Optimizing Virus Production Media for Cell-based Vaccine manufacturing

Cell-based Vaccine Manufacturing

There has been increasing interest in moving from egg-based vaccine manufacturing to cell culture-based methods. Cell-based vaccine manufacturing is quicker, efficiently scalable and offers more control over the manufacturing environment. There is also the advantage of fewer virus mutations and thus a more effective vaccine. This was certainly true with the 2017-2018 flu season. In a recent PBS article, “Flu vaccine grown without eggs provided measurably better protection this season, FDA says,” the FDA reported that the approved cell-culture based influenza vaccine performed about 20 percent better than the standard egg-based vaccines. This is due to the fact that the viruses have to adapt to grow in the eggs and with this adaptation can come mutations that render the vaccine less effective.

Vero Cells for Cell-based Vaccine Manufacturing

Vero cells have been very popular in the vaccine industry for over 3 decades. These cells have a long and successful history of safe vaccine production for many human and animal vaccines including polio, rotavirus, and smallpox.

Vero cells are adherent cells that require a surface to attach to in order to proliferate and produce viruses. Production scale-up with adherent cells is generally more challenging than with suspension cell cultures due to the surface area and media formulation requirements. Vero cells grow well in a variety of vessels including flasks, cell factories, and microcarriers in bioreactors, however they must be supported in attachment and need a robust media to ensure good growth and productivity.

Traditionally, Vero and other virus producing cells have been cultured in serum-containing medium. However, the vaccine industry has now moved toward serum-free media for Vero cell culture and vaccine production. Using media formulated without serum or animal components reduces risk of contamination with adventitious agents, streamlines regulatory documentation, and can increase consistency in culture.

Cell-based vaccine manufacturing media optimization

Current serum-free media formulations developed for Vero cell culture use high concentrations of plant-based hydrolysates to compensate for the removal of serum. However, the challenge with plant-based hydrolysates is that it is difficult to determine, with certainty, which exact components are providing which benefits to the cells. Thus, making it difficult to precisely optimize media components, attain chemical definition, and fully understand the best process for downstream purification. In addition, due to their undefined nature, plant-based hydrolysates can have variable performance.

At last week’s 2018 World Vaccine Congress in Washington DC, improvements to vaccine manufacturing were discussed. One of the areas of improvement was in chemically defined media for vaccine manufacturing. A poster, “Blood-free chemically defined virus production media for Vero cells,” was presented that provided a complete solution. In the poster, authors describe a study in which they were able to use a chemically defined virus production media (OptiVERO) containing recombinant human transferrin and recombinant human albumin to replace both serum and plant-based hydrolysates.

In the study, OptiVERO was compared to FBS and plant-based hydrolysates for VERO cell growth kinetics (Figure 1) and virus production (Figure 2). Based on the data presented, OptiVERO was able to support VERO in both 2D and 3D cultures. The new media also demonstrated equivalent production of flavivirus Dengue and Zika when compared to virus production serum-free medium and FBS. In influenza, OptiVERO demonstrated significantly better production capacity than that of virus production serum-free medium. This study demonstrates that Vero cells can thrive in a chemically defined media.

Figure 1: Growth performance in the blood-free chemically defined virus production media

Optimizing virus production media growth
(A) WT VERO cultured in FBS demonstrated a robust expansion capability, doubling 40.07 ± 4.16 times in 50 days. Cells expanded in OptiVERO exhibited a 42.99 ± 5.68 doublings while VP-SFM + 4 mM glutamine was less robust with 36.15 ± 4.44 doublings. (B) The VERO subclone line doubled in the EMEM + 10% FBS 40.96 ± 5.05 and in VP-SFM + 4 mM glutamine 39.71 ± 3.87 times. The OptiVERO was significantly more robust than either media, with cells undergoing 49.53 ± 4.28 doublings in 50 days. (C) VERO cells cultured in OptiVERO readily attached to plastic microcarriers and expanded rapidly within the first 4 days of spinner culture. (D) OptiVERO also exhibited a rapid induction of VERO cell growth on microcarriers within the first 4 days of spinner culture. ***, ** p ≤ 0.001 or 0.01 compared to VP-SFM and ###, ## p ≤ 0.001 or 0.01 compared to FBS, respectively.

Figure 2: Virus productivity in the OptiVERO media are comparable to VP-SFM and FBS.

Optimizing virus production media productivity
*** 0 1 2 3 4 5 6 7 8 9 10 Days Post Inoculation ## ## ### The OptiVERO media was subjected to productivity tests using a strain of Dengue and Zika flavivirus in WT VERO. For influenza, the genetically modified VERO subclone was utilized. WT VERO cells were grown for 3-4 days prior to infection. During the virus propagation phase, cultures were fed with a 45% glucose solution and the pH of the cultures were maintained at neutral using a 7.5% sodium bicarbonate solution. (A) FBS demonstrated a lag in the production of Dengue virus compared to VP-SFM and OptiVERO while (B) Zika virus did not demonstrate any significant differences between the media. Influenza virus demonstrated a higher degree of sensitivity to different media compared to the flavivirus Dengue and Zika. (C-D) VP-SFM failed to expand a strain of Influenza A and exhibited a significant delay in a second strain while OptiVERO was able to expand both of these virus strains very efficiently. (E-F) Influenza B strains 1 and 2 exhibited similar patterns in VP-SFM as this media induced a significant delay in the time to peak titer while OptiVERO was able to efficiently expand these virus types.

To see study details and all the data, please click on the poster below:

Formulation of a blood free-and-chemically-defined virus production media for vero cells

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