12_ANIMAL CELL TECHNOLOGY_707

145

146

Discussion 147
Franek:
Schill: What are your ideas on the mechanism of how ferric citrate
influences the sialylase?
Kempken: I do not think it is ferric citrate which influences glycosylation. In
continuous culture, we chose to decrease the ferric citrate
Schill: concentration as we did not want to limit the cultures by glucose or
another energy source. It is not the effect of the ferric citrate, but
Kempken: rather of increasing the specific productivity.
Schill: In principle, you could use a very low perfusion rate and could
Butler: verify a glyco-pattern that is similar to the end of a batch culture.
Schill: You could then use a very high perfusion rate and verify a pattern
that you have in an early stage of a batch fermenter.
We established two steady-states. The higher one was close to the
exponential growth rate. To establish a steady-state you need to
allow 7 residence times to stabilise the system, and with
mammalian cells this can take months. So the restriction to doing
this is time.
I agree, but you might then find a compromise between your
required product structure and the productivity of the system.
Specific productivity is not linked to the specific growth rate in this
system.
In establishing your steady-states, do you know what your limiting
nutrient was and, if so, have you considered that the limiting
nutrient might affect the interpretation of the data?
I think ferric citrate was the limiting factor in the cultures, but it
was still at measurable levels, ie not that low. We do not know the
affinity of the cell for ferric citrate, which could be higher as
normally transferrin is used as a transport factor.

EFFECTS OF DIFFERENT PRODUCTION SYSTEMS ON GLYCOSYLATION
PATTERN OF MURINE MONOCLONAL IgA

SCHWEIKART, F.1; LÜLLAU, E.2; JONES, R.1 ; HUGHES, G.J.1
1Dept. of Medical Biochemistry at the Medical Center, Faculty of Medicine,
University of Geneva, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland;
2Biomedical Research Institute, Glaxo Wellcome Research and Development
SA, 14 chemin des Aulx, CH-1228 Plan-les-Ouates, Switzerland

Introduction

Immunoglobulin A is one of the key components in mucosal immune defence in
mammals. Monoclonal IgA was produced under
protein free conditions by a murine hybridoma
cell line (ZAC3) in a 0.5 1 hollow fibre (HFR), a

2.15 1 continuous stirred tank (CSTR) and a 2 1

fluidized bed reactor (FBR). The IgA was

directed against the LPS-antigen of vibrio
cholerea. IgA from the three different production
systems was purified by DEAE anion exchange,
hydroxyapatite and size exclusion chromato-

graphy [1]. The glycosylation pattern of the alpha

chain was examined by two different approaches

outlined in Fig. 1.

Approach 1 gives information on glycosylation at

specific sites by analysing selected peptides from

a peptide map. The heterogeneity of the

glycostructures at a given site can be examined.

In a second approach HPLC profiling of the total oligosaccharide content was performed

after PNGase F cleavage of the whole IgA and labelling of the released
oligosaccharides. This was followed by analysis of the separated, derivatized
oligosaccharides by matrix-assisted laser desorption time of flight mass (MALDI-TOF)

spectrometry. This gives quantitative information about relative amounts of certain

structures.

Murine IgA glycosylation

Mouse has 4 potential N-glycosylation sites. Only two of them were originally

found to be glycosylated [3]. One is located in domain and the other in the

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150

tailpiece of domain (329NVS). The other potential sites for N-glycosylation are

99NCS (domain ) and 314NFT (domain ). Glycosylation at a third site has been

described for an chain expressed in an another myeloma cell line[4]. Myeloma IgA's

were shown to contain no N-acetylgalactosamine and therefore no O-linked

oligosaccharides [2, 5]. The light chains contain no N-linked glycosylation sites in the

constant region.

Results

All IgA’s examined carry a large variety of different N-

linked oligosaccharide structures. The glycosylation of

the chain varies according to the IgA production

system.

IgA chain from all three bioreactors is shown to

carry N-linked oligosaccharides only at position 38NVS

(domain ) (Fig.2). The expected site in the tail

piece (329NVS, domain ) is surprisingly not

glycosylated : the corresponding tryptic peptide was

found in a nonglycosylated form in the peptide map

and it could be identified by Edman sequencing and

precise mass assignment. A MALDI-TOF mass

spectrometric analysis of the glycosylated tryptic

peptide (peak 1) is shown in Fig. 3. The spectrum

reveals a great variety of structures
all of which could be independently
confirmed by following the second
approach. Here, by achieving a
monoisotopic resolution, a more
detailed and accurate analysis of the

oligosaccharide structures could be
made. N-linked oligosaccharides

Some m/z values and the corresponding proposed structure are indicated. After
treatment of the glycopeptide with PNGase F an average mass of 4291 could be determined (not

shown); the nominal mass of the peptide moiety A small portion of the peptide

appeared with a mass difference of 16, presumably due to the oxidised in the position

37. This mass difference makes a differentiation between e.g. NeuAc~NeuGc or Hex~Fuc in the

glycosylated peptide impossible. More detailed information are only available by following the
approach 2.

151

were released and labelled with PMP (Fig.4). The UV-profiles (Fig. 5) represent the

compositions of chain oligosaccharides derived from IgA's produced in the three

bioreactor systems.

The proportion of di- and oligosialylated

structures decreased in the following order:

CSTR>FBR>HFR; the HFR derived IgA

contained almost no sialylated

oligosaccharides. The proportion of

monosialylated structures decreased in the

same order but to a lesser extent. Selected

HPLC-fractions were analysed by

MALDI-TOF mass spectrometry. The

monosaccharide compositions were deduced

from the very accurate monoisotopic mass

determination of the whole PMP-labelled
oligosaccharide. The error in mass
determination varied between 0.005 and

0.02%. By measuring mass differences after

digestion with exoglycosidases a further

confirmation of several structures could be

obtained. High mannose structures were

deduced after treatment;

sialylated structures either by direct

measurement of Cl-methylated compounds or after sialidase treatment. At least 21
different oligosaccharide structures could be deduced. A further 20 additional, less
abundant oligosaccharide structures could be detected. HFR IgA contained more of a

GlcNac bisected hybrid structure (peak 1, 2) than FBR IgA. In CSTR IgA no such

structure could be detected. IgA's from all reactor systems contained a quite high

proportion of high mannose structures (HFR peak 3,4; CSTR peak 5,6). Otherwise

mainly complex oligosaccharides of the biantennary type and only low amounts of

triantennary complex structures were found. N-Glycolylneuraminic acid was present on a

large number of oligosaccharides from CSTR derived IgA. Many of the oligosaccharide
structures were fucosylated.

The large number of different structures suggests that many of them are incompletely
processed or truncated. Although ammonium in the cell culture medium is known to be

152

an important factor influencing the sialylation level of expressed glycoproteins, in these

studies no significantly higher or longer-lasting ammonium concentration

was observed during the fermentation in the three production systems. Our work

indicates that different production systems profoundly influence the glycosylation pattern

of a glycoprotein. Although these systems were operated under conditions as similar as

possible (protein free, pH, they represent quite different environmental conditions

for the cells. Given that specific oligosaccharide structures can inhibit bacterial adhesion

at mucosal surfaces [6], the different glycosylation patterns seen with different

production systems may influence the efficacy of antigen-specific IgA when used for

therapeutic intervention [7].

References

[1] Lüllau, E., Schweikart, F., Huser, M., von Stockar, U. and Hughes, G. J., Biotech. Bioeng.,
submitted

[2] The numbering of amino acid residues was taken over from that used in the SWISS-Prot entry
P01878

[3] Robinson, E. A. and Appella, E., Proc. Natl. Acad. Sci. USA.77, 4909-4913 (1980)
[4] Taylor, A. K. and Wall, R., Mol. Cell. Biol., 8, 4197-4203 (1988)
[5] Lipniunas, P. et al., Arch. Biochem. Biophys., 300, 335-345 (1993)
[6] Boren, T., Falk, P., Roth, K. A., Larson, G. and Normark, S., Science, 262, 1892-1895 (1993)
[7] Weltzin, R., Hsu, S. A., Mittler, E. S., Georgakopoulos, K. M. and Monath, T. P., Antimicrob.

Agents Chemother., 38, 2785-2791 (1994)

Acknowledgement

This work was supported by research funds from the Swiss National Science Foundation,
SPP Biotechnology Priority Program

IDENTIFICATION OF ALTERED GLYCOSYLATION AS THE MAJOR

DIFFERENCE BETWEEN INTRACELLULARY ACCUMULATED AND

SECRETED PROTEIN PRODUCED IN BACULOVIRUS-INFECTED

INSECT CELLS.

GRYSSELL RODRIGUEZ1*, HARALD. S. CONRADT2 and VOLKER
JÄGER1.

1Cell Culture Technology Dept., 2Protein Glycosylation Dept., Gesellschaft für
Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124
Braunschweig, Germany.
* Permanent address: Centro de Inmunología Molecular, POBox 11600
Habana, Cuba.

Introduction

The baculovirus expression vector system (BEVS) has become widely used for the

production of recombinant proteins due to its ability to express large amounts of foreign

gene products inserted under the transcriptional control of the polyhedrin or p10

promoters. Another advantage is that insect cells perform most of the post-translational

modifications of proteins observed in higher eukaryotes. However, the intracellular

accumulation of substantial amounts of protein can be observed frequently for numerous

recombinant proteins. Recombinant human a glycoprotein bearing

two N-glycosylation sites, was selected as a model protein. Isolated from human
cerebrospinal fluid, serum, plasma and urine its biological significance as a

prostaglandin-D synthetase remains disputed (3). This work is focussing on the

comparison of intracellularly accumulated and secreted produced by IPLB-SF21AE
and High Five™ insect cells and the identification of the intracellular accumulation of

recombinant protein, apparently associated to the glycosylation process.

Results and Discussion

Comparison and characterization of intra- and extracellular protein expressed
in insect cell lines

SDS-PAGE analysis (Figure 1) revealed that protein from insect cells runs

slightly faster than the protein secreted by recombinant BHK21 cells or isolated from

hemofiltrate of patients. This difference in the electrophoretic mobility implies smaller

N-glycan structures than those found in protein from recombinant BHK21 cells or
hemofiltrate (1,3).

Two insect cell lines, High Five and Sf21, were infected with a recombinant baculovirus for

expression of For both cell lines the pattern of intracellular protein was characterized

by three bands at the same positions than those of the secreted protein (Figure 2).

In addition, intracellularly accumulated protein revealed a band with a higher molecular

weight of 28 kDa, which was of increasing intensity with time post infection.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

154

Another band with a molecular weight of about 24 kDa was detected in protein from Sf21
cells. In our opinion this band corresponds to an intermediate stage in the processing of the
secreted protein inside the cell, but not to an intracellularly accumulated form., because the

intensity did not alter after infection.
The extracellular protein produced by both cell lines was represented by three bands with
molecular weights of about 25.6 kDa, 22.7 kDa and 21 kDa. From 4 days post infection

onward, after the majority of cells had died due to viral infection, a smeary band appeared
near to 28 kDa. This smear was proved to become finally a clear band (data not shown).
The presence of this band in the supernatant, at the same position than the intracellular band
with 28 kDa, suggests an accumulation of the corresponding protein inside of the cell,
which is released into the supernatant only after cell lysis.

Oligosaccharide processing, limiting step in the protein secretory pathway

The intracellular accumulation of substantial amounts of recombinant protein has been
reported frequently for baculovirus-infected insect cells (2, 4, 5). A detailed carbohydrate

analysis of both intracellular and secreted IgG was carried out recently (5). In contrast to

the secreted glycoprotein, the N-glycans of intracellular IgG from insect cells included

more than 50% of high mannose type structures, indicating a significant level of incom-

plete oligosaccharide processing for a fraction of the intracellular immunoglobulins. It

was hypothesized that these intracellular immunoglobulins may not reach the subsequent

cellular compartments in which carbohydrate trimming takes place, probably due to

retention or slow secretory pathway processing of some immunoglobulins in the

endoplasmatic reticulum or Golgi apparatus of baculovirus-infected cells.

Using protein as a model we wanted to check if glycosylation was playing a crucial

role in the protein accumulation. For that purpose it was checked if glycosylation was

responsible for the differences in the SDS-PAGE patterns between intra- and extracellular

protein.

155

Both protein fractions, intra- and extracellular were digested with N-Glycosidase F enzyme.

After enzymatic digestion, the electrophoretic pattern of recombinant intra- and

extracellular protein changed dramatically. All bands except the lowest one representing the

non-glycosylated form of protein disappeared and the remaining band became more

intensive (Figure 3).

These results clearly demonstrate that the different bands from intracellular protein

correspond to different glycosylation stages of the same peptide, and provide an evidence

about a possible bottle neck in the secretion of protein, apparently due to a problem

during the glycosylation process.

Conclusions
SDS-PAGE/western blotting of protein from supernatant as well as from lysed virus-
infected insect cells (IPLB SF21 AE, BTI Tn5Bl-4) revealed the presence of an

intracellularly acumulated species, which was demonstrated to have a molecular

weight different to those observed in the supernatant.

Enzymatic digestion with N-Glycosidase F has proved that the difference between the
intra-and extracellular protein pattern was due to different glycosylation stages of the

recombinant protein.

The results suggest the presence of a bottle neck in the glycosylation pathway of

baculovirus-infected insect cells secreting protein.

Acknowledgements
We want to acknowledge the financial support from ESACT for G. Rodriguez to
participate in this conference.

References
1. Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G. and Conradt, H.S. (1995) Construction of

stable BHK-21 cells coexpressing human secretory glycoprotein and human Gal GlucNAc-R

sialyltransferase NeuAc is preferentially attached to the Gal Gluc NAc ) Man

of diantennary oligosaccharides from secreted recombinant protein. Eur. J.

Biochem. 232, 232-718.
2. Hasema, C.A and Capra, J.D. (1990) High-levels production of a funtional immunoglobulin heterodimer

in a Baculovirus Expression System. Proc. Natl. Acad. Sci. USA. 87, 3942-3946. protein in
3. Hoffmann, A.,Nimtz, M.,and Conradt, H.S. (1997) Molecular characterization of

human serum and urine: a potencial diagnostic marker for renal disease. Glycobiology 7, 499-506.
4. Hsu, T.A, Eiden, J.J., Bourgarel, P., Meo, T. and Betenbaugh, M.J. (1994) Effect of co-expressing

chaperone BIP on funtional antibody production in the baculovirus system. Protein Expr. Purif. 5, 95-603.
5. Hsu, T.A., Takahashi, N., Tsukamoto, Y., Kato, K., Shimada, I., Masuda, K., Whiteley, E.M., Fan, J.-Q.,
Lee, Y.C and Betenbaugh, M.J. (1997) Differential N-Glycans patterns of secreted and intracellular IgG

produced in Trichoplusia ni cells. J. Biol. Chem. 272, 9062-9070.

HOW AMMONIUM DOMINATES THE METABOLISM OF
IN VITRO CULTIVATED MAMMALIAN CELLS

Aziz Çayli1, Manfred Wirth2 and Roland Wagner1
1Cell Culture Technology Department, 2Department of Gene Regulation and Differentia-
tion, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124
Braunschweig, Germany

Introduction

The accumulation of ammonium ions during cultivation of mammalian cells used for the
production of recombinant biopharmaceuticals affects growth rate and productivity
(Backer et al. 1988; Butler and Spier, 1984; Glacken, 1988; Ito and McLimans, 1981;
Jensen and Liu, 1961) and the quality of the synthesized product, particularly with regard
to glycosylation pattern (Gawlitzek et al., 1995, 1997, Maiorella, 1992; Thorens and
Vasselli, 1986). Recently we have reported that the intracellular content of UDP-activated
N-acetyl hexosamines (UDPGNAc) is substantially elevated under higher ammonium
concentrations in the medium (Ryll et al, 1994). Ammonium ions are channeled into the
pathway of UDPGNAc formation at the amination step of fructose-6-phosphate (Frc6P) to
form glucosamine-6-phosphate (GlcN6P). UDPGNAc is then formed by a bifurcated
pathway whereby UTP and N-acetyl glucosamine-1-phosphate (GlcNAclP) condense to
UDPGlcNAc which subsequently is transformed to UDPGalNAc. We could show that 15N
of UDPGNAc, a precursor of carbohydrates in glycoproteins, is found in the N-glycans
(Valley, 1996) resulting in the formulation of the hypothesis, that the effects of ammonium
ions on protein glycosylation are mediated via the ammonium-induced elevation of the
UDPGNAc pool (Grammatikos et al., 1998, Ryll et al., 1994). Here, we present the
glucosamine-6-phosphate isomerase (GPI) as the key enzyme responsible for ammonium
incorporation. Moreover, we propose two strategies to inhibit its activity. Firstly, a strat-
egy based on metabolic engineering by antisense RNA expression and secondly a strategy
based on process control by the addition of an inhibitor to the culture.

Results

The enzyme. The glucosamine-6-phosphate isomerase has been identified as the key
enzyme accepting ammonium ions and elevating the intracellular UDPGNAc content. GPI

was extracted from BHK21 cells and purified to homogeneity by affinity chromatography
(Calcagno et al, 1984). The enzyme is a hexamer with a molecular weight of approxi-

mately 180 kd. Sequence analysis of the amino terminus revealed a high similarity to the

human form of the enzyme. The reaction is reversible but the equilibrium is far on the side
of Frc6P. We could show that the enzyme exclusively accepts but no glutamine.

Glutamine is used by the glucosamine-6-phosphate synthase (GPS) which catalyzes the

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

158
same reaction and which is controlled by an end product inhibition of UDPGNAc (Fig. 1).
We assume that GPI is cut out for the role to control the stationary intracellular concentra-
tions of intermediate metabolites of the UDPGNAc pathway by regulating GlcN6P in vivo
where never high ammonium concentrations are reached. However, under in vitro condi-
tions in the presence of high ammonium concentrations, this regulation system causes
serious problems on the synthesis of UDPGNAc followed by an effect on the expression
of carbohydrate structures.
The inhibitor. GPI was characterized with respect to effectors to be used as modulators in
cultivation processes of mammalian cells. Mannose-6-phosphate (Man6P) was shown to
be a strong inhibitor whereas glucose-6-phosphate, glutamine and N-acetyl-glucosamine-
6-phosphate (GlcNAc6P) act as activators. By using mannose in appropriate concentra-
tions in combination with glucose as carbon source we have shown that the formation of
higher UDPGNAc concentrations could be prevented in the presence of ammonium in the
culture medium indicating that mannose which is intracellularly transformed to Man6P is
an efficient inhibitor in bioprocesses to control N-glycan expression of glycoproteins and
glycolipids.

Antisense strategy. Due to the fact that synthesis and regulation of UDPGNAc can be
exclusively performed by the GPS alone we planned to inhibit GPI by antisense RNA
expression. Two different plasmids were constructed using human cDNA and directed to

159
the 5´- and 3´-end of the enzyme under the control of the SV40 early promotor (Table 1).
In addition a further plasmid harboring the 5´-end in sense direction with the same
promotor was used as control.

As a result the transfected cell lines showed a significant decrease in the GPI enzyme
activity (Fig. 2). Plasmid 1 and 2 revealed an enzyme expression of only 30 and 50% of
the control, respectively. Cotransfection of both plasmids, however, resulted in 90% of
repression. Only 10% of the expression found in the control cells could be detected.

160

Conclusion

Glucosamine-6-phosphate isomerase has been identified as the ammonium accepting
enzyme resulting in an elevated UDPGNAc content under high ammonium concentrations.
GPI was efficiently inhibited using two different strategies: The addition of mannose to
culture media and the genetic modification of the cells using antisense RNA expression.

References

Backer, M.P., Metzger, L.S., Slaber, P.L., Nevitt, K.L., Boder, G.B. 1988. Large Scale production of
monoclonal antibodies in suspension culture. Biotechnol. Bioeng. 32: 993-1000.

Butler, M., Spier, R.E. 1984. The effects of glutamine utilisation and ammonia production on the growth of
BHK cells in microcarrier cultures. J. Biotechnol. 1: 187-196

Calcagno, M., Campos, P.J., Mulliert, G., Suastegui, J. 1984. Purification, molecular and kinetic properties of
glucosamine-6-P isomerase (deaminase) from Escherichia coli. Biochim. Biophys. Acta. 787: 165-
173

Gawlitzek, M., Valley, U., Wagner, R. 1995. Effects of Ammonia and Glucosamine on the Glycosylation

Pattern of Recombinant Proteins Expressed from BHK-21 Cells. In: Animal Cell Technology

'Developments towards the 21st Century'; Beuvery, E.C., Griffiths, J.B., Zeijlemaker, W.P., Eds.;
Kluwer Academic Publishers: Dordrecht, The Netherlands, pp. 379-383
Gawlitzek, M., Valley, U., Wagner, R. 1997. Ammonium ion/glucosamine dependent increase of oligosac
charide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol.
Bioeng, in press
Glacken, M.W. 1988. Catabolic Control of Mammalian Cell Culture. Bio/Technology 6: 1941-1050.
Ito, M., McLimans, W.F. 1981. Ammonium Inhibition of Interferon Synthesis. Cell Biol. Int. Rep. 5: 661-666..
Jensen, E.M., Liu, O.C. 1961. Studies of Inhibitory Effect of Ammonium Ions in Several Virus-Tissue Culture
Systems. P.S.E.B.M. 107: 834-838.
Maiorella, B.L. 1992. In Vitro Management of Ammonia's Effect on Glycosylation of Cell Products Through
pH Control. US Patent, Number 5,096,816.
Ryll, T., Valley, U., Wagner, R. 1994. Biochemistry of Growth Inhibition by Ammonium Ions in Mammalian
Cells. Biotechnol. Bioeng. 44: 184-193
Thorens, B., Vassalli, P. 1986. Chloroquine and ammonium chloride prevent terminal glycosylation of
immunoglobulins in plasma cells without affecting secretion. Nature 321: 618-620.
Valley, U. 1996. UDP-N-acetyl-hexosamines as central metabolites in growth and potein glycosylation of
recombinant BHK cells. Thesis, Technical University Braunschweig.

Discussion 161
Singhvi:
Cayli: Do you have any idea of the rates of UDP molecules that go by the
two different pathways of synthetase and isomerase? Would this
Konstantinov: be reduced by inhibiting the isomerase and by how much?
Cayli:
Normally the disproportionately high level of UDPGNAC is due to
Konstantinov: the activity of isomerase because in animal cell culture we do have
Cayli: 4-5 mM ammonium. These high ammonium levels drive the
reaction to UDPGNAC. I do not have any data on the synthetase
activity. I guess if the isomerase was inhibited, the concentration
of UDPGNAC should be very low.

You mentioned that you had tried mannose in your culture
medium. Could you comment on whether this affected
glycosylation?

There are 2 steps: first, adding mannose to the culture medium and
measuring UDPGNAC; second, the analysis of the glyco-
structures. We have just measured the UDPGNAC pool in the cell
because it is much quicker. We just optimised the mannose level in
the medium and just checked the decrease of UDPGNAC in the
cells. We have no data yet on the structures of the proteins.

You need mannose for glycosylation. So I was wondering, if you
add it to the medium whether it helps glycosylation by a different
mechanism?

We have no data on the effect of pure mannose on protein
glycosylation. So what you say is possible.

STUDY OF HUMAN RECOMBINANT GM-CSF PRODUCED IN DIFFERENT
HOST SYSTEMS USING MONOCLONAL ANTIBODIES

M. ETCHEVERRIGARAY, M. OGGERO, M. BOLLATI, R. KRATJE

Instituto de Tecnología Biológica (INTEBIO). Facultad de Bioquímica y
Ciencias Biológicas. Universidad Nacional del Litoral
Ciudad Universitaria - C.C. 530 - (3000) Santa Fe. ARGENTINA

Abstract

GM-CSF is a glycoprotein that activates growth and differentiation of hemopoietic

progenitor cells, usefull to reverse or prevent chemotherapy secondary leukopenias. In spite

of the differences in the chemical structure, due to the variable glycosydic content of the

forms produced in bacteria, yeast and mammalian cells, all of them have biological activity.
Independently, antigenicity of non glycosylated recombinant human proteins may have

relevance in the choice of the host system for the production of factors for clinical use. To
study the ui)ndTeosiorabbtlaeinimthmeugnleycroesspyolantseedinhothrme roenseu,ltCs HofOthdehtfhre-rapcye,llws ewuesreedmthaenifpoulllaotwedinbgy
strategy:
genetic methods to produce human GM-CSF under the control of the adenovirus major late
promoter. ii) A panel of GM-CSF MAbs was produced from hybridoma cells obtained in
our lab. We analyzed the specificity of the MAbs taking into account their neutralizing
capacity. Some of them neutralized the in vitro biological activity of the hormone, with
different affinities for GM-CSF from several sources. Patients under treatment with the

commercial non-glycosylated GM-CSF develop antibodies and undesirable responses.

Competition experiments between the neutralizing MAbs and these human antibodies would

demonstrate the importance of the immune response in the choice of the host system for its
production.

Introduction

Granulocyte macrophage colony stimulating factor (GM-CSF) was expressed in different
systems: bacteria, yeast and mammalian cells. All of them have biological activity. Despite
the fact that the low levels that can be achieved by recombinant mammalian cells are
disappointing for the production of GM-CSF, there are important reasons to support and
improve this host system. It was observed that the therapeutical administration of non-

glycosylated or partially glycosylated GM-CSF produces activation of the immune
system [1,2] and development of undesirable responses that undoubtly cause the inefficacy
of the treatment together with many disagreeable side effects. The recognition of antibodies
specificity in patients sera under treatment with GM-CSF may be important in the choice

of the appropriate host system for the production of this hormone.

Materials and Methods
Expression vectors for CHO cells were obtained from Genargen S.R.L., Buenos Aires,
Argentina. CHO dhfr - cells were transfected by lipofection to produce human GM-CSF

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164

under the control of the adenovirus major late promoter. The vector contained the gen for
dihydrofolate reductase (dhfr) to allow the selection of stable transfectants. Among the
several cell lines achieved, the clone was selected for further experiments, taking into
account its higher productivity [3].

Non-glycosylated GM-CSF was the E. coli derived GM-CSF International Standard
(NIBSC, 88/646, UK), and the E. coli derived GM-CSF purchased from Schering-Plough
Co., Ireland, that was used as internal standard previously valorized against the
International Standard. Glycosylated GM-CSF was the CHO derived hormone prepared
in our lab.

The GM-CSF MAbs obtained in our lab are shown in Table I.

To test the biological activity of E. coli and CHO derived GM-CSF, we used the factor-
dependent cell line TF-1 (ATCC CRL-2003). The proliferation assay was optimized in our
lab for GM-CSF valoration. TF-1 cell proliferation was determined measuring
dehydrogenase-enzyme´s activity as marker for the biological activity, using a commercial
colorimetric kit (Cell Titer 96™, Promega, USA).

MAbs were evaluated for their ability to inhibit the proliferation of GM-CSF stimulated
TF-1 cells, i.e. to neutralize in vitro GM-CSF activity. For this purpose we used either six
different MAbs: ascites 2E11, 8B10, 15G5 and 32H6, and purified 1B8 and 7E10 MAbs
by protein A affinity chromatography . Serial dilutions of the antibodies were tested both
with glycosylated and non-glycosylated GM-CSF. The results were expressed as
neutralization percent.

Results and Discussion

The isotype MAbs IB8 and 7E10 were purified with 82% and 70% recovery,

respectively. The MAb 32H6 was heated at 56°C for 30 minutes. The IgM isotype

ascites were used without previous treatment.

Fig. 1 shows an example of the different behaviour of neutralizing and non-neutralizing
antibodies. The neutralizing effect of the purified MAbs 7E10 and IB8 is shown in Fig. 2.

The behaviour of MAb 7E10 may be related with heteroclytic antibodies [4], i.e. antibodies
of higher affinity with a related molecule than with the immunogen (Table II). In contrast,
the other antibodies that reacted with the glycosylated hormone correspond to cross-reactive
antibodies, being E. coli derived GM-CSF the immunogen. It must be taken into account

165

that antibodies present in patients´ sera may be of both types and play an important role in
the development of undesirable responses: loss of response in future treatments and cross-
reaction with the own hormone. The choice of host systems for the production of clinicals
has to consider not only the biological activity of the product but also the immune system,
wich plays, in this case, an important role and once it is stimulated there is no possibility
to abolish its effect.
References:

1- Wadhwa, M., Bird, C., Fargerberg, J., Gaines-Das, R., Ragnhammar, P., Mellstedt, H. and Thorpe R. (1996).
Clin. Exp. Immunol. 104:351

2- Gribben, J. G., Devereux, S., Thomas, N. S. B., Keim, M., Jones, H. M., Goldstone, A. H. and Linch, D. C.
(1990). The Lancet 335:434

3- Bollati, M., Kratje, R. and Etcheverrigaray, M. (1997). Encuentro de Investigadores Jóvenes. UBA. Argentina.
4- Mäkelä, O. (1965). J. Immunol. 95:378

METABOLIC ENGINEERING

MODIFICATION OF HYBRIDOMA CELLS METABOLISM

J.J. Cairó, C. Paredes and F. Gòdia*
Departament d'Enginyeria Química. Facultat de Ciències. Edifici C.
Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona. Spain.

E. Prats, F. Azorín, LI. Cornudella
Departament de Biologia Molecular i Cel.lular. Centre d'Investigació i
Desenvolupament. CSIC. Jordi Girona Salgado, 18-26. 08034 Barcelona.
Spain.

* To whom correspondence should be addressed.

SUMMARY

Two different genetic modifications of hybridoma cells are studied. First, cells are
transfected with the glutamine synthetase gene, and important modifications in their
physiology can be observed, that are reflected on the corresponding analysis of the
intracellular metabolic fluxes. For the modified cell line the need for glutamine feeding
in the culture has been eliminated and the ammonium production suppressed. Moreover,
the glucose uptake rate has been reduced to half with respect to the parental strain,
while maintaining a similar growth pattern. Second, antisense RNA techniques were
employed to inhibit partially the expression of two glycolytic enzymes in order to create
some rate limiting steps in the glycolytic pathway. The preliminary results obtained with
the glucose transporter and enolase genes show the potential of such approach.

INTRODUCTION.

Typical batch cultures of hybridoma cells have a number of drawbacks :
low cell concentration, low level of product expression, limited viability of the cells and
complex nutrient requirements. The design of more efficient processes for monoclonal
antibody production and the development of optimized operation strategies1 has to be
based in a much better knowledge of the cell metabolism. Many authors have

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

168
demonstrated that the metabolism of several cells can be redirected to the obtention of
desired products by using the metabolic engineering approach2.

Hybridoma cell cultures typically produce lactic acid, ammonia and some

amino acids when metabolizing the carbon and energy sources, glucose and glutamine.
The byproduct generation from its metabolism represents a poor exploitation of these
substrates and can inhibit cell growth due to lactate and ammonium accumulation3. In
order to overcome the high glutamine uptake rate, the generation of high levels of
ammonium as well as the high glycolytic flux and the lactate generation two approaches
have been performed. First approach consists in the expression of the glutamine

synthetase in a hybridoma cell line. Second approach is based on the use of antisense

RNA techniques to partially inhibit the glucose transporter and the enolase gen.
Antisense control of gene expression has become widely used method for specifically
interfering with gene function4.

MATERIALS AND METHODS

Cells : KB-26.5 murine hybridoma, producing an against antigen of red cells.

Medium : DMEM supplemented with 2% foetal calf serum.

Viable cell number : haemocytometer, viability as trypan blue exclusion.

Batch and continuous cultures : Spinner flask, 250 ml working volume, 40 rpm, 37°C,

Continuous cultures at a dilution rate of , average cell population

cell/ml. Glutamine and Glucose feeding decreasing stepwise one at a time5.

Glucose and Lactate : YSI 2700
Ammonium concentration : flow injection analysis system6.

Glutamine and other amino acids concentrations : HPLC.
Glutamine Synthetase : pCMGS.gpt7 kindly provided by Celltech. Selection in glutamine

free medium. Clone selected by serial dilution.

Antisense constructions : 450 bp fragment downstream the ATG codon of both glucose

transporter gene GLUT1 and enolase gene. Fragments were cloned in antisense

orientation into the BamHI site of plasmid pcDNA3. Selection by resistance against

Neomycin.

Transfection : lipofection method.

Stoichiometric model : As described in [8].

RESULTS A N D DISCUSSION

Hybridoma cell line KB-26.5, like all tumoral cells, can be considered as
an energetically efficient system, since is able to take energy from different sources
(glucose and glutamine), but this efficiency exists as long as cells are in their ideal
surroundings, as in body tissue. The body provides the cells with an extremely controlled

environment, in which the inefficiency observed when cells are in culture does not exist.

Glucose and glutamine concentrations in batch and fed-batch cultures are usually much

169
higher than the physiological ones found in tissues, and cells will consume tar greater
amounts of both substrates, due to the high transport and glycolysis and glutaminolysis
rates, and produce excessive amounts of lactic acid and ammonium. This point was
corroborated in continuous culture experiments performed in two different ways:
maintaining a constant glucose concentration in the feed medium while decreasing the
glutamine concentration stepwise, and maintaining glutamine concentration constant
while decreasing glucose stepwise. The results of this series of experiments are
summarized in Figure 1a and 1b. It can be observed that as the glutamine concentration
found in the medium increases (so, more excess of glutamine is provided to the cell
metabolism), higher amounts of glutamine are consumed and ammonium produced. The
same behaviour can be observed for glucose and lactate. Thus, when excessive amounts
of glutamine are added to the hybridoma culture, glutamine is not efficiently used for
cell growth, but rather to produce glutamine-related by-products such as ammonium,
alanine and proline. On the other hand, as glucose level in the exhaust medium
increases, more lactate is produced. Thus, as this cellular level was the same, it seems
that there is not a real need to consume those great amounts of glucose and glutamine
in order to keep the cell growing.

In order to overcome the high glutamine uptake rate and the generation
of high levels of ammonium, the glutamine synthetase gene has been expressed in the
KB-26.5 cell line. The physiological consequences of this expression can be seen in
Figure 2. Transfected cell line show a lower growth rate than the original one but

170
specific production rate of ammonium is zero. Similarly specific glucose uptake rate is
reduced to a half of that of the untransformed cell line, and the Lactate/Glucose molar
yield has slightly decreased.

To elucidate which are the metabolic reasons tor this physiological change
a stoichiometric model was constructed to estimate the intracellular fluxes. In brief it
consists in the derivation of a mass balance for each considered metabolite including the
transport rates across membranes and their production or consumption rates by the
intracellular reactions considered. All these data makes possible the estimation of the
intracellular fluxes by means of a least squares procedure.

The simplified results obtained in the estimation of the corresponding intracellular fluxes
are presented in Figure 3. For the non modified hybridoma cells, the glucose and
glutamine high consumption rates generate high amounts of lactate and ammonium as
it has been stated before. A possible explanation of these results could be a low
efficiency in the shuttle system for mitochondrial reoxidation of the large amounts of
NADH produced in the glycolytic pathway. As a consequence NADH should be
regenerated via lactate dehydrogenase using pyruvate as a substrate rather than through
the electronic transport chain. Thus, the pyruvate amount that can be incorporated into
TCA cycle is very small and the rapid glutaminolysis rate collapses and unbalances the
TCA cycle. Simultaneously the great amount of ammonium generated from glutamine
and other amino acids deamination overflows the cell capacity and it is excreted as tree
ammonium, alanine and proline. Moreover as alanine is produced in large amounts the
availability of pyruvate for the cell metabolism decreases (alanine is formed from
pyruvate and glutamic acid).

171

The results obtained with the transfected cell line show similar trends with
respect to NADH regeneration via lactate production. The main difference is found in
the glycolytic flux, that in the transfected cell has been reduced to half of that for the
parental strain (Figure 2). The effect of the glutamine synthetase expression on the
ammonium excretion could he explained as a result of a change in the way that the cells

use in order to eliminate the excess of ammonium ions generated from glutamate
metabolism, not requiring neither ammonium excretion nor animation of pyruvate to
alanine. Last factor could be the reason for the reduction of the glycolytic flux as a
consequence of a lower need for pyruvate in the transfected cell line. The carbon
circulation w i t h i n the TCA cycle is not collapsed by glutamic acid. On the other hand,
it can be seen from Figure 3 that the exchange between mitochondrial and cytoplasmic
malate has been inverted for the transfected cells when compared to the parental ones.

In order to overcome the high glucose uptake rate and the generation of
high levels of lactate, an antisense R N A approach has been used. It is generally
assumed that the mechanism of inhibition by nuclear-derived antisense R N A entails
sequence-specific hybridization of antisense RNA transcript to the target mRNA in the
nucleus that blocks protein synthesis4. Under optimal conditions an antisense RNA

172
approach could inhibit absolutely protein translation. Thus under suboptimal conditions
it can be used to decrease the amount of the target protein. If that protein is an enzyme
its is possible to lower the flux through the enzymatic reaction that it catalyses lowering
the amount of enzyme. In the present work this possibility has been used in order to
create a rate-limiting step in the glycolytic pathway. Two points in the glycolytic
pathway were chosen as a target; first the transporter protein for glucose (GLUT 1) and
second the enzyme enolase. This enzyme catalyses the conversion from 2-
phosphoglyceric acid to phosphoenolpyruvic acid. The physiological consequences of
this approach can he seen in Figure 4. Both cell lines show higher growth rates than the
control. This fact can he explained by the fact that integration in the genome occurs
randomly and there will he some random effect or by the fact that cells carrying
antisense construct have some advantage over non-carrying ones. Specific glucose
uptake rate has decreased about 20% for enolase construction and 45% for glucose
transporter construction. From these values it is likely to suppose that both constructions
are able to generate a rate-limiting step in the glycolytic pathway. The molar yield for
lactate/glucose remains almost unchanged showing that these approach does not affect
to the cytosolic NADH regeneration.

CONCLUSIONS

When glucose and glutamine are added in excess to the culture medium they are
consumed at high rates, with low efficiency for the cells, and generation of by-products.
The use of the glutamine synthetase gene allows the reduction of ammonia production
and unexpectedly decreases glycolysis rate to one half. Some potential decrease of the
glycolysis rate could he introduced generating a rate-limiting step by means of antisense
RNA techniques. Further work is required to corroborate these results, assess the
stability of the transformed cells and the consequences in product formation.

173

Acknowledgements

CellTech Therapeutics kindly provided the glutamine synthetase gene. This work was
supported by CICYT (project BIO94-0288) and was carried out in the framework of the
Centre de Referencia de Biotecnologia.

References

1 .Bibila, T.A., Robinson, D.K. (1995) In Pursuit of the Optimal Fed-Batch Process for Monoclonal Antibody
Production, Biotechnology Progress 11, 1-13.

2.Bailey, J.E. (1991) Toward a Science of Metabolic Engineering, Science 252,1668-1675.

3.Ozturk, S.S., Riley, M.R., Palsson, B.O. (1992) Effects of ammonia and laclate on hybridoma growth,
metabolism, and antibody production. Biotechnology and Bioengineering 39,418-431.

4.Pestka S. (1992) Antisense R N A . History and Perspective in R. Baserga and D.T. Denhardt (eds.), Annals
of the New York Academy of Sciencies, Vol 660. New York Academy of Sciencies. New York pp. 251-262.

5.Sanfeliu A., Paredes C., Cairó J . J . , Gòdia F. (1997) Analysis of Glucose and Glutamine Metabolism of
Hybridoma Cells by Continuous Culture Experiments, in M.J.T. Carrondo, B. Griffiths and J.L.P. Moreira
(eds.), Animal Cell Technology. From Vaccines to Genetic Medicine, Kluwer Academic Publishers,
Dordrecht, pp. 785-789.

6.Campmajó, C., Cairó, J.J., Sanfeliu, A., Martínez, E., Alegret, S., Gòdia. F. (1994) Determination of
ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis. Cytotechnology
14,177-182.

7.Bebbington.C.R., Rentier, G., Thompson, S., King, D., Abrams, D., Yarranton, G.T. (1990) High level
expression of a recombinant antibody from myeloma cells using a glulaminc synthetase gene as an amplifiable
selectable marker. Bio/Technology 10,169-175.

8.Sanfeliu, A. (1995) Producció d’ Anticossos Monoclonals Mitjancant el Cultiu in vitro d’ Hibridomes en
Bioreactors : Anàlisi de la Fisiologia i Metabolisme Cel.lular, Ph. D. Thesis, Universitat Autònoma de
Barcelona.

174 How do the changes in metabolism influence the specific
Discussion productivity of the antibody?
We do not have quantitative data to answer your question. We
Franck: focus on genetic modification and metabolic pathway changes,
Godia: rather than productivity.
Singhvi:
Godia: Why did you choose the two enzymes for your anti-sense targets?
Was it random or based on preliminary work?
Cayli:
Godia: The rationale was that the glucose transporter was a clear target as
it is at the beginning of all processes. We also knew, by means of
DNA probes, which was the glucose transporter active in our cell
line and we could make it more easily than other ones. For the
inulase we tried to select one enzyme that was well down the
glycolytic pathway and so it would not affect the initial steps.
What kind of gene did you use for your anti-sense construct? Is it
from the hybridoma or another cell line? Also, are your anti-sense
clones stable?
I cannot tell how stable the clone is because we have only had it for
one month but we have to check for this. We obtained the gene
from a cDNA library using the oligonucleotide primers and by
PCR.

CLONING AND EXPRESSION OF A CYTOSOLIC SIALIDASE FROM CHO
CELLS IN A GLUTATHIONE S-TRANSFERASE (GST)-ENCODING
EXPRESSION VECTOR

M. BURG and J. MÜTHING
University of Bielefeld, Faculty of Technical Sciences,
Institute of Cell Culture Technology,
P.O.Box 100131, 33501 Bielefeld, Germany

1. Introduction

Many proteins, produced by recombinant DNA technology for clinical and therapeutical
purpose, are glycosylated in their native state. Sialic acids, which are terminally linked
to the oligosaccharide chains, play an important role in determining the in vivo fate of a
glycoprotein in the circulatory system concerning its activity [1] or hepatic clearance
rate [2]. Recent publications
revealed cytosol-derived sialidase
activity in cell-free supernatant of
Chinese hamster ovary (CHO) and
hybridoma cells [3] in bioreactor
cultures with potential for
glycoprotein desialylation [4].
Detailed studies published by our
laboratory showed the effect of
CHO cell derived sialidase towards
sialylated oligosaccharides of
recombinant human antithrombin
III [5,6] (Fig. 1). In continuation of
this work, we attempted to clone
and express the cytosolic sialidase
of CHO cells in E. coli strains. In
order to distinguish sialidases of the
CHO donor cells from the E. coli
host, we used an expression vector encoding for glutathione S-transferase (GST). A
fragment with almost the entire open reading frame was generated for subsequent
cloning.

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O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 175-179.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

176
2. Materials and Methods
POLYMERASE CHAIN REACTION (PCR) EXPERIMENTS

Reverse transcriptase PCR (RT PCR)
Single strand cDNA was synthesized by reverse transcription of CHO-K1 total RNA

with oligo (dT) primers
using the GeneAmp®

RNA PCR kit (Perkin

Elmer). Double strand
cDNA fragments with
almost the entire open
reading frame of the
cytosolic sialidase and in

frame restriction sites were
amplified using the
primers shown in Fig. 2

and a mixture of Klen

Therm DNA polymerase
and Accu Therm DNA
polymerase (Gene Craft). The samples were subjected to a temperature cycle step of

94°C (40 sec) and 68°C (2 min) for a total of 35 cycles, followed by a 7 min extension
step at 72°C after the final cycle.

SUBCLONING

The Original TA Cloning® kit (Invitrogen) was applied to subclone the PCR product in

the pCR® 2.1 vector by direct insertion and transformation of E. coli F’. The

generated plasmids, isolated with the Quantum Prep Plasmid Miniprep kit (BioRad)

from the cultivated subclones, were tested by restriction analysis. The inserted fragment

was cut off the ligated vector, isolated by gel extraction and used for further cloning

experiments.

CLONING AND EXPRESSION

The pGEX-2T plasmid vector of the GST Gene Fusion System (Pharmacia) was
employed for cloning experiments. This vector encodes for GST at the amino terminus
of the generated protein. The plasmid encoding the cytosolic CHO cell sialidase was
constructed by inserting a BamHI-EcoRI cDNA fragment into the multiple cloning site
of the vector. E. coli M15 harbouring pREP4 was transformed with the products of
ligation. The clones were screened by restriction analysis and two were sequenced by

the dye termination method with an EBI 377 (Perkin Elmer) sequencer from both insert
directions and with two additional internal nested primers (worked out by IID Biotech

Bioservice GmbH) for double strand sequencing of the whole insert. The clones were

177
induced by IPTG and the expression was assayed by SDS-PAGE running the total
protein extracts on 8-25 % gradient gels (Pharmacia).
3. Results
AMPLIFICATION OF A cDNA FRAGMENT BY RT PCR
We amplified a cDNA fragment of the cytosolic sialidase derived from CHO cells
encoding different restriction sites for cloning by using the designed primer sequences.
Fig. 3 A (lane 2) shows the generated RT PCR product on a 2% agarose gel. The
fragment was used for the subsequent cloning experiments.
INSERTION OF THE RT PCR PRODUCTS IN THE pCR® 2.1 VECTOR
To eliminate enzymatical modifications the product was subcloned in the pCR® 2.1
vector. Several M15 pREP4 transformands were produced and plasmid isolations of the
subclones (scl) scl1, scl2 and scl3 were tested by restriction analysis. The insert is
flanked by a BamHI and a EcoRI restriction site and encodes for one ScaI site (Fig. 3
B). The sc12 contains the successfully ligated plasmid pMBU4-2 (Fig. 3 B, lane 2).

CLONING AND EXPRESSION OF THE CYTOSOLIC SIALIDASE FROM CHO
CELLS IN pGEX-2T
The BamHI-EcoRI fragment (Fig. 3 C, lane 1) was cut off the pMBU4-2 plasmid and
cloned into the pGEX-2T plasmid vector, which encodes for GST. The screening of the
transformands revealed four clones (cl1, cl2, cl6 and cl8) with the constructed plasmid

178
encoding the CHO cell sialidase sequence, identified by its PstI restriction sites (Fig. 4).
Sequencing of the clones cll and cl2 ruled out base replacements or amplification
errors. SDS PAGE of the total protein extracts after induction revealed a new protein at
about 70 kDa (Fig. 5). This correlates with the molecular weight of a fusion protein
composed of the GST (29 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa).

4. Discussion
Regarding the published cDNA sequence of the CHO cell derived cytosolic sialidase [7]
and with reference to the cloning and expression data of the cytosolic sialidase of rat
cells [8], we successfully cloned a CHO cell sialidase cDNA fragment with the almost
entire open reading frame in a pGEX-2T expression vector. The GST-sialidase fusion
protein was found to be expressed in four M15 pREP4 clones, identified by SDS-PAGE.
Next we will purify the GST-sialidase fusion protein by affinity chromatography on
Gluthatione Sepharose 4B (Pharmacia), cleave the cytosolic sialidase from the GST by
thrombin digestion (using the thrombin specific recognition site encoding by the pGEX

179

plasmid), and utilize the isolated sialidase to produce specific antibodies against
cytosolic CHO cell sialidase.

5. References

[1] Chavin, S. I., Weidner S. M., (1983) Blood clotting factor IX, J. Biol. Chem. 259: 3387-3390
[2] Briggs, D. W., Fisher, J. W., George, W. J., (1974) Hepatic clearance of intact and desialylated

erythropoietin, Am. J. Physiol. 227: 1385-1388
[3] Gramer, M. J., Goochee, C. F., (1993) Glycosidases activities of the 293 and NS0 cell lines, and

of an antibody-producing hybridoma cell line, Biotechnol. Bioeng. 43: 423-428
[4] Gramer, J. M., Goochee, C. F., Chock, V. Y., Brousseau, D. T., Sliwkowski, M. B., (1995)

Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an
extracellular CHO cell sialidase, Biotechnology 13: 692-698
[5] Munzert, E., Müthing, J., Büntemeyer, H., Lehmann J., (1996) Sialidase activity in culture fluid
of chinese hamster ovary cells during batch culture and its effect on recombinant human
antithrombin III integrity, Biotechnol. Prog. 12: 559-563
[6] Munzert, E., Heidmann, R., Büntemeyer, H., Lehmann, J., Müthing, J., (1997) Production of
recombinant human antithrombin III on 20-L-bioreactor scale: corellation of supernatant,
neuraminidase activity, desialylation, and decrease of biological activity of recombinant
glycoprotein, Biotechnol. Bioeng., in press
[7] Ferrari, J., Harris R., Warner, T.G., (1994) Cloning and expression of a soluble sialidase from
chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases,
Glycobiology 4: 367-373
[8] Miyagi, T., Konno, K. , Emori, Y., Kawasaki, H., Suzuki, K., Yasui, A., Tsuik, S., (1993)
Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase, J.
Biol. Chem. 268: 26435-26440

6. Acknowledgements

This work was financed by the Deutsche Forschungsgemeinschaft (DFG),
Graduiertenkolleg ,,Zelluläre Grundlagen biotechnischer Prozesse“. We would like to
thank the ESACT for a bursary, which enabled MB to attend the 15th ESACT Meeting.

Construction of a novel CHO cell line coexpressing human
glycosyItransferases andfusion PSGL-1 - immunoglobulin G

B. Vonach, B. Hess and C. Leist.
Novartis PharmaAG,
Biotechnology Development and Production,
CH-4002 Basel, Switzerland.

Introduction

The sialyl Lewisx (sLex) determinants serve as ligands in the selectin-mediated
adhesion of leukocytes to activated endothelium or platelets. The most frequently
used mammalian host cell lines (CHO, BHK) for the production of recombinant
glycoproteins are incapable to produce sialyl Lewisx oligosaccharides. Therefore,
we have constructed CHO cell lines expressing human N-acetyl-D-
glucosaminyltransferase and 1,3-fucosyl-transferase III. Stable clones were
obtained which were subsequently transfected with a plasmid encoding human
PSGL1-lgG. The recombinant PSGL-1 molecule was recognized by specific anti-
sialyl Lewisx antibodies that only bind to the correctly glycosylated protein.
Furthermore, the recombinant PSGL-1 bound to P-selectin, its natural ligand,
showing that it was properly glycosylated.

Expression of recombinant human glycosyltransferases

Plasmids used:

pGNT-his, coding for the N-acetyl-D-glucosaminyltransferase and the
enzym histidinol-dehydrogenase

pFucTIII-zeo, coding for the 1,3-fucosyltransferase III and zeocine
resistance

pPSGL-1-lgG1-hyg, coding for the P-selectin glycoprotein ligand 1-
immunoglobulin G1 (Fc region) fusion protein and hygromycin resistance

CHO SSF3 cells were first cotransfected with pGNT-his and pFucTIII-zeo. pGNT-
his expressed the dominantly acting hisD gene of Salmonella typhimurium
encoding the enzym histidinol-dehydrogenase. The presence of this gene
allowed the cells to survive in media without histidine supplemented with 1mM L-
histidinol. Positive transfected clones were picked and grown in 96-well plates
until analysis.

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 181-183.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

182
Immunostaining and FAGS analysis
The presence of the sLex determinant on the surface of cotransfected cells
(CHO SSF3 /GNT/FucTIII) was verified by immunostaining using a specific
mouse anti-sialyl-Lex IgM (Becton Dickinson) and an anti-mouse-lgM-peroxidase
conjugated antibody (Jackson Dianova). The specificity of the reaction was
shown by the absence of signal in untransfected cells, indicating that these
CHOSSF3 cells normally lack the enzyms N-acetyl-D-glucosaminyltransferase
and fucosyl-transferase III. When analysing cotransfected cells a brown DAB
(Diamino-benzidin tetrahydrochloride)/ cobalt and nickel chloride precipitate was
clearly detectable by microscopy (enlargement:20x).
The presence of membrane-bound expressed sialyl Lex was checked on the
surface of cotransfected CHO cells (CHO SSF3/GNT /FucTIII) by FACS analysis.
The cells were treated with the specific mouse anti-sLex IgM followed by the
fluorescence dye DTAF. Untransfected cells were used as negative control. The
samples were analyzed by flow cytometry on a FACStar Flow Cytometer (Becton
Dickinson). The fluorescence of the positive clone was above 97% indicating that
the newly constructed cell line was almost pure (Fig.1).

Expression of PSGL-1-lgG1 in the newly constructed cell line
CHO SSF3/GNT/FucTIII cells were transfected with the plasmid pPSGL-1-lgG1-
hyg, coding for the P-selectin glycoprotein ligand 1 - immunoglobulin G fusion
protein. The recombinant PSGL-1 molecule was recognized by specific anti-sialyl
Lewisx antibodies that only bind to the correctly glycosylated protein.

183
We also evaluated binding of recombinant PSGL-1-lgG to P-selectin, the natural
ligand. The immobilized P-selectin only bound minor amounts of recombinant
PSGL-1-lgG purified from the supernatant of B5 cells (only transfected with the
plasmid pPSGL-1-lgG1-hyg) (Fig.2). However, the binding was significantly
increased when adding recombinant PSGL-1-lgG purified from the supernatant
of H4 cells (transfected with the plasmids pGNT-his, pFucTIII-zeo and pPSGL-1-
lgG1-hyg). There was no detectable difference between the proteins obtained
from cells grown in the absence or presence of fetal calf serum.

Conclusion
The CHO SSF3 cell line used as host for our DNA transfections has been
previously adapted for growth in suspension cultures in serum-free media. The
recombinant CHO SSF3 cells efficiently returned to growth in serum-free
suspension culture after extended culture in monolayers using serum-
supplemented media. This indicated that the preadapted cell phenotype was
stable after transfection and selection of heterologous genes. Furthermore, the
recombinant PSGL-1-lgG protein expressed by CHO SSF3 previously
cotransfected with plasmids encoding both the N-acetyl-D-glucosaminyl-
transferase and 1,3-fucosyltransferase presented a "human-like" glycosylation
pattern, which was specifically recognized by anti-Lewisx antibodies. The
recombinant PSGL-1 bound to P-selectin, its natural ligand, showing that it was
properly glycosylated. The transfection of mammalian cell lines with plamids
encoding human glycosyltransferases of defined specificity provides a tool to
generate novel stable host cell lines for the production of recombinant proteins
with tailored glycosylation pattern.

PRODUCTION OF DEFINED GLYCOSYLATION VARIANTS OF SECRETED

HUMAN GLYCOPROTEIN THERAPEUTICS BY COEXPRESSION WITH HUMAN

RECOMBINANT GLYCOSYLTRANSFERASES IN BHK-21 CELLS

Schlenke, P., Grabenhorst, E., * Costa, J., Nimtz, M., and Conradt, H.S.
Dept. of Protein Glycosylation, GBF, Mascheroder Weg 1, D-38124
Braunschweig, Germany, *IBET, ITQB, Apartado 12, 2780 Oeiras, Portugal

1. Abstract
To investigate the stability of a BHK-21 A cell line transfected with human EPO
(huEPO) and the human 2,6-sialyltransferase ST6N (huST6N), the cell line was
cultivated over a time period of 18 days in a 2.5 L perfused bioreactor in protein- and
serum-free medium and under physiological stress after addition of ammonium. The
stability of gene expression and the activity of the newly introduced huST6N and the
endogenous 2,3-sialyltransferase ( 2,3-ST) were analyzed during the different
cultivation conditions by RT-PCR and in vitro assays. Furthermore, the purified secreted
huEPO was analyzed by western blot and the liberated N-glycans were characterized by
HPAEC-PAD analysis. The results confirmed the stability of the cell line with respect to
the expression of the newly introduced human proteins even under physiological stress.
The elevated ammonium concentration led to reversible changes on the EPO N-glycans.

2. Introduction
When expressed from mammalian host cell lines, the glycan moieties found on
recombinant human glycoprotein therapeutics depend on the cell line used for
production, the polypeptide and the culture conditions. Several common motifs found on
proteins from natural sources like the terminal 2,6-sialylation of N-glycans which is
characteristic for human serum proteins or the Lex or sLex structures are not synthesized
from the most frequently used host cell lines BHK or CHO because these cell lines do
not express the specific glycosyltransferases. The differences in the glycan structures
between recombinant proteins and the natural human counterparts are of relevance for
glycoproteins produced for clinical applications because these motifs are involved in
numerous biological phenomena, e.g.: in-vivo half-live, inflammatory processes,
antigenicity.
We have shown that it is possible to alter the glycosylation potential of host cell lines by
rransfection with plasmids encoding glycosyltransferases not expressed from the parent
cell line (1,2). By coexpression of human glycoproteins with glycosyltransferases it is
possible to obtain stable cell lines producing reproducible human-type glycosylated
products. In addition, coupled with analysis of the resulting products by mass
spectrometry, the system offers a reliable approach to investigate the in vivo substrate

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186

specificity of glycosyltransferases. By using this system together with in vitro assays the
substrate specificity of the human fucosyltransferase III was elucidated (3).
To investigate the stability of a BHK-21A cell line transfected with huEPO and the
human 2,6-sialyltransferase, the cell line was cultivated in a 2.5 L perfused bioreactor
in protein- and serum-free medium with or without addition of ammonium and
ammonium/mannose. It is known that ammonium which accumulates during cell culture
due to amino acid metabolism and degradation of glutamine affects the glycosylation of
proteins (8,9).
The BHK-21A cells used for the reactor experiment differ from BHK-21B cells by the
high amount of terminal GalNAc structures found on the N-glycans (1). Due to the fact
that the endogenous 2,3-sialyltransferases is unable to act on these structures, N-
glycans produced from this cell line are undersialylated. This would result in a rapid
hepatic clearance of the corresponding proteins in humans.
We could show that the transfection of the BHK-21A cell line with the huST6N
increases the sialylation state of the coexpressed recombinant huEPO because the human
enzyme was shown to be able to act in vivo on terminal Gal as well as on terminal
GalNAc structures (2).

3. Material and Methods
Cell line and culture conditions: A BHK-21A cell line transfected with huEPO and

huST6N was cultivated in a 2.5 L perfused bioreactor for 3 days in protein- and serum-

free standard medium (SMIF 6, Gibco-BRL), followed by a cultivation period of 5 days

in SMIF 6 supplemented with 15 mM After return to standard conditions for 3

days the perfusion was started with standard medium supplemented with 15 mM

and 4 mM mannose. The process was finished after a cultivation period of 3 days in

standard medium (see Fig. 1). Total cell number and viability was determined as

described (4). During the different culture conditions, the supernatant and cells were

harvested for further investigations.

EPO purification, characterization and analysis of N-glycans: Western analysis of the

secreted protein was performed before and after removal of the N-glycans by PNGase F-

treatment. The secreted EPO was purified from the concentrated cell culture super-

natants by immunoaffiniry chromatography (2). After the purity of the preparation was

verified by SDS/PAGE and Coomassie staining, the protein was digested with trypsin

and the resulting peptides were separated by RP-HPLC. The N-glycans were liberated

from the peptides by PNGase F and separated by RP-HPLC. The native and desialylated

oligosaccharides were desalted and analyzed by HPAEC-PAD as described previously

(5).
PCR analysis: Total cellular RNA was prepared by Tri-Reagent followed by cDNA

synthesis using standard protocols (6). The cDNA samples were normalized with respect

to the signals obtained after actin PCR, electrophoretic separation of the PCR products

in agarose gels and densiometric analysis of the resulting bands. The normalized cDNA

samples were used for PCR analysis with primers corresponding to the endogenous

2,3-ST (E. Grabenhorst, unpublished results) and the newly introduced huST6N,

187

resulting in PCR fragments of 774 bp and 699 bp, respectively. Analysis of the PCR

products was performed as described.

Preparation of cell extracts and sialyltransferase assay: After harvesting, cells were

washed with PBS, spun at 1000 x g and resuspended at a concentration of

cells/ml in ice-cold extraction buffer (10 mM MES/NaOH, pH 6.5, 2% Triton CF-54, 1

mM PMSF, 10 µg /ml aprotinin, 10 µg /ml leupeptin) and stored at -70 °C. Prior to

analysis the samples were thawed and incubated for 1h on ice.

Sialyltransferase activity was tested with an 8-methoxycarbonyloctyl glycoside type II

acceptor in reaction mixtures containing in a

volume of 100 ': 50 mM MES/NaOH buffer, pH 6.5, 27 cell extract, 90 nmol

acceptor, 5 nmol of CMP- Neu5Ac (60.000 cpm/nmol), 20 mM , 100 mM

NaCl and 0.2 nmol ATP.

The mixture was incubated at 37 °C for 5h, diluted to 1 ml with ice-cold and

applied to Sep-Pak cartridges. After washing with 15 ml of water the products were

eluted with 1.5 ml of methanol. The eluat was dried under vacuum and resuspended in

700 sialidase buffer (50 mM Na-acetate pH 5.0, 5 mM , 0.02% ). Aliquots

of 200 were incubated for 4h at 37 °C with a) 10 mU Vibrio Cholerae sialidase

(removes 2,6 and 2,3 linked sialic acid), b) 5 mU Newcastle disease virus sialidase

(removes only 2,3 linked sialic acid) and c) without sialidase. Subsequently, the

reactions were stopped by the addition of water and the mixtures were applied to the

Sep-Pak cartridges as described. After elution with 1.5 ml of methanol the incorporation

of Neu5Ac onto the acceptor was determined by liquid scintillation counting.

4. Results and Discussion
By cotranfection of mammalian host cell lines with plasmids encoding glycoprotein
therapeutics and glycosyltransferases not expressed from the parent cell lines, it is
possible to obtain therapeutics with human-type glycosylation characteristics (1,2).

In order to investigate the stability of a genetically engineered BHK-21A cell line

transfected with huEPO and the huST6N, the cell line was cultivated over a time period

of 18 days in a perfused 2.5 L bioreactor. During the cultivation in protein- and serum-
free medium and under physiological stress induced by the addition of 15 mM

ammonium with or without 4 mM mannose to the standard medium, the cellular growth
rate and viability was determined. Furthermore, the stability of gene expression and the

activity of the newly introduced huST6N and the endogenous α2,3-ST was investigated
using RT-PCR analysis and in vitro sialyltransferase assays, respectively. The secreted
huEPO was characterized by immunoblotting after purification by affinity

chromatographie. The liberated N-glycans were analyzed by HPAEC-PAD.
Cell growth: Ammonium is known as a harmful substance accumulating during cell

culture. Several authors described a reduction of the cell density due to elevated
ammonium concentrations (see ref. 7 for review). The degree of growth reduction by
ammonium varies between a wide range and probably reflects the different sensitivity of
the cell lines used for the studies as well as differences in the cell culture conditions. In

the present study, no significant effect of the elevated ammonium concentration on cell

188

growth rates or the viability of the BHK-21 A cell line (82-95%) was observed (see Fig.

1).
RT-PCR analysis: RT-PCR analysis of the transcripts encoding the newly introduced
huST6N and the endogenous 2,3-ST revealed a stable expression of the corresponding

genes during the cultivation period even under high ammonium concentration. The

analysis indicated a constant transcription rate of both, the huST6N and the 2,3-ST,

which was not affected by the culture conditions. Northern analysis revealed a l0fold

stronger signal for the huST6N compared to the endogenous enzyme (not shown).
Sialyltransferase assay: Similar to the transcription rate of the sialyltransferases, the
enzyme activity detected in cellular extracts remained stable during the production

process. A total sialyltransferase activity between 0.18 and 0.34 cells was

determined. With the acceptor substrate used for the assay, the human sialyltransferase

showed a significantly higher activity as the endogenous enzyme (about 83% of the total

activity).
Immunoblot: Western blotting analysis of the supernatants collected during the
different culture conditions confirmed a stable secretion rate of the recombinant EPO

during the cultivation process. The analysis revealed a reversible increased mobility of

the protein in SDS-PAGE when produced under elevated ammonium concentration.

Removal of the N-glycans from the protein by PNGase F-treatment eliminated the

differences indicating a variation in N-glycosylation being responsible for the observed

differences in mobility of EPO after addition of ammonium. The protein part as well as

the ratio of O-glycosylated and non O-glycosylated protein remained unaffected during

the different culture conditions.
Glycan analysis: Differences in the oligosaccharide structures produced under elevated

ammonium concentrations were also observed by HPAEC-PAD analysis of the

separated native and desialylated N-glycans. Predominantly tetraantennary structures

that were highly sialylated were detected when synthesized under standard conditions.

On the contrary, stress conditions (application of ammonium with or without mannose)

led to a decrease in sialylation state and to the formation of partially agalacto tetra-
antennary structures as shown by MALDT-TOF MS analysis of the glycans (data not

shown). The oligosaccharide structures observed during standard conditions were

regained after switching from the stress conditions back to the standard conditions again.
These results clearly indicate the stability of the genetically engineered BHK-21 A cell

line during the production process with respect to the expression of the recombinant

proteins even under high ammonium concentrations. As expected, the addition of
ammonium to the culture medium led to changes in the N-glycan structures found on the

secreted EPO. Thorens and Vassalli (8) reported the complete inhibition of sialic acid

transfer to terminal galactose residues of immunoglobulins secreted by plasma cells in

the presence of 10 mM Similar to the results obtained in the present study, no

influence of ammonium on the secretion rate was observed. The detection of truncated

glycan structures on the recombinant huEPO produced under elevated ammonium

concentrations is consistent with results published by Borys et al. (9). The authors

described the inhibition of glycosylation of mouse placental lactogen-I expressed from

CHO cells by increasing levels of ammonium in a pH-dependent manner. Their results

189

indicated that ammonium has the potential not only to affect the terminal sialylation but
the entire glycosylation process, resulting in truncated structures. The reversibility of the
effects observed after the addition of ammonium confirmed the stability of the BHK-
21A cell line investigated.
The results revealed that it is possible to obtain stable cell lines by transfection of BHK-
21 cells with glycosyltransferases not expressed from the parent cell lines. These cell
lines can be used for the production of human-type glycosylated therapeutics.

5. References

1. Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G., Conradt, H.S.: Construction of stable BHK-21

cells coexpressing human secretory glycoproteins and human --sialyltransferase,

Eur. J. Biochem. 232 (1995), 718-725

2. Schlenke, P., Grabenhorst, E., Wagner, R., Nimtz, M., Conradt, H.S.: Expression of human α2,6-
sialyltransferase in BHK-21A cells increases the sialylation of coexpressed human erythropoietin, In:

Animal Cell Technology, Kluwer Academic Publisher, Dordrecht, (1997), 475-480

3. Costa, J., Grabenhorst, E., Nimtz, M., Conradt, H.S.: Stable expression of the golgi form and secretory
variants of human fucosyltransferase III from BHK-21 cells, J. Biol. Chem. 272 (1997), 11613-11621

4. Gawlitzek, M., Valley, U., Nimtz, M., Wagner, R., Conradt, H.S.: Characterization of changes in the
glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions, J.

Biotechnol. 42 (1995), 117-131

190

5. Nimtz, M., Martin, W., Wray, V., Klöppel, K.-D., Augustin, J., Conradt, H.S.: Structures of sialylated
oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells, Eur. J. Biochem. 213
(1993), 39-56

6. Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular cloning: a laboratory manual, 2ndedn, Cold Spring
Harbor Laboratory, Cold Spring Harbor NY (1989)

7. Schneider, M., Marison, I.W., Stockar, U.v.: The importance of ammonia in mammalian cell culture,
Minireview, J. Biotechnol. 46(1996), 161-185

8. Thorens, B. & Vassalli, P.: Chloroquine and ammonium chloride prevent terminal glycosylation of
immunoglobulins in plasma cells without affecting secretion, Nature 321 (1986) 618-621

9. Borys, M.C., Linzer, D.I.H., Papoutsakis, E.T.: Ammonia affects the glycosylation pattern of re-
combinant mouse placental lactogen-I by Chinese Hamster Ovary cells in a pH-dependent manner,
Biotechnol. Bioeng. 43 (1993), 505-514

Discussion

Handa-Corrigan: How stable is your protein in terms of processing and freezing and
thawing?

Schlenke: You can freeze it several times without affecting the activity.

ANTISENSE RNA FOR THE ELIMINATION OF NEUGC RESIDUES
FROM RECOMBINANT GLYCOPROTEINS

A. GREGOIRE, A. VISVIKIS*, A. MARC, J-L. GOERGEN
Laboratoire des Sciences du Génie Chimique, CNRS-INPL, BP 172, F
54505 Vandoeuvre-lès-Nancy
* Centre du Médicament - Fac. Pharmacie, 30 rue Lionnois - 54000 Nancy

1. Abstract
In order to inhibit the CHO CNAH translation, responsible for the conversion of NeuAc
to NeuGc residues, fragments of different sizes from the mouse cnah cDNA have been
cloned as antisense fragments. It appeared that the different antisense species tested were
able to inhibit the CNAH translation in vitro. However, when decreasing the amount of
the antisense fragments by half, different efficiencies were found according to the
fragment's length or its sequence.
2. Introduction
CHO cells have become one of the most favoured way for the production of complex
recombinant proteins destined for human therapy (1,2,3). While, in rodent cells, the
CMP-N-acetylneuraminic-acid-hydroxylase (CNAH) converts CMP-N-acetylneuraminic
acids (CMP-NeuAc) to CMP-N-glycolylneuraminic acids (CMP-NeuGc) (4), in adult
human cells this enzyme is absent and thus NeuGc-bearing proteins can produce a strong
immune response when injected (5). The recent cloning of the mouse cnah gene (6) can
be used, in an antisense strategy, to stop the production of NeuGc residues bearing
proteins in CHO cells. In this study, different pieces of the mouse cnah cDNA have been
used to generate antisense RNAs, which have then been tested for their ability to inhibit
the CNAH expression in vitro.
3. Materials and Methods
Plasmid constructions : Classical molecular biology techniques were performed
according to Sambrook et al., 1989 (7). The 400nt antisense fragment is a XbaI/SacI fragment
isolated from the pBSCNAH (plasmid containing the mouse cnah cDNA, kindly given by T.
Kawano) and subcloned in the pGEM3Z vector. The 800nt antisense fragment was amplified
by PCR, using the Eurogentec TAQ DNA polymerase, and cloned into the pCR2.1 vector
issued from the "TA cloning kit" (Invitrogen).
In vitro transcription & translation : In vitro transcription reactions were
performed using the "Riboprobe in vitro Transcription System". In vitro translation
reactions were performed using the "Rabbit Reticulocyte Lysate System" and
chemiluminescent detection was possible due to the "Transcend™ non-radioactive
Translation Detection System" (Promega).

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

192
4. Results & Discussion
4.1 ANTISENSE RNA PRINCIPLE AND STRATEGY
Since initial experiments (8, 9) showed that artificial antisense oligonucleotides may be
used to interfere with gene expression, there has been a growing body of literature on
antisense nucleic acids. As a first attempt, the lack of generally applicable rules for the
design of efficient antisense led us to test, in vitro, the ability of different antisense
RNAs to inhibit the translation of the mouse cnah mRNA. We thus tested the efficiency
of antisense RNAs of different lengths by cloning fragments from the mouse cnah
cDNA. Three different antisense RNAs could then be produced : a full length (FlAs of
ca. 1700nt), a 800nt and a 400nt, schematically represented on fig.l.

4.2 TRANSCRIPTION AND TRANSLATION INHIBITION TESTS
The mouse cnah cDNA and the antisense fragments were transcribed by either T7 or T3
RNA polymerase and the RNAs were loaded on an agarose gel (fig. 2). The RNA
lengths could be verified by comparison with 4 control RNAs of 250, 1525, 1065 and
2346nt. The band intensity reflected the amount of RNA obtained, allowing an
estimation of the sense and antisense RNAs quantities necessary for the in vitro
translation tests.

As shown in fig. 3, lane CNAH, a 66Kda protein was synthetised during the translation
reaction in the tube containing the cnah mRNA. The next step was then to inhibit
CNAH expression adding antisense RNAs in the reaction. The first in vitro translation
antisense experiments were performed with a same amount of sense and antisense RNAs.
In presence of any of the antisense RNAs, no band corresponding to the CNAH protein
appeared (fig. 3A, Lanes FlAs, 800As and 400As), indicating that the 3 antisense RNAs

193
are able to stop the cnah mRNA translation. With one half of antisense RNA, the full
length and the 400nt antisense fragments were still very efficient (fig. 3B, Lanes FlAs
and 400As), whereas a decreased inhibition was observed when the 800nt antisense RNA
was present (fig. 3B, Lane 800As). The 800nt antisense RNA is less efficient than the
shorter 400nt piece, probably because : (i) of a particularly stable secondary structure
adopted by the 800nt RNA; (ii) the 800nt RNA begins to hybridize to the cnah mRNA
just at the ATG codon, which is not the case for the 2 other antisense RNAs beginning
their hybridization earlier on 5' (fig. 1).

5. Conclusion

We were able to inhibit the in vitro mouse cnah mRNA translation by RNA antisense
technology. Three different mouse antisense RNAs were tested, and all of them
efficiently inhibited the CNAH translation. However, probably due to the region it
hybridizes to the mRNA, the 800nt antisense was less efficient. The shorter and most
efficient antisense (400As) will then be tested for its translation inhibition ability in
vivo in CHO cells.

6. References

1. Archer, R., Wood, L. (1992) in R.E. Spier, J.B. Griffiths, C. Macdonald (eds.),: Animal Cell Technology:
Developments, Processes and Products , Butterworth-Heinemann, pp. 403-408.
2. Lubiniecki, A., Arathoon, R., Polastri, G., Thomas, J., Wiebe, M., Garnick, R., Jones, A., van Reis, R.,
Builder, S. (1989) in R.E. Spier, J.B. Griffiths, J. Stephens, P.J. Crooy (eds.), Advances in Animal Cell
Biology and Technology for Bioprocesses., Butterworth-Heinemann, pp.442-449.
3. Cole, E.S., Lee, K., Lauziere, K., Kelton, C., Chappel, S., Weintraub, B., Ferrera, D., Peterson, P.,
Bernasconi, R., Edmunds, T., Richards, S., Dickrell, L., Kleeman, J.M., McPherson, J.M., Pratt, B.M.
(1993) Biotechnology 11, 1014-1024.
4. Kawashima, I., Ozawa, H., Kotani, M., Suzuki, M., Kawano, T., Gomibuchi, M., Tai, T. (1993) J.
Biochem. 114, 186-193.
5. Muchmore, E.A., Milewski M., Varki, A., Diaz, S. (1989) J. Biol. Chem. 264, 20216-20223.
6. Kawano, T., Koyama, S., Takematsu, H., Kozutsumi, Y., Kawasaki, H., Kawashima, S., Kawasaki, T.,
Suzuki, A. (1995) J. Biol. Chem. 270, 16458-16463.
7. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Cold Spring Harbor Laboratory Press.
8. Paterson, B.M., Roberts, B.E., Kuff, E.L. (1977) Proc. Acad. Sci. USA 74, 4370-4374.
9. Zamecnik, P.C., Stephenson, M.L. (1978) Proc. Natl. Acad. Sci. USA 75, 280-284.

CELL PROLIFERATION

PROLIFERATION CONTROL
APOPTOSIS

INTRACELLULAR FATTY ACID COMPOSITION AFFECTS CELL YIELD,
ENERGY METABOLISM AND CELL DAMAGE IN AGITATED CULTURES

M.BUTLER, N.HUZEL, N.BARNABÉ, L.BAJNO AND T.GRAY
Department of Microbiology, University of Manitoba, Winnipeg, Canada R3T 2N2

Abstract

Continuous passage of cells in serum-free media requires the presence of micronutrients and

growth factors to compensate for the lack of serum. Fatty acid supplementation is essential

to ensure an adequate composition of the structural lipid components of the cell. We have

shown that the unsaturated fatty acids, oleic and linoleic independently enhance

cell yield and Mab productivity. The cellular content of the fatty acids gradually increased

during continuous culture passage with no evidence of regulatory control. Most of the fatty

acid accumulated in the polar lipid fraction and the unsaturated/ saturated fatty acid ratio of

all cellular lipid fractions increased significantly. This caused a substantial decrease in the

rate of glutamine metabolism and an increase in the rate of glucose metabolism. The changes

in energy metabolism were reversed when the cells were removed from fatty acid-

supplemented medium. The most plausible explanation for this effect is an altered rate of

transport of glutamine via the cell membrane. An observed change in the phospholipid

composition of the membrane also caused a significant protective effect on the cells in

agitated cultures. The life-span of fatty acid-loaded cells showed a improvement

compared to controls in cultures stirred at high rates of agitation.

Introduction

The unsaturated fatty acids, linoleic acid and oleic acid have been shown to be

essential for the repeated passage of hybridomas in serum-free cultures. In previous reports

(1,2) we showed that linoleic or oleic acid enhances significantly the cell yield and monoclonal

antibody productivity ofa B-lymphocyte hybridoma (CC9C10). However, continued culture

passage with the unsaturated fatty acids leads to a lipid-loaded state in which cells maintain

a high capacity for growth but a decreased capacity for antibody production. A similar

differential effect of fatty acids on product secretion and cell growth has been reported

previously for the secretion of cytokines from human peripheral lymphocytes (3) and for

recombinant protein productivity from BHK cells (4).

The mechanism of growth-promotion of these fatty acids may be related to their importance

in the synthesis of cellular membranes (5,6) which may have a significant effect on membrane

fluidity (7). We now present data to show that the fatty acids cause significant changes in the

phospholipid composition of the cell membrane. This may lead to altered rates of transport

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198

of key energy substrates into the cell and reduced fragility under conditions of culture
agitation.

Materials and methods

Cell line: The murine B-lymphocyte hybridoma (CC9C10), which secretes a monoclonal
antibody against insulin, was obtained from the ATCC (No HB123). The cells were

adapted to a serum-free medium over several passages and grown for at least 10 passages in
this medium prior to the described experiments. Insulin was one component of the serum-free

formulation. However, substitution with a recombinant analogue of insulin-like growth factor
was equally effective with respect to growth promotion. Growth yields in the

presence of either insulin or recombinant IGF were enhanced by 20%.
Culture: The basal medium consisted of DMEM: Ham‘s F12 (1:1 v/v) supplemented with a
serum-free mixture of hormones and micronutrients (1). The cultures were also
supplemented, where indicated, with fatty acid-free bovine serum albumin
complexed with a specific concentration of oleic or linoleic acid. Total cell concentrations

were determined by a Coulter counter. Viable cell concentrations were determined by

counting a cell suspension diluted 1:1 v/v with 0.2% trypan blue using a Neubauer

haemocytometer.

Results

1. The effect of fatty acids on growth and antibody production
Cells were adapted from a serum-supplemented to a serum-free medium and grown for at least
10 passages before there was a noticeable decrease in cell yield. The original cell yields were

restored by the addition of oleic or linoleic acid to the serum-free cultures at 25-50 µM. Cell
yields were enhanced by as much as 300%. The doubling time in the presence of the fatty
acids was around 17 h. Ten other fatty acids were tested for growth promotion in the serum-
free medium but none produced similar effects to these unsaturated fatty acids.
Cultures of hybridomas grown over several passages in media supplemented with linoleic acid

(25 µM), oleic acid (25 µM) or an equimolar mix of oleic/ linoleic (25 µM) showed a
consistent enhancement of cell yield compared to fatty acid-free control cultures. Growth
yields were greatest in cultures containing an oleic/ linoleic acid mix >linoleic acid >oleic acid

>control. The effect of growth enhancement was reversible. When cells that had been
passaged continuously in the presence of fatty acids were re-introduced into unsupplemented
medium, the growth advantage over control cultures was lost within 2 to 3 passages.

The Mab yield increased significantly to 90 µg/ml at the first passage of growth in the
presence of fatty acids. However, the yield of Mab gradually declined over the subsequent

3 passages of growth with fatty acid until there was no difference in Mab yield (60 µg/ml)
from the control culture. The effect of removing the fatty acids was to temporarily restore
the higher Mab yield of the cells previously grown in fatty acid. However, this advantage was
eroded after 4 passages in the absence of fatty acids.

199
2. Metabolic state of cells grown with fatty acids
The results of the growth experiments suggest a model set out in Table 1. Initially (state 1)
the cells were starved of critical fatty acids following continuous growth in serum-free medium
without a fatty acid supplement. State 2 arises following a brief exposure to linoleic (or oleic)
acid (25-50 µM). State 3 arises from prolonged exposure to fatty acids and can be partially
reversed to state 2 following the removal of fatty acids from the growth medium.

The fatty acid composition of each of these states is significantly different. In state 1,82% of
cellular fatty acids could be accounted for by palmitic, oleic and stearic acids. The linoleic
acid concentration was low (<5%). In state 2 the linoleic content increased to 70% and in
state 3 to 90%, whilst the content of the other fatty acids decreased progressively over this
period to 5%. Further evidence of metabolic differences between cells in these 3 states comes
from the analysis of energy metabolism. We have shown by radioactive incorporation
experiments that the oxidative metabolism of glucose increases (x2) and the oxidative
metabolism of glutamine decreases (x8) as cells progress from state 1 to state 3. Cells in state
3 grown in the absence of fatty acid revert to state 2 and demonstrate a partial reversal of this
oxidative pattern. In all states the rate of fatty acid oxidation was low in relation to the total
utilisation of fatty acid and in relation to glucose and glutamine oxidation (8).

200

3. Regulation of fatty acid uptake
It would appear that the cells have a requirement for linoleic and/or oleic acid but are unable

to regulate the rate of uptake to maintain optimal intracellular metabolism (state 2 in Table

1). In order to verify this , we determined the rate of incorporation of radioactively labelled

fatty acids from the media into 3 intracellular lipid fractions (Fig. 1).

The pattern of fatty acid utilisation was determined for cells grown in the presence or absence

of linoleic acid for at least 5 passages. Most of the fatty acid was incorporated into the polar

lipid fraction (>74%). This fraction included the phospholipids derived from cell membranes.

There was no significant difference in the total incorporation of linoleic acid between control

and fatty acid-grown cells. This suggests that there is no regulatory mechanism to prevent

an over-accumulation of fatty acid into the cell. A similar result was obtained with respect to

the incorporation of oleic acid into these lipid fractions.

4. Uptake of energy substrates in the presence of fatty acids
The metabolic profile of cells grown in linoleic or oleic acid was found to be significantly

different from control cells. The possibility that the changes were associated with an altered

membrane permeability to substrate uptake was investigated by short-term radioactive uptake
experiments. The rates of cellular uptake of glucose and glutamine were determined from
radioactive incorporation into cells over a 3 min incubation period. In separate incubations

glucose was added at concentrations up to 100 mM and glutamine in

concentrations up to 20 mM (Fig. 2).

Hyperbolic curve fitting indicated Michaelis-Menten-type kinetics of substrate uptake with
respect to concentration. This data shows a significant difference in glutamine uptake between

the linoleic acid-grown and control cultures but no significant difference for glucose uptake.

201

The kinetic parameters of substrate uptake were determined by statistical analysis using the
soft-ware program, HYPER (Table 2). The was no apparent difference in the Km values
for glucose transport in the linoleic acid-grown cells. However, the Km for glutamine uptake
in linoleic acid-grown cells at 23±5 mM was almost an order of magnitude higher than the
value of 2.7±0.5mM for control cells. At the glutamine concentration (6 mM) used in the
standard growth medium the glutamine uptake rate was 2.5-fold lower in linoleic acid-grown
cells compared to control cells. This indicates that the incorporation of the unsaturated fatty
acids into the membrane lipids caused a significant decrease in the transport of glutamine.

5. The effect of culture agitation on fatty acid grown cells
Suspension cells agitated in spinner flasks are susceptible to shear damage caused by gas
entrainment. We determined that a measurable rate of loss of cell viability occured in spinner
flasks at an agitation rates between 470 - 630 rpm (tip speed 120-165 cm/sec). At the higher
value (630 rpm) the viable cell concentration decreased to zero after 1.5 h, whereas at the
lower value (470 rpm) the viable cell concentration decreased to half the original value within
1-3 h. Fig 3a shows the time-dependent decrease of viable concentration of linoleic acid-
grown and control cells over a period of 5 h. This is a representative of a series of curves
generated within the agitation range from which we determined the half-life of the viable cell
population. Fig. 3b shows that the half life values determined for the linoleic acid-grown cells
were significantly higher than those of control cells up to 550 rpm. At the higher agitation
rates the half-lives were extremely short and no significant difference was found for the values
for the two cell types. At 470 rpm the determined half-life for linoleic acid-grown cells was
x3 the equivalent value of control cells. This result indicates that the effect of the fatty acid
is to improve the robustness of the cells significantly in agitated culture.