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.

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

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 ∼10(4)), 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.

Safety of MDCK cell culture-based influenza vaccines.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This problem is compounded by the fact that the pandemic-driven situation in 2009 has roughly doubled the overall vaccine demand. Modern cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the increasing worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumors have fomented uncertainty among the general population and physicians, and also discredit officially accepted test results and product licensing. This article provides an overview of the safety profile of the cell culture technology, of the cells and of the final vaccine product.

Theory and practice of conventional adventitious virus testing.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) For decades conventional tests in cell cultures and in laboratory animals have served as standard methods for broad-spectrum screening for adventitious viruses. New virus detection methods based on molecular biology have broadened and improved our knowledge about potential contaminating viruses and about the suitability of the conventional test methods. This paper summarizes and discusses practical aspects of conventional test schemes, such as detectability of various viruses, questionable or false-positive results, animal numbers needed, time and cost of testing, and applicability for rapidly changing starting materials. Strategies to improve the virus safety of biological medicinal products are proposed. The strategies should be based upon a flexible application of existing and new methods along with a scientifically based risk assessment. However, testing alone does not guarantee the absence of adventitious agents and must be accompanied by virus removing or virus inactivating process steps for critical starting materials, raw materials, and for the drug product.

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

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 10(34). Residual MDCK-DNA is < or =10 ng per dose and the ss-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base 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.

[Safety of cell culture-based influenza vaccines]. / Sicherheit von Influenza-Impfstoffen auf Zellkulturbasis.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This situation is compounded by the fact that the present pandemic-driven situation has roughly doubled the overall vaccine demand virtually "overnight". Modem cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the exploding worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumours raised uncertainty among physicians and the general population, and also discredit officially accepted assessments and product licensing by the relevant authorities. The present article provides an overview on the cell culture technology and on the safety profile of the cells and of the vaccine product.

[Prion safety of medicinal products using the example of an influenza vaccine produced in a cell line]. / Prionen-Sicherheit von Arzneimitteln am Beispiel eines Zellkultur-Impfstoffs.

Prions are pathogenic proteins and are the cause for spongiform encephalopathies. Pathogenic prions differ from physiologically common non-pathogenic prions only in their sterical structure. Upon infection by a pathogenic prion protein, a series of reactions is initiated in which common non-pathogenic prion proteins are transformed into pathogenic prions. Animals, mainly ruminants like cattle, sheep and goats are susceptible to prions, but also man. Prions are very robust and it is difficult to inactivate them. During the production processes of pharmaceuticals, the risk for contamination by infectious prions can be reduced by careful choice of animal material, the replacement of animal material and by appropriate production procedures. For instance biologicals like influenza vaccines can be produced by a permanent canine cell line, whose prion safety has been proven by useful methods (standard scrapy cell assay). Mandatory guidelines ensure that the risk for contamination by pathogenic prions has to be considered and excluded in the production of bio-pharmaceuticals.

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

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.

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.