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Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography
A S Lau1, Y S Lie, L A Norling
1Genentech, Inc., South San Francisco, CA 94080, USA.
Abstract:
Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.
Insights
A new quantitative PCR method can measure retrovirus removal during pharmaceutical manufacturing. This technique distinguishes viral inactivation from physical clearance, crucial for ensuring drug safety and product registration.
Area of Science:
- Biotechnology
- Virology
- Pharmaceutical Manufacturing
Background:
- Chinese hamster ovary cells are used for pharmaceutical protein production and express retrovirus-like particles.
- Validating the clearance of these retroviral particles is essential for pharmaceutical product safety and registration.
- Xenotropic murine leukemia virus (X-MuLV) is a common model virus for these validation studies.
Purpose of the Study:
- To develop a method that can quantify both infectious and non-infectious X-MuLV.
- To differentiate viral inactivation from physical removal during chromatography processes.
- To provide a robust tool for validating retroviral clearance in pharmaceutical manufacturing.
Main Methods:
- Development of a quantitative competitive reverse transcription PCR (qRT-PCR) assay.
- Quantification of X-MuLV particle RNA (pRNA) in chromatography pools.
- Comparison of X-MuLV pRNA levels in load and elution pools to determine removal extent.
Main Results:
- The developed qRT-PCR method accurately quantifies X-MuLV particles, regardless of infectivity.
- The method successfully distinguishes between viral inactivation by low pH buffers and physical removal by chromatography.
- It provides a quantitative measure of X-MuLV removal by purification processes.
Conclusions:
- The quantitative PCR method is a powerful tool for assessing retroviral clearance in pharmaceutical manufacturing.
- It complements traditional cell-based infectivity assays by enabling the evaluation of physical removal.
- This method enhances the validation of manufacturing processes for ensuring pharmaceutical product safety.
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