapies require microbial testing including, mycoplasma, sterility, and endotoxin analysis prior to final product release. Since the cells are the final product, they cannot be put through a sterile filtration step or undergo a harsh sterilization step. Both of these options would harm cells and thus negatively impact the product itself. As a result, in-process and final product microbial testing is a critical part of meeting the stringent regulatory requirements for Cell Therapy products. However, microbial testing can be difficult due to the large sample volume required and lengthy turnaround times associated with traditional culture based testing methods. Cell Therapy products often have a shorter shelf life, so it is vital that these tests be done quickly and that the product release time is as short as possible. A quick turnaround time ensures that the sample tested is representative of the final Cell Therapy product. Additionally, with several Cell Therapy products, the product volume available for testing is also limited.

Traditional testing methods for mycoplasma, sterility and endotoxin can take several weeks to over a month, which doesn’t fit the needs of the quick turnaround time for cell therapies. This is why rapid microbial methods, including rapid mycoplasma tests, provide an important solution to a significant challenge in Cell Therapy manufacturing.

Rapid Microbial Methods

Rapid microbial methods can detect microbial contamination faster than traditional testing methods. Rapid microbial methods also require lower sample volumes for testing because of increased assay sensitivity. The 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. The quick turnaround time and lower sample volume requirements also permit close to real time in-process testing, thereby enabling process analytics and quality by design initiatives. Quality by design is particularly important considering the high value of these therapies. QbD ensures that all critical quality attributes are met and having in-process information ensures better manufacturing control and decision making during manufacturing.

Rapid microbial methods must be qualified in-house using the sponsors’ facilities, equipment, consumables, cells, media and matrices to meet regulatory testing requirements. Often companies see this as a costly or time consuming burden and choose instead to send samples out for testing at independent labs. The problem with this is that even if the lab uses rapid microbial testing, such as PCR, the shipping, testing of samples, and report creation, negates any time savings that could have been gained from the rapid testing. To fully realize the benefits that rapid microbial methods offer, the testing should be done in-house. 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.

Abbreviated qualification of a commercially available, rapid mycoplasma test, reduces the cost and burden of in-house testing qualification

In a recently published paper titled, “Strategy for an Abbreviated In-House Qualification of a Commercially Available Rapid Microbiology Method (RMM) for Canadian Regulatory Approval”(Cytotherapy December 2017), authors present a timely and cost effective solution to the challenge for in-process and final product release mycoplasma testing of their investigational Cell Therapy product, an autologous mesenchymal stromal Cell Therapy product under review for treatment of patients with knee osteoarthritis.

The authors worked with Health Canada to create an abbreviated qualification plan for use of Roche CustomBiotech’s MycoTOOL PCR Mycoplasma Detection Kit for in-process and final product release mycoplasma contamination testing. According to the European Pharmacopoeia (EP), chapter 2.6.7, it is stated, “…Where commercial kits are used for part or all of the analytical procedure, documented validation points already covered by the kit manufacturer can replace validation by the user. Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user…”. By incorporating the supplier’s kit validation information, based on the validation requirements of the EP 2.6.7 in the subchapter “Validation of Nucleic Acid Amplification Techniques (NAT) for the Detection of Mycoplasmas: Guidelines,” authors were able to significantly reduce the amount of effort for their product specific validation that needed to be done in order to implement the kit. Authors only had to show that the kit worked as intended in their cell culture medium and matrix and with their lab and staff in their facility. This combined with a mycoplasma-free history at their facility created an abbreviated qualification plan approved by Health Canada. Results showed using the MycoTOOL PCR Mycoplasma Detection Kit provided them with data that met Health Canada requirements and provided a more cost-effective testing solution that met their quicker turnaround timelines.

According to the study, they were able to reduce their cost 3-fold and reduce testing turnaround time from over 12 days to as little as 48 hours when compared with outsourcing PCR based testing to a GMP lab. The paper provides full details of both the qualification methods used and the results of the study.

I was fortunate to be able to interview Dr. Sowmya Viswanathan, corresponding and senior author on the study about her work. She provided great insight into the motivating factors for changing their testing processes, the benefits they saw, leveraging these gains for future products.

Primary Drivers for Switching to an in-house Commercially Available Rapid Mycoplasma Test

When we spoke about the primary motivations for qualifying a commercial mycoplasma testing kit in-house vs. outsourcing, Dr. Viswanathan explained that to meet regulatory testing requirements an in-process sample and a final product release sample must confirm no mycoplasma contamination. Authors were initially outsourcing the testing to a company in the United States for PCR based mycoplasma testing, but due to shipping, testing time and audited report generation; it would take 2-3 weeks to receive results. They needed both in-process and final product release testing, but found that the in-process results provided little value beyond the regulatory requirement, since they often didn’t receive these results until it was time to release the product. In order to get in-process information, they would also send samples to a local, un-licensed lab to ensure results were negative so they could proceed while they waited for the official results.

Ultimately the cost and turnaround time was too great to continue in this manner and authors were forced to look for another solution. This led them to seek qualification of the MycoTOOL PCR Mycoplasma Detection Kit. They selected this kit because it has been approved by the FDA, EMEA, and PMDA for in-process and final release testing in manufacturing to show the absence of mycoplasma for several clinical products. They were able to leverage these approvals and validation information generated by the kit manufacturer to demonstrate certain key parts of the qualification. Therefore, they didn’t need to re-qualify the kit itself, instead they only needed to demonstrate that the sensitivity was still intact using their medium and matrix and in their hands. As such, they were able to do a condensed validation focusing only on sensitivity/limit of detection and ruggedness.

Challenging the perception that in-house rapid microbial method qualification is difficult

I asked Dr. Viswanathan about the perception that in-house qualification is difficult to navigate with regulators. She told me that their experience was the opposite of this. She said that she found regulators to be very open to the idea and that they saw the benefits of a more sensitive, rapid and relevant test. Of course they want to know that the method is at least as valid as the compendial methods, which authors were easily able to prove. In fact, the data showed that sensitivity increased with PCR testing. In addition, the reduced turnaround time permitted release testing to be conducted much closer to the time of final product release and therefore provided data that more closely represented the actual purity of the product at the time of release.

She did say that working with the regulators in advance was very helpful. They were able to develop a protocol for qualification in advance and were able to ensure that the information provided would meet the requirements of the regulators without doing more than was necessary.

Key Benefits of implementing the In-house commercially available Rapid Mycoplasma test

We then talked about the key advantages that they saw after implementing the commercial PCR Mycoplasma Detection Kit. Dr. Viswanathan said that it made a huge difference. The primary driver and ultimate benefit was the reduced cost. They were able to complete mycoplasma testing for a third of the cost of outsourcing, even taking into consideration labor and other facility costs.

While the cost savings were certainly compelling, there were other important benefits to moving to in-house testing. Turnaround time, which dropped from a couple weeks to as little as 48 hours, now permitted close to real time in-process information. In addition the ability to test closer to release strengthened the safety profile. They were also not at the mercy of another lab or shipping delays to get the results they needed and this put them in control of timing for sampling and results.

Future Opportunities for Rapid Microbial Methods

Looking to the future, I asked Dr. Viswanathan about future implementation of this strategy. She said that this qualification was done for a product in clinical Phase I/II, but felt that the same approach would be applied for commercial manufacturing with similar benefits of cost savings and reduced turnaround time. For new products, they will have to at least do matrix interference testing, but this would be true for any of the testing methods. Dr. Viswanathan also sees this as an important strategy to be used for both sterility and endotoxin testing. She sits on the Articles Council of Canada’s Mirror Committee for International Organization for Articlesization (ISO TC276) on Analytical Methods and Bioprocessing, and is on the steering and working committee of an international Articless Coordinating Body (SCB) that conducted a workshop in April to look at how rapid microbial methods can be expanded and standardized industry wide https://www.standardscoordinatingbody.org/.

Lastly, I asked her what advice she would give to other advanced Cell Therapy product peers who are looking to adopt a rapid method for mycoplasma testing. She said that inertia and reluctance to change is strong, but the financial burdens forced them to evaluate other options. She is so glad that it did because the qualification was not overly arduous; in fact it was very straightforward. The new method provides an easier, quicker and cheaper solution. She advised to push past the inertia and to not to be afraid of the regulatory qualification. Her experience was that regulators are open to the change and they recognize the benefits of rapid microbial methods.

About Dr. Sowmya Viswanathan

Dr. Viswanathan is an Affiliate Scientist at the Krembil Research Institute (University Health Network) and an Assistant Professor at the Institute of Biomaterials and Biomedical Engineering and at the Department of Medicine (University of Toronto).  Her research interest is focused on using anti-inflammatory approaches to target osteoarthritis (OA), including using proprietarily-enhanced mesenchymal stromal cells (MSCs) in modulating inflammatory responses and re-programming monocytes/macrophages.  Dr. Viswanathan’s lab is focused on bioprocess optimization and translation of these cell-based therapies into clinical investigations.  Dr. Viswanathan is a co-Principal Investigator of a recently approved trial using autologous MSCs to treat OA patients, a North American first.  Dr. Viswanathan is the recipient of a Young Investigator’s Operational grant from The Arthritis Society (2015).

As a translational scientist, Dr. Viswanathan serves as a national regulatory and cell manufacturing consultant for projects across Canada.  Dr. Viswanathan chairs the Cell Therapy Stakeholder Group, a bilateral group that engages Health Canada on Cell Therapy related policy issues.  Dr. Viswanathan is a member of the Stem Cell Oversight Committee that advises the Governing Council of CIHR.  Dr. Viswanathan is a founding member, and team leader of the Manufacturing Committee of a network of Pan-Canadian cell manufacturing facilities called CellCAN.  Dr. Viswanathan is leading international efforts to standardize MSC nomenclature and create reference material(s) for MSC research; she sits on the Articles Council of Canada’s Mirror Committee for International Organization for Articlesization (ISO TC276) on Analytical Methods and Bioprocessing, and is on the steering and working committee of an international Articless Coordinating Body (SCB), and has recently been appointed as formal liaison between ISO TC276 and the International Society of Cellular Therapy.  Dr. Viswanathan is a co-editor of a translational book on MSCs “Mesenchymal Stromal Cells: Translational Pathways towards Clinical Adoption.”

At the Cell Therapy Program at the University Health Network, Dr.Viswanathan has overseen clinical translation of over 13 cell-based therapies ranging from immunotherapies, lysosomal storage disorders to acute kidney failure into Health Canada approved clinical investigations.  She leads a multidisciplinary team specializing in clinical-grade cell processing and manufacturing, regulatory and ethics submissions, and implementation of clinical trials.  She provided clinical translational and regulatory input to enable clinical trials for two companies, ExCellThera Inc. and Replicel LifesSciences Inc.