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Encyclopedia of Physical Science and Technology EN007K-319 July 2, 2001 17:53

442 Hybridomas, Genetic Engineering of

liquid flow. The pitched-blade or marine type impellers or via a the surface of silicone tubing introduced into the
are particularly suitable. culture vessel.

XXII. SERUM AND SERUM-FREE
B. Culture pH
MEDIUM FOR ANTIBODY
The optimal pH for hybridoma growth is around pH 7.0– PRODUCTION FROM HYBRIDOMAS
7.4 and maximum cell growth occurs if this is maintained.
A pH probe can be used to control the sparging of cul- The growth of mammalian cell lines requires a chemi-
tures with CO 2 or by low volume additions of a concen- cally defined liquid basal medium which is traditionally
trated sodium bicarbonate solution. Bicarbonate is used in supplemented with 10% (by volume) bovine serum to pro-
preference to sodium hydroxide because of the danger of vide supplements of growth factors. Although bovine (or
over-shooting the set-point with the stronger alkali. These other animal) serum provides excellent growth support for
additions are normally governed by a computer-controlled cells in culture, there are significant disadvantages in us-
pump or gas valve to a preset pH value. Acid (e.g., HCl) ing serum as a an additive to hybridoma cultures. These
may also be added from an external source. However, this include:
is usually not required as lactic acid is normally produced
by cell metabolism and causes the culture pH to decrease Batch-to-batch variation in composition. To generate a
during growth. The culture pH in a bioreactor is often supply of bovine serum for use in cell culture, the
controlled by the rate of CO 2 gas flow into the culture. An serum is extracted from a herd of cows and pooled.
electronic pH controller regulates the on/off function of a Each batch is then identified by the supplier and can be
control valve. sampled by the buyer for suitability in an industrial
process. However, the composition of each batch
varies in undefined ways depending upon the diet of
C. Oxygen
the cows. This variation can cause significant
The supply of oxygen to satisfy cell metabolism is one of differences in the growth-promoting characteristics of
the major problems associated with fermenter scale-up. the serum, and ultimately causes significant differences
The oxygen consumption rate of hybridoma cells varies in productivity of the cell culture process.
6
from 0.06 to 0.6 mmol/liter/hr for a culture at 10 cells/ml. A high protein content that hinders product purifica-
In small cultures (T-flasks, etc.), the oxygen demand can tion. The cells grown in a bioreactor secrete the
be satisfied by gas diffusion from the head space through product of interest (normally a protein) into the culture
the culture surface. However, with increasing culture vol- medium. If the culture medium contains serum, its
ume, the surface to volume ratio decreases. At cultures protein concentration is already high. Serum has a
of 1 L and above, the surface/volume ratio is generally protein concentration of 60–80 mg/ml and so the basal
too low to satisfy the overall oxygen demand at this cell level of protein in a 10% serum-supplemented culture
concentration. medium is 6–8 mg/ml. In comparison, the
Because the solubility of oxygen is low and a contin- concentration of a protein secreted by the cells being
uous supply of oxygen is required to satisfy the cellu- cultured typically reaches 100–150 µg/ml and
lar metabolism at higher culture volumes,. The maximum therefore a difficult purification process is required to
concentration of oxygen in culture media which is in equi- separate the product from the serum protein (called
librium with air is 0.22 mM at 37 C—referred to as “100% downstream processing). The monoclonal antibody of
◦
air saturation.” Growth of many animal cell lines has been interest may well be mixed with any other antibodies
found to be optimal at dissolved oxygen levels (DO) be- present in the serum and these are virtually impossible
low the maximum oxygen solubility and corresponding to to separate. The disadvantage of an impure or poorly
20–50% of air saturation. This DO level may be main- purified product such as a monoclonal antibody is that
tained by a control system that incorporates an oxygen there may be unwanted side reactions connected with
probe and input of sparged air or oxygen. its use as a diagnostic or therapeutic agent that reduces
An added problem is that excessive gas sparging may its effectiveness.
cause cell damage particularly in cultures with a large The potential for product contamination. The threat of
surface to volume ratio. This problem may be offset by the contamination arises from unwanted viruses and
use of chemical protectants such as the polymer, Pluronic mycoplasma that may be present in serum as well as
F-68, or by alternative methods of introducing oxygen the undefined and uncharacterized prion agents of
into the culture. The alternative methods may include gas bovine spongiform encephalopathy (BSE, or “Mad
sparging in a media reservoir not in contact with the cells Cow Disease”). Because of the concern over the