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Published on: November 1, 2018
Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical
Liming Shi1, Qi Chen, Lenore A Norling
1Process Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA.
Abstract:
Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.
Insights
A new real-time quantitative PCR (Q-PCR) method accurately detects Xenotropic murine leukemia virus (X-MuLV) in pharmaceutical manufacturing. This sensitive Q-PCR assay can replace traditional cell-based infectivity assays for viral clearance validation.
Area of Science:
- Biotechnology
- Virology
- Pharmaceutical Manufacturing
Background:
- Chinese hamster ovary cells, used in pharmaceutical production, can express retrovirus-like particles.
- Validating retroviral clearance is crucial for pharmaceutical product safety and registration.
- Xenotropic murine leukemia virus (X-MuLV) is a model virus for these clearance studies.
Purpose of the Study:
- To develop and validate a quantitative PCR (Q-PCR) method for detecting and quantifying X-MuLV.
- To compare the Q-PCR method with traditional cell-based infectivity assays for viral clearance studies.
- To assess the suitability of the Q-PCR method for process validation in pharmaceutical manufacturing.
Main Methods:
- Development of a real-time quantitative PCR (Q-PCR) assay for X-MuLV particle RNA (pRNA).
- Testing the sensitivity, dynamic range, and reproducibility of the Q-PCR method.
- Comparing X-MuLV clearance results obtained by Q-PCR and cell-based infectivity assays during chromatography and filtration.
Main Results:
- The Q-PCR method offers accurate and reproducible quantification of X-MuLV pRNA over a 100,000-fold dynamic range.
- The assay is approximately 100-fold more sensitive than cell-based infectivity assays, with a quantification limit of 1.5 pRNA copies/µL.
- Protein and cellular DNA did not impact X-MuLV quantification by Q-PCR.
- Q-PCR results for X-MuLV clearance during chromatography and filtration were comparable to cell-based assays.
- Both methods confirmed anion exchange chromatography (QSFF) and DV50 viral filtration as robust retroviral removal steps.
- Combined use of Q-PCR and cell-based assays distinguished viral removal from inactivation by Protein A chromatography.
Conclusions:
- The developed Q-PCR method is a sensitive, accurate, and reproducible tool for X-MuLV quantification.
- This Q-PCR assay can serve as a valuable substitute for cell-based infectivity assays in validating viral removal procedures.
- The method facilitates and potentially reduces the cost of viral clearance evaluations for biologic product development.
