Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification

Area of Science:

• Biotechnology
• Molecular Biology
• Pharmaceutical Manufacturing

Background:

• Continuous cell lines are crucial for pharmaceutical protein production.
• Viral contamination poses a significant risk to drug safety and requires rigorous validation of removal processes.
• Current viral clearance validation relies on cell-based infectivity assays, which can be time-consuming and costly.

Purpose of the Study:

• To develop and validate a real-time quantitative PCR (qPCR) method for assessing viral clearance during biopharmaceutical manufacturing.
• To evaluate the removal of simian virus 40 (SV40) using qPCR during chromatography and filtration steps.
• To compare the efficacy and advantages of the qPCR method against traditional cell-based infectivity assays.

Main Methods:

• Real-time quantitative PCR (qPCR) utilizing Taq DNA polymerase and a dual-labeled fluorogenic probe.
• Automated DNA quantification using the PRISM 7700 sequence detection system.
• Application of the qPCR method to evaluate SV40 DNA clearance during chromatography and filtration procedures.

Main Results:

• The developed qPCR method provides accurate and reproducible quantification of SV40 DNA.
• SV40 clearance determined by qPCR closely aligns with results from cell-based infectivity assays.
• The qPCR method demonstrated higher sensitivity, reliability, sample throughput, and lower cost compared to cell-based assays.

Conclusions:

• The real-time qPCR method is a highly effective tool for evaluating viral clearance in biopharmaceutical manufacturing.
• This qPCR method offers significant advantages over cell-based assays, facilitating process validation and reducing costs.
• The method has the potential to be applied to the clearance evaluation of various model viruses, streamlining biologic product development.