transgenes of interest, and their products show post-translational

modifications compatible with in-human use [3]. A number of

different modes of operation are currently employed, from micro-

carrier systems to different variants of perfusion processes. How-

ever, despite an increasing interest of perfusion technologies in

mammalian cell culture, the classic fed-batch cultivation of suspen-

sion cell lines in stirred tank bioreactors is currently the most widely

used technique, especially at larger scales [4, 5]. As a result, a

number of “off-the-shelf” solutions ranging from optimized cell

culture media to complete expression platforms providing the host

cells, required consumables, and the recipes for bioreactor cultiva-

tion were developed. Using these technologies, secreted recombi-

nant proteins can be produced with relative ease in volumetric

concentrations ranging from hundreds of milligrams per liter for

difficult to express proteins to several grams per liter of culture

medium for easy to express proteins, making the approach attrac-

tive both for pure research and biotechnological use.

The workflow for cell cultivation in bioreactors generally starts

by thawing of cells expressing a recombinant protein of interest.

The generation of such cell populations is described elsewhere

[6]. Cryovials of the cell populations are frozen and are referred

to as cell bank. Following thawing of the cell bank, the cells are

cultivated in vessels of increasing size, typically starting with small

shake flasks, with the aim of maintaining the cells in logarithmic

growth (the seed train) until they are available in sufficient amounts

to inoculate the production bioreactor(s) [7].

In order to reduce heterogeneity in the cell population, the

cells used for manufacturing of biologics are usually clonally

derived, meaning a cell population started from a single cell. Such

a cell population is often referred to as “clone,” although there is

significant heterogeneity even in such a population.

Cells are then cultivated under tightly controlled conditions for

1–3 weeks with nutrients added in the form of feeds either at

pre-defined intervals or based on consumption by the cells. Animal

derived component free (usually abbreviated ADCF or ADF) and

fully chemically defined media (usually abbreviated CD) and feeds

have largely replaced previous media including bovine or calf serum

or hydrolysates, as they reduce the risk of introduction of patho-

gens, show lower lot-to-lot variability and simplify downstream

purification and testing [8]. In recent years, single-use technologies

have found widespread application both in production and research

as they allow to skip time- and labor-intensive cleaning and sterili-

zation steps, reduce turnaround time for equipment and aim to

improve reproducibility [9]. Importantly, the risk of cross-

contamination with material of a previous batch is completely

removed. The general operation and the required equipment only

differ in minor details between single- and multi-use approaches

(see Note 1).

2

Ange´ lique Schmid et al.