Advanced therapies encompass a diversity of cell- and gene-based therapeutic platforms that are intended for human use. The exceptional promise of many cell and gene therapies to change the standard of patient care across a variety of disease indications has driven significant financial investment in the field and many products are now progressing through the clinical pipeline towards market approval. While approved products have already had a profound impact on patients, new scientific progress, clinical advances targeting large indications, and an evolving and supportive regulatory environment provide greater opportunity for these therapies in the future.  Despite the forward progress, a stumbling point for cell and gene therapies has been that the condensed clinical development timelines, complex manufacturing processes, short shelf-life of the products, and supply chain challenges make it difficult for developers to ensure timely testing of quality and safety attributes.

Mycoplasma Testing

One area of focus for the industry is microbial testing, specifically for Mycoplasma species, which are among the most ubiquitous contaminants of cell cultures. Contamination poses a potential threat to patients receiving infusions of cell and gene therapy products, which is why these products must demonstrate the absence of mycoplasmas  as part of product safety in clinical evaluation and subsequent commercial translation. However, traditional culture and indicator test methods, considered the gold standards for mycoplasma detection, require large sample volumes and long incubation times (up to 28 days) that can often exceed the fresh product shelf-life (24–48 hours for non-cryopreserved product) and even exceed timelines for patient administration for cryopreserved product. This lengthy turnaround time is at odds with the need for test results to inform go or no-go treatment decisions. Rapid microbiological methods such as nucleic acid amplification techniques (NATs), with precedence for regulatory approval, are increasingly being incorporated by developers as alternatives to the traditional compendial culture methods for mycoplasma detection owing to their lower sample volume requirement, ease of use, sensitivity and rapid turn-around time.  Unlike traditional mycoplasma culture testing that is commonly outsourced to specialized contract testing labs, NAT-based rapid mycoplasma methods are being implemented in-house.  In-house rapid mycoplasma testing allows developers to realize the full benefits these methods offer for same-day turnaround time, added control, and often, reduced cost.

Qualification of Rapid Mycoplasma Test

Previously, the authors of the publication we will review here were able to successfully obtain approval from Health Canada to use an end-point PCR method based on a commercial kit (MycoTOOL Mycoplasma Detection Amplification Kit; Roche Diagnostics) for in-house mycoplasma detection in their mesenchymal stromal cell (MSC) therapy product for a phase 1/2 osteoarthritis clinical trial (NCT02351011). An overview of that publication was reviewed on the Cell Culture Dish, please see “In-house Rapid Mycoplasma Test Qualification Provides a Cost-effective, Quick Turnaround Solution for Cell Therapy Product Release Testing”. Real-time PCR as a basis for rapid mycoplasma detection is an attractive solution since it is less time-consuming than endpoint PCR (no gel electrophoresis steps) with a larger dynamic range of detection (>2-fold changes in gene expression). Additionally, a cell-type independent recovery control (RC) can be utilized within the assay to mitigate false-negative results.

In their most recent paper published in Cytotherapy (November 2021), In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells, the feasibility of a commercially-available real-time PCR mycoplasma kit (MycoTOOL Mycoplasma Real-Time PCR Kit; Roche Diagnostics) was leveraged for their in-house method they evaluated for their investigational MSC product, using the same qualification strategy that proved successful for the end-point PCR method.

With patient consent, the biobanked bone marrow-derived MSCs (MSCs(M)) from their previous clinical trial (NCT02351011), were re-analyzed by real-time PCR to conduct an abbreviated in-house qualification of the kit for mycoplasma detection. The rationale for an abridged qualification strategy is supported by Ph. Eur. guidelines (section 2.6.7), which states that the end-user need not repeat the validation performed by the manufacturer. Therefore, the developers chose to evaluate several key parameters specific for their application (limit of detection, ruggedness, and matrix interference) rather than performing an in-depth validation strategy to qualify the commercial kit. The authors also tested the use of a DNA carrier, polyadenylic acid (poly(A)) included in the manual sample preparation kit (QC Sample Preparation Kit; Roche Diagnostics), to improve the recovery of mycoplasma genomic DNA (gDNA) from low cell density samples (≤45 000 cells/mL), which can lead to inconsistent DNA recovery efficiency in real-time PCR assays.

Assay Sensitivity/Limit of Detection (LOD)

LOD was evaluated with two mycoplasma species (M. arginini and M. hominis) representative of contamination routes for their application since the kit manufacturer already validated LOD against a panel of 10 mycoplasma species. The standard culture method is capable of detecting mycoplasma at or below 10 colony forming units (CFU)/mL and thus the real-time PCR kit must demonstrate performance of at least equivalent sensitivity. MSCs(M) from three donors were spiked with 10-fold dilutionsof certified mycoplasma gDNA reference standards (400 GCs/mL, 40 GCs/mL, 4 GCs/mL and 0.4 GC/mL of M. arginini and 2400 GCs/mL, 240 GCs/mL and 24 GCs/mL for M. hominis), and LOD was determined by the lowest mycoplasma gDNA dilution detected in all technical replicates by real-time PCR. Under the stringent criterion of 95% detection for qualification purposes, an acceptable LOD ≤40 GCs/mL (equiv. to 10 CFU/mL) and ≤240 GCs/mL (10 CFU/mL) for M. arginini and M. hominis, respectively, was achieved. LOD determined without (–) and with (+) the addition of poly(A) during DNA extraction demonstrated that gDNA recovery from lower cell density MSCs(M)  samples was enhanced by the addition of poly(A) in the extraction procedure.

Matrix Interference

Matrix interference or specificity is a measure of the ability of the assay to detect a target in the presence of matrix components, which constitute any substances present in the test samples that may interfere with target amplification. The final cell product MSCs(M) and culture medium represent dual sources of matrix interference. To separately test matrix interference from the cell product and the culture medium, the spent culture media (representative of an in-process cell-free sample) and the MSCs(M) cells suspended in DPBS (representative of the final cell therapy product formulation) were used in the real-time PCR assay.

Mean crossing point (Cp) values (calculated by the LightCycler® 480 software for signal quantification) in MSC(M) samples spiked with mycoplasma gDNA were used to assess the presence of matrix interference. If matrix components interfere with the assay, a decrease in Cp values would be expected in diluted samples compared to undiluted samples. Different MSC(M) dilutions with and without poly(A) addition confirmed the absence of matrix interference from either the cells or the culture medium.

Assay Ruggedness

Ruggedness was evaluated by testing replicate samples on different days. Data analyzed from 18 real-time PCR runs (nine runs each for species) showed an inter-assay %CV<4 and an intra-assay %CV<3 for both M. arginini and M. hominis, which met the acceptable criteria of %CV<5 across all runs as representative of a reliable and reproducible assay.

Successful Qualification Criteria

Taken together, the authors of the study reviewed herein found their mycoplasma method based on the Roche CustomBiotech MycoTOOL Mycoplasma Real-time PCR Kit met the minimum qualification criteria used previously for the authors’ Health Canada-approved endpoint PCR kit for mycoplasma detection of the same investigational MSC product. The assay demonstrated robustness, had a limit of detection of 10 CFU/mL for two commonly found mycoplasma species, with no detectable matrix interference in the test conditions. Moreover, the results showed comparable sensitivity to the MycoTOOL endpoint PCR kit, while providing the added benefit of time-savings in the assay-to-data turnaround time (<6h vs. ~48hrs). As well, evaluation of poly(A) addition showed that its inclusion could enhance mycoplasma gDNA extraction in their samples with low cell numbers (≤45000 cells/mL).  While not yet vetted by regulatory authorities, the authors anticipate the assay will likely be acceptable based on their experience with the endpoint PCR in-house qualification. Though each qualification approach needs to be discussed and agreed on in collaboration with the appropriate regulatory agencies where compendial methods are required for each product, the value of the real-time PCR method approach extends beyond testing low cell density samples.  This approach gives other developers interested in leveraging the commercial real-time PCR kit for cell-free samples, such as viral vector production or raw materials, a starting point.  Additionally, evidence is provided by other cell and gene therapy developers that have also qualified the same real-time PCR kit, taking a risk-based approach leading to a similar qualification strategy that was successful in allowing their real-time PCR mycoplasma detection method to replace compendial culture methods for product lot release.

Summary

The validation and adoption of rapid microbial methods for cell and gene therapy quality testing is an important evolution that circumvents the limitations presented by the traditional compendial culture methods. The sensitivity and rapidity of next-generation NAT methods can provide assurances for patient safety in a much shorter timeframe. Additionally, these methods can be readily applied to in-process testing, so critical to gaining valuable process insight and implementing Quality by Design approaches.  Regulatory bodies have demonstrated their willingness to work with cell and gene therapy developers to navigate the path towards qualifying alternate assays for product quality testing.   Past regulatory acceptance of the MycoTOOL Mycoplasma Real-Time PCR Kit after appropriate validation supports this trend.

Disclaimers
MycoTOOL Mycoplasma Real-Time PCR Kit and QC Sample Preparation Kit: For use in quality control/manufacturing process only.
LightCycler^® 480 Instrument: For Life Science Research Use Only. Not for use in diagnostic procedures.

Trademarks
MycoTOOL and LIGHTCYCLER are trademarks of Roche.

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