12_ANIMAL CELL TECHNOLOGY_707

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4.3. Carboxy-SNARF-1-AM staining of hybridoma cells in batch culture
Carboxy-SNARF-1-AM staining of Pef hybridoma cells growing, in batch culture showed an
increase in pHi during exponential growth (48 hrs), probably due to an increase in the percentage
of S-phase cells. Decrease in pHi at the decline phase was, however, a manifestation of both an
increase in G1-cells and apoptosis. The reduction in pHi occurred prior to the identification of
apoptosis by fluorescent microscopy. In hybridoma cells over expressing bcl-2 in batch culture
there was an initial slight increase followed by a continuous but slow decline in pHi with time
which coincided with an accumulation of cells in G1-phase of the cell cycle
5. Conclusion
Acidification and phosphatidylserine externalization demonstrate early events in apoptotic cell
death. Intracellular acidification is linked to cell growth inhibition and to apoptosis. Monitoring
acidification and phosphatidylserine externalization by flow cytometry provide earlier
identifications of apoptosis compared to other techniques and therefore can be used to control
bioreaction processes.
6. Acknowledgments
This work is funded by the E.C. Framework IV Programme

7. References
1. Al-Rubeai M., Emery A.N. & Chalder S. Flow cytometric study of cultured mammalian cells. J.Biotechnol.

1991, 19:67.
2. Singh R.P., Al-Rubeai M , Emery A.N. Apoptosis: Exploiting novel pathways to the improvement of cell culture

processes Genetic Engineer and Biotechnologist 1996, 16:227-251.

SESSION ON

INTEGRATED BIOPROCESSING IN ANIMAL CELL TECHNOLOGY

This area still represents the main line of activity of many of the ESACT members and
thus it provides the organisers with a large choice, so that, as in the earlier meeting in
Vilamoura, it was considered that no invited speaker was required. Indeed, as in the past,
the meeting organising committee had a very difficult task in choosing only ten oral
presentations out of the sixty two submitted abstracts. In order to decrease the unfairness
of the process and to balance out the oral and poster sessions, there was an attempt to
balance presentations along three topics of research and development:

1- Physical environment, cells and products - at the cross roads between the
physical environment which is becoming more controllable and the cell physiology,
yielding more reliable production:
2- Monitoring and control - even though sensors (on line and off line) have not
yet delivered as much as is expected of them, the relevance of this area for quality
assurance and economic production is becoming more firmly established;
3- Integrated processes and scale-up - an area which plenty of congress
organisers want to present but which is only slowly yielding results for public
discussion, as companies and teams become more assured of what they are doing and
risk presenting.

From the overall presented papers, a few can be briefly described to give the flavour of
the session.
Konstantin Konstantinov (Bayer, Berkeley, CA, USA) presented a production process
model for high-density perfusion cultures showing that dynamic, profit based
optimisation of feeding is essential for the efficient manufacturing of therapeutic
proteins; it is only to be expected that other protein manufacturers will add depth to this
trend setting process of economically pushing animal cell technology. The use of the
single round infective Semliki Forest Virus which targets a broad range of host cells for
the transient expression of proteins at high level was described by Horst Blasey
(GlaxoWellcome, Geneva, Switzerland) for the production of 15mg of the 5HT3 receptor
protein in an 11.5 litre batch reactor. Stephanos Grammatikos (Karl Thomae, Biberach,
FRG) provided industrial credibility for the use of intracellular ribonucleotide pools to
indicate the metabolic status of the cell. Monitoring of nucleotide ratios such as UTP to
UDP-N-acetylhexosamine in very large scale (up to 10000 1) can be used to predict the
behaviour of a culture up to two days before any hint of physiological changes is given
by traditional cell number/cell viability estimations - optimisation and economic
modelling are envisaged. At the other extreme, Nicolas Kalogerakis (NYSU, Buffalo,
NY, USA) described a dielectrophoresis-based cell separation/filter being worked out on
a very small scale using microfabrication tools which is under test for separating viable

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264

from non viable cells in perfusion systems, and speculated on the inherent capability of
this filter for the preferential removal of apoptotic bodies from bioreactor effluent
streams. Pedro Cruz (IBET, Oeiras, Portugal) described the importance of optimising the
conditions in the bioreactor in order to obtain high quality virus like particles from
baculovirus infected insect cells.
The use of sonoperfusion to keep single mammalian cells inside continuously stirred
tank reactors was described (A. Miller, Mons, Belgium). The effect of bioreactor
parameters on less commonly described receptor production (CD 13 in HL60 cells) was
presented by T. Papoutsakis (U.S.A.).
Given the increasing relevance of fed batch production, feeding and monitoring strategies
have been presented for the more common industrial cell types (CHOs, insect cells).
Control of proteolytic activities during fermentation were described for both mammalian
and insect cells. Indeed, the winner of the poster prize (courtesy of Promega) was

Ernesto Chico for work performed at GBF with insect cells; for these cell types, L.
Häggström (Royal Inst. Technol., Stockholm, Sweden) used osmotic shock to increase
growth rate and final cell density. Amongst the various control strategies described, a
couple of imaginative imaging processes were presented by groups in Stuttgart and
Berkeley, whereas the group of R. Kemp (U. Wales, UK) presented a viable cell monitor
based on a dielectric spectroscope and proposed on line heat flux measurement for animal
cell culture modelling in what might be an upscaleable fed batch system.

The recurrent utilisation of internal settlers in continuous culture was elegantly
presented by the group of A. Marc (Nancy, France) for the production of properly
glycosylated gamma interferon. Different groups evaluated fluidized bed systems for
production of therapeutic proteins (M. Biselli, Jülich, Germany; J. Shiloach, NIH, MD,
USA; H. Katinger, Vienna, Austria) while the advantages of temperature control namely
to temperature lower than 37° C, were presented by G. Kretzmer (Hannover, Germany)
and the Californian teams at Bayer and Genentech.

Different options for the purification of proteins were presented, ranging from the
expanded bed adsorption (again from Genentech, J.T. Beck) as well as the direct protein
sequestration concepts put forward by A. Lydiatt (Birmingham, UK); even though
regulatory issues might hinder the use of such concepts they deserve more work to prove
their feasibility. Integrated approaches and the influence of the chromatographic steps
used upon structure and activity of proteins were also covered (S. Fletcher, Sidney,
Australia; H. Vogel, GBF, Germany) whereas displacement chromatography was tested
for DNA preparation (R. Freitag, ETH Lausanne, Switzerland).

Appropriately for systems meant for production of proteins, different processes of virus
clearance and validation and other safety issues (General safety issues) were also
covered in what was a very holistic and expert dominated session.

M. Carrondo
Chairperson

PHYSICAL ENVIRONMENT
CELLS
AND

PRODUCTS

DIAUXIC CELL BEHAVIOR ENABLES DETOXIFICATION OF CHO CELL
CULTURE MEDIUM DURING FED BATCH CULTIVATION

Holger Lübben and Gerlinde Kretzmer,
Institut für Technische Chemie, 30167 Hannover,Germany

Introduction
In fed batch cultures usually the feeding of the main substrates as glucose and
glutamine leads to an increase in cell count and product concentration, but very often a
dramatic decrease in cell viability due to the enormous toxification of the cell culture
broth with lactate and ammonia at high cell density is observed.
Especially the excretion of large amounts of lactate is known to be responsible for the
growth inhibiting effect, finally ending in abrupt cell death. Using a special feeding
technique the recombinant CHO celline SSOA2 producing human antithrombin III is

able to grow on its waste product lactate as a carbon source and completely detoxifies
the cell culture broth from this major metabolite demonstrating a functional
gluconeogenesis of mammalian cells in vitro. Inducing the diauxic cell behaviour a new
way for extended cultivation spand, increased biomass and product titre as well as
optimal viability is generated.

Material and Methods
Different feeding techniques have been applied :
In the first experiments a pulsewise feeding of different substrates has been applied
(glucose and glutamine, asparagine and glutamine). The pulses were given at small
portions to the cultivation (1g/L for glucose and 100 mg/L glutamine) in order to
prevent excessive metabolisation as have been described earlier.
Later on a continuous feeding technique of glutamine and asparagine was developed.
The intension was to keep the glutamine concentration constant at 200 mg/L during the
feeding phase. Although asparagine is a key substrate and essential for the occurrence
of the lactate consuming ability it was not possible to measure the asparagine
concentration on-line. For that reason the feeding mix consists of a molar ratio of 4:1
glutamine to asparagine, respectively 0.1 m gln and 0.025 m asn. This ratio is equal to
the ratio of the specific consumption rates. The nutrient flow rate was controlled either
manually (210 mg/L.d) or by means of on-line FIA analysis (PID-controller).
Gluconeogenesis is not very common in mammalian cell culture. In this metabolic state
previously excreted metabolites as pyruvate, citrate and lactate are remobilized

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

268

(Figure 1). To enable several steps in the gluconeogenetic pathway, energy in form of
ATP is required. Without the amino acid precursors for energy production no
gluconeogenesis is possible. The functionality of TCA cycle is maintained by
anaplerotic input of glutamine via 2-oxoglutarate and aspartate via oxaloacetat. The

following figure illustrates a hypothesised mechanism of the pathway during the
gluconeogentic phase. The proposed reactions are in accordance with the biochemical
literature and the experimental measured data.

Huge amount of excreted succinate (1g/L) was observed and could not be explained by a
simple mass balance of the reactions that fill the TCA cycle. For explanation reasons
some of the reactions in the TCA must be reversed to some extend.
At oxaloacetate two directions are hypothesised. First: A part of oxaloacetate enables
the gluconeogenetic pathway by reversed reduction to malate, hence leaving the
mitochondrium. Another part is needed for maintaining the flux within the TCA for
further oxidation of acetyl-CoA.
The excretion of succinate is thought to be due to the reversed function of the
mitochondrial malate dehydrogenase. Excreted succinate is supposed to originate from

269

glutaminolysis, but also, in order to close the carbon balance, from lactate which
maintains the TCA cycle running via acetyl-CoA.

Results and discussion
The application of various feeding techniques induces different metabolic behaviour.
Applying a pulsewise feeding of glucose and glutamine an excessive metabolisation of
both nutrient sources was observed, although just small portions were given to the
culture. The velocity of glucose and glutamine consumption is enormous. Glycolysis
and TCA are completely unbalanced. As a consequence high excretion rates of TCA
metabolites (citrate and succinate) as well as lactate were observed. Already in the
beginning of the feeding phase cell growth is inhibited due to the large lactate
production and consequently no further recombinant product was obtained. Obviously
the relation between productivity and growing is due to the genetic construction. A
combination of glucose and glutamine feeding is not a successful way for process
optimization. In order to prevent an ongoing lactate production in the feeding phase a
pulsewise feeding of glutamine and asparagine was established. In this case the cells
change their metabolic behaviour. As far as glucose has been depleted they switched
from glycolysis to gluconeogenesis. This diauxic behaviour is well known in
procaryontic systems and is here indicated by the consumption of the excreted waste

270
product lactate and a reduced growth rate (0.715.d-1 during glucose metabolization vs.

0.308.d-1 in the lactate consuming phase).
The ability to use lactate as a carbon source resulted in a prolonged exponential growth,

mainly by reducing the inhibition of cellular growth by lactate. The cell count as well as
the product titre is increased (40% more biomass; 30% more ATIII). See Figure 3.
Although the glutamine demand in the gluconeogenetic phase is reduced (qGln= 0.061
(mg/(106ce.d)] compared to 0.176[mg/(106ce.d)] in the glucose consuming phase), a

manual feeding of 100 mg/(L.d) was not sufficient because of the increasing cell
number.
For this reason a continuous feeding of asparagine and glutamine was applied to
prevent the limitation of these amino acids. Due to the availability of these anaplerotic

amino acids the specific growth rate in gluconeogenetic phase was increased up to
0.44.d-1 and a further optimization was achieved (cell count: 4.11.106ce/ml, product
concentration: 36.45mg/L). The re-metabolisation of lactate is almost complete and
results in a high viability up to 160 hours. The results of different feeding techniques on
cell growth and productivity are summarised in Table 1.

271

Conclusions
For aims of process optimisation an increased cell and product concentration could not
be achieved by simple glucose and glutamine feeding due to the growth inhibiting
tension of their waste products lactate and several organic acids. The experiments

pointed out that any significant accumulation of lactate has to be avoided. By applying
an intelligent feeding technique we could induce a so far not often seen re-
metabolisation of the toxic waste product lactate for biosynthesis purposes and therefore
denote this phase as gluconeogenesis. The induction of the gluconeogenitic phase was
achieved by a continuous feeding of TCA-cycle anaplerotic amino acids glutamine and
asparagine. The utilisation of waste products diminishes the growth inhibiting effect.
As a consequence of the removement of lactate a prolongation of cell growth was
achieved, because of the occurrence of a second exponential phase when the cells grow
on lactate as a carbon source. Although in this phase the growth rate was decreased the
overall number of cells was improved. Increased biomass correlates with an increase of
product titre.

EFFECTS OF POLYSACCHARIDE DERIVED FROM TEA ON GROWTH OF

HUMAN CELL LINES IN SERUM FREE CULTURE

H.KAWAHARA, M.MAEDA-YAMAMOTO, K.OSADA, K.TSUJI
National Research Institute of Vegetables, Ornamental Plants and Tea,
MAFF, 2769 Kanaya, Shizuoka 428, JAPAN

1. Introduction

Serum free culture of mammarian cells is more advantageous method that culture

environment can be designed freely for various bio-assay than usual culture method
containing serum. We try to investigate various effects of food components on cell function

of human immune cell lines. In such a case, if culture medium containing serum is used, it
is difficulte to prove the function of those components because of unknown elements in
serum [1]. Serum free culture is supposed that it is effective for our system.

Growth factors such as insulin, trasferrin, ethanolamin, sodium selenite for serum
free culture were known well [2]. However, every cell llines don't always grow in serum
free culture containing such growth factors.

In this report, we studied that tea polysaccharides added in serum free media effect
on cell growth of human immunological cell lines.

2. Materials and Methods

2.1. CELL LINES AND MEDIA

Human immunological cell lines (Burkitt lymphoma RAJI, human T lymphoma PEER,

human eosinophil cell line EoL-1, human basophil cell line KU812, human chronic

granulocytic leukemia K-562) were cultured in ERDF basal medium containing 10%FBS.

Each cell lines was sure of growing in ERDF medium supplemented with insulin,

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

274

10µg/ml transferrin, 20µM ethanolamine, 25nM sodium selenite(ITES). Cells were cultured
in ITES-ERDF serum free medium for 2 days before testing. Each cell lines was sure of
growing in ITES-ERDF medium. Cell numbers were measured by trypan blue dye exclusion
method.

2.2. PURIFICATION OF HEMICELLULOSE FROM JAPANESE GREEN TEA

Hemicellulose from the Japanese green tea (Camellia Sinensis var. 'yabukita') was obtained
as follows. The extract from powdered green tea with acetone was mixed with 0.5N NaOH
and 0.1%NaBH4. After shaking the mixture for 2 hours at room temperature, it was
centrifuged at 5000xg for 30 min. The supernatant was obtained and adjusted to pH4.5
with acetic acid solution. Then, adding 7% trichloroacetic acid to the solution, it was
centrifuged. The supernatant was dialyzed against water for 3 days, and the solution was
mixed with ethanol to separate the polysaccharides fraction. After centrifugation, the
precipitate was dissolved in water and lyophilized. The lyophirized powder was dissolved
in distilled water again, the solution was added to culture medium.

2.3. PREPARATION OF pH OF CULTURE MEDIUM

ERDF basal medium adjusted to six point of pH6.0, 6.5, 7.0, 7.3, 7.5, 8.0 were applied to
this experiment. Acidified or neutralized medium of pH range from 6.0 to 7.3 was performed
with gas. Basic medium at pH7.5 or 8.0 was prepared by adding 1N NaOH to the
medium.

3. Result and Discussion

We acquired several kinds of polysaccharides
from tea by using various purification
methods. In these extracts, the polysaccharide
having the highest activity of cell growth was
named TH, and used following experiments.

We tried to add the TH at various
concentration to ITES-ERDF medium.
Figure 1 shows the maximum dose of TH for
5 kinds of cell lines. When the TH was added

275

to ITES-ERDF medium at 10µ g/ml of final concentration, all kinds of cell lines except
KU812 could grow in this serum free media. If TH over 10µg/ml was added to ITES-
ERDF medium, the viability of cell was significantly decreased on microscopic observation.

As shown in Figure 2, though lag of the cell growth was slightly prolonged by
adding TH, saturated cell density of ITES-ERDF-TH was about 2 times higher than that
of ERDF containing only ITES.

We investigated that the effect of pH of serum free culture medium on viability of
the cells. As shown in Figure 3, the viability of the cells cultured in ITES-ERDF-TH was
kept on the higher level than that of the cells cultured in ITES-ERDF. Specially, on the
higher pH than pH7.4, addding the TH to ITES-ERDF medium is more effective than the
culture medium without TH.

These results suggested that the TH had a effect to stabilize something change of
medium with cultivation rather than to stimulate the cell growth like a growth factor.

4. References

1.Shinmoto,H., Kobori,M., Tsushida,T., and Shinohara,K. (1995) Protein-free culture of human macrophage
like UM cell line, in E.C.Beuvery, J.B.Griffiths and W.P.Zeijlemaker (eds.), Animal Cell Technology,
Kluwer Academic Publishers, Dordrecht, pp. 1147-1151.

2.Murakami,H. (1989) Serum-free media used for cultivation of hybridomas, Monoclonal Antibodies: Production
and Application, Alan R. Liss, Inc. pp. 107-141.

METABOLIC DEMANDS OF BTI-TN-5B1-4 (HIGH FIVE™) INSECT CELLS
DURING GROWTH AND AFTER INFECTION WITH BACULOVIRUS

ERNESTO CHICO1,2 and VOLKER JÄGER1
1Gesellschaft für Biotechnologische Forschung mbH
Mascheroder Weg 1
D-38124 Braunschweig, Germany.
2Center of Molecular Immunology
POBox 16040
Habana 11600, Cuba.

Introduction
• Because of its high productivity BTI-Tn-5Bl-4 (High Five™) cell line has become

an attractive alternative to Spodoptera fugiperda cells for recombinant protein
production using the Baculovirus expression system.

• In this work we present the main metabolic demands of this cell line and discuss the
major challenges for growing these cells at high cell density for infection and
subsequent recombinant protein production.

Experimental conditions
High Five cells were cultivated in Ex-Cell 401 serum-free medium fortified with
glucose, asparagine and glutamine. Suspension cultures were carried out in Techne
spinner flasks as well as in membrane aerated bioreactors for studying the oxygen

demand. For infection, a recombinant baculovirus was used expressing human
protein. Synchronous infection of the cell population was achieved using a MOI of 5.

Results and Discussion

General Metabolic Pattern of High Five cells
Fig. 1 shows the general metabolic behaviour of High Five cells during a batch culture

which is characterized by following observations: The exponential growth phase ends
with the almost simultaneous depletion of Gln and Asn. From all of the amino acids
measured only the concentrations of Ala, Asn and Gln change substantially throughout

the culture time. Alanine accumulates during the exponential phase of growth and its
concentration remains constant after the depletion of asparagine and glutamine. Final

values of 10 - 20 mM of can be found at the end of a batch cultivation of High

Five cells. These values are extremely high in comparison with most insect as well as

mammalian cell lines.

Baculovirus infection does not affect the general metabolic behaviour of High
Five cells (data not shown).

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

278

Specific uptake rates of glutamine and asparagine (qGln and qAsn) during growth and
infection are presented in Fig. 2. Whereas qGln shows a clear trend to reach a
saturation in the experimental concentration range, qAsn still increases at
concentrations higher than 10 mM of Asn. This fact is important for the design of
feeding strategies for High Five cells. Although the general metabolic behaviour of

279

cells is not affected during baculovirus infection the cell specific consumption of
Asn and Gln is significantly higher than during exponential growth.

Oxygen demand
The specific oxygen uptake rates of High Five and IPLB Sf21 AE cells were measured
in membrane-aerated bioreactors. Results in Table I indicate that High Five cells have
the highest cell specific oxygen consumption rate reported in the literature for insect
as well as mammalian cell lines.

High Five cells showed a 270% higher cell specific oxygen consumption rate when
compared to Sf21 cells. This difference is reduced to approximately 30% if the
oxygen consumption rate is expressed per volume of the cell population due to the

significantly bigger cell size of High Five cells (See Table I).

The overall oxygen uptake rate (OUR) of the bioreactor was measured online from the
DO controller response during growth and after infection (data not shwon). During
exponential growth the OUR correlated well with the cell density. This correlation is

only altered during the first 30 hours post infection (hpi), suggesting that there is a
peak of the cell specific oxygen uptake rate which is associated to cellular events
during the early phase of baculovirus infection.

Conclusions
High Five cells are characterized by a very active metabolism, by which Asn and Gln
are consumed at very high rates. The use of feeding strategies, aimed to keep the
concentrations of these amino acids in a region of lower qAsn and qGln, is strongly
recommended to avoid nutrient limitations without extensive medium exchange. Due
to the high specific oxygen demand of High Five cells, the growth of this cell line up
to high cell densities could be limited by the transfer capabilities of current animal cell
bioreactor systems. An additional increase in the oxygen demand must be expected
during the first 30 hours post infection.

References

1. Eyer K, Oeggerli A, and Heinzle E (1995). Biotechnol. Bioeng. 45, 54-62.
2. Taticek RA, Hammer DA, and Shuler ML (1995) in: Shuler ML, Wood HA, Granados RR, and

Hammer DA (eds.), Baculovirus expression system and biopesticides, Wiley-Liss, New York, pp.
131-174

EFFECTS OF AMMONIUM AND LACTATE DURING CONTINUOUS
HYBRIDOMA FERMENTATIONS IN A FLUIDIZED-BED BIOREACTOR

H.Heine, M. Spies, M. Biselli, C. Wandrey
Institut für Biotechnologie 2,
Forschungszentrum Jülich GmbH, 52425 Jülich, Germany

Introduction

Cell immobilization in porous microcarriers in a fluidized-bed bioreactor is an efficient
method to reach very high cell densities in continuous fermentations [1]. High
medium fluxes have to be used to prevent cells from being limited by nutrient
exhaustion. Accumulation of inhibitory compounds like ammonium or lactate [2]
limits the degree of supplementation of the culture media.
The aim of this work is to compare kinetic effects of ammonium and lactate in
different fermentation systems. Hybridoma cells cultured in a fluidized-bed bioreactor
using porous glass carriers have been compared with suspension batch culture in
spinner vessels. It turned out that significant effects of three dimensional environment

in porous carriers occur.

Material and Methods

The cell line used is a mouse-mouse hy-
bridoma secreting a monoclonal anti-
body of the subclass Suspension
cultures were carried out in batch
spinner flasks. The immobilized culti-
vations were performed in a fluidized-
bed bioreactor (Fig. 1) with 60 ml carrier
and 250 ml total volume. The carriers

have a size
of 400-710 µm and are coated with ge-
latine [3]. The perfusion rate was adjust-
ed to after startup phase. Suspended cells were counted using a hemacytometer
with Erythrosin B-staining, immobilized cells were lysed using a 100 mM citric acid
buffer, with subsequently staining of nuclei with crystal voilet. The culture medium
consists of a 3:1 mixture of DMEM and Ham´s F-12, supplemented with insulin,
amino acids, transferrin, BSA, fatty acids, vitamins and trace elements (all Boehringer
Ingelheim, Germany). For the ammonium experiments was added to final
concentrations of 2, 4, 6, 8, 10, 12, 14 mmol/1. For the lactate experiments Na-lactate
was added to a final concentration of 2, 4, 8, 12, 16, 20, 25 mmol/1.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

282
Results and Discussion
ADDITION OF AMMONIUM:
In continuous immobilized culture addition of ammonium results in a reduction of
immobilized cell density whereas suspended cell density and antibody titer remain
constant (data not shown). This indicates an increasing growth rate due to more space
on the carrier (Fig. 2a). Though the growth rate increases in immobilized culture the
cell cycle distribution remains nearly constant at every ammonium concentration. But
there is a significant difference between suspended cells and immobilized cells in the
fluidized bed bioreactor. Indicating a faster proliferation, the greater part of the
immobilized cells are in the S-phase compared to the suspended cells. The specific
antibody production rate is coupled to growth (Fig. 2a). The metabolic rates increase
by factor six (data not shown). Suspended cells in batch culture show a different

response to ammonium addition (Fig. 2b), their growth rate remains nearly constant
whereas the specific productivity slightly decreases. Nevertheless the growth rate in
immobilized culture is lower than in suspension culture and only at the highest
ammonium concentrations growth rate is higher than in batch culture. The specific
productivity of immobilized cells in continuous culture is much higher than in batch
suspension culture.
ADDITION OF LACTATE
The addition of lactate has no influence on immobilized cell density, suspended cell
density and antibody titer. The growth rate and specific productivity remain more or
less constant (Fig. 3a). Due to the lower immobilized cell density in the continuous
experiment compared to the ammonium experiment the initial rates are higher
compared to Fig. 2a. In suspended spinner culture there is also no effect on the growth
rate and the specific productivity (Fig. 3b).

283

Summary
Our experiments show an influence of the three dimensional environment of the cells
during the addition of ammonium. Ammonium leads to decreasing immobilized cell
densities and increasing metabolic rates in porous carriers. In suspended batch culture
growth rate remains constant whereas cell specific productivity decreases slightly. The
addition of lactate shows no effect of the three dimensional environment. In both cases
the cell cycle distribution shows differences between immobilized cells and suspended
cells in the fluidized bed bioreactor.
References
[1] Thömmes, J. et al:The influence of dissolved oxygen tension on the metabolic
activity of an immobilized hybridoma population, Cytotechnology 13 (1993), 29-39.
[2] Miller, W.M., Wilke, C.R., Blanch, H.W.: Transient responses of hybridoma cells
to lactate and ammonia pulse and step changes in continuous culture, Bioprocess
Engeneering, 3 (1994), 113-122
[3] Lüllau, E. et al: Immobilization of animal cells on chemically modified carrier, in:
Spier, Griffith (eds.): Animal cell technology, Butterworth-Heinemann,(1992), 469-
475
Acknowledgement
The authors thank the European Commission Directorat General XII for the
sponsoring of this work.

SERUM CONCENTRATION AND pH AFFECT THE CD13 RECEPTOR
CONTENT OF HL60 CELLS CULTURED IN STIRRED BIOREACTORS

C. L. MCDOWELL AND E. T. PAPOUTSAKIS
Dept. of Chemical Engineering, Northwestern University
2145 Sheridan Road, Evanston, IL 60208-3120, USA

Keywords: antigen expression, agitation, pH, serum concentration

Abstract
The effects of serum medium concentration and culture pH on CD 13 receptor content
and mRNA levels of HL60 (human promyelocytic leukemia) cells were examined using
flow cytometry and Northern blotting. Increasing the serum concentration from 5% to
10% increased the CD 13 receptor content and mRNA levels of HL60 cells cultured at
80 rpm in a 2 L bioreactor. When the agitation rate was increased to 300 rpm, 10% FBS

increased the HL60 apparent growth rate, but decreased the CD 13 receptor content and

mRNA levels. Decreasing the culture pH from 7.4 to 7.2 (at 80 rpm) did not affect the
HL60 apparent growth rate, but increased the CD 13 receptor content. Unlike the results

of the serum experiment, changes in CD 13 content in response to culture pH were not
correlated with changes in CD13 mRNA levels.

Introduction
Large-scale culture of freely suspended cells is necessary for the production of whole
cells for use in somatic therapies. Industry is now making use of agitated bioreactors, as
they offer uniform culture conditions and the ability for growth to high cell densities.
However, the high agitation rates and/or sparging often needed to satisfy the oxygen
demand of high cell concentration cultures generate fluid-mechanical forces that affect
cell physiology and/or injure cells. It is important to understand not only how
hydrodynamic forces due to agitation affect cells, but also how other bioreactor
parameters, such as serum medium concentration and pH, affect cell physiology. Of
particular interest is how bioreactor parameters affect receptor concentration on the cell
surface, which has implications in the production of whole cells for somatic therapies.
Somatic therapies involve interaction, through their surface receptors, of the transferred
cells with other cells in the human body. Therefore, cells used in somatic therapies must
be cultured in a manner that maintains the expression of key surface receptors. In
addition to the requirement of intact and functional surface receptors, cells used in
somatic therapies must be cultivated in numbers sufficient for treatment
(7,9). Cultivation in agitated bioreactors could produce the cell numbers sufficient for
treatment, but parameters such as agitation rate, serum medium concentration, and pH
could affect the expression of key surface receptors. Therefore, it is necessary to
understand how agitation, serum medium concentration and pH affect cellular receptors.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

286
This work examines the effects of serum medium concentration, agitation rate, and pH
on the surface content of the CD 13 receptor (aminopeptidase N) of HL60 (human
promyelocytic leukemia) cells.

Materials and Methods
CELLS AND CULTURE MEDIUM
HL60 cells (cell line ATCC CCL240) were cultured in IMDM supplemented with 5% or
10% fetal bovine serum (FBS), sodium bicarbonate, and sodium pyruvate.
BIOREACTOR CULTURES
Experiments were carried out in two Setric Genie 2 L bioreactors (Toulouse, France),
with an initial volume of 1.35 L. Temperature was maintained at 37°C. Dissolved
oxygen concentration was maintained above 70% of air saturation. The pH was
maintained at 7.4 or 7.2 by injection into the headspace. In the pH experiment, the
cultures were grown at 80 rpm. In the FBS experiment, once the cultures reached mid-
exponential growth, the agitation rate of both was increased to 300 rpm. Every 8-12
hours, samples were taken to determine cell concentrations, viability, CD13 receptor
content, and CD 13 mRNA levels.
FLOW CYTOMETRIC ANALYSIS OF SURFACE ANTIGENS
Samples from each experiment were analyzed in the same session using a Becton
Dickinson FACSscan flow cytometer. Ten thousand cells were analyzed per sample.
NORTHERN BLOT ANALYSIS
Total RNA was purified using RNA STAT-60 (Tel-Test Inc., Friendswood, TX, USA).
Northern blots and autoradiography were carried out as described previously (8). The
amount of RNA was controlled by rehybridization of the blots with the glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) probe. Probes were labeled with
using random hexamer primers. CD13 cDNA was provided by Dr. A. Thomas Look
(St. Jude's Children's Research Hospital, Memphis, TN, USA). GAPDH cDNA was
received from ATCC.

Results
EFFECT OF 5% VERSUS 10% FBS AT 80 AND 300 RPM
As shown in Figure 1a, the cell concentrations of the 5% and 10% FBS cultures were
similar before the agitation rate increase to 300 rpm. After the agitation rate increase to
300 rpm, however, the 10% FBS culture had a cell concentration higher than the 5%
FBS culture. The viability of both cultures remained above 90% for the duration of the
experiment (data not shown). Prior to the agitation rate increase, the 10% FBS culture
had a higher CD 13 receptor content as compared to the 5% FBS culture (Figure 1b).
After the agitation rate increase to 300 rpm, however, the CD 13 content of the 10% FBS
culture continued to decline, while the decline in the 5% FBS culture was arrested. The

287
differences between the 5% and 10% FBS cultures in terms of CD 13 content correlated
with differences in CD 13 mRNA levels (Figure 1c).
EFFECT OF CULTURE pH 7.0 VERSUS 7.4 AT 80 RPM
Decreasing the culture pH from 7.4 to 7.2 had no affect on the HL60 apparent growth
rate (Figure 2a). It also had no effect on cell viability, as the viability of both cultures
remained at or above 90% (data not shown). However, decreasing the pH from 7.4 to
7.2 increased the CD13 receptor content, on average, by approximately 25% (Figure
2b). Unlike the FBS experiment, despite the differences in CD13 receptor surface
content there were no differences in CD13 mRNA levels (Figure 2c).

Discussion
THE PROTECTIVE EFFECT OF FBS AT 300 RPM
FBS protected HL60 cells from damage under conditions of agitation at 300 rpm. As
shown in Figure la, the 10% FBS culture had a higher cell concentration as compared to
the 5% FBS culture. These results are consistent with experiments in the literature,
which showed that serum protected cells cultured in agitated bioreactors (2, 3, 6).
FBS DECREASES CD 13 RECEPTOR CONTENT AT 300 RPM
At 300 rpm, 10% FBS leads to a decrease in CD 13 receptor content. As shown in
Figure 1b, the 10% FBS culture exhibited a continued decrease in CD 13 content at 300
rpm, while the 5% FBS culture exhibited an arrest in the decline of CD 13 content. The
behavior of the 10% FBS culture is consistent with experiments that showed that HL60
cells cultured in 10% FBS exhibited a decrease in CD 13 content in response to agitation
at 270 rpm, as compared to control cultures at 80 rpm (4). The behavior of the 5% FBS
culture is consistent with previous experiments demonstrating that HL60 cells cultured
in 5% FBS exhibited an arrest in the decline of CD13 content in response to agitation at
300 rpm, as compared to control cultures at 80 rpm (5). These results show that at 300
rpm, 10% FBS reduced the transduction of fluid-mechanical forces that affect CD 13
receptor content. In contrast, the 5% FBS culture sensed the fluid-mechanical forces
due to agitation and responded by arresting the decline in CD 13 receptor content.
FBS INCREASES CD 13 RECEPTOR CONTENT AND mRNA LEVELS AT 80 RPM
As shown in Figure 1b, the 10% FBS culture had a higher CD13 receptor content at 80
rpm as compared to the 5% FBS culture. This observation is consistent with studies
that showed that cytotoxic T lymphocytes cultured in serum had a higher CD38 and
CD57 receptor content as compared to cells cultured in the absence of serum (10).

As shown in Figure 1c, the higher level of CD 13 receptor content in the 10%
FBS culture was correlated with higher levels of CD 13 mRNA. This higher level of
CD 13 mRNA could be due to either an increase in the rate of CD 13 mRNA
transcription, or a reduction in the rate of CD13 mRNA degradation. These results have
implications for the culture of human hematopoietic cells with animal sera, or
autologous serum or plasma in stirred tank reactors.

288

289

290
pH AFFECTS CD 13 RECEPTOR CONTENT BUT NOT CD 13 mRNA LEVELS
Decreasing the culture pH from 7.4 to 7.2 increased the CD13 receptor content by
approximately 25% (Figure 2b). This behavior is consistent with experiments that
showed that a decrease in pH from 7.4 to 7.0 increased the atrial natriuretic peptide
receptor content on bovine aortic endothelial cells by approximately 100% (1).

In contrast to the FBS experiment, higher CD13 mRNA levels in the pH 7.2
culture were not correlated with higher CD 13 mRNA levels (Figure 2c). Since culture
pH affected the CD 13 receptor content, but not mRNA levels, another receptor
processing step must be affected. Culture pH could affect CD 13 receptor protein
synthesis, trafficking to the cell membrane, internalization, degradation, or recycling.

Acknowledgments
This work was supported by a National Science Foundation Graduate Fellowship
awarded to C.L.M. and a Predoctoral Biotechnology Training Grant (NIH GM08449).

References
1. Katafuchi, T., Hagiwara, H., Ito, T. and Hirose, S. (1993) A Dramatic pH Dependent

Alteration in ANP Receptor Density: a Note for Using Cultured Cells, American
Journal of Physiology 264, C1345-C1349.
2. Kunas, K. T., and Papoutsakis, E. T. (1989) Increasing Serum Concentrations
Decrease Cell Death and Allow Growth of Hybridoma Cells at Higher Agitation
Rates, Biotechnology Letters 11,525-530.
3. Kunas, K. T., and Papoutsakis, E. T. (1990) The Protective Effect of Serum Against
Hydrodynamic Damage of Hybridoma Cells in Agitated and Surface-Aerated
Bioreactors, Journal of Biotechnology 15, 57-70.
4. Lakhotia, S., Bauer, K. D., and Papoutsakis, E. T. (1993) Fluid-Mechanical Forces in
Agitated Bioreactors Reduce the CD13 and CD33 Surface Protein Content of HL60
Cells, Biotechnology and Bioengineering 41, 868-877.
5. McDowell, C. L., and Papoutsakis, E. T. (1997) Increased Agitation Intensity
Increases CD 13 Receptor Surface Content and mRNA Levels, and Alters the
Metabolism of HL60 Cells Cultured in Stirred Tank Bioreactors (submitted 1997).
6. Michaels, J. D., Petersen, J. F., Mclntire, L. V., and Papoutsakis, E. T. (1991)
Protection Mechanisms of Freely Suspended Animal Cells (CRL 8018) from Fluid-
Mechanical Injury. Viscometric and Bioreactor Studies Using Serum, Pluronic F68
and Polyethylene Glycol, Biotechnology and Bioengineering 38, 169-180.
7. Rosenberg, S. A., Anderson, W. F., Blaese, M., Hwu, P., Yanelli, J. R., Yang, J. C.,
Topalian, S. L., Schwartzentruber, D. J., Weber, J.S., Ettinghausen, S. E., Parkinson,
D. N., and White, D. E. (1993) The Development of Gene Therapy for the Treatment
of Cancer, Annals of Surgery 218, 455-464.
8. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY.
9. Scheding, S., Franke, H., Brugger, W., Kanz, L. and Schmitz, S. (1995) How Many
Myeloid Post-Progenitor Cells Have to be Transplanted to Completely Abrogate
Neutropenia After High-Dose Chemotherapy and Peripheral Blood Progenitor Cell
Transplantation? Blood 86, 224a.
10.Trimble, L., Perales, M., Knazek, R., and Lieberman, J. (1996) Serum Enhances the
Ex Vivo Generation of HIV-Specific Cytotoxic T Cells, Biotechnology and
Bioengineering 50,521-528.

Discussion 291
Wurm:
Papoutsakis: Have you looked at the protease activity in the supernatant under
Wurm: those various conditions?
Papoutsakis:
No.
Barteling:
Papoutsakis: It might give an explanation for your various levels. If you reduce
Bernard: serum, you might increase hydrodynamic damage and release more
Papoutsakis: proteases, which would affect protein on the cell.

Al- Rubeai: We have not studied this but did show that increased damage
increased the receptor level, which goes against the idea of
Papoutsakis:
proteolytic attack.

Could the serum effect be due to a reduction in shear which you
could mimic by the addition of pluronic?

The effect of serum is nothing to do with shear, but is complicated
by the presence of fluid mechanical forces.

The signal which you receive is by facs, I presume. Did you
calibrate this for actual C13 by another method?

Yes, you can do an enzyme assay. There is a complication in that
the culture environment might be changing in glycosylation and,
therefore, the recognition of a particular receptor. We have been
looking for a suitable receptor to look at the effect of glycosylation
in the culture medium but it is practically impossible. The only one
which comes close is CD34, but this is very complicated.

When you look at changes in cell size, do you think that there is a
possibility that the concentration of CD 13 has not changed but the
amount has?

No, if you normalise it by cell size, you see the same thing.

FOUR REGULATION-FRIENDLY SERUM-FREE MEDIA FOR MAMMALIAN
AND INSECT CELLS

Thomas W. Irish, Susan E. Lenk, Lisa A. Bugner, and Karen J. Etchberger
JRH Biosciences, Inc., 13804 West 107th Street, Lenexa, KS 66215 USA

Abstract

Mammalian and insect cell lines are used commercially to produce products for
both the human and animal health markets. Due to regulatory issues surrounding
the use of animal-derived raw materials in culture media, JRH Biosciences has
developed four regulation-friendly media formulated using raw materials that are
recombinant, synthetic or non-animal-derived. Cells can be transferred directly
from serum-containing cultures into these media without extensive adaptation or
extended lag phases. EX-CELL™Vero SF supports the growth of Vero cells in
stationary flasks as well as on microcarriers. EX-CELL™293-S supports the growth
of 293 cells as single cells in suspension cultures. EX-CELL™302 was designed
for the culture of CHO cells in suspension systems. In each of these media, cells
achieve doubling times and final cell densities comparable to 10% FBS controls.
EX-CELL™420 was optimized for growth of Sf9 and Sf21 insect cell lines,
supporting cell densities of cells/ml (viability >95%) for more than 10 days.

Materials and Methods

EX-CELL™302: CHO-K1 cells (ATCC CCL 61) previously adapted to
EX-CELL™301 were transferred to 250 mL spinner flasks with a seeding density

of cells/mL in 100 mL of EX-CELL™302 (~60 rpm). Daily cell counts and
viabilities were determined by trypan blue exclusion.

EX-CELL™420: Sf9 cells (ATCC CRL1711) were transferred directly into

EX-CELL™420 as attachment cultures from an ampule of ATCC supplied cells.

Cells grown in the serum free control medium were adapted from attachment

cultures grown in Hink's TNM-FH + 10% FBS. Cells in both serum-free media were

adapted to suspension culture in 125 mL shaker flasks at 130 rpm. For growth

curves, cells were seeded at either cells/mL or cells/mL in 50 mL

volume of media. Daily cell counts and viabilities were determined by trypan blue

exclusion. Virus production and activity assays were performed

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O.-W. Merten et at. (eds.). New Developments and New Applications in Animal Cell Technology, 293-302.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

294

by Invitrogen (Carlsbad, CA). Sf9 cells grown in EX-CELL™420 and Grace's + 10%
FBS were infected with a pVL941 CAT clone HTS at an MOI of 5. Viral liters were
determined by plaque assays were performed on the culture supernatants.

production by Sf9 cells grown in EX-CELL™420 and Grace's +
10% FBS was determined by spectrophotometric activity assays.

EX-CELL™293-S: 293 cells (ATCC 1573) were weaned to EX-CELL™293-S from

cultures grown in MEM + 10% DBCS. For adaptation to serum-free medium, the

cells were passaged as attachment cultures into EX-CELL™293-S + 1% FBS for

one passage followed by two passages in EX-CELL™293-S. Cell dissociation was

accomplished with trypsin followed by protease neutralization with ultrapure

soybean trypsin inhibitor (0.1 %). Cells were transferred to roller bottles and spinner

flasks at a density of Cell counts and viabilities were determined

by trypan blue exclusion.

EX-CELL™VERO-SF: Vero (ATCC CCL-81) and MDCK (CCL 34) cells were

adapted directly to EX-CELL™VERO-SF from cells grown in MEM + 10% FBS.

Serum-free attachment cultures were seeded at Cell dissociation

was accomplished with trypsin followed by protease neutralization with ultrapure

soybean trypsin inhibitor (0.1%). For growth curves, cells were counted at culture

days three though seven. Cell counts and viabilities were determined by trypan

blue exclusion.

EX- CELL™ 302

Data presented here demonstrates extended cellular growth in JRH Biosciences

EX-CELL™302 for nine (9) days in spinner batch cultures. Cultures of CHO-K1

cells achieved an approximate average maximum cell density of cells/mL

with exponential doubling times in the range of 20 hrs., maintaining viabilities

greater then 70%. EX-CELL™302 has been formulated without using animal

derived proteins. EX-CELL™302 does contain very low levels (> 50 ug/L total) of

recombinant proteins having a molecular weight less the 10 KD. CHO-K1 cells,

cryopreserved and adapted to serum free growth in JRH Bioscience’s EX-

CELL™301, were transferred directly into EX-CELL™302 without an adaptation

period. JRH Bioscience's "regulatory friendly" EX-CELL™302 can be safely used

for production of human therapeutical products because it does not contain animal

derived proteins, amino acids, or other components considered to be questionable.

295

296

Conclusions

EX-CELL™302 can achieve cellular densities in suspension batch cultures

greater then cell/mL.

• EX-CELL™302 can support batch cultures maintaining high viabilities for
greater then 9 days without a refeed.

• EX-CELL™302 does not contain any animal derived raw materials
considered to be regulatory questionable.

• Cells grown in serum free media can be directly transferred into EX-
CELL™302 without adaptation .

EX-CELL™420

Insect cells are commonly used for the expression of recombinant proteins and

virus production in a variety of applications including vaccines, diagnostics, and

biopesticides. Cells derived from the fall armyworm, Spodoptera frugiperda, have

been the cells of choice for baculovirus infection and protein production. EX-

CELL™420 is a complete medium developed and optimized for the serum-free

growth of Sf9 and Sf21 insect cell lines as either adherent or suspension cultures.

This medium contains no proteins detectable by standard methods. Cells can be

transferred directly from adherent cultures grown in serum-free or serum-containing

media into EX-CELL™420 suspension culture without an adaptation period. Sf9

cells cultured in EX-CELL™420 routinely achieve cell densities of cells/mL

with viabilities greater than 95% and can be maintained for more than 10 days at

these densities with no loss of viability. Cultures seeded at low density

cells/mL) over multiple passages do not exhibit the typical 24-48 hour lag phase

seen with many serum-free media. Sf9 and Sf21 cultures have been carried in EX-

CELL™420 for more than 20 passages. Sf9 cells grown in EX-CELL™420 were

infected with a pVL941 CAT clone HTS at an MOI of 5 and the resulting viral titer

at 72 h was pfu/mL, approximately half a log less than the 1.3 x 10

pfu/mL titer obtained from cells grown in Grace's + 10% FBS. Expression of IL-6,

CAT, and by Sf9 cells grown in EX-CELL™420 is comparable to

that obtained from cells grown in Grace's + 10% FBS.

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298

299

Conclusions

• Cells can be transferred directly to EX-CELL™420 from a serum-
containing medium without an adaptation period.

• Cells grown in EX-CELL™420 routinely achieve cell densities of
cells/mL with viabilities of >95%.

• Cultures can be maintained for more than 10 days with no loss of viability.

• Cultures seeded at low density do not exhibit the

typical 24-48 hour lag phase seen with many serum-free media.

• Virus production and protein expression in EX-CELL™420 is comparable
to that obtained in serum-containing media.

EX-CELL™Vero-SF

JRH Bioscience's EX-CELL™Vero-SF media has been formulated to support the
long term growth of the Vero cell line. This medium has demonstrated the ability
to grow both Vero cells and MDCK cells both of which are commonly used by the
human pharmaceutical industry to produce human and animal vaccines and protein
produces. Because this media is free of animal derived raw materials it is
considered to be regulatory friendly. Vero cells grown in EX-CELL™Vero-SF
media achieve culture densities comparable to densities achieved in a serum
containing media. MDCK cells grown in EX-CELL™Vero-SF media achieve
densities and growth rates greater then those achieved when grown in a media
containing serum. Both cells lines can be directly transferred into EX-CELL™Vero-
SF media from serum conditions without adaptation or lose of growth rates or
densities.

300

301

Conclusions

• Vero and MDCK cells can be grown in EX-CELL™Vero to densities
comparable to or higher then those achieved in a serum containing media.

• EX-CELL™Vero media does not contain any animal derived raw materials
making this media regulatory friendly.

• Both Vero and MDCK can be directly transferred into EX-CELL™Vero
media without adaptation.

• Population doubling are sustained over extended periods of time.

EX-CELL™293-S

The 293 cell line used for production of proteins, and adeno virus can be grown in
JRH Biosciences EX-CELL™293-S as a suspension culture. This media does
contain BSA as a protectant for cellular growth in spinner cultures and very low
levels of recombinant growth factors. Cells can be quickly adapted to serum free
growth in stationary culture and then transferred into suspension culture conditions.
Adaption of 293 cells acquired from ATCC requires three separate steps,
adaptation to serum free growth in static cultures, adaptation to suspension in roller
bottles and adaptation to spinner growth. 293 cells grown in JRH Biosciences EX-
CELL™293-S grow as single cells or small, loose, aggregates that can be easily
broken up by gentle pipetting. Doubling times and cellular densities achieve in
serum free growth are comparable to those achieved in a serum containing media.

302

Conclusions
• Cells can be transferred from serum containing media into low serum

conditions within two subpassages.
• 293 cells in low serum conditions (1% DBCS) will form loose cell

aggregates and grow as suspension cultures.
• Adaptation to serum free conditions require an additional one to two

subpassages for sustainable growth.
• 293 cells grown in EX-CELL™293-S will achieve culture densities of 1 x

in roller bottles 5 -7 days from a seed of

PROTEOLYTIC ACTIVITIES IN THE BACULOVIRUS-INSECT CELL
EXPRESSION SYSTEM

Georg Schmid and Andrea Bischoff
F. Hoffmann-La Roche Ltd., Pharmaceutical Research
Building 66/112A, CH-4070 Basel, Switzerland

Introduction

Proteolytic degradation of recombinant proteins produced in the baculovirus-insect cell
expression system is an often encountered phenomenon. The system encompasses
proteases of viral and cellular origin At least one viral proteinase (v-cath), a cathepsin L-
like cysteine proteinase, has been identified to date. The activities of cellular proteases
are often regulated by the cells' metabolic state and oxidative stress, heat shock or
amino acid (substrate) starvation induce formation of various stress proteins, including
proteases, that may have a significant impact on product quality in any given bioprocess.
We investigated the occurence of aspartic, metallo-, serine and cysteine proteinases in
non-infected and infected Spodoptera frugiperda (Sf9) insect cells using chromogenic
substrates, zymogram gels and SDS-PAGE analysis of degradation products of model
proteins. The secreted and intracellular activities were evaluated for spinner flask, lab-
and pilot-scale bioreactor cultures operated under different physiological conditions.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

304

305

306

MONITORING
AND

CONTROL

DEVELOPMENT OF PROTEINASE ASSAYS FOR IMPROVED CHO CELL
CULTURES

C. TANS, C. VANDER MAELEN, S. WATTIAUX-DE CONINCK*,
M.-M. GONZE AND L. FABRY
SmithKline Beecham Biologicals S.A., Rixensart - *F.U.N.D.P., Namur,
Belgium

1. Abstract
Proteolytic activity in CHO cell culture supernatants is a key parameter to be considered
towards an improved quality of secreted recombinant glycoproteins. In the present study,
synthetic substrates have been used to evaluate the proteolytic activity in parental and
recombinant CHO cell culture supernatants. Zymographic and isoelectric focusing
analysis showed that modifications in the peptidic sequence of a model secreted
glycoprotein are related to a protease activity detected in supernatants using the Z-Gly-
Gly-Arg-AMC substrate. Protease measured by the substrate was suggested to be a form
of plasminogen activator as its activity was inhibited by plasminogen activator inhibitor
type 1 and its optimal pH was in the range of pH 8.5.
Cell cultures performed at lower temperature, eg 29°C instead of 37°C resulted in a
decrease of the protease activity in supernatants as assayed using the Z-Gly-Gly-Arg-
AMC substrate (3 fold decrease). Consequently, lower cell culture temperature is
expected to be a key factor to improve the quality of recombinant secreted glycoproteins
in CHO cell cultures.

2. Introduction

The objective of the study was to evaluate the influence of CHO cell derived proteases
on the integrity of secreted glycoproteins.
The first step consisted of the characterization of proteases activity in CHO cell
supernatants able to modify a model recombinant glycoprotein (rec. glycoprotein):
potential synthetic substrate, inhibition profile, molecular weight.
During the second step, changes in the culture conditions and their effects on the protease
activity and rec. glycoprotein quality were evaluated.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

310

3. Material and methods

3.1. CHO CELL CULTURE

All cultures were carried out in serum-free medium. Parental (untransfected) CHO cell-
free supernatants were obtained from suspension batch culture after 72 hours culture at
37°C. Suspension batch culture of rec. CHO cells were performed at different
temperatures in spinner flask 1L.

3.2. INCUBATION OF THE PURIFIED GLYCOPROTEIN IN PARENTAL CHO
CELL CULTURE SUPERNATANTS

The purified rec. glycoprotein sample (4 % of N-terminal modifications) was incubated
in the presence of parental CHO cell culture supernatants (5 fold concentrated by
ultrafiltration with a cut-off limit of 10 kD) and 0.01 % merthiolate.
Prior to incubation, pH was adjusted to pH 7.2 with 5N HC1 or 5N NaOH.
Incubation time was 44 hours at 37°C.

3.3. ISOELECTRIC FOCUSING/WESTERN BLOTTING (IEF/WB),
IMMUNODETECTION TECHNIQUES

The glycoprotein samples were concentrated 15 fold by ultrafiltration (10kD cut off)
prior to diafiltration and IEF/WB. Gel electrophoresis was performed under denaturing
conditions as reported by O’Farrel (1975) and Lognonné (1994) in a vertical slab gel
unit; a pH gradient between 3 and 6 was used; migration was stopped after 30 000
Vxhours. After several washes in a fixing solution, gels were blotted to PVDF
membranes in the presence of SDS using a semidry transblot system. The blot was
incubated with a polyclonal rabbit anti recombinant glycoprotein antibody and then with
an anti-rabbit horseradish peroxidase conjugated antibody; the blot was developed with
3.3' diaminobenzamidine.
The IEF/WB were analysed with a densitometer/image analyser (Bio-Rad).

3.4. ZYMOGRAPHIC ANALYSIS OF PROTEOLYIC ACTIVITY IN CULTURE
SUPERNATANT

Supernatants samples migration was realised in a 7.5 % acrylamid gel polymerized in
presence of 0.2 % purified rec. glycoprotein at 4°C. After washes, gel was incubated at
37°C during 20 hours in 0.1 Tris-HCl buffer pH 8.5 and then stained with Coomassie
blue (Heussen and Dowdle, 1980).

3.5. ZYMOGRAPHIC DETECTION OF PROTEASE ACTIVITY AGAINST
SUBSTRATE Z-GLY-GLY-ARG-AMC

Supernatants samples migration was realised in a 7.5 % acrylamid gel at 4°C. After gel
washes, a membrane impregnated with Z-Gly-Gly-Arg-AMC was overlayed on the gel,

311
gel and membrane were incubated 3 hours at 37°C. Fluorescence picture was obtained
using Bio-Rad Glyco Doc Analytical Software.

3.6. PROTEASE ACTIVITY MEASUREMENTS

200 µl of CHO cell culture supernatants were incubated at 37°C in presence of 0.25 mM
synthetic substrate, 0.1 M buffer (Glycine - Tris - Acetate); the reaction is stopped after
2 hours.

4. Results and discussion

4.1. PROTEOLYTIC ACTIVITY IN PARENTAL CHO CELL CULTURE
SUPERNATANTS

Four synthetic substrates containing a basic residue at the cleavage site have been used to
detect the protease responsible for the N-terminal (N-term.) modifications of the model
rec. glycoprotein. Maximal activity was detected at basic pH for all synthetic substrates.
The results show that parental CHO cell culture supernatant contain proteolytic activity
which can be easily measured using fluorescent synthetic substrates.

312

4.2. DETERMINATION OF INHIBITION CHARACTERISTICS OF THE
PROTEASE RESPONSIBLE FOR N-TERM. MODIFICATIONS OF THE REC.
GLYCOPROTEIN

IEF/WB technique was used to analyse rec. glycoprotein isoforms and to evaluate the
extent of rec. glycoprotein profile changes.
Incubation of the rec. glycoprotein sample with CHO cell-free culture supernatant
resulted in the appearance of five bands 1, 2, 2’, 3, 5 (figure 2 : positive control). When
incubation occured in fresh culture medium, the five bands 1, 2, 2’, 3, 5 did not appear
(data not shown). N-terminal sequencing of bands 1, 2, 2’, 3, 5 in the IEF/WB profile
revealed that they contained more than 70 % of N-modified forms of the rec.
glycoprotein. The proposal was to use the relative amount of the bands 1, 2, 2’, 3, 5 in
the IEF/WB profile as obtained from the densitometric analysis to assess the N-terminal
integrity of the rec. glycoprotein.
Incubation of parental CHO cell supernatant in presence of AEBSF,p-aminobenzamidine
(data not shown) or human plasminogen activator inhibitor type 1 (hPAI-1) (figure 2)
showed that the relative amount of the bands 1, 2, 2’, 3, 5 in the IEF/WB profile
decreased significantly.
These data suggest that parental CHO cell culture supernatant contains protease(s) which
can modify the glycoprotein of interest at the N-term. domain. The protease(s)
responsible for the N-terminal modifications of the recombinant glycoprotein is a serine
protease inhibited by p-aminobenzamidme and could be a form of plasminogen activator.

313
4.3. ZYMOGRAPHIC ANALYSIS OF PROTEOLYIC ACTIVITY IN

PARENTAL CHO CELL CULTURE SUPERNATANT
Zymography was used to estimate the molecular weight of the protease(s) responsible of
rec. glycoprotein modifications in parental CHO cell culture supernatant (figure 3).
Proteolytic activity against the rec. glycoprotein was observed mainly at 76 kD.

4.4. ZYMOGRAPHIC DETECTION OF PROTEASE ACTIVITY AGAINST
SUBSTRATE Z-GLY-GLY-ARG-AMC IN PARENTAL CHO CELL
CULTURE SUPERNATANT

Z-Gly-Gly-Arg-AMC is described to be a good substrate to measure plasminogen
activator activity (Zimmerman & al, 1978).
Z-Gly-Gly-Arg-AFC was used in zymography to determine if Z-Gly-Gly-Arg-AMC
could be a good substrate to evaluate the activity of the protease responsible for the rec.
glycoprotein modifications (figure 4).
Z-Gly-Gly-Arg-AMC is cleaved at the same molecular weight than the protease
responsible for modifications of the rec. glycoprotein.
Other experiments (data not shown) revealed that

- Z-Gly-Gly-Arg-AMC cleavage in parental CHO cell supernatant is inhibited in
the same way as the rec. glycoprotein modifications

- rec. glycoprotein modifications are maximal at basic pH
- same zymographic results are obtained when analysing rec. CHO cell

supernatants
Z-Gly-Gly-Arg-AMC could be a good substrate to evaluate the activity of the protease
responsible for rec. glycoprotein modifications.

314

4.5. CHANGES IN CHO CELL CULTURE CONDITIONS TO REDUCE
PROTEOLYTIC ACTIVITY

Culture temperature was modified in order to decrease proteolytic activity and increase
rec. glycoprotein quality (figure 5-6).
Batch cultures conducted at different temperatures showed that culture temperature 29°C
after 48 hours reduced significantly protease activity (about 3 times for 37-29°C
compared to 37°C).
IEF profile confirmed the protease activity results : lower temperature could improve
protein integrity, best conditions are a shift of temperature from 37°C to 33°C after 48
hours.
Batch production at lower culture temperature could improve the glycoprotein quality in
CHO cell culture supernatant.

315

316

5. Conclusions

The study demonstrates that N-term. heterogeneity of a rec. secreted glycoprotein can
result from CHO cell derived proteases in the culture supernatant. The protease
responsible for the N-term. modifications was identified as a serine protease of 76 kD
with an optimal basic pH. Inhibition of the protease can be obtained by PAI-1
suggesting that the protease could be a form of plasminogen activator.
Specific assay (Z-Gly-Gly-Arg-AMC substrate) can be used to detect the protease in
CHO cell culture supernatant.
Batch production at lower temperature could improve the glycoprotein quality in CHO
cell culture supernatant.

6. Acknowledgements

This work was supported by a F.I.R.S.T grant nr 2369 from the Walloon Region of
Belgium

7. References

Heussen, C. and Dowdle, E. B. (1980), Anal. Biochem. 102, 196-202.
Lognonné, J.-L. (1994) 2-D analysis : a practical guide to principle critical parameters, Cell. Mol. Biol. 40 (1),

41-55.
O’Farrell, P.M. (1975) High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 250 (10),

4007-4021.
Zimmerman, M., Quigley, J. P., Ashe, B., Dorn, C., Goldfarb, R. and Troll, W. (1978) Direct fluorescent assay

of urokinase and plasminogen activators of normal and malignant cells : kinetics and inhibitor profile,
Proc. Natl. Acad. Sci. USA 75 (2), 750-753.

DIGITAL IMAGE ANALYSIS: QUANTITATIVE EVALUATION OF
COLORED MICROSCOPIC IMAGES OF ANIMAL CELLS

K. Falkner and E.D. Gilles

Institut für Systemdynamik und Regelungstechnik
Universität Stuttgart, 70550 Stuttgart, FRG
E-Mail: [email protected]

Abstract

A fully automated PC-based image analysis system was developed to count and
characterize animal cells in fermentation processes in a routine and reproducible way.
Chinese Hamster Ovary cells were used for the establishment of the system. Digital
image analysis provided data for statistical evaluation and graphical representation of
the cell population. This information improved the monitoring of the investigated

processes and therefore facilitates the development of adequate process control

strategies.

1 . Introduction

Fast and accurate online determination of the number of viable cells plays an important
role in fermentation processes with eucaryotic cells. Usually, the cells are stained, and
then viable and dead cells are counted. There are different possibilities for counting
cells [1], e.g. Coulter Counter, flow cytometry and the conventional method by using
the hemocytometer. In comparison to these methods, a digital image analysis (DIA)
system has several advantages. It is easy to handle, the system is comparatively
inexpensive and the results are operator independent. Fast characterization of the cells
improves the monitoring of the investigated processes and therefore DIA is an
important tool for the culture optimization. However, DIA can do much more than
evaluation of staining methods: quantitative and reproducible determination of shape,
size and morphology provides additional information about the state of the cells. DIA is
therefor superior to the conventional manual method.

2. Material and Methods

CELL LINE, MEDIUM AND DYES. Chinese Hamster Ovary (CHO) cells were

cultivated in RPMI 1640 Medium (LIFE TECHNOLOGIES) supplemented with 5%

fetal calf serum (LIFE TECHNOLOGIES) in culture flasks with 30 ml medium

317

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 317-319.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

318

at 37 °C in the presence of 5% The cell concentration was approximately

cells/ml and after 3 days a cell passage was done. For the determination of the number

of viable cells, the three different dyes Trypan Blue, Erythrosin B and Neutral Red were

used. Before staining, 1 ml cell suspension was centrifugated for 5 min at 1500 rpm and

subsequently resuspended in 0.5 ml PBS buffer (LIFE TECHNOLOGIES). Afterwards

0.4 % (w/v) dye solution prepared in 0.81 % NaCl and 0.6 % (w/v) was mixed

with the cell suspension. Then, viable as well as non-viable cells were counted using a

hemocytometer and a chamber (300µl) constructed for use with the DIA.

IMAGE ANALYSIS. For image acquisition, a CCD color video camera (DXC-950P
Power HAD, Sony) was coupled with an inverse microscope (Axiovert 10, Zeiss) and a
PC equipped with an IMASCAN framegrabber. For image analysis the MS-Windows
software package OPTIMAS (Stemmer) was used.

3. Results and Discussion

For the fast determination of cell density and viability in fermentation processes, a fully
automated DIA system was designed. The staining of dead CHO cells worked better
with Erythrosin B than with Trypan Blue. Erythrosin B was taken up faster than Trypan
Blue and the colouring was more intensive. Neutral Red was used to confirm the results
from staining with Erythrosin B. Fig. 1 shows an original Erythrosin B stained image
and the binary images resulting from image analysis. Table 1 shows the mean values of
extracted data of a 3 days old culture of CHO cells.

For a comparison of the DIA system to the manual counting method, the data from all

experiments were pooled for further statistical analysis. Table 2 shows that the DIA
method provides better results, but still the confidence interval is only a little smaller
than the confidence interval of the manual method. The variation of DIA data is mostly
explained by the presence of cell heaps. If there are only single cells in the cell

suspension, DIA is evidently superior to the manual counting method.