Abstract

Currently, the most commonly used methods for detectingMycoplasmaare the culture method and the indicator cell culture method. However, both approaches exhibit low sensitivity and are incapable of detecting low-concentration contamination. In addition, the detection period may extend up to 28 days, which is unsuitable for rapid screening and may delay timely contamination control measures. To address these limitations, aMycoplasmadetection method based on nucleic acid amplification technology (NAT) was developed following a comparative analysis of gene sequences from variousMycoplasmaspecies. The method was validated with respect to its detection performance and its applicability to biological product samples. DNA was extracted fromMycoplasma-contaminated samples using a magnetic bead-based nucleic acid extraction method. Universal primers were designed based on the highly conserved16S rRNAgene sequence ofMycoplasma, and amplification was performed using multiplex quantitative PCR (qPCR) with fluorescent probes. The limit of detection (LOD) was established based on statistics of 24 replicates. Method specificity and robustness were evaluated according to the guidelines set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2), while sample applicability was assessed in accordance with the European Pharmacopoeia (EP) <2.6.7>. The NAT-basedMycoplasmadetection method enabled rapid, qualitative identification ofMycoplasmacontamination. The validated LOD was 10 CFU/mL, and the method met predefined requirements for sensitivity, specificity, and robustness. To assess applicability, real biological product samples, including monoclonal antibodies, antibody fusion proteins, bispecific antibodies, and trispecific antibodies, were spiked with 10 CFU/mL of standardMycoplasmastrains. All spiked samples tested positive. These findings confirm that the NAT-basedMycoplasmadetection method is suitable for process contro