Ensuring Biotherapeutic Products Are Mycoplasma-Free

Mycoplasma contamination of manufacturing cell cultures is rare, but presents a threat for biologics manufacturers. Contamination sources can include raw materials used in the manufacturing of cell culture media, media supplements (especially if they are not animal origin-free), manufacturing personnel, and even the donors of cells used for cellular therapy products. Steps can be taken to reduce risk, but there have been reports of mycoplasma breaching sterile filtration processes, likely due to their deformability and unusually small size. And once in the cell culture environment, these bacteria can thrive without visible signs, enabling them to persist undetected for significant periods. The impact of an undetected mycoplasma contamination can be extreme, starting with rejection of the contaminated lot and potentially other lots associated with shared equipment and raw materials. Other consequences include cell culture plant shutdown, time-consuming investigations into how the infection occurred, and costly corrective and preventive actions, such as decontamination operations and intensive testing to confirm mycoplasma eradication. Clearly, timely mycoplasma detection systems are essential to ensure safe and efficient biologics manufacture. Seeking assurance in a changing environment Recognition of the issue resulted in regulations to guide detection of mycoplasma in bioprocesses. Early guidelines stipulated application of mycoplasma tests based on lengthy culture, particularly the broth culture followed by plating on agar method (1). This test – originally developed from a modification of a research tool developed by biologists studying mycoplasma – was hardly cutting-edge, but by default became the standard method. Today, it is recognized to have significant drawbacks: • Time-consuming – a negative result requires a minimum of 28 days culture, which delays lot-release and prevents prompt response to contamination events • Laborious – specific expertise in handling live mycoplasma is required • Expensive – typically, companies must outsource the test to a specialist service provider • Imperfect – false negatives may arise if a mycoplasma bacterium fails to give rise to a colony on the agar plate; false positives may occur if the positive controls contaminate the test sample The shortcomings of culture-based tests have become more acutely exposed as the pharmaceutical industry has evolved; the introduction of advanced therapeutics, such as autologous cell therapies, has had a particular impact. For example, a CAR-T therapy production cycle – from harvesting of patient cells, genetic modification and ex vivo expansion to reinjection – requires only five to seven days. Clearly, this is incompatible with a 28-day test. Furthermore, cell therapies don’t undergo the traditional viral inactivation and viral clearance steps that are required during purification of biologics manufactured in cell culture – steps that presumably would also inactivate any mycoplasma contamination that had been undetected in the cell culture. Evidently, sensitive and specific mycoplasma tests are essential to minimize or eliminate risk to patients from contamination that could occur during ex vivo manipulation and cell expansion. Thus, the emergence of cell and gene therapy products, and their need for rapid mycoplasma assays, has had a clear influence on regulatory acceptance of rapid methods (see “Evolving Regulatory Guidance: From 28-day Culture to 6-hour PCR”). A sign of increasing regulatory enthusiasm for rapid tests came in July of 2008, when the US Food and Drug Administration (FDA) convened a public workshop entitled “Rapid Methods for Detecting Mycoplasma Rapid Mycoplasma Testing Method for Lot-Release of Biotherapeutics Regulators require that the cell culture-based bioprocesses used for manufacturing of protein therapeutics, vaccines, and cell and gene therapy products be tested to ensure they are mycoplasmafree. Traditionally, testing was done using a culturebased, 28-day mycoplasma test. More recently, PCR-based alternatives have evolved, driven by an industry move to shorten lot disposition cycles and the emergence of cell-based therapeutics that require a more rapid test. Recent regulatory guidance allows manufacturers to select rapid tests – as long as they are validated to demonstrate sensitivity and specificity that are comparable to or that improve upon traditional tests. The big question: how should manufacturers respond? Produced by RAPID MYCOPLASMA TESTING EVOLVING REGULATORY GUIDANCE Figure 1. Rapid qPCR testing permits sampling, testing and decision-making throughout the bioprocess. Traditional testing methods are limited to analysis of samples from the beginning and end of the bioprocess, and therefore cannot provide actionable information regarding quality parameters at intermediate process stages. By contrast, rapid PCR-based tests allow repeated, real-time analysis throughout the process. COMMON QUESTIONS CASE STUDIES USER TIPS KEY FEATURES For more information thermofisher.com/mycoseq LINKSContamination in the Manufacture of Vaccines, Including Pandemic Influenza Vaccines, and Other Biological Products.” Discussions with industry and regulatory experts at that workshop framed expectations on the desired attributes of rapid mycoplasma tests and provided some guidance as to how a rapid nucleic acid-based test could be validated (2). Manufacturers have responded by developing increasingly sophisticated rapid nucleic acid-based tests. Such tests have advantages beyond lot-release testing to ensure product safety – they also enable effective risk mitigation (see Figure 1). Rapid testing permits in-process analysis at each step in the bioprocess, which means that manufacturing can be interrupted as early as possible if a contamination is detected, thereby preventing downstream propagation of the mycoplasma and consequent escalation of the problem. Additionally, negative results from in-process testing can enable conditional release of bioreactor harvests for downstream processing while the traditional culture-based tests are in progress – especially combined with in-process testing for mouse minute virus in CHO cell-based processes. Essence of a compliant test Regulators are now clear that PCR-based tests can be acceptable alternatives to the 28-day culture-based test, provided appropriate sensitivity and specificity are demonstrated in validation. But what should manufacturers look for in a nucleic acid-based test? In 2007, the European Pharmacopoeia released guidance on performance expectations and validation of nucleic acid-based mycoplasma detection methods (3). The guidance on sensitivity is 10 colony forming units (CFU) or genome copies (GC)/mL of test sample. Since then, that has been the limit of detection (LOD) target that is generally applied when the goal is to replace the use of the 28-day test. The expectation on specificity is that a test for mycoplasma should not detect non-mycoplasma species. That is critical in ensuring a positive result is definitely from mycoplasma and, given the ubiquity of non-mycoplasma bacterial DNA in the raw materials used for cell culture, this is an essential attribute. Another component of specificity that is sometimes overlooked is the ability of the method to detect mycoplasma after recovery from test sample matrices. A final expectation for an ideal rapid test is that it should be robust – that is, able to maintain performance despite test conditions that deviate from typical testing protocols and procedures. One test that meets these criteria is the Applied Biosystems™ MycoSEQ™ Mycoplasma Detection Kit from Thermo Fisher Scientific (see “MycoSEQTM assay:”). The assay has demonstrated sensitivity that meets or exceeds the sensitivity guidance of the European Pharmacopoeia. For specificity, the qPCR primers were designed to exclude detection of non-mycoplasma species and this capability has been demonstrated in multiple studies. Additionally, when combined with the PrepSEQTM-based sample preparation protocols – which incorporate background reduction and highly efficient nucleic acid extraction and purification – sensitivity and specificity are achieved from a variety of test sample matrices typically tested for mycoplasma. And finally, robustness: during the development of the MycoSEQ assay, robustness was confirmed by completion of a multi-variant design of experiments (DOE) to assess the impact of deliberate variations of experimental conditions. In fact, multiple customer studies have validated application of the MycoSEQ assay at a LOD of at least as sensitive as 10 GC or CFU/mL test sample (or both). Assay sensitivity of 1–3 GC per PCR reaction is well established and when combined with our protocol for lot-release testing, which tests the equivalent of 1 mL test sample per qPCR reaction, enables meeting or exceeding the European Pharmacopoeia guidance of 10 GC or CFU/mL.