Abstract
In this article, members of the USP Microbiology Expert Committee discuss the initiatives being taken to address the requirement of manufacturers of gene and cell therapies in the areas of microbial contamination control and testing. USP general chapters are in development addressing microbial risk assessment and mitigation and modern microbiological technologies for microbial contamination/ sterility monitoring suitable, in terms of time to results, for short-lived products. As these chapters are published on the USP website as in process revisions we encourage our stakeholders to comment.

Introduction
The field of cell and gene therapy is evolving rapidly. An increasing number of clinical studies are occurring every year leading to more regulatory submissions and concerns about manufacturing design and consistency for these modern therapies. Patients depend on access to these therapies as well as strict controls to assure prevention of microbiological contamination during the treatment lifecycle from patient to manufacturing back to patient. Cell therapy products are susceptible to diverse manufacturing processes, which may cover steps such as selection, genetic engineering of cells, expansion, purification and cryopreservation. Cell therapies are manufactured in small volumes, often as a single lot for a single patient, have unique manufacturing processes related to each product and may have many aseptic manipulations. This distinguishes them from large scale classical pharmaceutical products for which the general cGMP guidelines were initially intended. For most cell therapy products there is no terminal sterilization step but they are manufactured under conditions that would prevent microbial contamination so they can be categorized as “aseptically manufactured products”. The United States Pharmacopeia (USP) has been a leading source of current information and standards for the pharmaceutical industry for 200 years. The first two USP chapters written to support the cell and gene therapy industry were <1046> Cell-Based Advanced Therapies and Tissue- Based Products and <1047> Gene Therapy Products, authored by the Biologics-Advanced Therapies Expert Committee.

The 2020-2025 General Chapters-Microbiology Expert Committee (MEC) is currently developing new chapter proposals to provide foundational knowledge about microbiological control in cell and gene therapy manufacturing as well as modern methodologies for microbial contamination testing.

Patient-First Perspective
The origin of cells, the key raw material, for these therapies is the donor. The final administrated dose is given back to either the same patient (autologous) or to many other patients from donor cells (allogeneic). Thus, a primary concern is protecting the patient’s cells from microbiological contamination through the collection, processing, and administration lifecycle. Short turnaround times/shelf-lives are an expectation with patients that have a life-threatening illness so the manufacturing process and concurrent testing are expected to meet these demands compared to classical pharmaceutical products. Riskbased manufacturing process design and testing regimes take this short cycle time into account to assure patients access to the therapies.

Microbiological Controls
The new chapter content being developed by the USP MEC lays a foundation of risk-based approach to assess the manufacturing process and determine mitigation options for short shelf-life products that cannot be terminally sterilized nor aseptically filtered. Microbiological control of donor cells during collection is a primary starting point. The potential sources of microbial contaminants from the surrounding facilities and processes are then part of the risk assessment that can lead to a contamination control strategy with appropriate and practical mitigation steps aligned with <1211> Sterility Assurance.

Incorporating a Quality by Design (QbD) approach consistent with ICH Q8, Q9, and Q10, the changing landscape of cell and gene therapy manufacturing has been led by advances in process technologies that apply previously manual, ‘open’ (or exposed) operations performed in biosafety cabinets to new fully closed operations with single or multiple process steps performed by one instrument and utilizing single-use bag design.

Adequate microbial controls taking into account the reagents, process equipment, facility and type of manufacturing operations preventing the therapies from being contaminated during the manufacturing will promote patient safety as compared to overemphasis on final product testing. It is often said that “the product is the process” when referring to cell and gene therapy products and a basic tenet of quality is that it is not possible to “test quality into a product.” To this end, a new chapter is currently being drafted providing guidance on microbial contamination risk mitigations in the context of implementing a microbial control strategy program. As each cell and gene therapy manufacturing process is often unique, time constrained and include manual steps, traditional pharmaceutical controls and requirements may not be fully applicable and would require adaptation. Examples of risk-based approaches in the proposed chapter will provide support to the reader into categorizing the risks and will include mitigation actions to help balance contamination risks with a better utilization of resources on the most critical contamination control points and allow timely release of product to the patient. High risks related to an ISO 7 area surrounding an ISO 5 biosafety cabinet would include human interventions with environmental exposure of product (e.g., using large volume syringes for open transfer of material into bags). Less frequent environmental and personnel monitoring would need to be implemented to reduce human interventions. For fully automated systems operating in closed isolators without human intervention, personnel monitoring may be skipped and environmental monitoring significantly reduced.

The chapter will focus on several key microbial critical control points specific for cell and gene therapy products such as aseptic operator gowning and qualification, aseptic process simulation (media fill), material and personnel flow, cleaning and disinfection of equipment and working surfaces, quality and testing of aphaeresis material/blood/ cells or tissues, raw materials as well as culture media/solutions. Guidance to quality control testing of the final product will also be included as well as alternative rapid testing or release concepts (e.g., negative-to-date release) will be described. Sample volume size may often be a challenge for these products since they often have a very low final volume and may be a unique batch such as for autologous therapies. Excessive sample volume would consume precious material and too small volume would statistically make the test only capable of detecting gross microbial contamination. High contamination levels would likely be detected prior to the sterility test as abnormal turbidity observed in the cell culture or a sudden decrease in the yield and viability/size of mammalian cells. The chapter will provide a mathematical formula to define an adequate sample volume by estimating the probability of detection of contamination from a known product and sample volume.

Closed Systems
A revision to <1211> Sterility Assurance will be proposed that will improve clarity around the closed systems. Besides cell therapy manufacturing, other aseptic process steps have the option for being fully closed, such as manual operations for handling mammalian cells, which must be protected from extrinsic microbial contamination. The industry has long developed options for handling cells in closed systems such as isolators that are designed to be used for cell culture and processing and/or sterility testing. Some of these isolators have built-in decontamination systems which can be validated. Isolators, in the pharmaceutical or vaccines industry, used to be considered only for finish-fill operations, but now they are designed for multiple types of closed operations employing robotics, and can be alternatives to the use of biosafety cabinets.

Modern Quality Control Testing
New technologies require rethinking compliance. USP has supported rapid microbial methods using a risk-based approach in <1071> Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk Based Approach and has begun to publish proposed chapters for different modern microbiological methods that better fit the growing cellular therapy industry. These new chapters will propose methods for both growth-based and non-growth-based technologies, which generate analytical results in place of subjective evaluations based on turbidity or colony forming units and provide more rapid turnaround of results.

Harmonized compendial sterility testing as represented by <71> Sterility Tests specify sample quantities and minimum number of units to test are incompatible with small-volume cell and gene therapy products. Furthermore, final macroscopic examination of the microbiological growth medium for evidence of microbial growth after a 14-day incubation period often exceeds the short shelf-life of many of these products, especially if the final formulation cannot be cryopreserved. The USP is developing several chapters for rapid detection of microbial contamination for sponsors of cell and gene therapy products (and other short shelf-life products) to submit as release tests in regulatory applications in lieu of the classical sterility test. Two of these were published in the Pharmacopeial Forum and received favorable stakeholder comments. The first is for growth-based methods that measure the production of CO₂ from cellular respiration. These methods detect CO₂ production using colorimetric, fluorometric, or headspace pressure techniques. This procedure was described in <72> Respiration-Based Rapid Microbial Methods for the Release of Short Shelf-Life Products (PF 46(6)). The second is also for growth-based methods that measure ATP bioluminescence. These methods use the luciferin–luciferase reagent to generate an ATP bioluminescence reaction detected using either an imaging instrument to generate a signal that correlates to microbial colonies or growth in microbiological broth. This procedure was described in <73> ATP Bioluminescence-Based Rapid Microbial Methods for Release of Short Shelf-Life Products (PF 46(6)). Additional chapters in development rely on non-growth-based technologies such as solid phase cytometry and nucleic acid amplification detection.

Due to the nature of human-sourced cells, there is always a concern for other adventitious agents (besides bacteria and fungi) to be contaminants, such as mycoplasma, or viruses from autochthonous sources. Process-related viruses (i.e., vectors) are closely monitored during production and release of gene therapy products. Detection of exogenous viruses are part of quality control evaluation by screening donor source material for allogeneic therapies and animal-derived process raw materials. Depending on US or international regulations, donor source material for autologous therapies may not require screening because the therapy is returned to the donor.

The current compendial mycoplasma screening <63> Mycoplasma Tests consists of two techniques, a culture method and an indicator cell culture method, often referred to as the H-Stain (Hoechst Stain). These methods are frequently used in conjunction to test cell and gene therapy products as described in the 1993 FDA Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. The indicator cell culture method provides a result in 3-5 days, but has a higher detection limit than the agar and broth media culture method, which requires 28 days for a final result. This lengthy testing regimen often exceeds the short shelf-life of many cell and gene therapy products, so is not suitable as a product release test. Use of nucleic acid detection with polymerase chain reaction (PCR) amplification produces results quicker and is considered the current state-of-the-industry technology. There are many different types of nucleic acid amplification test techniques available including standard PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), and droplet digital PCR (ddPCR). USP is developing a new chapter that will describe different types of nucleic acid amplification methods that can be used to detect mycoplasma rapidly, often with same-day results that will be invaluable for ingredient, in-process, and finished product testing.

Adoption of rapid microbiological methods as product release tests is critical to ensure the safety of cell and gene therapy products, particularly for patient-specific therapies with short shelf-lives on the order of days or hours. Cell and gene therapy is still in its infancy, but recent advances in treatments such as CAR-T cell cancer immunotherapies require development of modern cutting-edge analytics to support delivery of these exciting products to patients. This will require collaboration between academia, industry, government regulators, and the pharmacopeial compendia. USP is committed to evaluating new technology, providing a clear mechanism to validate alternative methods in informational chapters, and adding new technologies supported by adequate scientific knowledge as general chapters to support development of these and other advanced therapy products representing the future of medicine.

References

1. https://www.grandviewresearch.com/industry-analysis/cell-therapy-market
2. https://www.fda.gov/news-events/press-announcements/statement-fda-commissionerscott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics
3. Cundell, T., S. Drummond, I. Ford, D. Reber and D. Singer 2020 Risk Assessment Approach to Microbiological Controls of Cell Therapies PDA J. Pharm. Sci. & Technol. 74 (2) 229-248; DOI: https://doi.org/10.5731/pdajpst.2019.010546