Quantitative competitive reverse transcription-PCR as a method to evaluateretrovirus removal during chromatography procedures

Chinese hamster ovary cells used for pharmaceutical protein production expr ess non-infectious retrovirus-like particles. To assure the safety of pharm aceutical proteins, validation of the ability of manufacturing process to c lear retrovirus-like particles is required for product registration. Xenotr opic murine leukemia virus (X-MuLV) is often used as a model virus for vali dation studies. Some chromatography procedures used for pharmaceutical prot ein purification utilize low pH (<pH 4.0) elution buffers which readily ina ctivate X-MuLV. Therefore, cell-based infectivity assays are unable to eval uate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by ch romatography, a quantitative competitive reverse transcription PCR method c apable of quantifying both infectious and non-infectious X-MuLV has been de veloped. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amo unt of X-MuLV pRNA. in the load pool and the product-containing elution poo l represents the extent of X-MuLV removal. This method is an extremely powe rful complement to cell based-infectivity assays as it allows physical remo val of X-MuLV by chromatography and filtration procedures to be distinguish ed from X-MuLV inactivation when buffers with the ability to inactivate ret rovirus are used. (C) 1999 Elsevier Science B.V. All rights reserved.