Biology Reference
In-Depth Information
The size of the equipment and the time over which each piece of equipment
needs to be occupied for a given production run will influence the design of a
manufacturing facility and the number of fermentations a manufacturing plant
can execute. The manufacturing facility is designed to comply with regulatory
guidelines for GMP manufacture (i.e., CFR parts 210 and 211 are used in the
US; guidelines differ slightly in other countries). Additional information on
process options can be found in [3] and [4].
Commercial manufacture
A commercial manufacture cell culture process consists of two parts: A seed
train is used to generate a sufficient number of cells with which to initiate pro-
duction in the production vessel; the required process conditions are imposed
on a production vessel to generate optimal quantity and quality of product.
Seed train
A seed train describes the expansion of cells from a frozen cell bank vial to the
inoculum of the production reactor. The volumes involved may range from 1 mL
to 10,000 L. The vessels involved may include T-flasks, shaker flasks, roller bot-
tles, Cellbags
®
(Wave Biotech,Bridgewater, NJ), and stirred tank reactors
(Fig. 6). The reactors may be operated in batch, fed-batch, or perfusion modes.
The purpose of each step of the seed train is to generate a sufficient number of
cells of known quality to enable inoculation of the subsequent step. The culmi-
nation of the seed train occurs at inoculation of the production reactor.
Given a cell bank vial containing 1
10
7
viable cells, the initial seed train
time to the production reactor may be estimated. First, the doubling time for
the cell line must be established. For mammalian cells, doubling time typical-
ly ranges from 16-48 hours, with 48 hours used as a conservative basis for
planning. Second, the requirements for cell number generated at the end of the
seed train must be established. For example, if the process is envisioned to go
towards commercial production with a 10,000-L production reactor and the
production reactor requires an inoculum density of 1
×
10
6
viable cells/mL and
×
an initial volume of 7,500 L, then 7.5
10
12
viable cells are required. The
number of cell doublings (n, or population doubling level) from vial thaw to
production reactor inoculation can be given by the formula:
×
n = LN(XS/X0)/LN(2)
n =LN(7.5
10
12
/1
10
7
)/LN(2)
×
×
n = 19.5
n~20
where LN = natural logarithm and XS final cell number = 7.5
10
12
and
×
X0 = initial cell number = 1
10
7
.
×
