From Research to Regulatory Practice: Yeasen and NIFDC Advance NAT-Based Mycoplasma Testing for Biologics

Mycoplasma contamination remains one of the most persistent safety concerns throughout biologics manufacturing—from cell banking and viral vector production to monoclonal antibodies, vaccines, and cell therapies. Because mycoplasmas lack a cell wall and often grow without visible signs of contamination, they can compromise product quality, reduce cell performance, and ultimately jeopardize patient safety.

To address these challenges, researchers from the National Institutes for Food and Drug Control (NIFDC) and Yeasen Biotechnology recently published a collaborative study entitled "Establishment of Nucleic Acid Amplification Technology for the Detection of Mycoplasma in Biological Products."

The study establishes a validated nucleic acid amplification technology (NAT)-based workflow that provides a practical reference for implementing rapid mycoplasma testing in biologics quality control.

Why Faster Mycoplasma Testing Matters

Traditional mycoplasma detection relies primarily on culture-based methods and indicator cell assays. While these methods have long been considered regulatory standards, they require extensive incubation periods and are increasingly incompatible with today's accelerated manufacturing timelines—particularly for cell and gene therapies (CGT), where products often have very short shelf lives.

Nucleic acid amplification technology (NAT) has emerged as an attractive alternative because it combines high sensitivity with dramatically reduced turnaround time, enabling faster quality decisions without sacrificing analytical performance.

Traditional Methods vs NAT

Feature	Conventional Culture	NAT-based qPCR
Time to result	Up to 28 days	<3 hours
Sensitivity	Moderate	≤10 CFU/mL
Throughput	Low	High
Suitable for rapid release	No	Yes

Regulatory Trends Are Moving Toward NAT

Global regulatory agencies increasingly recognize validated NAT methods as acceptable alternatives to conventional mycoplasma testing.

The European Pharmacopoeia (EP 2.6.7), USP <63>, and Japanese Pharmacopoeia all include nucleic acid amplification technologies following appropriate method validation. Although the Chinese Pharmacopoeia has not yet officially incorporated NAT as a standard method, it allows alternative approaches approved by national regulatory authorities. Recent technical guidelines for immune cell therapy products also encourage validated rapid testing strategies when conventional methods cannot meet clinical timelines.

This regulatory evolution is driving broader adoption of NAT across advanced therapy manufacturing.

Regulatory Agency	Requirement
FDA	Mycoplasma testing is recommended for raw materials, virus seeds, harvests, and other biological materials used in vaccine production.
Chinese Pharmacopoeia (ChP 2020)	Mycoplasma testing is required for MCB, WCB, and End-of-Production Cells (EOPC). Alternative validated methods recognized by national authorities may be used.
CDE (Cell Therapy Guidance)	Mycoplasma testing is recommended at critical in-process control points and for final product release.
European Pharmacopoeia (EP 2.6.7)	Validated NAT methods are accepted as an alternative to conventional culture methods.
USP <63> / Japanese Pharmacopoeia	NAT-based methods are accepted following method validation.

Key Takeaway

Rapid NAT methods are becoming the preferred solution for biologics manufacturers seeking both regulatory compliance and accelerated product release.   

The Collaborative Study

In this joint project, NIFDC provided regulatory expertise and method evaluation, while Yeasen contributed proprietary enzyme technologies, optimized reagent systems, and extensive validation data.

Together, the research team systematically evaluated the assay for:

• Analytical specificity
• Detection sensitivity
• Repeatability
• Robustness
• Anti-interference performance

The resulting workflow demonstrated reliable detection of multiple clinically relevant mycoplasma species across a variety of biologics sample types, including:

• Cell banks
• Viral vectors
• Recombinant proteins
• Vaccines
• Cell therapy products
• A Complete NAT Workflow

Beyond assay development, the collaboration also demonstrated a complete workflow suitable for routine laboratory implementation.

The recommended solution combines magnetic bead-based DNA purification with multiplex probe-based qPCR detection, allowing laboratories to obtain reliable qualitative results in less than three hours.

Figure 1. Workflow of mycoplasma detection using the Mycoplasma Real-time Quantitative PCR Detection Kit

Spotlight on Innovation: The MycAway™ Mycoplasma qPCR Detection Kit (2G)(Cat#40619)

Building on years of experience in molecular diagnostics and enzyme engineering, Yeasen developed the MycAwayTM Mycoplasma qPCR Detection Kit (2G) to support rapid NAT-based mycoplasma testing. 

The kit utilizes multiplex TaqMan probe chemistry with both target and internal control detection, providing high analytical confidence while minimizing false-negative results.

Performance	Validation Parameter	Kit Validation Results
Limit of Detection (LOD)	Mycoplasma strain detection limit	Tested against 10 mycoplasma strains required by regulatory pharmacopoeias at 10 CFU/mL. Across 24 tests, the detection rate was ≥95%.
Specificity	Sample matrix interference	Tested against 9 types of sample matrices and DNA diluents; no mycoplasma was detected.
	Cross-reactivity	Tested against 14 bacterial strains and 6 commonly used biomedical engineered cell lines; no mycoplasma was detected.
Robustness	Freeze-thaw stability	(Data not provided in source material)
	Heat-accelerated stability	Kit performance remained unaffected after 14 days at 37℃ and 30 days at 4℃.
	Instrument compatibility	Compatible with ABI 7500, ABI QuantStudio™ 5, Bio-Rad CFX96, and Roche LightCycler® 480.
Coverage	Covered mycoplasma DNA species	Database alignment via mycoplasma 16S rRNA sequences covers 183 species of the class Mollicutes.

Key Features

• Detects up to 183 mycoplasma species
• Detection limit ≤10 CFU/mL
• Total workflow <3 hours
• Multiplex FAM/Cy5 detection
• Internal control for inhibition monitoring
• Non-infectious positive control
• Validated according to EP 2.6.7, USP and JP requirements
• Manufactured under ISO 13485

The publication of this collaborative research represents another milestone in the adoption of standardized NAT-based mycoplasma detection and further demonstrates the growing role of domestically developed molecular technologies in supporting global biologics quality control.

Yeasen remains committed to advancing innovative quality control solutions for cell therapy, gene therapy, vaccines, and other next-generation biologics, helping laboratories accelerate testing while maintaining confidence in every result.

Related Product

Product Category	Catalog No.	Product Name	Specifications
Sample Preparation Kits	18461ES	HieffTM Magnetic Residual DNA Sample Preparation Kit (Bottled)	25T / 100T
	80511ES	48-Channel Automated Nucleic Acid Extractor	48-well throughput
Mycoplasma Detection Kit	40619ES	MycAwayTM Mycoplasma Real-time qPCR Detection Kit (Probe Method, 2G)	25T / 100T