Sterility & Mycoplasma testing with NGS

Sterility and mycoplasma assays are critical for quality control testing of pharmaceutical products. Traditional methods (e.g., 14-day culture method on agar) present significant challenges for some biopharmaceutical product release due to long incubation periods and high sample volume requirements. These methods are further limited because bacteriostatic or fungistatic substances within the sample can inhibit microbial growth, and additionally, many microorganisms cannot grow under standard laboratory conditions. This limits the effectiveness of pharmacopoeia-based methods in detecting environmental contaminants. As a result, traditional methods are often inefficient and less dependable for ensuring product safety. Rapid Microbiological Methods (RMMs) address these limitations by requiring smaller sample volumes and enabling faster and more comprehensive microbial detection, improving efficiency in sterility assurance.

Innovative therapies, such as Advanced Therapy Medicinal Products (ATMPs) and Cell and Gene Therapies (CGTs), are highly sensitive, complex, and often produced in limited quantities with short shelf lives, ranging from a few hours to a few days. Due to their small batch sizes and sensitive nature, traditional testing methods are not feasible, as they require large sample volumes and long incubation periods. These methods do not only delay product release, but they are also unable to meet the specific characteristic needs of these new therapies (e.g., AAVs, CAR-T cells, TIL, iPSCs, gene editing therapies, stem cell therapy). In contrast, Rapid Microbiological Methods, including NGS testing and PCR, offer a faster, more sensitive, and volume-efficient alternative for sterility testing.

Contaminants detected by NGS testing

Microbial contamination detection for sterility testing has advanced significantly to address the challenges of CGTs and ATMPs. Traditional sterility tests, which require 14 days for bacteria and 28 days for mycoplasma, are impractical for short-shelf-life products like CAR-T cell therapies, as these products are often administered before standard test results are available. Consequently, there is a growing demand for rapid, growth-independent assays – RMMs – to enable quicker and more efficient testing for raw materials, environmental monitoring, and final product release.

Alternative rapid microbial detection methods, such as ATP bioluminescence, flow cytometry, nucleic acid amplification, and solid-phase cytometry, have been developed. While these approaches improve detection speed, they still face limitations in sensitivity or accuracy.

PCR is a faster and highly sensitive method that amplifies specific DNA or RNA sequences, such as the 16S or 28S rRNA genes, which are highly conserved regions in bacteria and fungi.
NGS testing offers a sensitive and comprehensive solution by directly analyzing the genetic material of microorganisms, eliminating the need for microbial growth. This method enables the detection of a wide range of microbial contaminants, including bacteria, mycobacteria, fungi, and mycoplasma, while simultaneously providing immediate identification of these organisms.
When combined with PCR, NGS testing enables contaminant identification of the amplicons down to the species level. This targeted metagenomics approach accelerates the detection process (faster turnaround times) and enables the sequencing of specific genes, offering a detailed and accurate microbial profile. It offers unbiased pathogen detection in mixed microbial populations from complex samples like blood, urine, or cerebrospinal fluid.

Regulatory guidelines such as the European Pharmacopoeia (Ph. Eur. 2.6.1, Sterility, Ph. Eur. 2.6.7, Mycoplasma) and the United States Pharmacopeia (USP <63> Mycoplasma Tests, USP <71> Sterility Tests) acknowledge nucleic acid amplification techniques as alternatives to traditional culture-based methods, after thorough validation and demonstrating comparable sensitivities.