CAPILLARY-AIDED CELL CLONING
A Technique For One Step Cloning With High Probability Of Monoclonality
ADEKUNLE O. ONADIPE, HILARY K. METCALFE, PETER R. FREEMAN 1 & COLIN JAMES2
ABSTRACT
INTRODUCTION
METHODS
One of the issues for consideration in the manufacture of a therapeutic protein is the
requirement of a stable clonal cell line to ensure a consistent manufacturing process.
The use of a non-clonal cell line may result in an uneconomical process or, even worse,
variation in product quality and biological activity.
The capillary-aided cell cloning technique is a variation of the “spotting” technique
and relies on independent visual confirmation by two scientists of the presence of a
single cell in a 1µl droplet. This technique has been shown to permit the resolution of a
mixed cell culture to give monoclonal colonies. The technique is easy, robust, permits
the measurement of errors in observation and the data obtained can be subjected to
statistical analysis.
The high probability of monoclonality obtained using this technique means that only
one round of cloning is required during cell line development, instead of multiple
rounds of limiting dilution cloning. This results in time and cost savings in the
development of a cell line for therapeutic protein production. This technique has been
used to clone hybridoma, CHO and NSO cell lines.
The most common cell cloning method is limiting dilution,
which relies on a statistical distribution (Puck & Marcus,
1955). A limitation of this technique is that, while the seeding
of the cells follows a Poisson distribution, the number of
colonies observed does not (Coller & Coller, 1986; Underwood
& Bean, 1988). Therefore, to achieve an acceptable level of
probability of monoclonality, multiple rounds of limiting
dilution cloning are required. As the creation of a clonal cell
line is often a critical path activity during product
development, a technique that enables faster derivation of
clonal cell lines using one round of cloning is desirable. Hence
the development of the capillary-aided cell cloning technique,
a variation of the “spotting” technique (Clarke & Spier,
1980).
Capillary-aided Cell Cloning
One droplet (approximately 1µl) of a dilute cell suspension was dispensed into
individual wells in 48-well plates. Two scientists independently examined the
droplets microscopically and recorded the number of cells contained. Growth
medium was added to all the wells. The plates were incubated at 37°C for up to 12
weeks, to allow for the appearance of slow growing colonies. All the wells that
produced colonies were recorded.
Data Analysis
The observations of each of the scientists were summarised into three categories: no
cells, one cell or more than one cell. The observed outcome for each well was that it
showed either growth or no growth. These data were entered into a statistical model
that was used to estimate the probability of monoclonality of the colonies using
maximum likelihood.
RESULTS
1. Separation of a mixed culture of two cell lines
2. Statistical modelling of the experimental data
3. Cloning of other cell lines
To test the robustness of the capillary-aided cell cloning technique,
two very similar recombinant NS0 cell lines were mixed in equal
proportions. The cell lines were derived from the same NS0 cell
bank and used the glutamine synthetase (GS) expression system to
express two antibodies that differed only in minor changes in the
variable region. Two scientists independently confirmed that 321 of
the 2300 wells seeded contained one cell. After incubation,
colonies were found in 156 of these 321 wells. Validated ELISAs
specific for each antibody showed that each of the 156 wells
contained only one antibody (Table 1). No wells were positive or
negative for both antibodies. These results demonstrated that
capillary-aided cell cloning resolved a mixed culture of two cell lines
into monoclonal colonies
The structure of a statistical model was developed from two other CapillaryAided Cell Cloning experiments. Maximum likelihood was then used to fit this
model structure to the data from other experiments. There was a close
agreement between the observed data and those predicted by the model,
indicating no serious departures from the assumptions of the model
(Figures 1 & 2). The probability of monoclonality of a single cell-derived colony
in the mixed culture experiment was estimated as 0.9992.
Hybridoma, CHO and other NS0 cell lines have been cloned using this
technique with probability of monoclonality routinely > 0.99 (Table 3). This is
similar to the probability obtained from two rounds of limiting dilution
cloning.
Observation
Wells positive for antibody A
Wells positive for antibody B
Wells positive for both antibodies
Wells negative for both antibodies
Total
Number of
wells
94
62
0
0
156
Table 1. Monoclonality of colonies obtained from a mixed culture of two GS-NS0 cell lines
producing different antibodies after Capillary-Aided Cell Cloning.
Colonies were observed in 2 of 474 wells confirmed to contain no
cells (Table 2). This indicated that there was an error in the visual
observation by the two scientists. A statistical model was,
therefore, developed to quantify the error in the Capillary-Aided
Cell Cloning technique.
Observation
Wells scored as containing 0 cells
Wells that subsequently showed growth
Wells that subsequently showed no growth
Number of
wells
474
2 (0.4%)
472 (99.6%)
Cell
Line
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
500
400
300
200
100
0
0, 0
0, 1
1, 1
0, >1
1, >1
>1, >1
Paired observations of cells in droplets
Observed
Predicted
Figure 1. Experimentally observed data compared with data predicted by the statistical model for wells showing
cell growth after the cloning of a mixed culture of two GS-NS0 cell lines using the Capillary-Aided Cell Cloning
technique. The horizontal axis represents paired observations of the number of cells reported by two scientists.
NS0
NS0
NS0
NS0
NS0
NS0
NS0
NS0
NS0
NS0
NS0
CHO
CHO
CHO
CHO
CHO
HYBRIDOMA
Expression
system
GS
GS
GS
GS
GS
GS
GS
GS
GS
GS
GS
DHFR
GS
GS
GS
GS
-
Probability of
monoclonality
0.9998
0.9997
0.9998
0.9987
0.9885
0.9961
0.9986
0.9957
0.9987
0.9999
0.9998
0.9827
0.9915
0.9976
0.9983
0.9955
0.9997
Table 3. Probability of monoclonality of cell lines derived using one round of Capillary-Aided Cell Cloning.
500
CONCLUSIONS
400
One round of capillary-aided cell cloning can replace two rounds of
limiting dilution cloning to obtain a monoclonal cell line.
300
The probability of monoclonality from one round of capillary-aided
cell cloning was typically >0.99 (similar to two rounds of limiting
dilution cloning).
200
100
The model developed is robust and predicts results that show good
agreement with experimental data.
0
0, 0
0, 1
1, 1
0, >1
1, >1
>1, >1
The technique can be used routinely to demonstrate monoclonality.
Paired observations of cells in droplets
Table 2. Error observed during Capillary-Aided Cell Cloning
of a mixed culture of two GS-NS0 cell lines.
Observed
Predicted
Figure 2. Experimentally observed data compared with data predicted by the statistical model for wells showing
no cell growth after the cloning of a mixed culture of two GS-NS0 cell lines using the Capillary-Aided Cell Cloning
technique. The horizontal axis represents paired observations of the number of cells reported by two scientists.
Lonza Biologics, 228 Bath Road, Slough, Berkshire. SLI 4DY, U.K.
Tel: +44 (1753) 777000
Fax: +44 (1753) 777001
email: [email protected]
Website: www.lonzabiologics.com
Cell Type
Code
0, 0
0, 1
1, 1
0, >1
1, >1
>1, >1
Scientists’ reports
Both say no cells
One says no cells, the other says one cell
Both say one cell
One says no cells, the other says more than one cell
One says one cell, the other says more than one cell
Both say more than one cell
REFERENCES
§
§
§
§
Clarke JB and Spier RE (1980) Arch. Virol., 63: 1-9.
Coller HS and Coller BS (1986) Meth. Enzymol., 121: 412-417.
Puck TT and Marcus PI (1955) PNAS USA, 41: 432-437.
Underwood PA and Bean PA (1988) J. Immunol. Meth., 107: 119-128.
ACKNOWLEDGEMENTS
The authors thank the Cell Biology and Fermentation Development scientists at
Lonza Biologics who performed the clonings that provided the data for this work.
1Ship
2RSS
Cottage, Cadgwith, Helston, Cornwall TR12 7JX, U.K.
Centre for Statistical Education, Nottingham Trent University, Nottingham NG1 4BU, U.K.