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Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

Hu AY, Tseng YF, Weng TC, Liao CC, Wu J, Chou AH, Chao HJ, Gu A, Chen J, Lin SC, Hsiao CH, Wu SC, Chong P - PLoS ONE (2011)

Bottom Line: No significant difference was found in the cell growth in different sizes of bioreactors studied.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.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.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research and Development Center, National Health Research Institutes, Zhunan, Taiwan Authority.

ABSTRACT

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.

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Related in: MedlinePlus

Production of HA antigens produced either in SC or SF medium.Samples taken from the 7.5 L bioreactor during the infection period were analyzed for HA antigen content. The harvest of HA antigen reached a peak at 14.1 µg/mL on day 3 post infection, as determined by SRID assay. By contrast, the harvest from the SC medium was 12.7 µg of HA/mL.
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pone-0014578-g003: Production of HA antigens produced either in SC or SF medium.Samples taken from the 7.5 L bioreactor during the infection period were analyzed for HA antigen content. The harvest of HA antigen reached a peak at 14.1 µg/mL on day 3 post infection, as determined by SRID assay. By contrast, the harvest from the SC medium was 12.7 µg of HA/mL.

Mentions: Similar to the spinner flask results, MDCK cell density grown in the 7.5 L bioreactor was found to be higher in the SC medium (>6×106 cell/mL) than those obtained from the SF medium (2.3×106 cell/mL). But the virus yield and HA titers at the 3rd day harvest had very similar values (1024 HA titer/50 µL and >7×108 pfu/mL) obtained from the SC study. Samples taken from the 7.5 L bioreactor during the infection period were further analyzed for HA antigen concentration, and then compared with the values obtained from the SC study. The HA antigen concentrations were determined by the single-radial immunediffusion (SRID) assay [21] and shown in Figure 3. The HA concentrations increased with infection time. The concentrations of HA antigen in day 1 were under the detection limit. On the harvest day, the HA antigen concentration from the SF culture was found to be 14.1 µg/mL on average over three runs, whereas the average 12.7 µg of HA antigen/mL was found in the SC culture. It shows that the titer of HA antigen produced from SF medium was slightly higher than that from SC medium (p<0.05). Influenza H5N1 virus produced in the SF medium was further concentrated, sucrose-gradient purified, and formalin-inactivated as described in the Materials and Methods section. Based on the amount of HA found in the harvest, the average of the total recovery rate of HA yield was around 45%. In the antigenicity study, the experiments were performed on the H5N1 viruses produced from different culture (SC and SF) media and found to have the same reactive titer (400) with the NIBRG14 standard antibody obtained from the WHO reference laboratory. These titers show that high-yield MDCK cell-based influenza vaccine production is possible using the current SF bioreactor technology.


Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

Hu AY, Tseng YF, Weng TC, Liao CC, Wu J, Chou AH, Chao HJ, Gu A, Chen J, Lin SC, Hsiao CH, Wu SC, Chong P - PLoS ONE (2011)

Production of HA antigens produced either in SC or SF medium.Samples taken from the 7.5 L bioreactor during the infection period were analyzed for HA antigen content. The harvest of HA antigen reached a peak at 14.1 µg/mL on day 3 post infection, as determined by SRID assay. By contrast, the harvest from the SC medium was 12.7 µg of HA/mL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025921&req=5

pone-0014578-g003: Production of HA antigens produced either in SC or SF medium.Samples taken from the 7.5 L bioreactor during the infection period were analyzed for HA antigen content. The harvest of HA antigen reached a peak at 14.1 µg/mL on day 3 post infection, as determined by SRID assay. By contrast, the harvest from the SC medium was 12.7 µg of HA/mL.
Mentions: Similar to the spinner flask results, MDCK cell density grown in the 7.5 L bioreactor was found to be higher in the SC medium (>6×106 cell/mL) than those obtained from the SF medium (2.3×106 cell/mL). But the virus yield and HA titers at the 3rd day harvest had very similar values (1024 HA titer/50 µL and >7×108 pfu/mL) obtained from the SC study. Samples taken from the 7.5 L bioreactor during the infection period were further analyzed for HA antigen concentration, and then compared with the values obtained from the SC study. The HA antigen concentrations were determined by the single-radial immunediffusion (SRID) assay [21] and shown in Figure 3. The HA concentrations increased with infection time. The concentrations of HA antigen in day 1 were under the detection limit. On the harvest day, the HA antigen concentration from the SF culture was found to be 14.1 µg/mL on average over three runs, whereas the average 12.7 µg of HA antigen/mL was found in the SC culture. It shows that the titer of HA antigen produced from SF medium was slightly higher than that from SC medium (p<0.05). Influenza H5N1 virus produced in the SF medium was further concentrated, sucrose-gradient purified, and formalin-inactivated as described in the Materials and Methods section. Based on the amount of HA found in the harvest, the average of the total recovery rate of HA yield was around 45%. In the antigenicity study, the experiments were performed on the H5N1 viruses produced from different culture (SC and SF) media and found to have the same reactive titer (400) with the NIBRG14 standard antibody obtained from the WHO reference laboratory. These titers show that high-yield MDCK cell-based influenza vaccine production is possible using the current SF bioreactor technology.

Bottom Line: No significant difference was found in the cell growth in different sizes of bioreactors studied.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.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.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research and Development Center, National Health Research Institutes, Zhunan, Taiwan Authority.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus