Exploration of RPA-CRISPR/Cas12a-based mycoplasma detection technology for application in biopharmaceutical production
GAO Tianyu
GUO Qingcheng
PAN Zhiyuan
ZHU Weifan
ZHUANG Huangzhen
REN Yule
CHEN Yi
GUO Menghui
Liu Tao
WANG Chenguang
JI Lusha
QIAN Weizhu
HOU Sheng
LI Jun
SUN Ziqiao
WANG Xuekun
XU Jin
ZHANG Dapeng
GUO Huaizu
Abstract：Mycoplasma contamination poses a critical risk in biopharmaceutical production,severely com-promising the safety and efficacy of biological products such as monoclonal antibodies.Traditional detec-tion methods are often time-consuming,lack sensitivity,or are complex to operate,making it difficult to meet the fast quality control demands throughout the biopharmaceutical manufacturing process.To ad-dress this challenge,this study further expands the application potential of the RPA-CRISPR/Cas12a-based mycoplasma detection technology,which was previously developed,and evaluates its practicality and reliability.The optimization of the pre-mixed CRISPR system significantly enhances operational con-venience and ensures stability for up to 7 days at 4℃without significant performance degradation,provi-ding technical support for on-site detection.Semi-quantitative analysis allows for the rapid and accurate as-sessment of mycoplasma levels.Application testing spans the entire biopharmaceutical production process:in upstream processes,the system provides early contamination warnings during CHO cell cul-ture;in downstream purification steps,it monitors the efficiency of multiple-step contamination removal,highlighting its high sensitivity.Additionally,the method was successfully applied to mycoplasma detec-tion in cell banks,culture media,and serum,exhibiting high reliability in simulated contamination experi-ments.The RPA-CRISPR/Cas12a system integrates rapidity,sensitivity,user-friendliness,and multi-sce-nario adaptability,offering an innovative solution for mycoplasma monitoring in biopharmaceutical pro-duction.This advancement significantly enhances quality control efficiency and product safety,undersco-ring its broad application potential.
Keywords：mycoplasmaRPA-CRISPR/Cas12aprocess analytical technologybiopharmaceuticalsprocess monitoring
Publication Date：2026-06-25
Online Publishing Date：2026-08-28(First online date of this platform, not the publication date of the document)
Pages：11( 447-456,474 )