process looks like, a clear definition of the method is required [90]. This includes

descriptions of each stage of cell growth: inoculation and growth of initial and

precultures (volumes, time, temperature, pH, etc.), main culture system including

operating and control parameters (temperature, pO2, pH, static vs. agitated, aerobic

vs. anaerobic, volume of bioreactor, number of bioreactors, process mode, etc.)

[89]. Moreover, in-process controls and testing (purity, viability, yields, etc.) as

well as sterilization/preparation/cleaning procedures should be described. All used

materials (e.g., water, media, serum, additives, antibiotics) must comply with the

respective guidelines provided by the regulatory agencies. At a certain point during

the production process, the drug substance has to be harvested. A brief description

outlining the criteria for harvesting and the monitored process parameters should be

described. Method(s) used for separation of unrefined drug substance from the cell

broth (e.g., precipitation, filtration, centrifugation, etc.) must be provided. Each

harvest has to be tested for extraneous agents and characterized regarding identity

of the virus and infectious/total virus titers. Here, thresholds for acceptable titers

should be established [89,90].

5.9.4

PURIFICATION AND DOWNSTREAM PROCESSING

Detailed descriptions of purification and downstream methods can be found else-

where. For chemistry, manufacturing and controls (CMC) submissions, the used

material and methods for separation and concentration of the drug substance should

be described. The final purification step can be prepared from a single virus harvest

or several virus pools. Each step (e.g., inactivation, purification, stability proces-

sing, detoxification, etc.) must be outlined, including adopted or developed analy-

tical tests to show identity, purity, concentration, and levels of impurities. If final

formulation substances such as stabilizers or diluents are used, efficacy and safety

of the drug product cannot be impaired and verification of the stability should be

demonstrated. The use of antibiotics as antimicrobial preservatives is usually

strictly prohibited. Potential titer loss during filling, freeze-drying, and shelf life,

should be compensated by a higher titer in the final bulk vaccine. Final testing of the

vaccine should be performed on samples from each batch, analyzing sterility, sta-

bility, potency, and identity. Quantitative and qualitative analyses must include

thermal stability, endotoxin levels, residual host cell protein, residual agents/animal

serum proteins, infectious virus titer, or immunogenicity in vivo [89,90].

Regardless of the vaccine type, they all have to fulfil strict regulatory require-

ments throughout their manufacturing process. An understanding of those re-

quirements is critical for developers to manufacture a reproducible, consistent, safe,

and effective vaccine. Even though regulatory processes do not directly impact the

early development stage, choices for equipment, process steps, and in vitro and in

vivo testing design could be reconsidered as they may not fulfill the needed qua-

lifications. Professional consultation by knowledgeable qualified persons or reg-

ulatory consultants already at the research stage might streamline the vaccine

development process and alleviate stress and unnecessary delays with regulatory

submissions. With the SARS-CoV-2 pandemic this became very prominent as

those vaccines that became available very fast, were developed by industrial

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Bioprocessing of Viral Vaccines