Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification

• Published in: Biotechnology and bioengineering 2004-09, Vol.87 (7), p.884-896
• Main Authors: Shi, Liming, Chen, Qi, Norling, Lenore A., Lau, Allen S.L., Krejci, Sherrie, Xu, Yuan
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cells; Chromatography; Chromatography - methods; chromatography and filtration; Cricetinae; Cricetulus; Drug Contamination - prevention & control; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Leukemia; Miscellaneous; Murine leukemia virus; Online Systems; Pharmaceutical Preparations - isolation & purification; Pharmacology; Polymerase Chain Reaction - methods; Proteins; Real time; real-time quantitative PCR; Receptors, G-Protein-Coupled; Receptors, Virus - analysis; Receptors, Virus - genetics; Receptors, Virus - isolation & purification; Recombinant Proteins - isolation & purification; Recombinant Proteins - therapeutic use; Reproducibility of Results; retrovirus; Rodents; Sensitivity and Specificity; virus clearance; Viruses; Xenotropic murine leukemia virus
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20198

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 μL−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 chramotography (QSFF) and DV50 viral filtration are robust retrovirual 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. © 2004 Wiley Periodicals, Inc.