Real time PCR - European Pharmaceutical Review

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2009 PAT
a bioprocess further emphasises the
need of a thorough analysis of testing
and optimisation possibilities with
modern PAT methods.
The variety of procedural
alternatives for propagation,
expansion, differentiation,
preservation and characterisation of
hESC are indeed multi-parametrical
and need to be adapted, refined and
optimised to satisfy the demands
of the regulators, including the
expectations inherent in guidelines
of QbD and PAT.
Stem cell technology
The first step in the preparation of
hESC and hESC derived products is to
enzymatically digest the inner cell
mass of embryonic blastocytes 11-12 .
The isolated cell mass is placed on a
layer of growth-inhibited mouse
fibroblast feeder cells in tissue culture
dishes. After one or two weeks the
outgrown cells from the inner cell
mass are isolated, by dissection or
enzymatic treatment, and transferred
to new culture dishes for further
growth in an un-dissociated cellular
state. The feeder cells provide
differentiation inhibitors which hinder
the stem cells to differentiate.
The stability of the cell culture is
monitored through characterising
specific biomarker molecules,
typically cell surface proteins
(e.g. SSEA-3/4, Oct3/4 and Nanog)
which indicate that the cell mass
remains in the undifferentiated state 13 .
Additional characterisation and
control methods are appropriate to
use, such as gene array analyses, flow
cytometry other methods 14,15 .
If the production goal is to
produce undifferentiated stem cells,
the characterised cells are isolated
and further expanded under
controlled conditions (see Figure 1 on
page 34). Up to 100 passages have
been reported provided efficient
inhibition factors are supplied 10 .
If the goal of the production is a
particular cell type, a well defined
differentiation protocol ensues in a
stepwise procedure of propagation
Figure 2: The Hubka-Eder model where the general model is supported by a biological
systems entity to clearly depict how a biological system interacts and effects the other
systems in the transformation occurring during a production process.
and isolation of the cells. The added
differentiation factors and the culture
conditions applied stimulate the cells
to develop into progenitor cells and
matured cells from which the desired
cell type is isolated. This procedure
takes an additional two to four weeks.
Crucial for a successful result is
continuing characterisation of
biomarker molecules, now others that
are characteristic for the desired cell
type. Supporting the biomarker data
are other functionality tests for the
cell type. The cells are going through
several isolations by manual
microscopic selection and dissection.
Furthermore, the cells are
cryopreserved intermittently and
preservation and shipping procedures
require careful handling of the cells.
The flow chart in Figure 1 (see
page 32) summarises the steps in
the derivation of the stem cells
with accompanying analytical
characterisation. This chart of
procedural steps is the blue print
which should be transformed and
scaled-up to regular manufacturing.
Figure 3: The HE model adapted to a simplified manufacturing where embryonic stem
cells are the starting material. This example refers to a manufacturing process producing
a particular differentiated cell type.
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