Validation of a PCR coupled to a microarray method for detection of mycoplasma in vaccines.

The revised section of the European, United States, and Japan Pharmacopeias on mycoplasma testing provided guidance for the set up and validation of a nucleic acid amplification technique (NAT) as an alternative method to agar culture and indicator cell culture compendial methods. The CytoInspect™ method, based on Polymerase Chain Reaction (PCR) coupled to microarray analysis, has been selected for detection and identification of mycoplasma in vaccines. To replace compendial methods, the alternative method must demonstrate equivalence in both limit of detection (LOD) and specificity compared with compendial methods. Here, we summarize the validation of the CytoInspect™ method according to current pharmacopeia requirements. Validation of the robustness, sensitivity (at least 10 colony forming units/ml) and specificity of the CytoInspect™ method are demonstrated. Likewise, a comparability study was performed to compare the LOD for CytoInspect™ compared with the previously validated LOD for compendial culture tests.

Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Comparative analysis of the performance of residual host cell DNA assays for viral vaccines produced in Vero cells.

Residual host cell DNA (rcDNA) from continuous cell lines used for manufacturing of biological medicinal products has been considered as safety risk. Historically, several analytical methods have been used for rcDNA quantitation including hybridization assay, Threshold® assay and quantitative polymerase chain reaction (qPCR). Sanofi Pasteur has a wealth of experience in the development of methods quantifying rcDNA in vaccines. Here, we compared the performance of our in-house assays for quantifying rcDNA in viral vaccines produced in Vero cells. Vero alpha-satellite sequence qPCR was compared with the hybridization and Threshold® assays in terms of specificity, sensitivity and precision. The impact of viral inactivation with ß-propiolactone (BPL) on rcDNA, within the vaccine production process, was also assessed. We demonstrate that the quantity of rcDNA measured is influenced by the analytical method used. Vero cell DNA-specific qPCR assay was shown to be robust with a large dynamic range and no matrix interference on a range of products. The qPCR assay demonstrated greater sensitivity and specificity versus the hybridization and Threshold® methods. Vero alpha-satellite sequence qPCR is a specific and sensitive method for the assessment of the quantity of Vero rcDNA in the highly purified vaccines.