Antibody is a protein, synthesized and secreted by B-lymphocytes
(B-cell) that bind to antigens. Antibody is members of a family of molecules
(the immunoglobulin that constitute the humeral branch of the immune system)
and form approximately 20% of the plasma proteins in humans. Different
populations of immunoglobulin are found on the surface of lymphocytes,
in exocrine secretions and in extravascular fluids (Pharmacia Biotech,
Monoclonal Antibody (MAb) is antibody that binds only
to a specific antigen that compatible to its binding site (Xiao et
al., 2005). Thus, in MAb production, there is no need for further
purification in order to get the desired antibody as has been done for
polyclonal antibody. MAb has become indispensable tools in research, diagnostics
and therapeutics. They have gradually replaced the polyclonal antibodies
since hybridoma technology was introduced (Zola, 2000).
The growth rate of mammalian cells (hybridoma cells)
containing the antibody varies depending on the cell type, medium composition
including the growth factors and other environmental conditions such as
dissolved oxygen, carbon dioxide levels, pH and ionic strength (Butler,
1996; Constantino et al., 1995; Jung et al., 1992; Lee et
al., 1991). Moreover, it is understood that the specific growth rate
(Î¼) in hybridoma cells cultures starts to decline from the maximum
level at 20 h of cultivation and continuously doing so until the growth
eventually ceases (Doyle and Griffiths, 1998). Many studies had been conducted
to optimize those parameters in optimizing the production of MAb (Satoshi
et al., 2005; Heilmann et al., 2005; Lorea et al.,
2005; Guez et al., 2004; Tibor et al., 2004; Ralf and Thomas,
The main problem in producing MAb is cell apoptosis.
Media optimization provides a way to reduce the chance of apoptosis and
at the same time increase the hybridoma cell viability (Pakkanen and Neutra,
1994; Stoll et al., 1996). In this study, we had tried
to optimize the media components of the RC1 hybridoma cells cultures not
only to enhance the cells viability but also improve the production of
MAb, using the Central Composite Design (CCD) method.
MATERIALS AND METHODS
Design of Experiment (DOE)
Experiment was conducted at Animal Cell Engineering Laboratory of
IIUM and was designed by Response Surface Methodology (RSM) using a STATISTICA
Software (Statsoft, 2001). RSM is a set of techniques designed to find
the best value of response.
RC1 Hybridoma cell, a monoclonal antibody (IgG)-secreting cell line
was purchased from Japanese Cell Culture Stock and had been used in this
Media Preparation and Optimization
RPMI media in liquid form was used for this optimization process.
Formulation of media was first checked in order to determine the other
component which is required. The RPMI media without L-glutamine was used
and the three other components were added. The media was then taken into
hood with any other supplement or addition that was required. The bottle
was swabbed with 70% alcohol before uncap. For media optimization, 10
mL medium was prepared for 10 cm2 T-flask used. Design of experiment
was first done using STATISTICA to get the simulated value required for
each variable of the 16 Runs.
The percentage of serum, sodium bicarbonate and L-glutamine
needed to be added into each Run was designed as in Table
1. They were added according to the percentage volume of a total volume.
Serum was added after the pH of media was adjusted to 7.2 and the media
was then filtered.
Maintenance of Established Cell
The culture was examined carefully for any signs of contamination
or deterioration. The cell was suspended carefully to homogenize the cell
suspension. Five to seven milliliters medium was then removed and discarded according to the need. Fresh medium
was added up to 10-20 mL. The cell was dispersed into a single cell suspension
by repeated pipetting. Volume was then maintained or split into two flask.
The flask was then capped and stored in CO2 incubator.
|| Design of experiments of serum, sodium bicarbonate
All equipment such as 10 cm2 T-flask, pipette, required
media and inoculum were taken into hood. Nine milliliters media was transferred
into a labeled T-flask using pipette. One milliliter of the inoculum was
then taken and transferred into the T-flask making the volume in the T-flask
became 10 mL. The fresh media and the inoculum were suspended in order
to mix them well and then were incubated in 5% CO2 incubator
at 37 Â°C. The cells were counted and their viability was determined
by the trypan blue dye exclusion test. The counting result was then recorded
for reference purpose.
T-flask was put directly in hood from CO2 incubator. Cap
of T-flask was removed and it was held in the same hand that holds T-flask.
Inoculum in T-flask was suspended aseptically in hood for 1-2 min to homogenize
the hybridoma cell. Then, about 3 mL of cell was taken out using pipette
into the centrifuge tube. About 10 Î¼L of the cell in centrifuge tube
was taken for cell counting. The rest of the cell in the centrifuge tube
was centrifuged at 1000 rpm, 27 Â°C for 10 min. The supernatant was
collected for biochemical analysis.
RESULTS AND DISCUSSION
Media Optimization Result
All readings of the cell viability and the total cell number taken
from the 16 T-flask on day 4 were entered into the STATISTICA software
because most of the runs reach the highest cell viability in day 4 (data
not shown). All measured readings were first transferred into based 10
values as shown in Table 2. The screen parameter is
in percentage value.
From Analysis of Variance (ANOVA), it can be said that
L-glutamine and serum were the main factors affecting the growth of hybridoma
cell. The result was very significant since the p-value obtained for serum
and L-glutamine was very small (0.003964 and 0.000069, respectively).
From these values, it can be concluded that L-glutamine was a main factor
that increased the viability of the hybridoma cell, followed by the serum
|| Result from T-flask optimization
|| Critical value among the growth factors
|| Correlation between L-glutamine and serum on cell viability
Thus, the interaction between serum and L-glutamine in
affecting the viability needs further clarification. It can be said that
cell viability will increase as the percentage of both serum and L-glutamine
increased. However, L-glutamine had influenced more than serum in increasing
the cell viability. For example, when 14% serum was added in the RPMI
media, cell viability would only increase when L-glutamine was added.
Thus, the more serum added into the media, the more L-glutamine needs
to be added in order to get higher cell viability (Fig.
As shown in Fig. 2, cell viability
increased as the percentage of serum increased. However, the addition
of more NaHCO3 does not affect the cell viability. For example,
when adding 8% serum into RPMI media, the increase in the percentage of
NaHCO3 would only slightly increase the cell viability. This
indicates that serum plays more important role to boost cell viability
compared to NaHCO3.
In Fig. 3, it can be observed that
the cell viability increased as the percentage of L-glutamine increased.
The NaHCO3, however, does not affect the cell viability. For
example when 1% of sodium bicarbonate was added into the RPMI media, cell
viability has increased as the percentage of L-glutamine increased. However,
when 1% L-glutamine was added into the media, there was no effect on cell
viability even though there was an increasing in the percentage of NaHCO3.
The influence of L-glutamine on the cell viability was superior
compared to that of the NaHCO3.
|| Correlation between NaHCO3 and serum on
|| Correlation between NaHCO3 and L-glutamine
on cell viability
|| Pareto chart of relatively importance correlation between
independent screened parameters
Alternatively, STATISTICA had also provided the relatively
importance correlation between the independent screened parameters in
this research, which were serum, L-glutamine and NaHCO3 by
the Pareto chart (Fig. 4). This chart identified the
most significant parameter to the dependent variable, in the case of this
study, cell viability. The chart had also clearly showed that the L-glutamine
was the most important component that influenced the hybridoma cell viability
followed by serum and then combination between glutamine and serum.
At this point, it can be said that the main component
that contribute most to the high hybridoma cell viability was L-glutamine.
It has the largest effect (shown by p-value less than 0.05) on the increment
of hybridoma cell viability.
Optimized Value of Serum, NaHCO3 and L-glutamine
From the measured value of viability that was entered, STATISTICA
gave critical value which is also called optimized value in percentage
of serum, L-glutamine and NaHCO3 (Fig. 4).
It can clearly be seen that high percentage of serum was needed to get
high cell viability where 13.5% serum was required compared to the need
of only 1.68% of L-glutamine and 0.87% of NaHCO3. This optimized
value then can be used for the inoculation of the cell in a bigger bioreactor.
While many commercially available cell culture media
exist, none are able to meet the specific requirements of every cell line.
Optimization provides a way to increase the hybridoma cell viability which
at the same time increases the production of MAb. Moreover, the addition
of media component such as serum, L-glutamine and NaHCO3 must
be at sufficient amount in order to reduce the production cost.