Perfusion Cultures of Hybridoma Cells

for Monoclonal Antibody Production

FRANCISCO J. CASTILLO, LARRY J. MULLEN,
JOHN C. THRIFT, AND BARRY C. GRANT
XOMA Corporation
Berkelq, California 94710

INTRODUCTION

Perfusion cultivations of mammalian cells allow for the maintenance of long-

term, high-density, and high-productivity cultures.1,2Among the different perfusion
systems, hollow fibers have been used for the production of biologicals?“ including
monoclonal a n t i b o d i e ~ ,with
~ - ~ good results. However, few studies have gone beyond
simply describing the performance of a given cell line in a particular perfusion
system. These studies are of limited use for predicting the performance of other cell
lines, even in the few cases where the mass transfer characteristics of the bioreactor
are well understood.
Hybridoma cells have nutritional requirements, metabolic rates, and product
formation/secretion rates that are unique to each line even when derived from the
same parental myeloma. Often, these characteristics are functions of the culture
conditions, including the composition of the medium, the bioreactor type, and the
way it is operated. These characteristics need to be studied and quantitated under
reproducible conditions that will reflect actual production processes. Simple chemo-
stats may be valuable tools when utilized for this purpose by studying hybridomas
under continuous cultivation. The results obtained may then be used to predict the
performance of the corresponding cell line in a perfusion reactor operated continu-
ously for extended periods.
The objective of this work is to describe the cultivation of hybridoma lines in
industrial-scale perfusion culture, and to determine the value of chemostat-derived
data in the interpretation of the results obtained, as a method for predicting the
performance of hybridoma/bioreactor systems.

MATERIALS AND METHODS

The hybridoma cells used were all of murine-murine origin and are listed in
TABLE1. The media used consisted of Dulbecco’s Modified Eagle’s Medium/Ham’s
F-12 (1:l) containing 25 mM glucose and 4 or 6 mM glutamine and supplemented
either with 5% fetal bovine serum (FBS) or with 20 nM sodium selenite, 20 JLM
ethanolamine, 30 mg/L bovine transferrin, and 5 mg/L bovine insulin.1°
For chemostat experiments, jacketed spinner flasks (Wheaton, Celstir 3000 mL),
1500 to 2000 mL working volume, were used. The covers were perforated and fitted
with connectors and silicone tubing to allow continuous supply of medium and
72
CASTILLO et a[.: PERFUSION CULTURES 73

withdrawal of spent medium and cells. Ports were also made for removal of samples,
addition of gas (5-10% COz in air), gas exhaust, inoculation of cells, and other
additions. Gas inlets and outlets had 0.2-pm hydrophobic filters. The gases were
applied to the liquid overhead to poise the medium before inoculation. Agitation was
provided by magnetic floating stir bars rotating between 60 and 150 rpm. The
temperature was maintained at 37 "C by pumping water through the jackets with a
heating-circulator bath. Automatic addition of 7% sodium bicarbonate was used to
control the pH within the desired values.
Continuous perfusion runs were done in Acusyst-P hollow fiber systems (Endo-
tronics, Incorporated). Samples, 5 to 10 mL, were withdrawn with syringes. Total and
viable cells were counted in hemacytometers after diluting 1:l with 0.2% trypan blue.
The values of pH, pCO2, PO*, and HC03 were measured immediately after sampling
in a blood pH/gas analyzer (Corning). Glucose, lactate, and ammonia were deter-
mined by Sigma enzymatic procedure nos. 510, 876 UV, and 170 UV, respectively.
Glutamine was estimated enzymatically by a modification of a previously described
method."
Monoclonal antibody (MAb) concentrations were determined by HPLC meth-
ods.IZGlucose and glutamine consumption rates were calculated based on the media

TABLE1. Cell Lines Studiedu

Cell Line MAb Type Parental Myeloma
XM-A P3-NS1-1-Ag4-1
XM-D P3-NS1-1-Ag4-1
XM-G P3-NS1-1-Ag4-1
XM-I P3-X63-Ag8.653
XM-SM SP2/0-Ag14
"All splenocytes of BALB/c origin.

flow rates and residual values of the nutrients in the spent media. Oxygen consump-
tion rates were continuously calculated by the computer controlling the Acusyst
systems. The calculations were based on gas compositions and flow rates and on the
differences in oxygen partial pressures between the inlet and outlet of the bioreactors
measured in line by oxygen electrodes and in samples analyzed as described
previously. Consumption and production rates were normalized to express the values
per liter of bioreactor volume.

RESULTS AND DISCUSSION

The MAb productivity and the oxygen consumption rate (OCR) for cell line
XM-A in perfusion culture are shown in FIGURE 1. An apparent correlation between
MAb productivity and OCR seems to exist, with an average yield between 7 and 7.5
mg of MAb per mmol of oxygen. Similar correlations were observed with all the cell
lines studied and the corresponding OCRs and MAb yields are listed in TABLE2. As
can be seen, the OCR for line XM-I was lower in serum-free medium, but the MAb
yield remained very similar. Clearly, if the value of MAb/02 for a cell line is known,
14 ANNALS NEW YORK ACADEMY OF SCIENCES

-
L
g
+
i-

d
2 - 12 g
c
n -
o > 10 5-
Y

2 \
2 8
2 2
k m
- k 6
E Z
5 5 1 .
i 2?r
z
z
< O 4 2 .
2 - z -
z 2 %
-
<
9
c,
0
- 0
F
g
5 0

then its corresponding OCR in a perfusion system could be utilized to estimate the
productivity of the system.
The glucose uptake and lactate production rates during the perfusion culture of
line XM-A are shown in FIGURE2. The maxima in both cases were near 8 g/h and
lactate production matched glucose uptake, implying that most of the glucose was
utilized through glycolysis. Other lines produced less lactate than the glucose
consumed, indicating anabolic use of the hexose through the pentose phosphate
pathway or oxidation through the Krebs cycle. Glucose was not limiting because its
residual level was always above 1.0 g/L. As seen in TABLE3, cell line XM-A had the
highest glucose consumption rate and the lowest MAb yields. This contrasts with the
values obtained for cell line XM-D, which shares the same parental myeloma of line
XM-A. Cell line XM-I had higher glucose consumption and lower MAb yields in
serum-free medium. These results, seen also with other cells not discussed here, are
to be expected because in a serum-free medium the cells need to synthesize
metabolites no longer provided by the serum and therefore will have a higher
requirement for energy. The maximum yields of MAb per glucose are similar to some
reported by others.I3J4

TABLE
2. Oxygen Consumption Rates and MAb Yields in Perfusion Cultures
Oxygen Consumption
Rates MAb Yields
Cell Line (mmol/L. h) (mg/mmolO2)
XM-A 6 8.9
XM-D 12-16 13
XM-G 12-16 14
XM-I (serum-cont.) 3.4 3.2
XM-I (serum-free) 2.9 3.1
CASTILLO el al.: PERFUSION CULTURES 75

h
i
L
\
M
v

0 0 10 20 30 40 50 60
DAYS
FIGURE 2. Glucose uptake and lactate production rates of hybridoma line XM-A in perfusion
culture.

Glutamine uptake and ammonia production rates by line XM-A are shown in
FIGURE 3. During the production phase, no residual glutamine could be detected
and the same was observed with all the lines studied. This indicates that higher
concentrations of the amino acid in the media formulations may be needed. O n
molar ratios, the ammonia produced was less than the glutamine utilized, suggesting
anabolic utilization of part of the amino acid and ammonia recycling through
transamination reactions. The yields of MAb per glutamine for three of the lines are
shown in TABLE 4. These yields were lower than the 16 to 24 mg of MAb per mmol of
glutamine reported for hybridoma AB2-143.2.I5
In a perfusion system with cell retention, the specific growth rate (k) of the cells
will be equal to the bleed rate (harvest rate in the runs discussed here) divided by the
volume of the bioreactor.I6 During the production phase of these runs, p ranged
between 0.02 and 0.05 h-I.
The specific glucose and glutamine utilization rates as well as the specific MAb
productivities of the hybridoma lines were determined in chemostats at specific
' . ~ ~values of these rates
growth rates within the range of those used in p e r f ~ s i o n . ~The
for cell line XM-A are shown in FIGURE 4. MAb production in this line is clearly
growth-related. A pattern of productivity has been found frequently with murine/

TABLE3. Glucose Consumption Rates and MAb Yields in Perfusion Cultures

Glucose Consumption
Rates MAb Yields
Cell Line (mmol/L. h ) (mg/mmol glucose)
XM-A 72 1.45
XM-D 46 5.13
XM-G 44 5
XM-I (serum-cont.) 3 4.15
XM-I (serum-free) 4 2.3
76 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 3. Glutamine uptake and ammonia production rates of hybridoma line XM-A in
perfusion culture.

murine hybridomas irrespective of their parental myeloma.I7J8This pattern is by no

means unique and represents only one of several reported for hybridomas.1g-2*
Using these chemostat-derived data and the results from the perfusion run,
corresponding numbers for viable cell concentrations in the confluent bioreactors
were calculated. These values were then compared to potential total cell concentra-
tions calculated using an average cell diameter of 15 pm. As seen in TABLE 5, the
total cell concentration lies somewhere between 2.94 x lo8 and 5.66 x lo8 per mL.
The estimated viable cell concentrations in all three cases were very similar and were
approximately only 30% of the maximum total. Similar values have been reported for
other hybridoma in hollow fiber culture?2
Obtaining true representative samples of the extracapillary space of hollow fiber
bioreactors is difficult, especially after reaching confluency, and it is not possible to
accurately determine viable cell concentrations. The results presented here demon-
strate the value of using nutrient uptake14s23,24 or specific MAb productivity values
obtained in chemostats as practical noninvasive methods to estimate viable cell
concentrations. Other methods such as measuring the electrical resistance of a
culturez or nuclear magnetic resonance (NMR)22have also been used for the same
purpose. Although NMR is not practical for production systems, it is evolving rapidly
and will continue to produce valuable physiological data that are very difficult or
impossible to obtain by any other method.

4. Glutamine Consumption Rates and MAb Yields in Perfusion Cultures

TABLE
Glutamine Consumption
Rates MAb Yields
Cell Line (mmol/L. h) (mg/mmol glutamine)
XM-A 22.6 5
XM-D 22.6 10.4
XM-G 22.6 5
CASTILLO ef al.: PERFUSION CULTURES 77

I . I .

\
78 ANNALS NEW YORK ACADEMY OF SCIENCES

Available hollow fiber perfusion technology has distinct advantages for the
production of MAbs at several scales. However, understanding of the physiology of
hybridomas in high-density extended cultures is in its infancy. Only by growing the
cells in the corresponding system can the performance be unequivocally evaluated.
There may be factors, exogenous or autocrine,26 that affect the cells only when
present at the concentrations and under the conditions seen in confluent cultures.
Several hybridomas that showed high metabolic activity and were easily maintained
for months of operation in hollow fiber bioreactors nevertheless expressed specific
MAb productivities lower by one order of magnitude or more than those obtained in
conventional suspension cultures. However, when cells of the corresponding hybrido-
mas were recovered from the bioreactors and were cultivated in low-density suspen-
sion cultures, they had the original specific MAb productivities and yields (unpub-
lished results). Increases in specific MAb productivities for some hybridomas when

TABLE 5. Estimation of Cell Concentrations in Systems with Cell Retention (XM-A

Cell Line)
Viable Total
Estimate Based on: (X 1O8/mL) ( x t@/mL)
Diameter (15 em) no void volume: 5.66
47% void volume: 2.94

Glucose Consumption Rate 1.38-1.66 2429% of maximum total

(10-14 mmol/109 cells . day)

Glutamine Consumption Rate 0.95-1.55 17-27% of maximum total

(3.5-5.7 mmol/109 cells. day)

Specific MAb Productivity 1.58-1.73 28-31% of maximum total

(12-19 pg/109 cells . day)

Oxygen Consumption Rate 0.95 17% of maximum total

(4 mmol/109 cells . day; taken from
literature)

grown in perfusion cultures have also been r e p ~ r t e d .These

~ ~ . ~observations
~ indicate
that certain hybridomas, otherwise perceived to be stable, may have phenotypic
responses in high-density cultures that are of practical importance, but that cannot
be predicted a priori. This may apply to any mammalian cell intended for production
use.
Hollow fiber bioreactors, and other perfusion systems, will likely grow in impor-
tance as more data accumulate to demonstrate their practicality and reliability.
Important areas for development include understanding of mammalian cell physiol-
ogy in general and of the producing cells in particular, comparative studies of runs in
different systems, and fundamentals of the operation of the cell/bioreactor sys-
tems.29
CASTILLO et al.: PERFUSION CULTURES 79

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