B
I O
P
R O C E S S
TECHNICAL
Strategies for Producing
Commercial Cell Lines
One Biotech Company’s Experience
Sue Clarke, Janette Dillon, Ann Smith, and Elizabeth Sotheran
L
orantis Ltd. (Cambridge,
United Kingdom) is a
discovery company
developing products for
antigen-specific
immunotherapy, ASPECT, based on
the Notch signaling pathway, which
has been shown to play a key role in
regulating immune responses. The
Notch receptor and its associated
ligands represent a family of highly
conserved transmembrane proteins
expressed during embryonic
development in both vertebrates
and invertebrates (1). Binding of a
Notch ligand to a Notch receptor
activates signaling cascades that alter
the transcriptional program of the
target cell, ultimately determining
its fate. Our ASPECT platforms
exploit the company’s proprietary
position covering the Notch
signaling pathway in immune cells
and have broad clinical applications.
PRODUCT: CELL-CULTURED PRODUCTS
PROCESS FOCUS: PRODUCTION,
ANALYTICAL METHODS DEVELOPMENT
WHO SHOULD READ: PROCESS
ENGINEERS, MANUFACTURING,
Host Cells and Expression System:
KEYWORDS: CELL CULTURE, SERUMFREE MEDIA, ASSAYS, CHO CELL LINES
LEVEL: INTERMEDIATE
BioProcess International
The products in our current
preclinical pipeline are derived from
the Notch binding protein, Delta-1.
To evaluate its biological efficacy,
we chose to pursue several
approaches. One was to produce a
commercial cell line that secretes a
recombinant protein derived from
the extracellular domain of Delta-1
fused to an antibody constant
region. This report summarizes the
cell culture strategies we used to
speed up the development of a
suitable cell line — and ultimately
the time to clinic.
INITIAL CHOICES
ANALYTICAL PERSONNEL
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PHOTODISC (WWW.PHOTODISC.COM)
APRIL 2004
Production of a commercial cell line
involves several key decisions and
activities. First you must choose the
host cell type and expression system.
We decided to use the glutamine
synthetase (GS) gene expression
system from Lonza Biologics
(www.lonzabiologics.com) using
CHO-K1 cells. GS is the enzyme
responsible for biosynthesis of
glutamine using glutamate and
ammonium as substrates. When a
cell line expresses GS, it can survive
in a glutamine-free medium. The
activity of the GS gene can also be
selectively inhibited by methionine
sulphoxamide (MSX).
CHO-K1 cells produce
endogenous GS. But they can be
used to produce stable cell lines by
transfecting in a GS gene and using
glutamine-free medium plus MSX
(at sufficient levels to inhibit the
endogenous enzyme) to provide
selection pressure. Use of a
mammalian host cell should give
our protein the appropriate
posttranslational modifications.
The advantages of the GS gene
expression system are many. It is
used throughout the industry and
therefore comes with a wealth of
accumulated knowledge and
available experience. A fully
qualified master cell bank is
provided, which is a critical starting
material for the whole process. GS
has been used for licensed products,
Figure 1: Notch ligand signaling activty
pressure, maintaining segregation
and thereby reducing the risk of
cross-contamination and the
introduction of adventitious agents.
All materials are logged in by batch
number, allowing full traceability.
All such activities must be well
documented to provide traceability
and meet regulatory guidelines (2).
A summary of data is not only
needed for clinical
submissions; it is
also extremely
useful when
transferring the cell
line to a contract
manufacturer.
Plasmid DNA:
Table 1: Plating efficiency in four 96-well
plates
Expected
cells/well
10
5
1
Frequency of
observed cells/well
0
71
166
332
1
91
115
42
>1
222
103
10
and consequently, regulatory
authorities are familiar with it.
Although it carries an inherent cost,
that is money well spent for a startup company.
We continue to enhance the
quality of our activities by sourcing
raw materials, particularly those of
animal origin, from reputable
suppliers that can provide necessary
supporting documentation. The
most critical, of course, is a BSEfree source of fetal bovine serum
(FBS). Our batch was specifically
chosen to create stable cell lines. It
is low in IgG, which aids the
purification process, it supports
clonal cell growth, and it is dialysed
and confirmed to be glutamine free.
Tissue culture procedures for our
cell line are carried out in an
isolated room under positive
The second most
important starting
material is the
plasmid DNA.
Traditionally you
would want to
confirm that the
correct protein is secreted, usually
by transient expression, before
embarking on stable cell line work.
Because we are confident in both
the vectors and our proteins, we
now set up a stable transfection and
use a bulk culture supernatant for
the initial analysis at the same time
we dispense a duplicate culture into
96-well tissue culture plates (plate
out the culture) at a variety of lowdensity concentrations and leave the
cells to grow for at least three
weeks. While they grow, we fully
sequence the plasmid and confirm
the quality and biological activity of
the secreted protein. Running these
procedures in parallel can therefore
save time, but it does carry a risk
factor. Several methods are available
for the actual transfection, and we
have successfully used both
electroporation and a lipid carrier
such as Lipofectamine from Gibco
BRL (www.gibcoBRL.com).
ANALYTICAL METHODS
To be able to select the desired
clone in your cell line, you need the
right analytical methods to measure
the quantity, quality, and biological
activity of the product. If you are
working with novel proteins, that
can be challenging because standard
Circle Reader Service No. 128
CLONING
Figure 2: Typical growth profile
reagents may be unavailable.
Reagent production must therefore
be considered in the overall project
timeline.
Because our product is a fusion
protein, we have been able to use
the antibody constant region to aid
us in both the quantitative analysis
and subsequent purification. We use
standard Western blots as a
qualitative assessment for Delta, and
we had to make our own polyclonal
antibodies for that purpose.
The efficacy of the final molecule
is, of course, paramount. We made a
stable CHO reporter cell line to test
for that purpose. Cells are
engineered to overexpress a Notch2
membrane-bound protein and also
have a core binding factor 1
(CBF1)–driven luciferase construct
for easy monitoring of Notch
signaling (3). When the secreted
recombinant ligand or test product
is mobilized to an assay plate, the
Delta portion of this fusion protein
binds the Notch presented by the
reporter cells. That in turn allows
cleavage and activation of the
luciferase reporter gene (Figure 1).
Using the above assay, we
demonstrate good activation of the
reporter cell, with culture
supernatants in the 10–30 µg/mL
concentration range. Further
in vitro analysis of our later clones
demonstrated that our fusion
protein delivers an active Notch
signal and modulates the cytokine
profiles of mouse CD4+ T cells (4).
As mentioned earlier, the plated50
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AND
SELECTION
Cloning: We tried two different
APRIL 2004
out transfections are ready for assay
after several weeks of incubation.
Ideally you would assay several
hundred clones and take a
reasonable number of them on
through further selection and
cloning steps. However, that process
can be extremely labor intensive. A
more realistic task (given the limited
resources of a small biotech
company) would be to reduce
significantly the number of cultures
assessed. The final endpoint does
not change: You still need a highproducing, stable clone. Cloning
and selection strategies therefore
become vital.
Figure 3: Stability study
cloning and plating strategies to
reach our endpoint. The first
approach took about 12 months
from gene to final stable cell line.
We initially plated out the cells at a
nonclonal dilution and tested all the
wells (12 ⫻ 96). We then expanded
a selection of wells and performed
further assays, while preparing
cryopreserved stocks as back-up.
The best clone was subjected to a
limiting dilution cloning. The last
two steps were repeated, and four of
the resulting clones were selected as
candidates to adapt to suspension.
Each candidate can take up to two
months to clone, select, and freeze.
The second method we used
aimed to achieve a confidence of
clonality at an earlier stage. Table 1
shows the numbers of clones that
were microscopically observed when
plating the cells three to five days
posttransfection. It can been seen
from these results that the lower
dilutions — five and one expected
cells/well — gave us enough wells
that appeared as a single colony. The
probability that these single colonies
come from one cell is increased at
the lower dilutions. Transfection
efficiency also affects the actual
Figure 4: Analytical cloning
above. So we used analytical cloning
for both clones and found that
#0304 was 96% positive, but #0342
was only 56% positive for protein
production. Careful observation of
the plates microscopically gave us
more information. Had we needed
to, we could have subcultured the
clones at a lower confluency range
but higher productivity (Figure 4).
number of cells that survive in the
selection medium at this initial phase.
We therefore took the highest
producing clones from the lower
dilution plates to increase the chance
of clonality. In this experiment, we
performed one additional round of
limiting dilution cloning to select
the final clones. Note here that the
best producer in the static flask may
not be the best producer once
adapted to suspension, so a wider
choice is always the preference. This
second method reduced the timeline
by six months and still yielded a
good quality cell line.
Selection: Whenever possible,
selection of the optimal clone
should take into consideration the
eventual production process. So the
last stage added to both of the
above methods is taking the final
clones and adapting them to both
suspension and a protein-free
medium to make them more readily
scalable. Again, realistically only a
few can probably be handled.
We adapted the clones by
reducing the percentage of FBS in
the adherent culture to 2% and then
inoculating a 500-mL spinner or
shake flask with 4–5 ⫻ 105
cells/mL in protein-free Ex-Cell
325 medium from JRH Biosciences
(www.jrhbiosciences.com), giving a
final concentration of 1% FBS.
Then, additions of 325 medium
alone were made, so the cells slowly
(about three months) adapted to
the new culture system. Figure 2 is a
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APRIL 2004
typical growth profile of an adapted
clone followed for 17 days.
Once adapted, the final clones
must be tested for stability to ensure
that they are robust enough for a
manufacturing campaign —
normally at least 50–60 population
doublings. A stock culture is
maintained and defined in terms of
population doublings (5). At regular
intervals (about 10 weeks apart), a
normally prepared production vessel
set up from the stock flask is used to
assess productivity in relation to the
overall population generations.
Figure 3 shows that for a sixweek study, two of the clones
looked stable. However, when we
continued the study for a further
few weeks, clone #LC06-0342 lost
productivity.
Analytical Cloning: A faster method
for testing stability was to perform an
analytical cloning as an assessment of
population drift. Ideally, 100% of a
given population produces the target
protein. If a population has
nonproducers, they could outgrow
the producers, causing an instability
in the culture over time. An
analytical cloning therefore examines
the distribution of cells at a
concentration that permits all
apparently single-cell wells to be
tested for protein expression.
This method takes only three
weeks at most — two weeks for the
clones to grow and a week for
analysis — instead of the ten weeks
required by the method described
MAKE IT QUICK
In summary, building in quality from
the start and having the appropriate
assays available make it possible for a
small biotech company to produce a
suitable cell line for a phase I study.
Timelines can be significantly
reduced — even when resources are
limited — by the simple cloning
strategies described here.
REFERENCES
1 McKenzie, GJ; et al. Notch Signaling
in the Regulation of Peripheral T-Cell
Function. Seminars in Cell & Developmental
Biology 2003, 14: 127–134.
2 Center for Biologics Evaluation and
Research. Guidance for Industry: Q5D
Quality of Biotechnological/Biological Products
— Derivation and Characterisation of Cell
Substrates Used for Production of
Biotechnological/Biological Products. US Food
and Drug Administration, 1998;
www.fda.gov/cber/qdlns/qualbiot.txt.
3 Wong KK; et.al. Notch Ligation by
Delta 1 Inhibits Peripheral Immune
Responses to Transplantation Antigens By a
CD8+ Cell Dependent Mechanism. J.
Clinical Investigation, in press.
4 Young L; et al. The Notch Ligand
Delta 1 Selectively Enhances the Production
of IL-10 in Murine T Cells. Immunology
2002, 107, Supplement 1: 1–7.
5 McAteer, JA; Davis JM. Basic Cell
Culture Techniques and the Maintenance of
Cell Lines. In Basic Cell Culture, second
edition (University Press: Oxford, UK,
2001), p. 166. Corresponding author Sue Clarke is
head of manufacturing development;
and Janette Dillon, Ann Smith, and
Elizabeth Sotheran are development
scientists at Lorantis Ltd., 410
Cambridge Science Park, Cambridge
CB4 0PE, UK; 44-1223-702500,
fax 44-1223-702599; sue.clarke@
lorantis.com, www.lorantis.com.