Rapid PCR-Based Sterility Testing in Cell Therapy Manufacturing

Introduction

Cell therapies represent a revolutionary approach in modern medicine, offering potential treatments for various diseases. Nevertheless, the distinctive characteristics of these therapies pose considerable obstacles to production and quality assurance, especially when it comes to maintaining sterility due to their limited storage lifespan. This article explores the implementation of rapid sterility testing in cell therapy manufacturing, focusing on PCR-based methods for mycoplasma detection.

Background

Regardless of class, pharmaceutical products require extensive compliance testing during development and manufacturing to ensure their identity, purity, integrity, concentration, potency, stability, and sterility. While traditional sterility testing methods are effective for conventional pharmaceuticals, they present challenges for cell therapies due to the latter's limited stability and the need for rapid administration.

Mycoplasma contamination is a concern in cell culture facilities, with estimates suggesting that the contamination in the biologicals and raw materials may reach between 4% and 35% ⁽¹⁾. Traditional culture-based detection methods can take up to 28 days, which is incompatible with the production and administration timelines requirements. This time constraint has driven the need for rapid sterility testing methods that can deliver results within hours rather than weeks.

PCR-Based Rapid Sterility Testing

A well-validated Polymerase Chain Reaction (PCR) method allows for the detection of mycoplasma at low colony-forming units (CFUs) within hours of sample receipt at the laboratory, making it an ideal technique for sterility testing in cell therapy manufacturing ⁽²⁾. PCR is easy to perform, sensitive, specific, fast, reliable, efficient, and cost-effective.

Advantages Over Traditional Methods

• Speed: Results within hours instead of weeks.
• Sensitivity: Can detect as low as 10 CFUs per mL, meeting regulatory requirements.
• Specificity: Capable of detecting a wide range of mycoplasma species.

Implementation Considerations

1. Assay Selection: Various commercial rapid mycoplasma detection kits are available, employing techniques such as melting curve analysis of PCR products or fluorescence hybridization probe technologies.
2. Validation: It is crucial to confirm the specificity and limit of detection of the selected test on mycoplasma strains, as Pharmacopeias recommend. 
3. Sample Preparation: The type and volume of the sample used in the assay are critical. Samples should be prepared fresh whenever possible, as storage or freezing may reduce the number of viable organisms ⁽¹⁾. Cultures should have grown without antibiotics for at least three subcultures or two weeks, whichever is longer.
4. Integration into Manufacturing Process: Implementing rapid mycoplasma testing during cell expansion and final production stages, could allow for timely go/no-go decisions in case of contamination.

Alternative Rapid Detection Methods

While PCR-based methods are commonly used, other rapid detection techniques are under development.

- Fluorescence in situ hybridization (FISH) ⁽²⁾

- ATP detection assays using fluorescence microscopy and luminometry ⁽²⁾

- Long-read nanopore sequencing combined with machine learning ⁽³⁾

These methods offer additional options for rapid sterility testing, each with advantages and considerations.

Regulatory Considerations

The regulatory framework for rapid sterility testing methods in cell therapy manufacturing is evolving as agencies recognize the need for faster, more efficient testing approaches. However, this evolution comes with challenges and specific requirements. Key considerations include ⁽¹⁾:

• Adherence to FDA and EMA guidance on rapid microbiological methods (RMMs).
• Validation of methods to ensure accuracy and reliability.
• Compliance with current Good Manufacturing Practices (cGMP).

Several commercial rapid mycoplasma detection kits by PCR are available for implementation in quality control labs, extensively validated across hundreds of mycoplasma strains. 

The DNA extraction method and nucleic acid amplification steps must be verified with an appropriate number of replicates to ensure the detection limit is achieved in at least 95% of the replicates. Sample volume and type are also critical considerations during assay development.

Challenges in Implementation 

Implementing rapid sterility testing may present hurdles like:

• Initial costs for equipment and training
• Validation of the method for specific cell therapy products
• Ensuring staff competency in performing and interpreting tests
• Integration with existing manufacturing workflows
• Regulatory acceptance and compliance

Overcoming these challenges requires careful planning, investment, and collaboration with regulatory bodies. Partnering with third-party experts can significantly ease the implementation process.

The Neopharm Solution

Neopharm is committed to supporting the cell therapy industry by offering advanced PCR-based sterility testing services. Our rigorous validation processes ensure that each assay is thoroughly tested within the specific therapeutic drug matrix, achieving the precise detection limits required for regulatory compliance. 

1. Comprehensive Testing: Neopharm provides a wide range of analytical services, including chemistry, microbiology (including sterility), quality control, R&D support, ICH stability studies, method validation, and transfer, as well as Quality Assurance and Regulatory Affairs services.
2. Microbiology Capabilities: A dedicated state-of-the-art microbiology laboratory offers customary analyses, sterility testing, endotoxin testing, antibiotic testing, and bioburden assessments.
3. Commitment to Quality and Innovation: Our team of experts with diverse scientific backgrounds and technical proficiencies is ready to tackle complex challenges with precision and efficiency.

Conclusion

PCR-based rapid sterility testing techniques are crucial for detecting contaminating microorganisms that might take over a week to grow and detect using traditional methods. This approach is particularly vital in cell and gene therapies, where the stability of the drug product is limited, and administration to the patient must occur within hours of manufacturing.

Sources:

(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315024/

(2) https://www.rapidmicrobiology.com/news/rmm-for-cell-and-gene-therapies-a-rapidmicrobiology-special-focus

(3) https://www.technologynetworks.com/cell-science/articles/enhancing-t-cell-therapy-breakthroughs-in-rapid-sterility-testing-383463