Jens-Peter Gregersen

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine.

Abstract Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu®); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 1034. Residual MDCK-DNA is ≤10ng per dose and the ß-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.

A quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine

Abstract A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU®, Novartis Vaccines and Diagnostics) is produced using Madin–Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10−6 to 10−16 infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.

A risk-assessment model to rate the occurrence and relevance of adventitious agents in the production of influenza vaccines

Abstract Influenza vaccine production has traditionally relied on the use of embryonated chicken eggs for virus isolation and propagation, but recently, cell-culture-derived manufacturing methods have been introduced. During influenza vaccine production, by either conventional or cell culture methods, there is a risk of incidental contamination by adventitious agents. Thus, a risk-assessment model has been developed to qualitatively assess the potential risk of vaccine process contamination by viral pathogens. The model takes into account the basic growth characteristics of each virus, its ability to grow in different cell substrates and resistance to processing steps during vaccine manufacture. The risk-assessment model has been applied to various pathogens to determine potential risk and relevance in different manufacturing scenarios, using different cell substrates for virus propagation, including Madin–Darby canine kidney (MDCK) cells. Avian viruses, introduced via use of embryonated eggs for virus isolation, were found to present the greatest risk, irrespective of the substrate used for influenza virus propagation. The use of MDCK cells to propagate vaccine virus from egg-isolated influenza virus strains does not introduce a new or greater adventitious virus risk, compared with egg-based vaccine production. Indeed, the adventitious virus risk is potentially reduced as fewer viruses are able to grow in MDCK cells.

Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses

Abstract This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼104), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.

Comparison of the novel ResPlex III assay and existing techniques for the detection and subtyping of influenza virus during the influenza season 2006–2007

Influenza virus is a major cause of disease worldwide. The accurate detection and further subtyping of influenza A viruses are important for epidemiologic surveillance, and subsequent comprehensive characterization of circulating influenza viruses is essential for the selection of an optimal vaccine composition. ResPlex III is a new multiplex reverse transcriptase polymerase chain reaction (RT-PCR)-based method for detecting, typing, and subtyping influenza virus in clinical specimens. The ResPlex III assay was compared with other methods with respect to sensitivity and accuracy, using 450 clinical specimens obtained from subjects throughout Germany during the 2006–2007 influenza season. Samples were analyzed for the presence of influenza virus in Madin-Darby canine kidney (MDCK) cells by rapid cell culture using peroxidase staining and conventional cell culture confirmed by hemagglutination inhibition assay, a rapid diagnostic assay (Directigen Flu A+B test; BD Diagnostic Systems, Heidelberg, Germany), in-house real-time RT-PCR (RRT-PCR), and ResPlex III (Qiagen, Hilden, Germany). ResPlex III had the highest sensitivity for detecting influenza virus in clinical specimens, followed by in-house RRT-PCR (96% compared with ResPlex III). Conventional cell culture in MDCK cells, rapid culture, and quick test assays were substantially less sensitive (55%, 72%, and 39%, respectively). Virus subtyping results were identical using ResPlex III and the standard virological subtyping method, hemagglutination inhibition. ResPlex III is a quick, accurate, and sensitive assay for detecting and typing influenza A and B viruses and subtyping influenza A viruses in clinical specimens, and might be considered for a supplemental role in worldwide seasonal and pandemic influenza surveillance.

Inactivation of stable viruses in cell culture facilities by peracetic acid fogging

Looking for a robust and simple method to replace formaldehyde fumigation for the disinfection of virus-handling laboratories and facilities, we tested peracetic acid fogging as a method to inactivate stable viruses under practical conditions. Peracetic acid/hydrogen peroxide (5.8%/27.5%, 2.0 mL/m³) was diluted in sufficient water to achieve ≥ 70% relative humidity and was vaporized as <10 μm droplets in a fully equipped 95 m³ laboratory unit. High titers of reovirus 3, MVM parvovirus and an avian polyomavirus were coated on frosted glass carriers and were exposed to the peracetic acid fog in various positions in the laboratory. After vaporization, a 60 min exposure time, and venting of the laboratory, no residual virus was detected on any of the carriers (detection limit <1 infectious unit/sample volume tested). The log reduction values were 9.0 for reovirus, 6.4 for MVM parvovirus, and 7.65 for the polyomavirus. After more than 10 disinfection runs within 12 months, no damage or functional impairment of electrical and electronic equipment was noted.