Nucleic acid amplification techniques (NAAT) are recommended by pharmacopoeias in the United States, the EU, and Japan as an alternative to traditional culture and indicator cell methods used to detect Mycoplasma in biological products. Used for in-process controls and release testing, NAAT is an improvement over traditional methods that take up to 28 days, but attempts to balance broad species coverage and sensitivity favor one over the other.
Recently, Chinese researchers developed a NAAT that overcomes those challenges, according to a recent paper by scientists at the National Institute for Food and Drug Control, Yeasen Biotechnology, and Xi’an Jiaotong-Liverpool University.
Rather than surpassing existing technologies, “It optimizes the core pain points of mainstream multiplex NAATs,” encompassing their advantages and forming others, senior author Xiaoliang Sun, PhD, scientist, genomics division, Yeasen Biotechnology, tells GEN.
For example, for cell therapy products, Sun says this NAAT technique, “cuts Mycoplasma tests from a 28-day culture to several hours, enabling same-day batch release and avoiding cell product expiry scrap.” It also “accelerates finished-product release for short shelf-life recombinant proteins and monoclonal antibodies.”
The three pairs of primer-probe sets in this assay cover 183 Mollicutes species by targeting Mycoplasma-specific conserved regions. “This fills the detection gap [experienced by] some rare strains…and makes it suitable for trace contamination screening in biopharmaceutical production,” Sun points out.
To objectively assess this method, they compared it to traditional 165 rRNA degenerate PCR, strain-specific NAATs, and mainstream multiplex NAATs cited in the literature.
The analysis shows the new assay offers “single-copy detection sensitivity (validated by 10 pharmacopoeia standard strains), no cross-reactivity (validated with 14 non-Mycoplasma genera and six engineered cell lines), short amplicons of 100 to 200 bp, amplification efficiency of 95–105%, and excellent repeatability,” Sun says. This ensures specificity and improves detection consistency. Consequently, the method “meets the strain detection requirement of Chinese and European pharmacopoeia for full-process regulatory scenarios.
“The detection process is compatible with existing qPCR platforms without requiring special equipment and can be directly applied to full-process scenarios, such as raw material screening, cell bank verification, and finished product release,” Sun continues, “achieving more comprehensive compliance.”
There are limitations, though. “Detection performance for unrecorded Mycoplasma strains from extreme environments or highly variable subspecies has not been verified… leaving a potential detection gap,” he cautions. Additionally, “The use of three pairs of primer-probe sets increases the reagent cost per sample.” Therefore, scientists may best use this method for initial screening, followed by digital PCR confirmation.
For biopharmaceutical manufacturers, Sun says this assay’s main advantages are “comprehensiveness, stability, and practicability.” Future work is envisioned to expand the assay’s applicability parameters and robustness.
