Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath. Dilute the thawed cells slowly before you incubate them, using pre-warmed growth medium. Plate thawed cells at high density to optimize recovery. Always use proper aseptic technique and work in a laminar flow hood.

As a start, cryopreserved vials are thawed and then cells are cultivated using T-flasks for 5, 15 and 35 mL scale (e.g. T25, T75 and T175), roller bottles or shake flasks for 200 mL scale and bioreactors for 1, 5, 20, 80, 400 and 2,000 L scale. From 5 mL-scale until inoculation of the 10,000 L-scale manual control of cell growth in the range of 20–30 days has to be conducted consistently. Delays caused by unusual low growth rates, contamination of a scale etc. can further increase this time span. The higher the number of seed train steps, the more will the culture be prone to deviations. Moreover, deviation from standard growth rates can enforce the personnel to adapt the typically used seed train to the new growth characteristics. Analysing and optimizing existing seed trains as well as designing new seed trains offers the potential to decrease the time efforts and costs of seed trains. Moreover, failure rates of seed trains may also be reduced. Another important aspect of seed trains is that the quality of inoculum in the first cultivation steps has an impact on the cell performance during the production process. Therefore, keeping the cells in a good state is another criterion for seed trains or the design of new seed trains, respectively. 

Viruses are propagated in cell culture, grown either in roller bottles (as a monolayer) or suspension cultures, or bound to microcarriers. A typical pooled roller bottle batch volume is 500–700 L, and suspension culture is 1,000–2,000 L. There are several types of cells used for growing viruses for vaccine application: human diploid, Vero, Per.C6, MDCK, MRC 5, WI38, and 293P cells. Vero cells (developed from African green monkey kidney cells) are most commonly used for viral vaccine manufacturing. 

Most cell cultures for viral vaccine applications are grown in low-oxygen tension in the presence of ~5% CO2. Virus inoculation is done aseptically to cell culture that has grown for five to seven days. Virus harvesting is done after 24 to 72 hours of virus inoculation. Depending on the virus type, they either bud out of cells or lyse the cells and emerge in the extracellular culture fluid. In some cases, the cells need to be lysed by the addition of detergents or surfactants (for example, Tween® 20 nonionic detergent) to release the viruses. 

Clarification removes the cells or cell debris and harvests viruses. Zonal centrifugation is commonly used for primary clarification. Some manufacturers also use tangential flow filtration (TFF) under low shear conditions or normal flow filtration (NFF), in most case depth filtration, for clarification of viral vaccine. Attenuated viruses are fragile and shear sensitive. Microfiltration (MF) TFF devices (without screen) are preferred to minimize shear. Solid content in viral vaccine harvest is low, so normal flow filters also work well for such applications. Some attenuated live viruses tend to bind to cell surfaces or get trapped in lysed cell debris. This leads to their removal during clarification resulting in poor virus recovery. Because viruses are negatively charged, it is important to be aware of adsorptive effects on filter media. 

Nucleic acids are negatively charged large molecular components that interfere in virus purification. Carryover nucleic acid from lysed cells is a key contaminant in viral vaccine processes. Viruses propagated in human diploid cells or non-human cells (for example, viruses grown in dog kidney cell lines [MDCK]) pose a greater risk of nucleic acid carryover. Regulations require that carryover host cell nucleic acid content should be below 10 ng/dose of attenuated viral vaccine. Benzonase® endonuclease is commonly used to degrade the nucleic acids such as RNA and DNA of the host cells to as low as three to eight base pairs (<6 kDa). The virus harvest is treated with ~0.9 to ~1.1 units/mL of Benzonase® endonuclease at 30–34 °C for four to eight hours. 

After Benzonase® endonuclease treatments, the harvest is diafiltered using TFF (100–300 kDa ultrafiltration devices) operating at low crossflow to remove Benzonase® endonuclease and degraded nucleic acid components. The typical flux (for 300 kDa Biomax® ultrafiltration membrane) is 25 LMH at 1.5–3.0 psi transmembrane pressure (TMP) and 4-5 L/min/m² feed flow rate. 

Benzonase® endonuclease treatment is sufficient to bring most attenuated viral vaccines—measles, mumps, rubela, polio, rota, and yellow fever among others—to the desired level of purity during the concentration and diafiltration step. However, chromatography is normally required to bring new generation of viral vaccines like Japanese encephalitis virus (JEV) and dengue virus (DENV) to the desired level of purity. For example, sulfate ester covalently linked to a cellulose matrix can be used to purify JEV. The virus binds to matrix based on mixed-mode interaction with virus surface receptors or heparin-binding domain present on a few enveloped viruses. As viruses are negatively charged, anion exchange chromatography (Q or DEAE) works well in bind and elute mode or flow through mode. These operations run in mild conditions with low salt. Post chromatography, the eluted virus is concentrated by using 100–300 kDa TFF devices. Purity of the live viral vaccines is determined by measuring the removed contaminants (bovine serum albumin, ovalbumin, residual DNA, host cell protein, etc). Quality and quantity of virus in the purified bulk is determined by estimation of virus concentration based on HA titre, neutralizing antibodies, and CCID50 infectivity assay. 

Final virus vaccine bulk is comparable to that of water. During final filter, vaccine is filter sterilized using 0.22 µm sterilizing filtration. Many of the attenuated viral vaccines are finally formulated with different strains. These multivalent vaccines include rota, polio, and dengue. They are aseptically blended after sterile filtration. Most of the live attenuated viral vaccines do not need any adjuvants because they are naturally potent immunogens. Most of them are lyophilized (freeze-dried). Examples are measles, mumps, and rubella. 

The final formulation of attenuated viral vaccines contains a small amount of antibiotics (neomycin), excipient (human serum albumin, HAS), stabilizer (hydrolyzed gelatin, egg protein, sorbitol, sucrose) and buffering agents (NaCl, other salts). Most of these vaccines are administered subcutaneously except for rota virus and polio virus vaccines, which are isotonic solutions that are administered orally.