- OptiVERO – Formulation of a blood-free and chemically defined virus production media for VERO cells
OptiVERO – Formulation of a blood-free and chemically defined virus production media for VERO cells
Published on 10 May 2019
Atherly Pennybaker, Media Formulation and Product Applications Specialist, InVitria
Randy Alfano, PhD, VP Product Development, InVitria
Claire Huang, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention
Recent years has witnessed a major shift in the paradigm of vaccine manufacturing from more primitive manufacturing platforms, such as egg-based manufacturing of influenza, to more robust large- scale methods that utilize mammalian continuous cell line (CCL)-based systems due to production limitations, longer development times, and highly variable production yields. One CCL in particular, VERO, has been widely adopted and is utilized in the production of a multitude of different virus types . Current serum free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates . Although these media have demonstrated acceptable ability to support VERO cell growth and virus production compared to fetal bovine serum (FBS), the overall performance relies heavily on the hydrolysates as withdraw results in the cessation of cell proliferation . It is thought that protein hydrolysates act as a concentrated balanced nutrient mixture that can at least in part replace serum . However, current understanding of these complex mixtures cannot unequivocally provide evidence of the exact components responsible for the beneficial activity observed . Thus, media formulations that contain these complex mixtures have a poor degree of chemical definition which can potentially complicate both manufacturing efforts due to the variable performance derived from poorly controlled raw material inclusion in addition to hindering downstream discovery efforts to improve manufacturing yields.
To circumvent the lack of chemical definition in virus production media, we have utilized a recombinant human transferrin and albumin to formulate a chemically defined media that is optimized specifically for the VERO-based virus production in both 2D, and 3D culture. The efficacy of this chemically defined media, known as OptiVERO, is demonstrated in the production of 6 different virus in comparison to both serum and plant hydrolysate-containing media in both wild type VERO and a genetically engineered VERO subclone. The data presented here provides proof of principal that poorly defined plant hydrolysates and FBS can be replaced with recombinant proteins to produce a chemically defined, blood-free virus production media.
Methods and Results
Formulation of the blood-free chemically defined virus production media
To formulate OptiVERO, thirty-seven different components, including the recombinant albumin and transferrin, Cellastim S and Optiferrin, were screened to determine optimal formulations for VERO cells (Figure 1). A total of six candidates were formulated at small scale and confirmed via T-flask-based expansion versus EMEM + 10% FBS and VP-SFM + 4 mM Glutamine. From these early studies, a single formulation was selected based on formulation complexity and performance for further cell growth kinetics and virus production studies.
Growth performance and VERO morphology in the blood-free chemically defined virus production media
The OptiVERO media was subjected to a 14-passage test in T-75 flasks, comparing the population doublings at each passage to that of VP-SFM + 4 mM glutamine and EMEM + 10% FBS in both the WT VERO (A) and VERO subclone (B) lines (Figure 2).
The OptiVERO exhibited equivalent ability in inducing WT VERO expansion capability to FBS while VP-SFM + 4 mM glutamine was significantly less robust. For the VERO subclone line, OptiVERO had more population doublings than either control media. Trends in growth kinetics were also similar in 3-dimensional culture. VERO cells cultured in OptiVERO readily attached to plastic microcarriers and expanded rapidly within the first 4 days of spinner culture as evidenced by elevated glucose consumption and lactate production when seeded at low densities. VP-SFM + 4 mM glutamine and EMEM + 10% FBS, although supported VERO cell growth on plastic microcarriers, demonstrated a significantly lower ability to expand VERO cells in 3-dimensional culture.
Cells appeared to be morphologically normal in OptiVERO compared to VP-SFM and EMEM + 10% FBS at both low and high confluencies in T flasks (Figure 3).
Productivity of VERO cells expanded in the ACF chemically defined virus production media
The flavivirus Dengue and Zika in addition to 2 strains of influenza A and B were used as proof-of-concept virus production platforms to determine if the OptiVERO media was able to efficiently expand these virus types (Figure 4).
The OptiVERO media was able to produce equivalent virus titers compared to each control media in Dengue (A) and Zika (B). For influenza, the production of 2 strains of influenza A and B were compared to that of VP-SFM. Influenza A Strain 1 failed to propagate in VP-SFM while OptiVERO was able to support the expansion of this virus (C). The second strain of influenza A demonstrated a delay in the time to peak virus titer in VP-SFM versus OptiVERO (D) OptiVERO also exhibited a higher virus titer (E) or less time to peak virus titer (F) in influenza B versus VP-SFM.
Here, a blood-free chemically defined virus production media known as OptiVERO is compared in both VERO cell growth kinetics and virus production to FBS and VP-SFM. OptiVERO was found to have robust ability to support VERO in both 2D and 3D cultures. The new media also demonstrated equivalent production of flavivirus Dengue and Zika when compared to VPSFM and FBS. In influenza, OptiVERO demonstrated significantly better production capacity than that of VP-SFM, potentially suggesting the presence of a viral replication inhibitor is present in the di and tri-peptide hydrolysate component of this media. Taken together, these studies suggest that the use of undefined plant hydrolysates or FBS for virus production is unnecessary as chemically defined media can support VERO cell expansion and virus production.
- M. W. K. O. H. M. Barrett P, “Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines,” Expert Rev Vaccines, vol. 8, no. 5, pp. 607-618, 2009.
- S. A. Mizrahi A, “Partial replacement of serum by vegetable proteins in BHK culture medium,” ffournal of Biological Standardization, vol. 5, pp. 327-332, 1977.
- J. S. E. A. a.-R. M. Jan DC1, “Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture.,” Cytotechnology, vol. 16, no. 1, pp. 17-26, 1994.
- H. O. K. H. Franek F, “Plant Protein Hydrolysates: Preparation of Defined Peptide Fractions Promoting Growth and Production in Animal Cells Culture,” Biotechnol. prog. , vol. 16, pp. 688-692, 2000