Roller Bottles Research Articles

Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.

The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice-grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks' yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase.

Editorial: What Next for Democracy?

Rat conceptuses of pregnancy day 11 (embryonic age approximately 10-5 days) with embryos within the yolk and amniotic sacs and polar allantoic placental tissues were cultured by the 'New system' utilizing homologous serum in 60 ml roller bottles with 20% O2, 5% CO2 and 75% N2 gas environment (five conceptuses/10 ml serum). Embryonic growth and development was assessed by external morphology and somite counts, and by measurement of DNA, RNA and protein contents. The embryonic growth was considerably retarded beyond 24 h when culture was extended to 48 h. During continuous culture of conceptuses for 48 h, the partial pressure of O2 in the medium decreased gradually with simultaneous increase of pCO2 and decline of pH values. Glucose level was depleted considerably and small quantities of urea, uric acid and creatinine accumulated in the medium. The concentrations of electrolytes (Na+, K+, Cl-) and total Ca and P, and lactic dehydrogenase activity in the medium increased during the latter part of the 48 h culture period. The growth and differentiation of embryos were significantly improved by transferring conceptuses to fresh serum medium with 40% O2, 5% CO2 and 55% N2 gas phase after 22-24 h culture and continuing the culture for an additional 24 h. Embryos grown in vitro for 48 h developed 39 somites and showed a tenfold increase in DNA content and a five- to seven-fold increase in RNA and protein contents, over initial values.

Assessment of packed bed bioreactor systems in the production of viral vaccines

Vaccination is believed to be the most effective method for the prevention of infectious diseases. Thus it is imperative to develop cost effective and scalable process for the production of vaccines so as to make them affordable for mass use. In this study, performance of a novel disposable iCELLis fixed bed bioreactor system was investigated for the production of some viral vaccines like Rabies, Hepatitis-A and Chikungunya vaccines in comparison to conventional systems like the commercially available packed bed system and roller bottle system. Vero and MRC-5 cell substrates were evaluated for growth parameters in all the three systems maintaining similar seeding density, multiplicity of infection (MOI) and media components. It was observed that Vero cells showed similar growth in all the three bioreactors whereas MRC-5 cells showed better growth in iCELLis Nano system and roller bottle system. Subsequently, the virus infection and antigen production studies also revealed that for Hepatitis-A and Chikungunya iCELLis Nano bioreactor system was better to the commercial packed bed bioreactor and roller bottle systems. Although for rabies antigen production commercially available packed bed bioreactor system was found to be better. This study shows that different bioreactor platforms may be employed for viral vaccine production and iCELLis Nano is one of such new convenient and a stable platform for production of human viral vaccines.

Transdifferentiation of hepatocytes to biliary epithelial cells requires expression of reprogramming factor Oct3/4 (398.8)

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial are not known. Recently we reported that adult rat liver expresses reprogramming factors Oct3/4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor Oct3/4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary transdifferentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6‐8 days in culture. We found that Oct3/4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of Oct3/4 was blocked in hepatocytes using adenovirus containing Oct shRNA, there was a significant decrease in appearance of biliary cells (compared to controls) as assessed by biliary marker HNF1‐beta, strongly supporting the role of Oct3/4 in transdifferentiation of hepatocyte to biliary epithelial cells. Experiments to investigate mechanisms through which Oct3/4 might regulate transdifferentiation are under way.Grant Funding Source: 1. Pathology Post‐doctoral Research Training grant, 2. Center for chronic diseases of aging grant

Ecological roles and biotechnological applications of marine and intertidal microbial biofilms.

This review is a retrospective of ecological effects of bioactivities produced by biofilms of surface-dwelling marine/intertidal microbes as well as of the industrial and environmental biotechnologies developed exploiting the knowledge of biofilm formation. Some examples of significant interest pertaining to the ecological aspects of biofilm-forming species belonging to the Roseobacter clade include autochthonous bacteria from turbot larvae-rearing units with potential application as a probiotic as well as production of tropodithietic acid and indigoidine. Species of the Pseudoalteromonas genus are important examples of successful surface colonizers through elaboration of the AlpP protein and antimicrobial agents possessing broad-spectrum antagonistic activity against medical and environmental isolates. Further examples of significance comprise antiprotozoan activity of Pseudoalteromonas tunicata elicited by violacein, inhibition of fungal colonization, antifouling activities, inhibition of algal spore germination, and 2-n-pentyl-4-quinolinol production. Nitrous oxide, an important greenhouse gas, emanates from surface-attached microbial activity of marine animals. Marine and intertidal biofilms have been applied in the biotechnological production of violacein, phenylnannolones, and exopolysaccharides from marine and tropical intertidal environments. More examples of importance encompass production of protease, cellulase, and xylanase, melanin, and riboflavin. Antifouling activity of Bacillus sp. and application of anammox bacterial biofilms in bioremediation are described. Marine biofilms have been used as anodes and cathodes in microbial fuel cells. Some of the reaction vessels for biofilm cultivation reviewed are roller bottle, rotating disc bioreactor, polymethylmethacrylate conico-cylindrical flask, fixed bed reactor, artificial microbial mats, packed-bed bioreactors, and the Tanaka photobioreactor.

Comparative study of bluetongue virus serotype 8 production on BHK-21 cells in a 50L Biostat® STR single-use bioreactors vs roller bottles

Background Bluetongue is a major disease of ruminant livestock that can have a substantial impact on income and animal welfare. Bluetongue virus serotype 8 (BTV-8) first emerged in the European Union in 2006, peaking at 45,000 cases in 2008. Zoetis (formerly Pfizer Animal Health) licensed bluetongue vaccines (Zulvac 4 Ovis, Zulvac 1 Ovis, Zulvac 1 Bovis, Zulvac 8 Ovis and Zulvac 8 Bovis and combinations) able to prevent viremia, stressing the role of the vaccine as an aid for the epidemiological control of the disease. One important issue to be taken into account in the development of vaccines is their cost, especially in veterinary use. Vaccine production requires high-yield, stable bioproduction systems and implementation of new technologies. Mammalian cells are the substrate for production of most of the veterinary vaccines. BHK-21 cells are commonly used to produce bluetongue vaccines. As an example, the use of the BTV-8 vaccine is routinely produced in roller bottles (RB). The aim of this study is to investigate Single-Use Bioreactor technology as an alternative to RB. This technology combines the basic concept of allowing the cells to attach to a surface (microcarriers) with the advantages of suspension, which allows a better control of culture conditions and systematic and automatic culture process. Single use technology can also be an alternative to conventional production methods reducing facility complexity, possibility of the rapid expansion of the capacity of the production and to avoid the cleaning process and reduction of the risk of cross-contamination. Lower culture handling and more homogeneity can be achieved. Selection of appropriate culture conditions can be important to achieve consistent cell culture and virus production across sites and scales. Because characteristics like tank geometry and hardware (impellers, sparger) are not subject to change during scale-up, the scalability from 50L to 1000L in the BIOSTAT STR bioreactor can be an easy strategy for our production process.

Viral vector production in the integrity® iCELLis® single-use fixed-bed bioreactor, from bench-scale to industrial scale

Wild-type or recombinant viruses used as vaccines and human gene therapy vectors are an important development tool in modern medicine. Some have demonstrated high potential such as lentivirus, paramyxovirus and adeno-associated-virus (AAV). These vectors are produced in adherent and suspension cell cultures (e.g. HEK293T, A549, VERO, PER.C6, Sf9) using either transient transfection (e.g PEI, calcium phosphate precipitation) or infection (e.g. modified or recombinant viruses) strategies. Most of these processes are currently achieved in static mode on 2-D systems (Roller Bottles, Cell Factories, etc.) or on suspended microcarriers (porous or non-porous). However, these two systems are time-consuming (large numbers of manipulation, preparation of equipment, etc.) and hardly scalable. In regards to process simplification and traceability, Integrity® iCELLis® bioreactors offer a new solution for scalability and monitoring of adherent cell cultures. The Integrity iCELLis Bioreactor Integrity® iCELLis® bioreactors from ATMI LifeSciences were designed for adherent cell culture applications such as recombinant protein, viral vaccine and gene therapy vector production. Using PET carriers trapped into a fixed-bed, cells grow in a 3-D environment with temperature, pH and dissolved oxygen controls. The iCELLis technology can be used at small-scale (the iCELLis nano from 0.5 to 4 m2) and manufacturing scale (iCELLis 500 from from 66 to 500 m2) which eases process scale-up and its overall utilization.

Model-based design of the first steps of a seed train for cell culture processes

Concept Production of biopharmaceuticals for diagnostic and therapeutic applications with suspension cells in bioreactors requires a seed train up to production scale [1]. For the final process steps in pilot and production scale the scale-up steps are usually defined (e.g. a factor of 5 10). More difficult in this respect are the first steps, the transitions between T-flasks, spinner tubes, roller bottles, shake flasks, stirred bioreactors or single-use reactors, because here often scale-up steps are different. The experimental effort to lay these steps out is correspondingly high. At the same time it is known that the first cultivation steps have a significant impact on the success or failure on production scale. The concept for a model based design of the seed train consists of the following steps:

Seed train optimization for suspension cell culture

The purpose of a seed train is the generation of an adequate number of cells for the inoculation of a production bioreactor. This is time- and cost-intensive: From volumes used for cell thawing or cell line maintenance the cell number has to be increased. The cells are usually run through many cultivation systems which become larger with each passage (e.g. T-flasks, roller bottles or shake flasks, small scale bioreactor systems and subsequently larger bioreactors. Single-use systems may be applied and systems which are inoculated at a partly filled state and culture volume is increased afterwards by medium addition). The production bioreactor is inoculated out of the largest seed train scale.

Effect of Operating Conditions in Production of Diagnostic Salmonella Enteritidis O-Antigen-Specific Monoclonal Antibody in Different Bioreactor Systems

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.

Role of Bacterial Exopolysaccharides (EPS) in the Fate of the Oil Released during the Deepwater Horizon Oil Spill

Halomonas species are recognized for producing exopolysaccharides (EPS) exhibiting amphiphilic properties that allow these macromolecules to interface with hydrophobic substrates, such as hydrocarbons. There remains a paucity of knowledge, however, on the potential of Halomonas EPS to influence the biodegradation of hydrocarbons. In this study, the well-characterized amphiphilic EPS produced by Halomonas species strain TG39 was shown to effectively increase the solubilization of aromatic hydrocarbons and enhance their biodegradation by an indigenous microbial community from oil-contaminated surface waters collected during the active phase of the Deepwater Horizon oil spill. Three Halomonas strains were isolated from the Deepwater Horizon site, all of which produced EPS with excellent emulsifying qualities and shared high (97-100%) 16S rRNA sequence identity with strain TG39 and other EPS-producing Halomonas strains. Analysis of pyrosequence data from surface water samples collected during the spill revealed several distinct Halomonas phylotypes, of which some shared a high sequence identity (≥97%) to strain TG39 and the Gulf spill isolates. Other bacterial groups comprising members with well-characterized EPS-producing qualities, such as Alteromonas , Colwellia and Pseudoalteromonas , were also found enriched in surface waters, suggesting that the total pool of EPS in the Gulf during the spill may have been supplemented by these organisms. Roller bottle incubations with one of the Halomonas isolates from the Deepwater Horizon spill site demonstrated its ability to effectively produce oil aggregates and emulsify the oil. The enrichment of EPS-producing bacteria during the spill coupled with their capacity to produce amphiphilic EPS is likely to have contributed to the ultimate removal of the oil and to the formation of oil aggregates, which were a dominant feature observed in contaminated surface waters.

Efficacy and Safety of Cell-Associated Vaccines Against Marek's Disease Virus Grown in QT35 Cells or JBJ-1 Cells

The Marek's disease virus (MDV) vaccine strain CVI 988 usually is grown in primary chicken embryo fibroblasts (CEFs). We found that the strains could be grown also in the QT35 and JBJ-1 cell lines to titers in the same range as in the CEFs. Both cell lines are fibroblast-like cell lines, which can be grown in flat-bottomed tissue-culture flasks, roller bottles, and on microcarriers. For growth in QT35 cells it was necessary to adapt the virus to the cell line; for growth in JBJ-1 cells this was not necessary. We investigated the efficacy of experimental CVI 988 vaccines grown in QT35 cells and JBJ-1 cells. The efficacy studies were performed in accordance with European Pharmacopoeia (EP) monograph for live MDV disease vaccines. Groups of 1-day-old specific-pathogen-free chicks were vaccinated. Nonvaccinated control groups were included in the studies. Five to 7 days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of MD. The protection induced by CVI 988 grown in QT35 cells as well as JBJ-1 cells complied with the requirements of the EP that prescribe that the protection index should be at least 80%. The safety of the vaccines grown in QT35 cells and JBJ-1 cells was tested in a field study in commercial layer chickens. The vaccine virus was not safe after passaging in QT35 cells. This can be explained by the presence of fragments of the genome of MDV strains in the QT35 cell line. No signs of MD were noticed in the study in which CVI988 grown in JBJ-1 cells was tested. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Production of Cell Culture Based Anti- rabies Vaccine in Ethiopia

Prevention and control of rabies in the world will require international efforts to increase the availability and use of high quality cell-culture rabies vaccines for use in human and veterinary. An important aspect of activities to ensure such availability is transfer of technologies to developing countries for production of these vaccines. Methods for Rabies Virus manipulation have changed fundamentally from random attenuation to defined modifications. In 2001, WHO issued a resolution for the complete replacement of nerve tissue vaccines by 2006 with cell-culture rabies vaccines. However, sheep brain derived Fermi type rabies vaccine is still being manufactured and utilized for the majority of exposed patients in Ethiopia. Therefore, production of a safer and effective cell culture based anti-rabies vaccine is needed. Currently the Ethiopian government has heavily invested in upgrading the facilities required to produce a rabies vaccine in keeping with WHO recommendation. Rabies virus suspensions were obtained from vero cells cultivated on roller bottles after infection with the Pasteur virus strain (PV) and Evelyn Rokitniki Abelseth (ERA). Initially the titer of the obtained virus and multiplicity of infection of the viruses had to be optimized; therefore in rabies virus infected cultures, higher virus yields was obtained when infected with 0.001ERA virus/cell and incubated at 37°C in 5% CO2 for 96hr and 0.01PV/cell incubated at 37°C in 5% CO2 for 48hr. Based on the results it is conclude that, ERA virus 0.001ID/cell with incubation period of 96h and was selected as best titer for rabies vaccine production.

The feasibility of a novel bioreactor for vaccine production of classical swine fever virus

The performance of a new type of tide mode culture system was investigated in this study. This novel bioreactor provides two separated stages, liquid and gas, for cell growth requirements. The immobilized cells absorbed the nutrient from medium during the liquid stage and subsequently were exposed directly to fresh air to absorb oxygen during the gas stage. Operating with PK15 cells under optimal conditions, we obtained 2.3×109 cells in 500ml reactor. It is 30 times higher than the initial inoculum and about 11 times higher than the production by roller bottle. For the vaccine production of classical swine fever (CSFV), a high virus titer of 2.1×109 median tissue culture infective dose (TCID50) was yielded which provided exceed 300 doses per milliliter of CSFV solution. Therefore, this new cultural system performed well not only for cell production but also for virus yield. It should be a highly efficient production for the CSFV vaccine and have practical potential in other animal cell culture vaccine.

Establishment and characterization of immortalized human hepatocyte cell line for applications in bioartificial livers

An immortalized human hepatocyte cell line, HepLi5, was established via transfection of Simian virus 40 large T antigen (SV40 LT) into primary human hepatocytes. The morphologic and functional characteristics of HepLi5 cells were evaluated. The expression of SV40 LT in HepLi5 cells was detected by reverse transcription-PCR (RT-PCR) and western blotting. mRNA expression of liver-enriched genes, including glutamine synthetase, albumin, and cytochrome P450 was detected via RT-PCR in HepLi5 cells. Activity of CYP1A2, one of the drug-metabolizing P450 enzymes, was detected. Subcutaneous injection of HepLi5 cells into nude mice did not induce tumors within 3 months. Short Tandem Repeat results confirmed the authenticity of the cell line. Clinical-grade quantities of HepLi5 cells could be harvested using large-scale culture in roller bottles after which their cellular function was significantly enhanced. Therefore, the immortalized HepLi5 cells are a suitable cell source for applications in bioartificial livers.

Microbial Activities and Dissolved Organic Matter Dynamics in Oil-Contaminated Surface Seawater from the Deepwater Horizon Oil Spill Site

The Deepwater Horizon oil spill triggered a complex cascade of microbial responses that reshaped the dynamics of heterotrophic carbon degradation and the turnover of dissolved organic carbon (DOC) in oil contaminated waters. Our results from 21-day laboratory incubations in rotating glass bottles (roller bottles) demonstrate that microbial dynamics and carbon flux in oil-contaminated surface water sampled near the spill site two weeks after the onset of the blowout were greatly affected by activities of microbes associated with macroscopic oil aggregates. Roller bottles with oil-amended water showed rapid formation of oil aggregates that were similar in size and appearance compared to oil aggregates observed in surface waters near the spill site. Oil aggregates that formed in roller bottles were densely colonized by heterotrophic bacteria, exhibiting high rates of enzymatic activity (lipase hydrolysis) indicative of oil degradation. Ambient waters surrounding aggregates also showed enhanced microbial activities not directly associated with primary oil-degradation (β-glucosidase; peptidase), as well as a twofold increase in DOC. Concurrent changes in fluorescence properties of colored dissolved organic matter (CDOM) suggest an increase in oil-derived, aromatic hydrocarbons in the DOC pool. Thus our data indicate that oil aggregates mediate, by two distinct mechanisms, the transfer of hydrocarbons to the deep sea: a microbially-derived flux of oil-derived DOC from sinking oil aggregates into the ambient water column, and rapid sedimentation of the oil aggregates themselves, serving as vehicles for oily particulate matter as well as oil aggregate-associated microbial communities.

A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes.

Human ex vivo gene therapy protocols have been used successfully to treat a variety of genetic disorders, infectious diseases, and cancer. Murine oncoretroviruses (specifically, gammaretroviruses) have served as the primary gene delivery vehicles for these trials. However, in some cases, such vectors have been associated with insertional mutagenesis. As a result, alternative vector platforms such as lentiviral vectors (LVVs) are being developed. LVVs may provide advantages compared with gammaretroviral vectors, including the ability to transduce large numbers of nondividing cells, resistance to gene silencing, and a potentially safer integration profile. The aim of this study was to develop a simplified process for the rapid production of clinical-grade LVVs. To that end, we used a self-inactivating bicistronic LVV encoding an MART (melanoma antigen recognized by T cells)-1-reactive T cell receptor containing oPRE, an optimized and truncated version of woodchuck hepatitis virus posttranslational regulatory element (wPRE). Using our simplified clinical production process, 293T cells were transiently transfected in roller bottles. The LVV supernatant was collected, treated with Benzonase, and clarified by modified step filtration. LVV produced in this manner exhibited titers and a biosafety profile similar to those of cGMP (current Good Manufacturing Practices) LVVs previously manufactured at the Indiana University Vector Production Facility in support of a phase I/II clinical trial. We describe a simple, efficient, and low-cost method for the production of clinical-grade LVV for ex vivo gene therapy protocols.

Role of reprogramming factors in transdifferentiation of hepatocytes to biliary epithelial cells

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial are not known. Recently we reported that adult rat liver expresses reprogramming factors Oct3/4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factors play a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid cultures. In this system, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6–8 days in culture. When we looked at the expression of reprogramming factors in this system, we found that Oct3/4, Nanog, and Klf4 are upregulated at day 6 in this system as assessed by mRNA levels suggesting the possible involvement of reprogramming factors in transdifferentiation. Further analysis of protein levels by Western blot, immunohistochemical staining, and intervention studies by blocking reprogramming factors are in progress to elucidate the role of reprogramming factors in transdifferentiation.

Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination

Severe climate change and global warming may impact significantly on vector-borne disease including Japanese encephalitis (JE) infection in human and animals. Thus, veterinary authority requires large quantity of diagnostic tools to survey vector-borne diseases. New producing method having a relation with JE antigen is needed to substitute conventional sucrose-acetone extraction method using suckling mouse. So, we developed new manufacturing method using polyethylene glycol (PEG) precipitation. Japanese encephalitis virus (JEV) was propagated in roller bottle containing Vero cell and inactivated with two kinds of inactivating reagents. Viability of the supernatant of bulk containing antigen was checked using Vero cell after inactivation. The supernatant did not show hemagglutination (HA) activity with goose erythrocytes. The antigen inactivated by binary ethylenimine (BEI) and concentrated by PEG precipitation method was found to be 2048 HA, but the antigen inactivated by 0.3% formaldehyde solution and concentrated by PEG precipitation method did not show HA titer. The antigen prepared from mice brain using sucrose-acetone extraction method showed 256 HA titer. This BEI inactivation method does not evoke animal welfare problem and can replace the conventional method that required biological hazardous reagents and suckling mice in preparing HA antigen. This new BEI inactivation method was safe in producing HA antigen against JEV in laboratory and can reduce environmental contamination of acetone.

Large pilot scale cultivation process study of adherent MDBK cells for porcine Influenza A virus propagation using a novel disposable stirred-tank bioreactor

Influenza is one of the major viral diseases and has tremendous importance for human and animal health worldwide. The etiologic agent, Influenza A virus belongs to the family Orthomyxoviridae and is a single stranded (-) enveloped RNA virus with round shape and a diameter of 90-130 nm. Its genome consists of 8 segments which code for up to 11 proteins. The major surface proteins and antigenic determinants are hemagglutinin (HA) and neuraminidase (NA). Pigs are of special importance for Influenza. They are susceptible for a number of Influenza A viruses from different species (human, avian, porcine); in the case of multiple infections of swine new and dangerous virus strains can be assembled by reassortment of genes (antigenic shift). The strain which was used in this study is part of a trivalent inactivated vaccine against porcine Influenza (IDT Biologika GmbH). The cultivation of Influenza virus for the production of vaccines is often still done in embryonated hens eggs, although there are a number of cell lines available which propagate the virus, e. g. VERO or MDCK cells. Egg production processes are limited in terms of capacity whereas cell culture based processes are more flexible and easy to scale up when facing pandemic situations (H5N1 in 2005 and H1N1pan in 2009/10). Currently, adherent MDBK cells are used for the production of porcine Influenza A viruses at commercial scale (IDT Biologika GmbH). This process is performed in roller bottles so far using a biphasic process scheme (cell growth and virus propagation phase) and is thus limited in capacity, yields and process control. The main goal of this study was to explore options for direct transfer of this process into a novel disposable stirred-tank bioreactor (BIOSTAT CultiBag STR, Sartorius Stedim Biotech GmbH) using microcarriers for the attachment of MDBK cells. The advantage of this system is its scalability up to 1000 l, better process control and presumably higher virus yields. The use of disposables has positive effects on costs and efforts but also for cleaning validation in a multi-purpose facility. Based on the results of this study further optimization steps can be done and the implementation of such systems in GMP routine vaccine production can be evaluated.