VVP

Volumetric virus productivity

virions/L/d

wv

Working volume

L

u

y

x

Velocity gradient

s⁻¹

5.1

INTRODUCTION

This chapter focuses on general aspects of virus production in small-scale vessels

and bioreactors. It provides experimental, practical, and theoretical aspects that

are essential for cell-culture−based upstream processing (USP) of viral vaccines.

It is not the intention of the authors, however, to cover all basics of animal cell

culture. Moreover, the literature and citations are not exhaustive as the authors

wanted to highlight only selected aspects of viral vaccine manufacturing. The

examples shown mainly concern studies that were performed at laboratory scale.

Nevertheless, most of the covered aspects certainly should also hold for large-

scale manufacturing. Regarding details of large-scale processes, see the case

studies described in chapters 9 to 12.

5.2

BASIC DESIGN OF A VIRUS PRODUCTION PROCESS

Cell-culture−based production of viral vaccines is a complex process that involves

several steps until the final product is obtained (Figure 5.1). First, a vaccine can-

didate is considered. Then a set of cell lines permissive to infection is screened to

identify the most promising host for high-yield virus production. In a next step, the

generation and testing of master and working banks of both virus and cell lines is

pivotal. To initiate production, cryopreserved cells from the working cell bank are

thawed to inoculate the first of a series of precultures of increasing volume (in-

oculum train). Cells successfully maintained in exponential growth are subse-

quently transferred to a stirred tank reactor (STR), followed by cultivation in

successive STR runs at increasing volumes (seed train) until the required amount of

cells is generated to start the production run. Finally, in the production bioreactor,

once the target cell concentration is reached (cell growth phase), the seed virus is

added to initiate the virus infection phase. Often, this involves a dilution step or a

complete medium exchange to provide the required substrates and to reduce levels

of potential inhibitor concentrations that might interfere with virus production.

Virus replication and release into the extracellular medium often involves cell death

and cell lysis. In a next step, the virus harvest is collected and cells and debris are

removed by clarification. For subsequent virus purification, host cell nucleic acids

(mainly deoxyribonucleic acid [DNA]) contaminating the virus harvest are de-

graded enzymatically through addition of a nuclease. In downstream processing

(DSP) the level of particulate contaminants, residual nucleic acids, host cell proteins

and other compounds of the virus harvest is reduced by a purification train typically

comprising several filtration, concentration, and chromatography steps. In a few

cases, when virus particles accumulate within cells or aggregates are formed,

a homogenization step is required that may involve freeze-thawing or use of

Upstream processing for viral vaccines

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