Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Suspension adapted Vero cell cultures in IHM03 had a cell doubling time of 40-44 h.

“The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40-44 h.”

• Also stated in: § 3.1. Adaptation of adherent Vero cells to grow in suspension in serumfree media · plain

  “The Vero cells started to grow in suspension only in serum-free IHM03 medium after 5-8 weeks of adaptation process to reach a doubling time in a range between 40 and 44 h.”

All Vero cell samples tested, including adherent and suspension adapted cells at various passages, had 100% concordance with the Vero DNA control sample in STR analysis.

“Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability.”

• Also stated in: § 3.3. Genetic stability of adherent and suspension adapted Vero cells over the culture passaging and maintenance process · hedged

  “Electropherogram results of all samples showed 100% concordance with the Vero DNA control sample, indicating that the adherent and suspension adapted Vero cells retained their genetic stability after passaging and/or adapting to grow in suspension in serumfree IHM03 medium for many passages.”

Suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as adherent Vero cells grown in commercial serum-free media.

“The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media”

Much higher cell density (8 × 10^6 cells/mL) was achieved in a batch culture when three volumes of culture medium were replaced during the batch culture process.

“Much higher cell density (8 Â 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process.”

• Also stated in: § 3.2. Growth of suspension adapted Vero cell in batch cultures · plain

  “The data in Fig. 2 showed the viable cell density in shake flask cultures reached 8 Â 10 6 cells/mL after 3 complete medium replacements during the batch culture process.”

Suspension adapted Vero cells at passage number 163 were not found to be tumorigenic under the conditions of the study.

“Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic.”

• Also stated in: § 3.4. Tumorigenicity of suspension adapted Vero cells · plain

  “In conclusion, under the conditions of this study, the test article, suspension adapted Vero cell at a passage number of 163, was not found to be tumorigenic.”

Vero cells started to grow in suspension only in serum-free IHM03 medium after 5-8 weeks of adaptation process.

“The Vero cells started to grow in suspension only in serum-free IHM03 medium after 5-8 weeks of adaptation process to reach a doubling time in a range between 40 and 44 h.”

More than one log of increase in VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10^6 cells/mL.

“More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 Â 10 6 cells/mL.”

Adherent Vero cells were successfully adapted to grow in suspension in serum-free and animal component-free medium IHM03 developed in-house.

“Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house.”

Suspension cell culture showed better productivity of VSV than adherent Vero cell culture.

“The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture.”

• Also stated in: § 3.5. Production of vesicular stomatitis virus (VSV) in adherent and suspension adapted Vero cell flask cultures · plain

  “The data in Fig. 4 showed that the volumetric virus productivity of the suspension adapted Vero cell was higher than the titer achieved by the adherent Vero cell in T-flask.”

• AssumptionRests on an assumption: VSV is a representative model for viral productivity of other vaccine viruses.

The culture media used to grow cells in the adherent culture stage did not affect the time required to adapt adherent cells to grow in suspension.

“Experimental data indicated that the culture media (starting media) used to grow the cells in adherent culture stage did not affect time required to adapt the adherent cells to grow in suspension.”

Concentrations of lactate (about 20 mM/L) and ammonia (4-5 mM/L) were at normal ranges observed in other types of cell cultures.

“While the concentration of lactate (about 20 mM/L) and ammonia (4-5 mM/L), the two common inhibitory metabolites, were at normal ranges observed in other types of cell cultures.”

Mixing IHM03 with other high performance commercial media such as HEK GM did not improve maximum cell density or cell doubling times.

“In addition, mixing IHM03 with other high performance commercial media such as HEK GM (Xell, Bielefeld, Germany) did not either improve the maximum cell density in the batch culture or cell doubling times.”

VSV productivity achieved in the 3L bioreactor culture was similar to the titer obtained in small shake flask cultures infected at 1 × 10^6 cells/mL.

“Fig. 6 showed that the VSV productivity achieved in the 3L bioreactor culture was similar to the titer obtained in the small shake flask cultures infected at 1 Â 10 6 cells/mL (Fig. 4)”

Volumetric productivity improved from 2.3 × 10^9 TCID50%/mL to 8.9 × 10^9 TCID50%/mL, or 3.8 folds, when cell density at infection increased from 0.87 × 10^6 cells/mL to 2.5 × 10^6 cells/mL.

“The data in Fig. 5 showed the volumetric productivity improved from 2.3 Â 10 9 TCID 50% /mL to 8.9 Â 10 9 TCID 50% /mL, or 3.8 folds of increase, when the cell density at infection increased from 0.87 Â 10 6 cells/mL to 2.5 Â 10 6 cells/mL.”

Volumetric virus productivity declined in adherent Vero culture when cell density at infection increased to 1.0 × 10^6 cells/mL from 0.30 × 10^6 cells/mL.

“while the volumetric virus productivity declined in the adherent Vero culture when the cell density at infection increased to 1.0 Â 10 6 cells/mL from 0.30 Â 10 6 cells/mL in the adherent cultures.”

All positive control mice injected with 18C1-10T cells developed tumors at the injection site.

“All of the positive control mice (Group 2) developed tumors at the injection site as expected, although tumors were not found in other sites examined.”

Formation of cell aggregates was mainly due to attachment of cells to the surface of shake flask and formation of a cell ring.

“Later work showed that the formation of cell aggregates was mainly due to the attachment of cells to the surface of shake flask and formation of a cell ring.”

The doubling time of 40-44 h of suspension adapted Vero cells in IHM03 was longer than the about 24 h reported in serum-containing and actively growing adherent culture.

“The doubling time (40-44 h) of suspension adapted Vero cells in IHM03 medium was longer than the about 24 h reported in serum-containing and actively growing adherent culture [15].”

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Suspension adapted Vero cells grew to a density slightly higher than 2 × 10^6 cells/mL in shake flask cultures.

“The suspension adapted Vero cell grew to a density slightly higher than 2x10 6 cells/mL in shake flask cultures (Fig. 2).”

Analysis of residual nutrient components in spent media revealed substantial amounts of glucose (almost 20 mM/L) and amino acids at the end of culture.

“Analysis of residual nutrient components in the spent media taken from the batch culture process revealed that there were still substantial amounts of glucose (almost 20 mM/L) and amino acids in the spent media at the end of culture.”

The maximum cell density in batch cultures was not significantly improved by feeding with Cell Boost 5 or HEK TF.

“The maximum cell density in the batch cultures was not significantly improved by feeding the batch cultures with Cell Boost 5 (Hyclone, Logan, UT) or HEK TF (Xell, Bielefeld, Germany) (data not shown).”

Virus productivity in the bioreactor was slightly better than in the infected control shake flask cultures with cells either from the bioreactor or from the shake flask with medium replacement.

“The virus productivity in the bioreactor (Bioreactor) was slightly better than the infected control shake flask cultures (BioR-CTL-6.8E6and SF-CTL-6E6) with the cells either from the bioreactor or from the shake flask with medium replacement”

Further increase in cell density to 5.0 × 10^6 cells/mL at infection resulted in no further noticeable increase in VSV production.

“However, further increase in the cell density to 5.0 Â 10 6 cells/mL at infection resulted in no further noticeable increase in the VSV production, indicating a potential limitation in nutrient availability.”

Far fewer cell aggregates were observed in bioreactor culture due to the reduced ratio of bioreactor wall surface to the culture volume.

“Far fewer cell aggregates were observed in bioreactor culture due to the reduced ratio of bioreactor wall surface to the culture volume.”

VSV production in the bioreactor and control shake flask cultures all peaked at 22 h post infection.

“Data in Fig. 8 showed that the VSV production in the bioreactor and control shake flask cultures all peaked at 22 h of post infection.”

VSV production increased with increasing cell density in suspension shake flask cultures.

“In addition, the VSV production increased with the increasing cell density in the suspension shake flask cultures”

Production kinetics of VSV in the 3L bioreactor culture was faster than in the shake flask control culture.

“The data in Fig. 6 also revealed that the production kinetic of VSV in the 3L bioreactor culture was faster than in the shake flask control culture.”

Suspension adapted Vero cells maintained their genetic stability after passaging and/or adapting to grow in suspension in serum-free IHM03.

“Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability.”

• Also stated in: § 3.3. Genetic stability of adherent and suspension adapted Vero cells over the culture passaging and maintenance process · hedged

  “Electropherogram results of all samples showed 100% concordance with the Vero DNA control sample, indicating that the adherent and suspension adapted Vero cells retained their genetic stability after passaging and/or adapting to grow in suspension in serumfree IHM03 medium for many passages.”

The bioreactor provided a possible favorable condition for virus production.

“indicating a possible favorable condition in the bioreactor for the virus production.”

It is possible to increase volumetric productivity of VSV through infection at higher cell density.

“These results indicated that it is possible to increase the volumetric productivity of VSV through infection at higher cell density.”

Availability of nutrients might not be a limiting factor when the culture was infected at a low cell density such as 1 × 10^6 cells/mL.

“indicating the availability of nutrients might not be a limiting factor when the culture was infected at a low cell density such as 1 Â 10 6 cells/mL”

A higher cell density was achieved by Thomassen et al. when their culture was operated under a recirculation mode with cells retained in the bioreactor and 5 culture volumes of fresh medium per day circulated.

“Similarly, a higher cell density was achieved by Thomassen et al. [21], when their culture was operated under a recirculation mode, where cells were retained in the bioreactor while 5 culture volumes of fresh medium per day was circulated.”

• cites [21]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Thomassen et al. found that growth of Vero cells in stirred culture was not inhibited by lactate and ammonia at the above concentration ranges.

“Thomassen et al. [21] also found the growth of Vero cells in stirred culture was not inhibited by the lactate and ammonia at the above concentration ranges.”

• cites [21]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Perfusion was an effective means to increase maximum cell density of the culture.

“These data showed that the perfusion was an effective mean to increase maximum cell density of the culture.”

Perfusion culture combined with infection at high cell density was a feasible approach to increase volumetric virus production.

“This clearly demonstrates that the perfusion culture combined with infection at high cell density was a feasible approach to increase the volumetric virus production.”

Cell viability was always higher than 98% during both batch culture processes with and without medium replacement.

“The cell viability was always higher than 98% during the both culture processes.”

When Vero cells were cultured in serum-free media, especially in stirred cultures, the doubling time was usually longer than 24 h.

“Therefore, it appears that, when the Vero cells were cultured in serum-free media especially in stirred cultures, the doubling time of Vero cells was usually longer than 24 h [9,3,4].”

• cites [9]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [3]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [4]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Shear stress in suspension culture, especially without microcarrier supports, might also reduce the cell growth rate.

“Also shear stress in the suspension culture, especially in the culture without supports of microcarrier, might also reduce the cell growth rate.”

The lack of further increase in VSV production at 5.0 × 10^6 cells/mL indicated a potential limitation in nutrient availability.

“However, further increase in the cell density to 5.0 Â 10 6 cells/mL at infection resulted in no further noticeable increase in the VSV production, indicating a potential limitation in nutrient availability.”

The increased cell doubling time of suspension adapted Vero cells in serum-free cultures was more likely and partially due to the removal of serum.

“The increased cell doubling time of suspension adapted Vero cells in serum-free cultures was more likely and partially due to the removal of serum, as the removal of serum usually increased the cell doubling time, such as from 31 to 43 h [17,19]”

• cites [17]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [19]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Accumulation of unknown metabolites in the cellular microenvironment was probably responsible for the density-dependent inhibition of cell growth observed under the optimized environmental condition.

“Accumulation of unknown metabolites in the cellular microenvironment was probably responsible for the densitydependent inhibition of cell growth observed under the optimized environmental condition [15].”

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.