high-pressure cell lysis (not shown). For inactivated vaccines, an inactivation step is

introduced either before or after DSP. Next, the buffer used to recover the virus in

the last step of purification is replaced by a buffer suitable for the intended ad-

ministration, and additional compounds, i.e., adjuvants, are added for formulation

of the vaccine bulk (finishing). Subsequently, the primary containers are filled with

the vaccine and, in case a solid form is required, a lyophilization step may be

performed. Lastly, the containers are capped and sealed. For the final product,

further steps such as labeling, packaging, and storage, among others are required.

Inoculum

train

Seed train

Cell growth

N

N-1

N-2

Virus

harvest

N

Cell line

selection

Cell

banking

Vaccine

candidate

Seed virus

banking

Purification

Final

product

Finishing

Infection

N

Blend-Fill-Pack

Nuclease

treatment

Viral inactivation

Buffer exchange

& formulation

Upstream processing

Downstream processing and finishing

µ-F

CFF

C1

Clarification

DF

Concentration

Cvir

Cs

Cx

Time

Production

bioreactor

C2

FIGURE 5.1 Overview on steps of a cell-culture−based virus production process. Cells

undergo expansion through a series of precultures until production scale in a bioreactor.

Compared to other biopharmaceutical products produced with animal cell culture, up-

stream processing of viral vaccines is typically a two-phase process: 1) Cell growth to

the target cell concentration; 2) infection, virus replication, virus release together with

cell death and cell lysis, and finally virus harvest (upstream processing). After inactivation,

the virus harvest is subjected to a purification train (downstream processing), followed

by formulation and filling (finishing). In some cases, virus inactivation is done after

downstream processing (not shown). Abbreviations: N, number of cell expansions for

production; DF, depth filtration; µ-F, microfiltration; CFF, cross-flow filtration;

C1: chromatography step 1; C2 chromatography step 2. Arrows color indicates no

virus (black), active virus (red), or inactive virus (green) flows. Arrow size indicates

internal (small) and external process flows (large). Discontinuous arrows represent a

waste flow. Cvir: virus concentration; Cs: substrate concentration; Cx: cell concentration

over process time for cell growth and virus production phase. Created with Biorender

( www.biorender.com).

82

Bioprocessing of Viral Vaccines