Process development of chromatography-based purification on pandemic influenza virus-like particle based vaccines

Since 2013, the H7N9 avian influenza virus is considered a threat to global public health. The development of the H7N9 avian influenza vaccine is one of the most effective strategies to prevent influenza pandemics. Virus-like particles (VLPs) influenza vaccines is non-infectious viral structural proteins. Not only to retain the ability to produce neutralizing antibodies against to viral surface protein, but also safer than the conventional inactivated vaccines. In our previous study, we successfully expressed three structural proteins, hemagglutinin (HA), neuraminidase (NA) and matrix (M1) from influenza A / Taiwan / 1/2013 (H7N9) of the VLP in insect cells. In this study, we present a downstream purification method for the VLP platform. The purification process involves microfiltration, chromatography (using ion exchange, affinity and gel filtration combinations), concentration, diafiltration and sterile filtration steps. In this study, 600 ml of the harvest from the baculovirus expression system was used. The characteristics of VLP volume were examined by HA assay, SDS-PAGE and negative staining transmission electron microscopy (TEM). The overall recovery of HA protein was approximately 38%. In the evaluation of immunized mouse, such virus particles have been shown the HI titer \u3e256. This study demonstrated the chromatographic-based purification process can provide an effective VLP vaccine production for the preparation of the H7N9 influenza pandemic

Low-cost cell-based production platform for seasonal and pandemic influenza vaccines

Influenza-related illnesses have caused an estimated over million cases of severe illness, and it has about hundred thousands of deaths worldwide annually. Traditionally these vaccines are produced in embryonated chicken eggs. However, in the case of a pandemic outbreak, this egg-based production system may not be quickly enough to meet the surging demand. The efficacy associated with egg-based vaccines are low in recently years. The raising concerns with egg-derived vaccines is resulting in the spurred exploration of alternatives. MDCK cells are becoming as an alternative host to embryonated eggs for influenza virus propagation. Although MDCK cells were considered to be a suitable host for the virus production, their inability to grow in suspension still limits the process of scale-up and their production capability. Please click Additional Files below to see the full abstract

Simple and robust downstream purification process for cell-derived influenza vaccines

New emerging influenza viruses with pandemic potentials were occurred in recent years, e.g. H5N1 in 1997, H1N1 in 2009, and H7N9 in 2013. The demand of producing pandemic influenza vaccines for human use with quick supply is high. For the cell-based pandemic influenza vaccines, we proposed a flow-through chromatography purification process. This process has only involved few purification steps and is easy to operate. Vero- and MDCK- cell derived avian influenza viruses including H5N1 and H7N9 were purified efficiently by the process proposed. The presented purification process consisted of clarification, inactivation, concentration, anion exchange chromatography (Capto Q), size exclusion and adsorption chromatography (Capto Core 700), diafiltration and sterile filtration. In the chromatography steps, cell DNA and protein were removed remarkably, and the virus were flowed through these columns. The flow rate was set as fast as 250 cm/min. The loading volume of virus solution was up to 50 times of column volume (CV).The DNA was removed over 90% after using Capto Q column, and was further removed by Capto Core 700 column. The overall removal rate of cellular DNA was more than 99%. The HA recovery rates of H5N1 and H7N9 influenza virus from Vero and MDCK cells were 20 to 40%. The DNA concentration of all purified bulks met the regulatory requirement of 10ng per dose. The developed purification process is simple and efficient, and it is suitable for purification of various influenza virus strains and can be used for the pandemic influenza vaccine production

Stability evaluation of inactivated influenza H7N9 vaccines derived from adhesion and suspension MDCK cells

In recent years, cell-based manufacturing processes for influenza vaccine production has gained a great interest over the egg-based process. We have adapted MDCK cells to grow in chemical-defined medium and found this newly suspension MDCK cell line (sMDCK cells) is very suitable for the production of influenza virus. However, the property of purified antigens from sMDCK cells remains unclear. In this study, a stability program of influenza H7N9 vaccine (NIBRG268 vaccine strain) produced by sMDCK cells was investigated, and the data were compared with the vaccine derived from adhesion MDCK (aMDCK) cells in serum-free medium. The H7N9 bulks (with different storage time) derived from sMDCK and aMDCK cells were stored at 2-8°C for some times, and a number of parameters were used to monitor the H7N9 vaccine antigen stability was evaluated at different periods (1, 2, 3 and 6 months). The monitoring parameters are including virus structure, HA titer, HA content, total protein level, antigenicity, and immunogenicity. The sMDCK-derived H7N9 bulk showed similar virus structure to that aMDCK-derived H7N9 bulk, and there was no obvious change after further 6 months of storage. Furthermore, HA titer, HA content and total protein level of sMDCK- and aMDCK-derived H7N9 bulks were stable after 6 months of storage. sMDCK- and aMDCK-derived H7N9 bulks displayed similar antigenicity detected by hemagglutination inhibition (HI) test using standard serum. Finally, the results of HI and neutralization tests showed that sMDCK- and aMDCK-derived H7N9 vaccines were similar in immunogenicity in BALB/c mice vaccinated with 0.2 μg of H7N9 vaccine with an adjuvant of aluminum hydroxide. These results indicate that sMDCK-derived H7N9 bulk has good stability data similar to the aMDCK-derived H7N9 bulk. Thus, the newly developed suspension MDCK cells show a great market potential over the traditional vaccine manufacturing methods

Emulsified Nanoparticles Containing Inactivated Influenza Virus and CpG Oligodeoxynucleotides Critically Influences the Host Immune Responses in Mice

Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity.After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span(R)85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 microg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2).Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness

Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 108 TCID50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes

Production of Inactivated Influenza H5N1 Vaccines from MDCK Cells in Serum-Free Medium

BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production

Anaerobic in-tank membrane bioreactor for wastewater treatment

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