12_ANIMAL CELL TECHNOLOGY_707

31

32

33

4. Conclusions
We have examined three proteins stably expressed in D. melanogaster S2 cells
under the transcriptional control of the Mtn promoter. In each case we observed that the

addition of 1% DMSO 24 hours prior to induction with (see figs. 1-4) resulted in

significant increases in productivity. In addition, there was no negative effect of 1%

DMSO on cell viability or growth. While the mode of action of DMSO in D.
melanogaster S2 cells is not understood, we propose that the effect is linked to
stimulating the specific Mtn promoter used in these studies. This is based on the

following observations. First, we have seen DMSO dependent increases in Mtn

controlled mRNA. Second, DMSO did not affect expression from the constitutive D.
melanogaster actin promoter. Third, DMSO did not effect recombinant protein
expression in recombinant CHO containing constitutive promoters. Finally, Waalkes and
Wilson observed that DMSO increased metallothionein expression in cultured liver cells

(2).

5. References
Reid S., and Greenfield P.F. 1992. Improved production of

recombinant protein by the baculovirus expression system. In Animal Cell Technology:
Basic & Applied aspects, pp. 419-424. H. Murakami et al. (Eds), Kluwer Academic
Publishers.

and Wilson M. J. 1987. Increased synthetic capacity for
metallothionein in cultured liver cells following dimethyl sulfoxide pretreatment.

Experimental Cell Research 169:25-30.

and Swetly P. 1982. Interferon production in human hematopoietic cell
lines: Response to chemicals and characterization of Interferon's. Journal of Interferon
Research 2:261-270.

A., Johansen H., Sweet, R., and Rosenberg, M. 1988. Efficient

expression of foreign genes in cultured Drosophila melanogaster cells using hygromycin
B selection. Invertebrate and Fish Tissue Culture, pp. 131-134. Kuroda, Y., Kurstak,
E., and Maramorosch K., eds., Springer-Verlag, Berlin.

van der Straten, A., Oto, E., Maroni, G., and Rosenberg, M. 1989.
Regulated expression at high copy number allows production of lethal oncogene product
in Drosophila Schneider cells. Genes & Development 3:882-889.

Shea, C., Sant R. G. 1997. Intravesical dimethyl sulfoxide (DMSO) for
interstitial cystitis-a practical approach. Urology 49:105-107.

STUDIES OF THE HEPATITIS C VIRUS RECOMBINANT
PROTEIN PRODUCTION AND ITS EFFECTS ON SF-9 INSECT
CELLS METABOLISM

CÉLINE. M. CHARON
University of Westminster, School of Biological & Health Sciences, 115 New
Cavendish Street, London W1M 8JS, UK.

ABSTRACT. The recombinant hepatitis C protein BHC 11 was produced in serum-free

medium using the baculovirus expression vector (BEV). Protein synthesis in suspension

culture of Spodoptera frugiperda Sf-9 insect cells was achieved in controlled and
uncontrolled oxygen environment. The same multiplicity of infections (MOI = 0.1 and

MOI = 1) and time ofinfection were used in each experiment and

compared. N-glycosylation of the protein was investigated with different lectins.

1. Introduction

Product productivity depends on different parameters such as, the type of medium used
[1], the MOI and the time of infection. The effect of dissolved oxygen tension (D.O.T)
on recombinant protein production is controversial. Maintaining a constant level of
D.O.T can improve [4] or decrease product accumulation [4, 6]. In some cases the level
of D.O.T was shown to have no effect on transient protein production [3, 5]. The
presence of nutrients is also important and the limiting factor for protein synthesis is
still unknown. The following experiments compare the protein productivity in small
scale culture (100 ml) and larger scale culture (1 L). In the latter the D.O.T was kept at
30%, the pH was monitored and nutrient consumption rates were determined.

2. Materials and Methods

2.1. SUSPENSION CULTURES

In small scale culture insect cells were grown in 100 ml SF-900 Gibco medium
using 250 ml shaken flasks (128 rpm). Larger scale culture experiments were performed
in a 2L stirred (70 rpm) tank bioreactor (LSL Biolafitte) and maintained under 30%
D.O.T. The medium (1 L working volume) was aerated by sparging air. pH remained ~6.

35

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 35-37.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

36
Viable cell density was assessed in a Neubauer counting chamber (BDH) using the
trypan blue exclusion test [7].
2.2. PROTEIN ANALYSIS
The recombinant protein BHC11 (97 kD) was extracted at 24, 48, 72, 96 and 120 hours
post infection (hpi). Transient protein expression was studied on western blots and
quantified by ELISAs (Murex Diagnostics). N-glycosylation studies were carried out
using two biotinylated lectins Galanthus nivalis and Concanavalin A (Vector
laboratories) for ECL western blot analysis (Amersham).
2.3. BIOCHEMICAL ANALYSIS
Substrates (glucose & glutamine) and product (lactate) were analysed under 30% D.O.T
as previously described [2].

3. Results
3.1. PROTEIN PRODUCTION IN UNCONTROLLED OXYGEN ENVIRONMENT
Concentrations of protein BHC11 per unit volume Erleruneyer flask with MOIs of 1 (338

were found at 72 hpi (Figures 1 & 2). Western blot
analysis of BHC11 with an MOI = 1 showed accumulation of the product at 48, 72 and
96 hpi.

3.2. PROTEIN PRODUCTION IN STIRRED TANK BIOREACTOR
Protein production was studied for each MOI under 30% D.O.T. The volumetric
productivity with an MOI of 1, produced at 48 hpi and 72 hpi was 38

37

The specific glucose consumption rate and the specific

glutamine consumption rate in the first 48 hours of
infection with an MOI of were higher than those recorded with uninfected cells or Sf-9

cells infected with an MOI of 0.1. The lactate concentration remained low (2 mM) in

infected and uninfected batch cultures.
Glycosylation studies showed that the recombinant protein BHC11 was not N-
glycosylated. Western blot analyses also revealed that the recombinant protein BHC11
was more stable when using an MOI of 1 rather than 0.1 (results not shown).
Recombinant protein production in batch fermentation under 30% D.O.T was limited

compared with non-oxygen limited shaken flask cultures.

4. References

1. Akhnoukh R., Kretzmer G and Schügerl K. (1996): On line monitoring and control of the cultivation of

Spodoptera frugiperda Sf-9 insect cells and production by Autographa californica virus vector.

Enz. Microb. Technol. 18, 220-228.

2. Charon C. (1995): Infection of the insect cells Spodoptera frugtperda SF-9 with the 2r1esctomcebnitnuaryn.t baculovirus
BHC11 in serum-free medium. In: Animal cell technology: Developments towards the Beuvery E.C.,
Griffiths J.B and Zeijlemaker W.P. (Eds). Kluwer academic publishers. Dordrecht, pp 131-135.
3. Hensler W.T and Agathos S.N. (1994): Evaluation of monitoring approaches and effects of culture conditions on

recombinant protein production in baculovirus-infected insect cells. Cytotechnology. 15, 177-186.
4. Jain D.. Ramasubramanyan K., Gould S., Lenny A., Candelore M., Tota M, Strader C., Alves K., Cuca G., Tung

J.S., Hunt G., Junker B., Buckland B.C and Silberklang M (1991): Large scale recombinant protein production

using the insect cell baculovirus expression vector system: antistatin and receptor. In: Spier R.E.,

Griffiths J.B., Meignier B (Eds). Production of biologicals from animal cells in culture. Butterworth - Heinemann
Press. Oxford, pp 345-351.

5. Kamen A.A., Bédard C., Tom R., Perret S and Jardin B. (1996): On-line monitoring of respiration in

recombinant-baculovirus infected and uninfected insect cell bioreactor cultures. Biotech. Bioeng. 50, 36-48.
6. Reuveny S., Kim Y.J., Kemp C.W and Shiloach J. (1993): Effect of temperature and oxygen on cell growth and

recombinant protein production in insect cell cultures. Appl Microb Biotechnol. 38, 619-623

7. Summers M.D and Smith G.E. (1987): A manual of methods for baculovirus vectors and insect cell culture

procedures. Texas Agricultural Experiments Station, and Texas A&M University, College Station. Bulletin No

1555. pp 1 0 - 1 8 .

THE HUMAN MULTIDRUG TRANSPORTER (MDR1)
EXPRESSED IN THE BACULOVIRUS-SF9 INSECT CELL
SYSTEM

1, 2SZABÓK, 1BAKOS,É., 1,2WELKER,E., 1VÁRADI,A.,
3GOODFELLOW,H.R., 3HIGGINS,C.F., AND 2SARKADI,B.
1Inst. of Enzymology, Hung. Acad. Sci., and2Natl. Inst. of
Haematology and Immunology, Research Group of the Hung.
Acad. Sci., Budapest, Hungary; 3Inst. of Molecular Medicine,
University of Oxford, UK.
NIHI, Budapest, Daródczi u. 24., H-1113, Hungary

Introduction

Overexpression of the human multidrug transporter (MDR1 or P-glycoprotein) is
responsible for the phenomenon of multiple drug resistance in various cancer cell
types. MDR1 is an integral plasma membrane protein which acts as an ATP-
dependent efflux pump to reduce the intracellular concentration of diverse
hydrophobic compounds [1]. MDR1 was described as a phosphorylated
glycoprotein, but no clear role for phosphorylation in the transporter activity of
MDR1 has yet been established [2,3,4,5]. The “linker” region represents the only
documented target for phosphorylation both in vitro and in vivo. We have
constructed MDR1 mutants in which the serines and/or threonines in the linker
region of the protein were replaced by alanincs (to prevent phosphorylation) or
glutamic acid residues (to mimic the charge of permanent phosphorylation). In
one set of mutants the three serine residues phosphorylated in vivo (Ser 661, 667
and 671) were replaced to generate 3A and 3E mutants. In the other set all the
eight serines and threonines providing possible additional phosphorylation sites
in the linker region were replaced ( 8A and 8E mutants), in order to investigate
the possible role of secondary phosphorylations in this region. The wild-type and
mutant transporters, expressed using the baculovirus-Sf9 insect cell system, were
tested for their expression level and correct membrane insertion, and
characterized by analyzing their ATP and drug affinity in terms of drug-
stimulated ATPase activity.

39

O. - W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 39-41.
© 1998 Khuwer Academic Publishers. Printed in the Netherlands.

40

Summary of the results

The results presented here indicate that phosphorylation of serine residues in the
linker region of the MDR1 modulates its interaction with the certain transported
hydrophobic substrate molecules. There is no measurable effect of
phosphorylation on the ATP-concentration dependence of the multidrug
transporter [6].

1. A similar high expression level of all the phosphorylation site variants
could be achieved as that of the wild-type MDR1 protein, and the normal
membrane insertion pattern was also preserved for all these mutants expressed in
the baculovirus-Sf9 insect cells system.

2. The maximum level of drug-stimulated MDRl-ATPase activity was
similar for the wild-type and the mutant proteins using several MDRl-interacting
compounds.

3. The half-maximum activation of the MDRl-ATPase activity of the
mutants 3E and 8E, which mimic the phosphorylated MDR1, and the wild-type
MDR1, known to be phosphorylated in Sf9 membranes, was achieved at
significantly lower concentrations of verapamil, rhodamine 123 and vinblastine
than that for the nonphosphorylatable mutants 3A and 8A. Fig. 1A shows the
data obtained by varying the concentration of verapamil for the 3A and 3E
mutants.

4. The ATPase activation in the wild-type MDR1 and its phosphorylation
mutants was indistinguishable by calcein-AM or valinomycin.

5. The kinetic analysis presented here may be interpreted to mean that
MDR1 has multiple drug-interacting sites, which are characteristically modulated
by the phosphorylation of the serine residues in the linker region of this protein.
In the non-phosphorylated state these sites show an apparent negative
cooperativity or significantly different drug affinities, while in a fully
phosphorylated form of the enzyme this phenomenon can not be observed. Fig.
1B shows data from Fig. 1A analyzed using a simple Michaelis-Menten kinetic
approach and presented in a linearised (Lineweaver-Burk) plot. Fig. 1C presents
a Hill plot analysis of the same data points.

6. The dependence of the MDRl-ATPase activity on ATP concentration
was identical in the wild-type and the mutant proteins, and

7. Hill-plots indicated that more than one ATP-binding/interacting sites
are present in the enzyme and these sites interact with a positive cooperativity.

8. The 8A and 8E mutants showed a similar behaviour in all the above
experiments as their 3A or 3E counterparts, indicating that the phosphorylation of
one or more of the targeted 3 serine residues is fully responsible for the drug-
interaction effects observed, and there is no additional role of serine or threonine
phosphorylation in this region in terms of drug-stimulated ATPase activity, even
if three serines are already replaced by alanines.

Acknowledgements
Participation of this work at the meeting was supported by ESACT.

41

References
1. GGoetrtmcsamnann, ,UM.A.M., .C, ahnadmPbaesrtsa,nT, .IC..(,1A99m3b) uAdnknaar.,RSe.vV..B, iPoacshtaenm,.I6.,2a, n3d85G-4o2tt7esman,
2. M.M. (1995) J.
3. GottesmBiaone,neMrgM. .B, ioHmryecmybnra.,27C,.5A3..-6S1chocnlein, P.V., Gemann, U.A., and Pastan, I. (1995)
GGoeromdfacnAGlnlnoo,nwtaUt,c. sAHRme..,Rva.C.n,G,hSMaeman.reMbdtei.n.r2si(,,91,9TA69.C.06,7.),-R6JA.u4Bme9itobzl,u.dCSk.h,aerCm, a.Sl2.lVa7g1.,,h1aL7n0i,c8h-Rt1,.7. T1G6.,roCsa, rdPa.,reMllic,
4. Higgins, C.F. (1996) J. Biol. Chem. 271, 13668-13674 C.O., Pastan, I., and
5 Naughton, P.A., and

6. Szabó, K., Bakos,É., Welkcr.E., Müller.M., Goodfellow.H.R., Higgins, C.F., Váradi, A., and

Sarkadi, B. (1997) J. Biol. Chem. 272, 23165-23171

GLUTAMINE SYNTHETASE TRANSFECTED CELLS MAY AVOID SELECTION BY
RELEASING GLUTAMINE

P.BIRD, E. BOLAM, L.CASTELL*, O.OBEID#, N. DARTON and G.HALE.
Therapeutic Antibody Centre, Sir William Dunn School of Pathology, University of Oxford
Old Road, Headington, Oxford, 0X3 7JT, UK. Tel +44-1865-744845 Fax +44-1865-741291.
*Cellular Nutrition Research Group, Dept of Biochemistry, University of Oxford, South Parks
Road, Oxford OX1 3QU. #Dept. of Human Nutrition, The Royal London Hospital, Whitechapel,
London El 1BB.

1. Abstract

Glutamine synthetase (GS) is a popular marker for selection of plasmid expression in mammalian cell

lines. Positive selection is maintained simply by omitting glutamine (gln) from culture medium. We have

studied the stability of GS transfectants which produce humanised monoclonal antibodies. Spent culture

medium of GS transfectants contained up to gln which we conclude is released from the cells.

Using a new assay to detect intracellular Ig, cells were detected with low levels of Ig after a short time of

culture in medium with as little as added gln and also in hollow fibre cultures fed with gln free

medium. These variants comprised up to 50% of the cell population and secreted very little antibody. It

may be hard to maintain selection pressure in practical large scale cultures particularly in hollow fibre

systems where, with a high cell density, the local gln concentrations may be substantial.

2. Introduction

The Therapeutic Antibody Centre (TAC) was set up 7 years ago in Cambridge as a centre for the small
scale production of therapeutic monoclonal antibodies available for clinical trials under DDX certification.
Two years ago the centre moved to a new purpose-built facility in Oxford. In total more than 600g of
therapeutic antibody has been produced to treat more than 3000 patients. Seven humanised monoclonal
antibodies have been grown in the TAC using hollow fibre based production in seven Acusyst machines
(Cellex) and an Acusyst P3X where six hollow fibre devices are incorporated in a single flowpath.
Humanised antibodies have been expressed in CHO or NS0 cells using either dihydrofolate reductase or
glutamine synthetase for plasmid selection.

Glutamine synthetase (GS) is a popular marker for selection of plasmid expression in mammalian cell
lines. Positive selection is maintained simply by omitting glutamine (gln) from culture medium
(Bebbington et al 1992). The plasmid for transfection is licensed by Lonza. Using this plasmid to transfect
NS0, we have produced four different humanised IgG1 antibodies. When these cell lines are grown in
hollow fibre we expect to see an increase in antibody concentration from 0.1-0.4 mg/ml in saturated cell
supernatants in small scale culture to 1-4 mg/ml in the Acusyst harvest. Each cell line has a characteristic
product concentration. One of our GS transfectants (cell line 1) showed considerable variation in mean
product concentration from day 50-100 post inoculation in three different runs (1.0, 1.7, and 3.1 mg/ml)
whilst another (cell line 2) failed to show any significant increase in concentration of product when grown
in hollow fibre (maximum 0.3 mg/ml) compared with small scale tissue culture (0.1 mg/ml). This
variability in hollow fibre production by these two cell lines has been examined.

We looked for the presence of low or negative antibody producing cells in each cell line by analytical

43

O.- W Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 43-49.
© 1998 Kllnver Academic Publishers. Printed in the Netherlands.

44

cloning in medium containing 0 or 1 mM gln. Additionally we developed an assay to detect intracellular
immunoglobulin (ICIg) in individual cells by permeabilising the plasma cell membrane with saponin prior
to incubation in FITC conjugated anti- human Ig and assaying individual cell fluorescence on a FACScan.
A considerable amount of cell death occurs in hollow fibre cell culture. Our hypothesis was that the death
of high Ig producing cells may release intracellular synthesised gln that can be used by lower Ig producing
cells that have downregulated both GS and Ig synthesis. Therefore we have measured gln concentrations in
small scale tissue cultures and in samples taken from the extracapillary side of hollow fibre cultures of
these lines.

3. Methods
3.1. MEDIA.

Two types of gln free media were compared. Media 1 was IMDM (Sigma I-2762) with the standard Lonza

recommended nucleoside, glutamic acid (glu) and asparagine (asn) additions. Media 2

was a special TAC formulation of IMDM lacking gln but with nucleosides and a higher level of glu (2600

and asn Dialysed fetal calf serum (Gibco ultra low Ig) was added to both media at 5%.

The gln content of the FCS was below post dialysis final).

3.2. INTRACELLULAR IMMUNOGLOBULIN (ICIg) ASSAY

Actively growing cells were washed into PBS and fixed in 4% formaldehyde/PBS for 15 min and then
permeabilised with 0.5% saponin ( Sigma S-2149)/0.5% BSA/PBS for 30 min. Cells from hollow fibre
cultures were separated first over Histopaque to enrich for live cells. Fixed cells were incubated in optimal

dilution of FITC conjugated sheep anti- human IgG (Fc specific) in permabilising buffer for 30 min. After
washing the cells were resuspended in PBS/BSA/azide to close the membrane pores and fluorescence

assayed on a Becton Dickinson FACScan. A sample of line1 cells grown in gln free medium was used as a
positive control and non transfected NS0 cells as a negative control in every assay. During analysis of gated
'live' cells, the % positive cells (using a marker to define NS0 as <2% positive), the % strong positive cells
(using a marker to define line 1 cells as >95% positive), and the geometric mean of FL1 were recorded. An
additional control of staining positive cells with FITC conjugated anti-rat Ig gave similar intensityof FL1
staining as NS0.

3.3. GLUTAMINE ASSAY BY HPLC.

Cell culture supernatants samples were stored frozen. Deproteinisation was performed with an equal
volume of 10% sulphosalicylic acid. Amino acid derivatives of o-phthalaldehyde were separated on a

Hichrom spherisorb reverse phase ODS C18 column as in Mann et al (1988). Peak areas were integrated
and amino acid concentrations calculated from peak areas by reference to the area of an internal standard
homocysteine peak.

4. Results and Discussion.
4.1. INTRACELLULAR IG ASSAY.

4.1.1 Assay validation

This assay distinguished high Ig producing cells (1.1A: line 1 cells d 236 in culture, from
untransfected negative controls (1.1B: NSO cells d 42, by their intensity of green (FL1)

fluorescence. Premixing positive and negative cells (1.1C) gave similar % of positive cells and FL1
geometric means to that seen when cells were mixed after staining (LID). Additionally, in a number of cell

stability tests a drop in the FL1 geometric mean and % strong positive cells by ICIg analysis has been seen

to precede a drop in secreted Ig production by about 10 days, (for example see figure 2) suggesting that

this assay is an early indicator of cell instability.

45

4.2. THE DEVELOPMENT OF LOW IG PRODUCING LINE 1 CELLS IN MEDIA WITH VARYING
GLN CONCENTRATIONS.

Line1 cells were passaged twice weekly in 1 ml of TAC media with gln added back from

Saturated supernatants of passed cells were collected and analysed for Ig concentration by ELISA and

percent of strongly positive intracellular Ig cells was also assayed with time (figure 2). At all gln

concentrations tested, low Ig producing cells developed as detected by reduced Ig production and

intracellular Ig levels. Only in gln free medium were no low producers detected over 120 days of culture.

As little as gln was sufficient to allow low Ig producers to survive in line 1 cultures. (Line 2 cells

developed low Ig producing cells in the complete absence of added gln (data not shown).

46

4.3 GLUTAMINE CONCENTRATIONS IN CELL CULTURES.

4.3.1 In hollow fibre culture
Three different hollow fibre cultures of line1 sampled on days 153 and 214 respectively contained 50 - 90

gln in the extracapillary (cell growing) side. Considering that there is continuous dialysis across the

hollow fibre it could be that the cells are bathed in higher local concentrations of gln. We have shown that

line 1 cells can lose productivity in as little as gln (figure 2) and therefore local production of gln in

the hollow fibres may affect productivity.

4.3.2 In flask cultures
Supernatant from line 1 cell cultures in TAC medium were collected over a growth period of 15 days. Cell

numbers and viability were correlated with gln levels in the supernatant (figure 3). Gln levels rose from 0
to over on day 10 of culture and this increase coincided with the loss of cell viability suggesting

that release of cell synthesised gln on cell death is a likely cause of the gln detected in these cultures and

also in the hollow fibre cultures. When these cell cultures were repeated in standard media with lower asn

and glu concentrations, lower levels of gln(maximum were detected in the media. We are

currently trying to find optimal levels of media constituents that allow high Ig production but low gln

accumulation.

47

5. Conclusions

A number of factors will affect GS transfectant stability, such as integration site, copy number, and

product. GS transfectants exist which retain stability in the presence of gln. Our GS transfectant cell line1

is stable for over 200 days in gln free medium when passaged twice a week in flask culture but not in the
presence of or more gln nor in long term hollow fibre culture. We have shown here that GS
transfectants can release up to gln into culture medium in association with cell death and that

hollow fibre cultures of GS transfectants can also develop high levels of gln on the cell side. We propose

that the loss in productivity of this transfectant in hollow fibre culture results from the level of gln
produced locally. This gln related instability may limit the growth of similar GS transfectants in hollow
fibre devices.

6. Acknowledgements

This work was funded by MRC and Leucosite inc.. We wish to thank Hilary Metcalfe and Sue Bright at
Lonza for their helpful discussions and Andy Beevers for his help.

7. References

Bebbington, C.R., Renner.G., Thomson, S., King, D., Abrams,D., and Yarranton G.T.. (1992) High level
expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an
amplifiable selectable marker, Biotechnology 10, 169-175.

Mann G.E., Smith S.A., Norman P.S.R., and Emery P.W. (1988). Fasting refeeding and diabetes modulate
free amino acid concentrations in the rat exocrine pancreas: role of transstimulation in amino acid efflux,
Pancreas 3, 67-76.

48 Your data are very consistent to the original CellTech data which
Discussion showed that if glutamine was present, the stability decreased. They
Ozturk: suggested using methionine sulphoxamine. Have you tried it?
Bird:
Yes, we tried it for cell line 2, but unsuccessfully. Possibly the
Ozturk: product is a bit more unstable for cell line 2. Also, as glutamine is
Bird: by-passing the need for glutamine synthetase and MSO blocks this
enzyme as it is in such low concentrations, the fact that glutamine
Griffiths: is around may by-pass that inhibition.

Petrie: In your hollow fibre system you are getting 3 g/l antibody. Does
Bird: the membrane cut-off affect productivity as the yield looked very
high?

We inoculate a 200 ml culture at cells and they grow in the
presence of foetal calf serum, so you get a very high cell
concentration within the EC side. This is what leads to the very
high antibody concentration. The MW cut-off is 10,000 of the
kidney dialysis membranes which we use.

I am wondering whether there is a straight-forward nutritional
explanation for the glutamine effects which you observe. Whether
an amino acid is essential or non-essential depends upon the cell
density. The higher the density, the fewer the amino acids that are
essential. Above 50 million/ml, glutamine ceases to be essential.
So at the cell densities you are using, there is no advantage for the
cell to have glutamine synthetase. In fact, it maybe a disadvantage,
especially with all the asparagine and amides that are present. So it
may well be just a question of the advantage that glutamine
synthetase has for the cell is completely lost at high densities.

Have you observed loss of glutamine synthetase activity also at low
density where there is less cell death? Also, have you observed this
with other cells, such as CHO and non-NS0 cells?

Cell line 1 is a very stable producer in small-scale culture in
glutamine-free medium. We have cultured them for nearly 1 year,
passaging them twice weekly, and the ICIG levels remain high.
Each transfectant will very slightly in its characteristics, but at low
cell density there is not a problem. With CHO cells, we have not
seen this problem.

Berthold: 49

Bird: I always struggle with the naming of cell lines when they are not
Berthold: apparently genetically stable. Assuming that the de-selection of
Bird: what you see is due to genomic diversity, would it be possible to
Aunins: have alternative strategies to somehow constrain these cell lines to
Bird: more mono-clonality? Do you have steps to ensure mono-clonality
because it is well known, for these cell lines in particular, that the
chromosomes are not a set but a histogram, so you have all types
of de-selection possibilities?

The easy answer is, no, we have not. We can routinely check for
low IG producers. We have varied the media to see if we could
get lower glutamine levels.

Do you use single-cell cloning from time to time?

Yes, we set up each run from a master cell bank containing a
minimum number of cell passages before freezing, although of
course the hollow fibre runs are long.

I would have thought that you will eventually come to a steady-
state even with low producing clones present. Have you run your
experiments out to see ifyou get a steady-state population?

This is seen clearly in cell line 2, with or without glutamine present.
The percentage of high versus low producers changes very slowly,
but you do get to a steady-state where the amount of glutamine
produced by the high-producers is just enough and they are
growing with a constant division time.

STUDY OF DIFFERENT CELL CULTURE CONDITIONS FOR THE
PRODUCTION OF A “RESHAPED” MAB IN NSO CELLS.

A.J. CASTILLO1, A. FERNANDEZ1, T. BOGGIANO1, P. PUGEAUD2,
I. W. MARISON2.
1) Center of Molecular-Immunology (CM), P.O. Box 16040, Havana City
11600, Cuba, 2) Institute of Chemical Engineering and Bioengineering,
Swiss Federal Institute of Technology, CH-1015, Lausanne, Switzerland.

Abstract:

The recombinant cell line R3/T16 expresses a “reshaped” Mab against epidermal
growth factor receptor (EGF-R) in NS0 myeloma. In this study the ability of two culture
media to mantain good levels of growth and production of this cell line under serum
reduced conditions have been compared and the effect of supplementation of
aminoacids and vitamins at 1% of fetal calf serum was investigated.
Furthermore, production and growth yields of R3/T16 cell line in cultures with different
macroporous microcarriers have been compared. Results showed better performances
for the immobilized cell cultures in fluidized bed reactor, suggesting that such systems
are suitable for a large scale production of this recombinant Mab.

Introduccion themain aims in developing commercial
processes is to lower production costs by
There are a number of problems increasing unit production. To achieve
associated with the presence of serum in this goal, high density systems capable of
culture medium used for production, volumetric scale-up and long term
including high cost, regulatory operation are required. These aims could
considerations and the difficulty in be obtained using culture systems with
removing serum proteins when purifying cell immobilization in macroporous
the product of interest. By these reasons microcarriers. Immobilization is also
the reduction of serum supplement or the reported to improve cell specific
use of serum-free medium for the culture productionrate [2].

of genetically engineered mammalian By these reasons overall aims of this
cells offers many advantages. Previously study were to optimize a basal medium
it has been shown that NSO cells are that could support good levels of growth
cholesterol auxothrophs, that renders and production of R3/T16 recombinant
very difficult their adaptation to serum- cell line at low levels of serum

free medium. However some authors supplement and to test different
have reported the use of serum-free macroporous microcarriers in order to
media to culture this cell line [1]. One of

51

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 51-53.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

52 and other metabolites were measured by
grow this cell line in medium with low RP-HPLC methods (4).
concentration of serum supplement.

Materials and methods Results

The R3/T16 cell line is a NSO myeloma Growth curves were carried out in 6 well

that produces an humanized monoclonal plates with a seed cell density of
antibody against EGF receptor [3]. cells/ml using FMX-1 and PFHM-II
Protein free media Turbodoma FMX-B1 serum free media supplemented with 8, 3
(FMX Std) and HP 1 (THP1) were and 1 percent of FCS. From this study
obtained from Dr. F. Messi, Cell Culture (Fig. 1) we selected Turbodoma medium,
Technologies and all other basal media due to its best support for cell growth and
and fetal calf serum (FCS) were production at low serum levels,
purchased from Gibco BRL. All other Kinetic studies in T-flasks were
reagents were acquired from Sigma. carried out in DMEM/F-12 medium
Previously to culture experiments for supplemented with 8 % FCS (control)
testing different medium supplements, and in THP1 medium supplemented with

cells were passaged at least 7 days in 9 and 1 % of FCS. It was observed an
order to adapt them. For each culture increase in specific glucose consumption
experiment in spinners or T- and lactate production rates (Table 1),
flasks cells were grown in duplicates. when reducing serum supplement.
Cultures in well plates were carried out Glutamine specific consumption rate was
in triplicates. higher in THPl medium with respect to
Cytopore I (Pharmacia Biotech) and control. However growth and production
Cultispher G (Hyclone) microcarriers parameters were very similar in all cases,
were tested in spinner flasks (Integra Critical aminoacids were determined and
Biosciences). One culture was done in supplemented following the criteria

the Cytopilot Mini fluidized bed reactor previously described by Stoll et al. [5].
(FBR) with 200 ml of Cytoline I Kinetic studies in T-flasks using
microcarrier (Pharmacia Biotech). In all THP 1 medium with 1 % of FCS and
cases seed density was cells/mL different supplements were done. Not
and culture medium was Turbodoma significative differences were observed
HP1 supplemented with 1 % of FCS. for growth and production behaviour
Cell count was peformed by trypan blue (Fig. 2) , as well as for specific
exclusion method. IgG concentration was consumption and production rates of
measured by an anti-human heavy chain different metabolites (data not shown)
ELISA developed at CIM. Aminoacids with different medium supplements.

53

very similar in THP1 supplemented both
with 9 or 1 % of FCS compared with
control medium (DMEM-F12 + 8%
FCS). Non positive effect was observed
respect to biomass, cumulative product
and key metabolic parameters when the
adding aminoacids and vitamins,
indicating that these metabolites are not

limiting steps when this cell line is

cultured under serum reduced conditions.

R3/T16 cell culture in FBR with Cytoline
I microcarriers showed the best
performances respect to determined

values of Qab. Further experiments with
longer culture time are required in order
to investigate full potentialities of this
type of system.

Values for overall production rates (Qab) References
and average IgG concentrations for
cultures with different microcarriers are 1.- Keen M. J. and Steward T.W. (1995). Adaptation
showed in Table 2. Best value for Qab of cholesterol-requiring NSO mouse myeloma cells to
was obtained for cell culture in FBR high density growth in a fully defined protein-free
using Cytoline I microcarriers. and cholesterol-free culture medium. Cytotechnology
17; 203-211.
Conclusions 2.- Lee G. M. and Palsson B. O. (1990).
Turbodoma culture medium showed Immobiliization can improve the stability of
better support for cell growth and hybridoma antibody production in serum-tree
antibody production at low serum medium. Biotechnology & Bioengineering 36; 1049-
supplement. Growth parameters were 1055.
3.- Mateo C., Moreno E., Amour K., Lombardero J.,
Harris W. and Perez R. (1997). Humanization of a
mouse monoclonal antibody that blocks the epidermal
growth factor receptor: recovery of antagonistic
activity. Immunotechnology 3; 71 -81.
4.- Stoll T., Pugeaud P., Von Stockar U. and Marison
I. W. (1994). A simple HPLC technique for accurate
monitoring of mammalian cell metabolism.
Cytotechnology 14; 123-128.
5.- Stoll T., Muhlethaler K., Von Stockar U. and
Marison I. W. (1996). Systematic improvement of a
chemically-defined protein medium for hybridoma
growth and monoclonal antibody production. Journal
of Biotechnology 45 (2); 111-123.

INCREASING MONOCLONAL ANTIBODY PRODUCTIVITY BY
SEMICONTINUOUS SUBSTITUTION OF PRODUCTION MEDIUM FOR
GROWTH MEDIUM

and K. ŠRÁMKOVÁ
Institute of Molecular Genetics, Fundamental Cytotechnology Laboratory,
CZ-14220 Praha 4, Czech Republic

Introduction

Hybridoma culture in perfusion mode, both in homogeneous suspension and in heterogeneous
systems, offers a daily harvest of monoclonal antibody (Mab) synthesized in a relatively small
working volume by a high-density cell population over a long period of time [1].
Because high perfusion rates are needed to provide sufficient

quantity of substrates and to remove toxic waste products, the simple variant of perfusion

culture has some disadvantages. The Mab concentration in the harvest fluid is generally lower
than that achievable in an optimized fed-batch culture. The volume of medium consumed per

mass unit of the product is relatively high, and the utilization of the substrates is incomplete.

Improvements to the perfusion process have been developed based on addition of
nutrient concentrates [2,3], bleeding of homogeneous perfusion cultures [2,4], or separate use
of concentrated medium for feeding and buffer solution for dialysis [3].
In this work we report on the development of a semicontinuous process in which an
efficient Mab batch production employs cells transferred from a cell growth modul run in
perfusion mode.
The philosophy of this strategy arises from the notion that optimal medium for Mab
production is different from optimal medium for growth. In this process we apply the
knowledge of suppression of apoptotic death by addition of some amino acids [5-10] to the cell
growth medium. Daily cell bleeds from the growth modul served to set up short-term
production cultures in batch mode, in the course of which the Mab concentration rose to
multiples of the starting value.

Material and Methods

Mouse hybridoma ME-750, an IgG2a producer, was cultured in DMEM/F 12/RPMI 1640
(2:1:1) medium supplemented with BME amino acids, 2.0 mM glutamine, 0.4 mM each of
alanine, serine, asparagine and proline, 15 mM HEPES, 2.0 g/1 sodium bicarbonate and with
the iron-rich protein-free growth-promoting mixture containing ferric citrate [11].The
values of the culture parameters were determined as described before [5-11].

The cell growth modul consisted of a strirred reactor with a working volume of 1 liter
equipped with a sedimentation cone serving as the cell-retention device. The perfusion-mode
hybridoma cultures were carried out at the dissolved oxygen tension of 10% air saturation. The
pH was kept at the value of 7.0.

55

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 55-57.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

56

Results and Discussion

Upon reaching viable cell density cells/ml the cell growth modul was run in perfusion
mode Cell bleed volumes (0.3 -0.4 1) were withdrawn daily when the viable cell
density increased to cells/ml. The cell growth modul was runnig for several weeks
under steady-state conditions (viability 90-95%, viable cell density cells/ml, residual
glucose 8-10 mM, residual glutamine 1-2 mM, ammonia 4-5 mM, lactate 15-20 mM and Mab

concentration 80-110mg/l).
Batch cultures were initiated by supplementing the cell bleed withdrawn from the

growth modul with a volume of fresh medium and with essential amino acids (Figs. 1 and 2).
Our novel strategy changes substantially the functions of perfusion and batch modes

in the process (Table 1).

Conclusions

1. A hybridoma perfusion culture can be maintained at steady state (viability >90%, cell density
up to cells/ml) for arbitrary time period, thanks to the enrichment of the medium with
apoptosis-suppressing amino acids, and to the application of a high cell bleed rate.
2. The perfusion cell growth modul with a working volume of 1.0 1 can serve as a source of

cells available daily for initiation of short-term batch cultures.
3. Due to high initial cell density, and substantial exhaustion of nutrients in the growth

medium, the batch cultures behave as if initiated at their stationary phase.

57

4. If the medium is sufficiently enriched with the building stones ofthe Mab molecule, i.e. the
essential amino acids, and with the main energy source, glutamine, the synthesis of Mabs
proceeds at high specific rate. The exhaustion ofglutamine, but not of glucose, acts as the stop-
signal for Mab synthesis.

5. The Mab concentration in the medium harvested from the batch cultures is several-fold
higher than in the medium harvested directly from the perfusion cell growth modul.

Acknowledgement. The work was supported by the Contract No. BIO4 CT95-0207 of the
Commision ofthe EC , DGXII/ Contract No. OK120 of the Czech State Budget, and the Grant
No. I/68957 ofthe Volkswagen Foundation.

References

1. Griffiths, IB. (1992) Animal cell culture processes - batch or continuous. J. Biotechnol. 22, 21-30.
2. Büntemayer, H., Wallerius, C., and Lehmann, J. (1992) Optimal medium use for continuous high density perfusion
processes. Cytotechnology 9, 59-67.
3. Pörtner, R., Lüdemann, I. and Märkl, H. (1997) Dialysis cultures with immobilized hybridoma cells for effective

production ofmonoclonal antibodies. Cytotechnology 23, 39-45.
4. Hiller, G.W., Clark, D.S. and Blanch, H.W. (1993) Cell-retention-chemostat studies ofhybridoma cells: Analysis
of hybridoma growth and metabolism in continuous suspension culture on serum-free medium. Biotechnol. Bioeng.
42, 185-195.

5. F. (1995) Starvation-induced programmed death ofhybridoma cells: Prevention by amino acid mixtures.
Biotechnol. Bioeng. 45, 86-90.
6. F., and K. (1997) Unbalanced media for hybridoma cell culture: an alternative reality, in M.J.T.
Carrondo et al. (eds.), Animal Cell Technology. From Vaccines to Genetic Medicine, Kluwer Academic
Publishers, Dordrecht, pp. 675-680.

7. F., and Chládková- K. (1995) Apoptosis and nutrition: Involvement of amino acid transport
system in repression ofhybridoma cell death. Cytotechnology 18, 113-117.
8. F. and K. (1996) Cell suicide in starving hybridoma culture: survival-signal effect of some
amino acids. Cytotechnology 21, 81-89.
9. F.,and K. (1996) Protection of B lymphocyte hybridoma against starvation-induced apoptosis:
survival-signal role of some amino acids, Immunol. Lett. 52, 139-144.
10. F.,and K. (1997) Amino acid signaling for growth or death, in K. Funatsu et al. (eds.), Animal

Cell Technology: Basic & AppliedAspects, Vol.8, Kluwer Academic Publishers, Dordrecht, pp. 131-135.
11. F., Vomastek, T. and J. (1992) Fragmented DNA and apoptotic bodies document the
programmed way of cell death in hybridoma cultures. Cytotechnology 9, 117-123.

EXPRESSION OF RECOMBINANT HUMAN INSULIN IN CHINESE
HAMSTER OVARY CELLS IS COMPLICATED BY INTRACELLULAR
INSULIN-DEGRADING ENZYMES

S.C.O. PAK, S.M.N. , M.J. SLEIGH* & P.P. GRAY

Department ofBiotechnology and the CRCfor Biophurmaceutical Research, University

of New South Wales, Sydney 2052 Australia; of Molecular Oncology,
Westmead hospital; *Peptide Technology Limited, Sydney 2009, Australia

Abstract

To investigate the role of insulin degrading enzymes in heterologous insulin gene
expression, Chinese hamster ovary (CHO) cells were transfected with a mammalian
expression vector carrying the cDNA for the human pre-proinsulin under the control of
the RSV promoter. Stable transfectants were isolated and characterised in regard to
insulin transcription, translation, processing and secretion. Levels of insulin expressed by
these cells were extremely low, peaking at around cells/24 hours. Analysis
of the secreted product indicated inefficient processing with less than 10% of the total
product being fully processed. degradation assays revealed insulin
degrading activity in the cytosol and in the conditioned medium of CHO cells. In both
cases the insulin degrading activity was significantly inhibited by the addition of
bacitracin, a peptide antibiotic and an inhibitor of insulin degrading enzyme (IDE). No
noticeable effects were seen with the addition of a general protease inhibitor, PMSF.
When transfected CHO cultures were supplemented with small quantities of bacitracin
insulin expression improved considerably. These results combined support the
hypothesis that IDEs are directly involved in inhibiting overexpression of recombinant
human insulin in transfected CHO cells.

Introduction

Insulin is a potent mitogenic protein naturally synthesised by endocrine islet cells of the
pancreas. Although insulin expression has been extensively studies in many different
mammalian cell lines, attempts to overexpress insulin in non-endocrine cell lines have
largely been unsuccessful (Groskreutz et al., 1994; Moore et al., 1983; Quin et al., 1991;
Vollenweider et al., 1992). The reason for this is currently unclear.

Insulin degrading enzymes (IDE) are neutral thiol metalloproteinases that are involved in
specifically degrading insulin molecules (reviewed by Duckworth, 1988). When insulin
binds to it's receptors on the cell surface a wide range of metabolic/anabolic pathways are

59

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 59-67.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

60
stimulated. This is followed by endocytosis of the receptor-ligand complex to form an
intracytoplasmic vesicle called an endosome. The pH in the endosoine rapidly drops to
5.6 resulting in dissociation of the insulin-receptor complex. Although some receptors
are degraded, the majority are recycled to the cell membrane. The internalised insulin, on
the other hand, undergoes rapid degradation by insulin-specific enzymes that are present
in the cytosol. A large number of cells (including the liver, kidney, muscle, pituitary and
fibroblast) are known to be rich in IDEs.
To investigate the role of insulin degrading enzymes in recombinant insulin expression in
non-endocrine cells, CHO cells were transfected with a mammalian expression vector
carrying the human preproinsulin cDNA under the control of the RSV promoter.
Consistent with previous reports, insulin expression and processing by recombinant
CHO cells was extremely poor. Further characterisation revealed considerable insulin
degrading activity in the cytoplasm and culture supernatant of CHO cells. In this report
we provide evidence to support the hypothesis that intracellular IDEs complicate
overexpression of recombinant human insulin in transfected CHO cells.

Materials and Methods

Cell Culture. Recombinant CHO cells expressing human insulin were generated by
transfecting CHO-K1 cells (ATCC CCL-61) with a plasmid (pRSVIns) carrying the
human pre-proinsulin cDNA under the control of the RSV promoter. The basal medium
used in this paper is a custom-made preparation from Gibco (USA) which consisted of a
1:1 mix of Dulbecco's Modification of Eagle's Medium (DMEM) and Coon's F12

medium supplemented sodium selenite and is refer to in this paper as serum-
free (SF) medium. Recombinant CHO-Ins cells were cultured in serum-free medium
supplemented with human apo-transferrin (Sigma, USA) and is referred as
SF+Tf.

Sample Collection. To assess the recombinant cell lines for the production of insulin,
conditioned medium was collected from confluent T-75 cultures. Cells were grown to
confluence in 10% FCS medium, rinsed twice with PBS and incubated in 10 ml SF+Tf
for 24 hours. Cell lysates were prepared by directly applying 10 ml of lysis buffer [140
mM NaCl, 4 mM KC1, 1 mM NaPO4, 20 mM Tris (pH 7.4), 1% NP-40, PMSF] to the
cells. Culture supernatant and cell lysates were clarified by centrifugation and stored at
-20 °C until required.

Analysis of insulin mRNA in CHO cells. Cytoplasmic RNA from CHO cells were
analysed for insulin mRNA by Ribonuclease Protection Assay (Ambion, Inc., Texas) or
by Northern blot as previously described (Hunt et al., 1996).

Analysis ofinsulin. Insulin in the cell lysate and in the culture supernatant was measured
by an automated Microparticle Enzyme Immunoassay (MEIA) courtesy of Mr Kris Tan
(Royal Prince Alfred Hospital, Sydney) and by an immunoenzymetric assay using a

61
MEDGENIX INS-EASIA kit (MEDGENIX Diagnostics). Total immunoreactive
insulin (IRI) was measured by a radioimmunoassay (RIA).

degrading assay. A confluent monolayer of cells from a T-flask
was detached and washed with ice-cold PBS before centrifuging at 2000 rpm for 2
minutes to pellet the cells. The pellet was then resuspended in 3 ml ice-cold Buffer A (10
mM Tris-Cl, 10 mM KC1, 0.06% pH 8.1) and homogenised for 20
minutes on ice. The homogenate was spun at 14,000 rpm for 60 minutes at 4 °C to pellet
cellular debris. A small aliquot of the supernatant was removed for determination of
protein concentration using a BioRad protein assay kit. A of the cytosolic
fraction was preincubated with of Buffer B (125 mM potassium
phosphate, 0.25% BSA, pH 7.4) and of a protease inhibitor (PMSF: 100 mM;

Bacitracin: 350 units/ml) for 20 minutes at 37 °C. A no protease inhibitor control was
also included for comparison. After the preincubation, of
insulin [ICN] was added to each tube and incubated at 37 °C for 0, 15, 30, 50 and 80
minutes. Reactions were stopped at the specified times by addition of ice-cold
40% TCA. Following incubation on ice for 30 minutes the tubes were centrifuged for 20
minutes at 14,000 rpm. Supernatant was discarded and the radioactivity in the pellet was
measured using a Packard Auto-Gamma 5650 counter. The decrease in the radioactive
counts in the pellet indicated the extent of insulin degradation.

Conditioned medium for insulin degradation assay was prepared by culturing CHO-K1
cells in roller bottle containing 10% FCS. Upon reaching confluence, cells were
rinsed twice with PBS and incubated in 50 ml of SF medium for 24 hours. After
centrifugation, culture supernatant was preincubated with or without of 350
units/ml solution of bacitracin for 20 minutes at 37 °C. Assays were then initiated by
addition of of insulin [ICN] to each tube. After incubation at

37 °C for 0, 15, 30, 60, 90 and 120 minutes, reactions were stopped and the radioactivity
in the pellet measured as described above.

Results and Discussion

Expression of Insulin by CHO-Ins cells

To generate stable insulin-expressing cells, of pRSVIns and
DNA were co-transfected into CHO cells and exposed to of active G418 for 6
weeks. Individual clones (78) were isolated by limiting dilution and tested for insulin
expression by RIA. Consistent with previous studies (Yanagita et al., 1992;
Vollenweider et al., 1992), levels of insulin expressed were extremely low with less than
7 ng of cells/24 hours being produced by the best clone (Figure 1, A). In an
effort to delineate the factors involved in the low level expression of insulin, cytoplasmic
RNA from CHO-Ins cells were analysed by Northern blot. Insulin-specific mRNA was
detected in ail the transfected cells but not in the control indicating active transcription of
the stably integrated genes (Figure 1, B).

62
Characterisation ofsecreted insulin
To determine whether CHO cells processed insulin correctly, CHO-Ins conditioned
medium was analysed using methods which distinguish mature (fully processed) insulin
from partially processed intermediates. Results indicate that over 90% of the total
immunoreactive insulin secreted by CHO cells is partially processed, mainly in the form
of proinsulin (Figure 2). In an attempt to improve furin processing, site-directed
mutagenesis was explored to change the DNA sequence at the C-peptide/A-chain
junction of the insulin gene from Leu-X-Lys-Arg to the optimal furin cleavage sequence
Arg-X-Lys-Arg. Although cells transfected with the mutated insulin gene processed
insulin well, the overall levels of insulin expressed remained below

degradation by CHO cell extract

To investigate the possibility of intracellular insulin degradation, known amounts of
radiolabelled were incubated with of cytosolic CHO cell extracts over
a period of 80 minutes in the presence and absence of protease inhibitors (Figure 3). In
the absence of any protease inhibitors, greater than 95% of the added insulin was rapidly
degraded as measured by a decrease in radioactivity of the TCA precipitated intact insulin
(a). In the presence of PMSF, a general protease inhibitor, degradation was only slightly
inhibited (b). However, in the presence of bacitracin, a peptide antibiotic and an inhibitor
of insulin degrading enzyme (IDE), insulin degradation was almost completely inhibited
(c). These results indicate that there is substantial insulin degrading activity in the cytosol
of CHO cells and that the majority of that activity is due to IDEs.

degradation by CHO culture supernatant

To test for insulin degrading activity in the conditioned medium of CHO cells, insulin
degradation by cell-free, conditioned medium was measured at 37 °C in the presence and
absence of bacitracin (Figure 4). In the absence of any protease inhibitors, the CHO
conditioned medium degraded 80% of the added within 2 hours of
incubation (a). On the other hand, when labelled insulin was co-incubated with
bacitracin, degradation was significantly inhibited (b).

Effect of Bacitracin on recombinant insulin secretion

degradation experiments have indicated that there is significant insulin
degrading activity in the CHO cell extract and culture supernatant. To determine whether
these enzymes are involved in inhibiting expression of recombinant human insulin in
CHO cells, transfected CHO cells were cultured in SF+Tf medium with various
concentrations of bacitracin (Figure 5). In the absence of any bacitracin, CHO-Ins cells
expressed 0.13 pmoles of cells/24 hours. In comparison, the same cells
cultured in the presence of bacitracin showed improved insulin expression proportional

63
to bacitracin concentration. When bacitracin was present at a concentration of 1 mg/ml,
insulin expression was shown to improve by around 250%.

Conclusion

Recombinant insulin expression has been extensively studied in a number of different
mammalian host cell lines. Whilst endocrine cells express insulin well, attempts to
overexpress insulin in non-endocrine cells have largely been unsuccessful. Although
insulin remains as the most well studied protein, the factors influencing its expression in
non-endocrine cells have not been fully established. In an effort to investigate the role of
IDEs in heterologous insulin expression, we transfected CHO cells with a plasmid
expressing the cDNA for human preproinsulin. Active transcription of the stably
integrated insulin gene was confirmed by Northern blot analysis (Figure 1. B).
Consistent with previous studies, the levels of insulin expressed were below
cells/ 24 hours (Figure 1. A). Analysis of the conditioned medium revealed inefficient
insulin processing as indicated by the presence of insulin and proinsulin at a ratio of 1:9
(Figure 2). The inefficient processing was thought to be due to the presence of a
suboptimal furin recognition sequence at the C-peptide/A-chain junction of the insulin
molecule. Thus to improve insulin processing site-directed mutagenesis was explored to
alter the furin recognition sequence. By changing one amino acid, from leucine to
arginine, it was possible to improve insulin processing considerably (up to 100%
efficiency). However, despite this improvement, the overall levels of insulin secreted did

not improve (data not shown).

Insulin-degrading enzymes (IDEs) are enzymes involved in switching off the insulin
signal by rapidly degrading internalised receptor-bound insulin. Considerable insulin
degrading activity was detected in the cytosol of CHO cells as measured by the

degradation of insulin by the CHO cell extract (Figure 3). Analysis of the
cell extract showed presence of IDEs as determined by bacitracin inhibition of
insulin degradation. It may be possible that these enzymes are rapidly degrading newly
synthesised recombinant insulin and thus prevent the release of intact insulin. Although
pulse-chase studies would provide concrete evidence for intracellular insulin degradation,
attempts were frustrated by the low levels of insulin expressed by these cells. Instead,
evidence for the involvement of IDEs was provided by monitoring insulin expression by
cells cultured in the presence and absence of bacitracin. In the presence of bacitracin,
intracellular IDEs were inhibited resulting in improved insulin expression in a manner
dependent on bacitracin concentration (Figure 5). Investigations are currently under way
to identify the sites of IDE binding and cleavage. It is believed that introducing
favourable mutations at these sites will reduce intracellular degradation and improved
insulin expression. Some evidence for this has already been supplied by Groskreutz and
colleagues (1994). These investigators obtained better insulin expression by altering the
amino acid at position B-10 from histidine to aspartic acid. By introducing further
mutations to the insulin gene it may be possible to engineer an insulin molecule that is
completely resistant to IDE degradation.

64

65

References
Duckworth, WC (1988) Insulin Degradation: mechanisms, products and significance. Endocrine

Reviews 9: 319-345
Groskreuiz DJ, Sliwkowski MX and Gorman CM (1994) Genetically engineered proinsulin

constitutively processed and secreted as mature, active insulin. J Biol Chem 269:6241-6245
Hunt SMN, Tail AS, Gray PP and Sleigh MJ (1996) Processing of mutated human proinsulin to

mature insulin in the non-endocrine cell line, CHO. Cytotechnology 21:279-288
Moore H-PH, Walker MD, Lee F and Kelly RB (1983) Expressing a human proinsulin cDNA in a

mouse ACTH-secreting cell. Intraccllular storage, proteolytic processing and secretion on
stimulation. Cell 35:531-538
Quin D, Orci L, Ravazzola M and Moore H-PH (1991) Intracellular transport and sorting of mutant
human proinsulin that fails to form hexamers. J Cell Biol 113:987-996
Vollenweider F, Imiinger JC, Gross DJ, Villa KL and Halban PA (1992) Processing of proinsulin by
transfected hepatoma (FAO) cells. J Biol Chem 267:14629-14636
Yanagita M. Hoshino H, Nakayama K and Takeuchi T (1993) Processing of mutated insulin with
tctrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell
lines. Endocrinology 133:639-644

66 You are working with human insulin. Did you try to incorporate
Discussion hamster-type insulin as the system might work differently?
Barteling:
Pak: We have not tried that but other groups have used rats and insulin
Ozturk: from various other species. They have all found similar results.
Pak:
What happens if insulin is not added to the cells? Can you select a
Sasaki: clone that is insulin independent?
Pak:
The isolation of insulin independent mutants has been tried
Aunins: successfully. However, the growth rates were considerably slower
and since the cell line was intended for recombinant
Pak: biopharmacological production, it was essential to get a fast
Ryll: growing cell.

Pak: You add insulin to stimulate CHO cell growth or to maintain
viability. Does bacitracin stimulate cell growth?

No, bacitracin at high concentrations will inhibit cell growth. I
included it in this talk to demonstrate that insulin-degrading
enzymes were involved in preventing insulin expression in CHO
cells. We have expressed ILGFI, and that escapes degradation and
stimulates cell growth.

I want to clarify a previous answer on insulin mutants. As I
understand it, you did your selection in just G418. Did you have

insulin present when selecting your clones?

It was present.

With insulin being a secreted protein targeted to the Golgi, how do
you envisage the intracellular cytosolic activity as being responsible
for the degradation?

Although the majority of the insulin-degrading activity is found in
the cytosol, there are insulin-degrading enzymes in the cell
membrane and also in the culture supernatant. Those enzymes
were also preventing the accumulation of insulin extracellularly.

Bader: 67
Pak:
Meade: You describe how the insulin is taken up by receptors and goes
into an endosome, and is then suddenly degraded by a cytosolic
Pak: enzyme. How is that possible? Why would insulin not permeate
through the membrane? What is your definition of cytosolic?

Although insulin is internalised through vesicles, these enzymes
have been shown to be permeable and do enter and cleave these
molecules.

I am interested to know why someone is interested in making
insulin in mammalian cells since E. coli produces it so well. We
tried to do it in milk and got 20 mg/ml. The animals get in trouble
but we had no problems in getting it secreted out. So does it just
happen in a tissue culture system rather than in a natural system?

Endocrine cells are quite capable of producing insulin but,
unfortunately, CHO cells are not endocrine, therefore they lack the
necessary functions to easily secrete insulin.

HIGH YIELD EXPRESSION OF RECOMBINANT PLASMA FACTORS:
USE OF RECOMBINANT ENDOPROTEASE DERIVATIVES IN VIVO AND
IN VITRO

U. SCHLOKAT1,3, A. PREININGER1, M. HIMMELSPACH1, G. MOHR1,
B. FISCHER2, AND F. DORNER
Departments of Molecular Cell Biology1 and Protein Chemistry2,
Biomedical Research Center, IMMUNO AG, Uferstr. 15, 2304 Orth/
Donau, AUSTRIA; for correspondence3: e-mail [email protected]; fax
+43-2212-2716; phone +43-1-20100-4144

1. Abstract
Posttranslational proteolytic processing steps are often required to render proteins
mature and functionally active. However, upon high yield expression of recombinant
forms of these proteins, proteolytic processing becomes severely l i m i t i n g .
In this report, the mammalian endoprotease Furin was used in order to achieve com-
plete processing of recombinant von Willebrand Factor and Factor X precursors.
Significant processing of rvWF was found to be mediated primarily by naturally
secreted 'shed' rFurin accumulating in the cell culture supernatant, rather than intra-
cellularly, upon coexpression. Coamplification of rvWF/rFurin resulted in increases
in rvWF yield but, at best, maintenance of rFurin expression, implying that rFurin
becomes lethal to its host cell upon overexpression. In order to establish a system
allowing for the processing of large quantities of recombinant protein precursor
molecules with comparably small amounts of rFurin molecules in vitro, C-terminal-
ly truncated, epitope-tagged rFurin derivatives were constructed. The fate of these
molecules upon expression was investigated and suitable candidates were purified to
homogeneity by a one step procedure. These molecules were used for in vitro pro-
cessing in solution, in reactions consisting solely of defined components, and allo-
wing for recycling and re-use of the rFurin. Initial attempts towards the development
of a downstream processing system employing matrix bound rFurin indicate that
complete proteolytic processing of large amounts of precursor molecules by small
quantities of rFurin is feasible.

2. Introduction

High yield expression of desired biopharmaceuticals is routinely achieved by ampli-
fication of the foreign gene/cDNA copies in their host cell. Unfortunately, posttrans-
lational modifications, such as or proteolytic pro-
cessing, which frequently are required for maturation and/or exertion of functional
activity, often become limiting at overexpression.

Human von Willebrand Factor (vWF) and Factor X (FX) are glycoproteins circula-
ting in the blood which exhibit pivotal functions in haemostasis: vWF mediates adhe-

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

70

sion of platelets to the subendothelium at the site of injury, is involved in platelet-pla-
telet interactions, and stabilizes circulating Factor VIII. The conversion of FX to FXa
is a mandatory reaction in the coagulation cascade ultimately leading to the formati-
on of the fibrin clot.
vWF and FX require proteolytic removal of a propeptide for maturation; in addition,
processing of the single chain precursor to the light and heavy chains is necessary in
order to render FX functionally active. Upon overexpression of recombinant vWF1
(rvWF) and these proteolytic cleavage reactions become limiting.
The mammalian endoprotease Furin3 has recently been shown to mediate processing
of a variety of plasma proteins, e.g. rFIX and rProtein C precursors, C-terminal to the
consensus cleavage site arg - x - lys/arg - arg.
Here, we describe the construction, expression, and fate of full length and truncated
rFurin molecules and their use in order to achieve complete processing of desired
precursor molecules in vivo upon coexpression as well as in vitro.

3. Results
3.1. PROTEOLYTIC PROCESSING IN VIVO: CO-EXPRESSION OF RvWF AND
WILDTYPE RFURIN

CHO cell clones expressing rvWF were established; these cells secreted
x day. As shown in Fig. 1, only propeptide free, completely proces-
sed rvWF molecules were detectable in the conditioned medium derived from indi-
vidual cell clones. Upon dihydrofolate reductase/methotrexate mediated amplificati-
on, rvWF expression increased to x day at At this
expression level, however, propeptide removal by the endogenous proteolytic machi-
nery had become insufficient: approximately 50% of the secreted rvWF molecules
still possessed the propeptide (Fig. 1 A).

In an attempt to improve propeptide removal in intermediate yield CHO-rvWF cells,
a CHO-rvWF cell clone, secreting x day, was further transfected
with a vector mediating human wild-type, full length rFurin expression. Cell clones
isolated from this transfection exhibited different degrees of rvWF precursor proces-
sing due to their varying degrees of individual rFurin expression (Fig. 1B).

A CHO-rvWF/rFurin cell clone exhibiting complete rvWF precursor processing in
24 hour supernatants was further characterized4. Unexpectedly, significant amounts
of rvWF precursor molecules were detectable upon frequent media changes, e.g.
every 8 hours, but the precursor was absent from subsequent 24 hour supernatants
(Fig. 2, top left panel)5. In contrast, 8 hour and 24 hour conditioned media derived
from CHO-rvWF cells did not reveal any significant changes in the rvWF precur-
sormature rvWF ratio when compared to each other (Fig. 2, top right panel).

In conditioned media derived from CHO-rvWF/rFurin cells, significant amounts of
rFurin were identified (Fig. 2, lower left panel). Even though Furin resides, via its
trans-membrane domain, mainly in the trans-Golgi network, a naturally secreted
form termed 'shed' Furin has recently been identified6-8. This form results from pro-
teolytic cleavage N-terminal to its transmembrane domain. The degree of rvWF pro-
peptide removal correlated well with the amount of 'shed' rFurin in the conditioned
media. Subsequent mixing experiments employing conditioned media containing
rvWF precursor molecules and shed rFurin confirmed that the 'shed' rFurin mediated

71
rvWF propeptide removal in vitro5, contrasting previous reports by others using an
experimentally truncated and slightly larger rFurin molecule 9,10. By titration western
blotting, rFurin levels were found to exceed endogenous Furin levels by at least a
factor of 100 (not shown). Since intracellular rFurin did not noticeably contribute to
processing despite this enormous overexpression, the enzyme might either be mis-
compartmentalized or non functional/silenced, as long as it is located intracellularly,
and activated or accessible only once it is extracellular11.

A variety of attempts to establish CHO-rvWF/rFurin high yield coexpressing clones
upon rvWF/rFurin/dhfr triple transfection and subsequent amplification failed. While
the rvWF yield could be improved, rFurin expression always uncoupled, resulting in
expression of propeptide containing rvWF molecules, although at higher yields (not
shown). Presumably, the failure to increase the rFurin yield any further resulted from
rFurin becoming lethal to its host cell upon overexpression12.
The ability to process large amounts of rvWF precursor therefore requires the esta-
blishment of an efficient in vitro processing procedure.
3.2. PROTEOLYTIC PROCESSING IN VITRO: USE OF RFURIN DERIVATIVES
In order to produce sufficient quantities of rFurin for purification, a variety of C-ter-
minally truncated rFurin molecules were constructed. To test the function of these
molecules, they were transiently expressed in 293 HEK cells, as summarized in Fig.
3. Removal of the transmembrane domain that anchors Furin in the membrane of the
trans-Golgi network resulted in enhanced secretion of rFurin molecules into the cell

72
culture supernatant. Interestingly, rFurin molecules with deletions affecting amino
acids N-terminal to amino acid residue 577, i.e. the 'middle' domain

, were strongly expressed intracellularly; however, secreted
forms of these derivatives were not detected in the conditioned medium, indicating
either blockage of secretion or, alternately, rapid degradation during the secretory
process.

Expression of resulted in the secretion of two molecular
species, visualized as a double band in western blots. The lower/faster band migra-
ted at a position identical to that exhibited by shed rFurin. Thus, it can be concluded
that the cleavage site in rFurin leading to shed rFurin is located in the vicinity of and
N-terminal to amino acid 708 (not shown). When permanently expressed in CHO
cells, trans membrane domain deleted rFurin molecules generally led to 10 to 20 fold
increased amounts of secreted rFurin compared to wt/shed rFurin expression.

In order to facilitate purification, poly-histidine epitopes were added to the C-termi-
ni of the deletion mutants and separated from the residual rFurin sequence by spacers
of varying length. Interestingly, one construct in which eight histidines were substi-
tuted with a threonine/isoleucine rich stretch of amino acids was not secreted any
longer. Thus, very few amino acids at the C-terminus may decide upon the molecu-
les' fate and/or intracellular destination.

cell clones were established, and
from serum free conditioned media was purified to homogeneity
on a matrix by a one step procedure as seen in Fig. 4A. The peak functio-
nal activity, determined by a fluorogenic Furin specific substrate test5, and the Furin
immunoreactive material in a western blot coincided with the 200mM and 500mM

73
imidazole eluates; Furin is the only protein band in these fractions, as visualized by
silver staining.

After dialysis to remove the imidazole, purified and
rvWF precursor were mixed in a buffer containing ions. Under these defined
minimal conditions, rvWF precursor was successfully processed (Fig. 4B). A mock-
purified preparation from unmanipulated CHO cells did not mediate rvWF precursor
processing.The polyhistidine tail allows for easy re-purification of the rFurin deriva-
tive upon completion of the processing reaction. Thus, this enzyme can be 'recycled'.
In this manner, small quantities of rFurin can be used for the maturation of large amo-
unts of recombinant target molecules.

Similarly, both rFX propeptide removal and the processing of the single chain pre-
cursor to the heavy and light chains were successfully performed by rFurin derivati-
ve in vitro. Strikingly, the rFX propeptide, which does not contain a typical Furin
site, is cleaved by a different endoprotease, as determined by expression in Furin
deficient CHO cells and subsequent purification2.

With the aim of establishing an industrial scale in vitro processing procedure, the
histidine tagged rFurin molecules were tested for their functional activity when
bound to the resin. In iFtiuarlinexsppeecriifmicenftlsuoeromgpelnoiycinsgubsmtraattrei5x bound
and a demon-
strated successful substrate conversion and, hence, the feasibility of this approach.
Optimization of the length and sequence of the spacer between the epitope and the
residual rFurin sequence, and choice of resin, e.g. tentacle-gels, will allow proteoly-

74
tic maturation of large amounts of incompletely processed recombinant protein pre-
cursors by small quantities of rFurin.

4. Summary
In high yield CHO-rvWF cell clones, removal of the propeptide from rvWF precur-
sors was incomplete. Additional wild-type, full length rFurin expression in these
cells improved processing. Unexpectedly, complete rvWF precursor processing was
mediated by a naturally secreted form of rFurin, termed 'shed' rFurin, in the condi-
tioned medium. The contribution of intracellular wild-type rFurin was limited despi-
te tremendous overexpression of this enzyme compared to endogenous Furin.
Attempts to further improve rFurin expression by amplification did not meet with
success, presumably due to toxicity of rFurin to its host cell upon overexpression. In
contrast, rvWF amplification could successfully be demonstrated.
In order to achieve complete processing of large amounts of rvWF precursor with
comparably small amounts of rFurin, the use of rFurin for in vitro processing was
investigated. A variety of C-terminally truncated and, hence, secreted, epitope-tagged
rFurin derivatives were constructed and expressed. The extent of the truncation, the
type and length of the spacer between the residual rFurin sequence and the epitope,
and the amino acids constituting the new C-terminus determined the fate of the
rFurin derivatives upon expression and their individual properties. Using a one step

75

procedure, rFurin derivatives were purified to homogeneity and used in in vitro
recombinant target protein precursor processing reactions consisting solely of defi-
ned components. Preliminary experiments have demonstrated the feasibility of resin-
bound rFurin mediated in vitro processing.

5. Acknowledgements

We are grateful to Wim van de Yen, John Creemers, and Stephen Leppla for materi-
al, Gary Thomas, Nabil Seidah, Anton Roebroek and Wolfgang Garten for discussi-
ons, and Carol P. Gibbs for critical reading of the manuscript.

6. References

1. Fischer B, Mitterer A, Schlokat U, DenBouwmeester R, Dorner F (1994). Structural analysis of rceom-
binant von Willebrand factor: identification of hetero- and homonmultimers. FEBS Lett. 351: 345 - 348.
2. Himmelspach M, Pfleiderer M, Fischer B, Antoine G, Gritschenberger W, Falkncr FG, Schlokat U,
Dorner F. Recombinant Human Factor X: High Yield Expression And Maturation By Furin Mediated In
Vitro Processing. Submitted.
3. Van de Ven WJM, Voorberg J, Fontijn R, Pannekoek H, van den Ouweland AMW, van Duijnhoven HLP,
Roebroek AJM, Siezen RJ (1990). Furin is a subtilisin-likc pro-protein processing enzyme in higher
eucaryotes. Mol. Biol. Rep. 14: 265 - 275.
4. Fischer BE, Schlokat U, Mitterer A, Reiter M, Mundt W, Turecek PL, Schwarz. HP, Dorner F (1995).
Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of
transformed CHO cells co-expressing rccombinant furin. FEBS Lett. 375: 259 - 262.
5. Schlokat U, Fischer BE, Herlitschka S, Antoine G, Preiningcr A, Mohr G, Himmelspach M, Kistncr O,
Falkner FG, Dorner F (1996). Production of highly homogeneous and structurally intact recombinant von
Willebrand Factor multimers by furin mediated pro-peptide removal in vitro. Biotechnol. Appl. Biochem.
24: 257 - 267.
6. Vey M, Schäfer W, Berghöfer S. Klenk HD, Garten W (1994). Maturation of the trans-Golgi Network
Protease Furin: Compartmentalization of Propeptide Removal, Substrate Cleavage, and COOH-terminal
Truncation. J. Cell Biol. 127: 1829 - 1842.
7. Molloy SS, Thomas L. VanSlyke JK, Stcnherg PE, Thomas G (1994). Intracellular trafficking and
activation of the furin proprotein convertase: localization to the TGN and recycling from thc cell surface.
EMBO J. 13: 18 - 33.
8. Vidricaire G, Denault JB, Leduc R (1993). Characterization of a secreted form of human furin endo-
protease. Biochem Biophys. Res. Comm. 195: 1011 - 1018.
9. Rehemtulla A, Kaufman RJ (1992). Preferred Sequence Requirements for Cleavage of Pro-von
Willebrand Factor by Propeptide-Processing Enzymes. Blood 79: 2349 - 2355.
10. Rehemtulla A, Dorner AJ, Kaufman RJ (1992). Regulation of PACE propeptide-processing activity:
Requirement for a post-cndoplasmic reticulum compartment and autoproteolytic activation. Proc. Natl.
Acad. Sci. USA 89: 8235 - 8239.
11. Anderson ED, VanSlyke JK, Thulin CD, Jean F, Thomas G (1997). Activation of the furin endopro-
tease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J.
16: 1508 - 1518.
12. Creemers JWM (1994). Structural and Functional Characterization of the Mammalian Proprotein
Processing Enzyme Furin. Ph.D. Thesis at the University of Leuven, Belgium.

76 The over-expression of furin seems to result in some sort of
Discussion secretion of the protease in the medium. Do you have any idea
Massie: why; for instance, is the Golgi saturated or is some other type of
event occurring?
Schlokat:
There is a cleavage site. It is not an auto-proteolytic cleavage by
Massie: furin. If you express wild type furin at some point it is a natural
Schlokat: process for furin to be secreted from the cell. In CHO cells even
Pak: without the expression of any additional furin, just endogenous
Schlokat: furin, you have secretion of furin-like activity which mediates the
processing of furin-like substrate in the conditioned medium. So it
is just part of a natural process.

Have you considered using an inducible system to over-express
furin at sufficient levels to overcome the toxicity effect?

It is an alternative but we think it is better to do it just in vitro.

My experience is that furin is expressed intracellularly. Could the
detection of furin in the supernatant be a result of cell lysis rather
than secretion?

I do not think so as there are such massive amounts of furin in the
supernatants, even when you grow cells at low densities where
there is no detectable lysis. It is not the wild type furin but a
shorter molecule. The trans-membrane domain of the wild type
furin molecules is missing. If what you suggest is happening, you
would see the whole molecule including the trans-membrane
domain. So I do not think it is lysis.

THE ROLE OF CELL CYCLE IN DETERMINING LEVELS OF GENE
EXPRESSION IN CHO CELLS

D.R. LLOYD, V. LEELAVATCHARAMAS, D.C. EDWARDS,
A.N. EMERY & M. AL-RUBEAI
Centre for Bioprocess Engineering, School of Chemical Engineering,
University ofBirmingham, Edgbaston, Birmingham B15 2TT, U.K.

1. Abstract

Understanding relationships between cell cycle and protein expression is critical as a

means to optimising media and environmental conditions for successful commercial

operation. Using flow cytometry and centrifugal elutriation together with
synchronisation techniques, the possible role of CHO cell cycle dependent features in
product expression was evaluated. The production of . was found to be time
dependent. Maximum specific productivity was achieved at the mid-exponential phase
of batch culture and was always associated with maximum cellular concentration of

protein and a high proportion of S phase cells. However, in experiments using sorted

S and phase cells the apparent correlation between S phase and productivity was

shown not to be causal since there was no significant difference in specific productivity

between the different cell cycle phases.

2. Introduction

Growth of mammalian cell cultures is determined by cell cycle duration, cell death rate
and the number of dividing (mitotic) cells. The cell cycle state of a culture can be
deconvoluted by flow cytometry. Although the duration of the cell cycle or its
individual phases may vary between different cell lines, cell cycle events always occur
in sequence and checkpoints ensure that if preceding events are not completed
correctly, the cell is arrested and will eventually die. Defining the relationship between
cell cycle phase and protein expression is crucial to understanding the dynamics of
product elaboration which, in turn, must be incorporated in culture protocol design for
successful commercial operation. The relationship between cell cycle and protein
expression varies with cell line and gene expressed. The aim of this work is to
determine the relative effects of cell cycle phase and culture environment on the
(specific) productivity of recombinant CHO cells.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

78
3. Materials and Methods

Recombinant CHO cells were grown in stirred batch suspension cultures at 37°C; CHO
320, producing human was grown in RPMI 1640 & 5% FCS & 1 µM
methotrexate (MTX); CHO tF70R, producing human tissue plasminogen activator
(t-PA), was grown in a proprietary serum free medium (Biopro 1, BioWhittaker,

Belgium). Both constructs use the SV40 promoter/enhancer.

Cultures examined were either synchronised by late exponential nutrient deprivation or
phase enriched fractions separated using a Beckman JE-6B centrifugal elutriation
system with a standard chamber.

Product was measured by ELISA; using Genzyme DuoSeT kit product number 80-

3932-00 for and Biopool Imulyse tPA kit product number 101005 for tPA.

The DNA content of propidium iodide (PI) stained, RNAse treated, ethanol fixed cells

was measured by flow cytometry (Coulter EPICS Elite). Cell cycle analysis was

performed using Multicycle software (Phoenix Flow Systems). Cell size was

determined either by Coulter principle using a Coulter Multisizer or as relative to flow
cytometric forward angle light scatter (FS). After staining formaldehyde fixed cells with
FITC conjugated anti-human intracellular was determined as

the ratio measurement of FITC: FS by flow cytometry.

4. Results

4.1. CHO 320 SYNCHRONIZED BY NUTRIENT DEPRIVATION

Intracellular interferon concentration & specific productivity profiles follow that of
percentage of S phase cells but also follow the growth curve. Thus, intracellular
interferon concentration and specific productivity are related both to the cell cycle and
culture growth phases.

4.2. APPLICATION OF CENTRIFUGAL ELUTRIATION TO THE STUDY OF
CELL CYCLE RELATED PRODUCTIVITY

Cultures separated into fractions on the basis of size, allowing preparation of phase
enriched samples which all have the same culture history and whose physiology has not
been disturbed by nutrient starvation or chemical synchronisation techniques.

4.3. PRODUCTIVITY OF ELUTRIATED FRACTIONS

Elutriated CHO tF70R fractions were cultured for 4 hours and the specific
productivity/cell/ hour was determined (Figure 1). Preliminary observations that

79
productivity was related to cell cycle phase proved groundless on further investigation.
No fraction was shown to have a specific productivity which was significantly different
(p=0.03) from that of any other fraction. Overall, the mean specific productivity was 43
fg/cell/hr. These results also indicate that the SV40 promoter in the construct has not
influenced the regulation of any cell cycle dependant protein production mechanism.

5. Conclusions
Gene expression (intracellular product concentration) may be partly cell cycle related
because it varies with cell cycle phase and with batch phase; the same was thought to be
true of specific productivity. However this work, using centrifugal elutriation, has
shown that the cell cycle phase of a population has no significant effect on the specific
productivity of that population. The apparent cell cycle association seen previously in
batch culture is thus shown to be a coincident effect of batch phase transition from lag
to exponential, rather than a causal relationship. Thus it must be the batch phase,
possibly due to the environmental conditions (medium condition), which is the major
determinant of recombinant CHO productivity. Further work will confirm these
observations and clarify the effect of different promoters on cell cycle/size related
specific productivity.
6. Acknowledgements
This work is supported by the BBSRC UK and EC Framework IV programme.

HETEROGENEITY WITHIN DHFR-MEDIATED GENE AMPLIFIED
POPULATION OF CHO CELLS PRODUCING CHIMERIC ANTIBODIES

No Soo Kim, Sang Jick Kim, and Gyun Min Lee
Department of Biological Sciences, Korea Advanced Institute of Science and Technology,
373-1, Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea

ABSTRACT
Recombinant Chinese hamster ovary (CHO) cells expressing a high level of chimeric antibody were
obtained by cotransfection of heavy and light chain cDNA expression vectors into dihydrofolate
reductase (dhfr) deficient CHO cells and subsequent gene amplification in medium containing stepwise
increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, and 1.0 µM. In order to determine
the clonal variability within amplified cell population in regard to antibody production stability, 20
subclones were randomly isolated from amplified cell population at 1 µM MTX and their antibody
production stability was characterized. During 8 weeks of cultivation in the absence of selective
pressure, the specific antibody productivity decreased significantly. However, the relative extent
of decrease in was varied among subclones, ranging from 30 % to 80 %, showing that clonal
analysis allows one to screen the clone with better stability. Southern and Northern blot analyes
showed that this decreased resulted mainly from the loss of amplified immunoglobulin (Ig) gene
copies and their respective cytoplasmic mRNAs. Furthermore, since the stability of subclones
regarding is not related with either their or Ig gene copies at the beginning of long-term culture,

their stability during long-term culture cannot be predicted based on theirinitial or Ig gene copies.

INTRODUCTION
The degree of gene amplification, in most cases, is proportional to the level of gene expression (Pensde
et al., 1992). When subjected to successive rounds of selection in medium containing stepwise

increments in methotrexate (MTX) concentration, recombinant CHO (rCHO) cells with specific
antibody productivity as high as were obtained by Page and Sydenham
(1991).
Not only the expression level but the stability of cell lines used is critical for large-scale production of
recombinant antibodies. When drug-resistant cells are propagated in the absence of the selective agent,
the amplified genes may be maintained or lost. Changes in rCHO populations after extended culture in
the presence and absence of selective pressure have been reported (Kaufman et al., 1985; Sinacore et
al., 1995; Weidle et al., 1988; Zettlmeissl et al., 1987). Most of these studies were based on the average

cell population. However, CHO cells become quite heterogeneous in regard to in the course of
dhfr-mediated gene amplification, though they are derived from a single parental clone (Kim et al.,
1997). Likewise, their stability during long-term culture may also be heterogeneous. If so, the clonal
analysis of the entire cell population allows one to screen the clone with better stability. However, it
requires extensive efforts. If the initial properties of clones such as specific growth rate (µ) and

gene copy numbers help predict their stability, lots of efforts in screening a stable clone will be saved.

MATERIALS AND METHODS

Cell line and Cell Culture
Parental CHO cells expressing a chimeric antibody directed against the S surface antigen of hepatitis B
virus (HBV) were made by cotransfecting the light and heavy chain expression plasmids into dhfr
CHO cells (DG44). Amplified 20 subclones investigated in this study were randomly isolated from

high producer CS 13-1.0 resistant at 1.0 µM MTX using limiting dilution method.
For long-term culture in the absence of MTX, 20 subclones were cultivated as monolayer in 6 well
tissue culture plates containing 3 mL of with 5 % dialyzed FBS in mixture,
humidified at 37°C. The viable cell concentration were determined by the trypan blue dye exclusion
method and samples for antibody, nutrient, and metabolic assays were taken every other passage and
kept frozen at -80 °C.

Chemical and Antibody Assays
Glucose and lactate concentration were measured using a glucose/lactate analyzer (Yellow Spring
Instruments, Model 2300 STAT) and secreted chimeric antibody was quantified using an ELISA

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O.-W. Merten el al. (eds.), New Developments and New Applications in Animal Cell Technology, 81-83.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

82

Flow Cytometry
Intracellular antibody content was determined using a FACScan flow cytometer (Becton Dickinson).
FITC-labeled goat anti-human IgGs specific for heavy chain was used to stain the cells.

Southern Blot Analysis
Chromosomal DNAs of subclones at the beginning and end of long-term culture were prepared using

protocols described in kit manual (NEB). Heavy and light chain cDNAs (1,600 and 900 bp,

respectively) were used as templates for heavy and light chain probes, respectively. The probes were

labeled by randomly primed incorporation of DIG-labeled deoxyuridine-triphosphate.

Prehybridization, hybridization, and subsequent colorimetric detection were conducted as described in

the DIG user's guide (BM). The gene copy number was determined by comparison of band intensities

on each Southern blot using a UltraScan laser densitomer.

Northern and Slot Blot Analysis
For Northern and slot blottings, cytoplasmic mRNA was isolated from each subclone at the beginning
and end of culture using NP-40 method. For analyses, 5 µg mRNAs of each subclone were loaded and
other procedures were performed as described in Southern blotting.

Evaluation of Specific Growth Rate, Specific Antibody Productivity, and Specific Glucose

Consumption Rate and Lactate Production Rates
Specific growth rate (µ), specific antibody productivity and specific glucose consumption

and lactateproduction rates were evaluated as described earlier.

RESULTS AND DISCUSSION

1. Changes in µ and of Subclones during Long-term Culture in the
Clonal analysis shows that CS13-1.0 cells became quite heterogeneous in regard to µ and

course of dhfr-mediated gene amplification, though they were derived from a single clone. No
significant change in µ was observed during long-term culture. However, the of all 20 subclones

significantly decreased during the culture. The average of 20 subclones at the end of long-term

culture was which is approximately 47 % of the initial average The
antibody production characteristics were varied among the subclones, though the of all 20
subclones decreased during the culture. The of A6, which was most stable, gradually decreased

for first 20 days and thereafter was stabilized at The of D1, which was
most unstable, gradually decreased throughout the culture and was at the end of
long-term culture.

2. Characterization of Subclones by Southern Blot Analyses
In order to investigate whether clonal variability with respect to production stability is related to the

variation of changes in immunoglobulin (Ig) gene copy numbers of subclones during culture, genomic

DNA isolated from 20 subclones at the beginning and end of long-term culture was characterized by
Southern blotting. With heavy and light chain probes, 2.3 kb and 7.1 kb major band were detected,
respectively. The relative gene copy numbers of all 20 subclones were summarized in following table.
In general, the decrease in of most subclones except for a few subclones was mainly due to the loss

of heavy and light chain gene copies during long-term culture. However, the extent of decrease in gene

copy numbers was varied significantly among the subclones, suggesting that the initial gene copy

numbers of subclones do not help predict their long-term production stability.

3. Characterization of Subclones by Northern and Slot Blot Analyses
Northern and slot blotting were carried out in order to quantify the relative heavy and light chain

mRNA content of subclones at the beginning and end of long-term culture. In Northern analysis, the

major bands of heavy and light chain mRNA were positioned at the similar size of 18s rRNA and

below 18s rRNA, respectively. On the whole, a trend obtained in the quantification of the heavy and

light chain mRNA content of subclones was similar to that in

4. Characterization of Antibody Production Stability of Subclones by Flow Cytometry
In order to assess the degree of population heterogeneity after long-term culture, the intracellular
antibody content of each subclone at the beginning and end of long-term culture was measured by flow
cytometry. The changes in intracellular antibody contents of stable A6 and B3 subclones after long-
term culture were insignificant. On the other hand, the intracellular antibody content of unstable D4
and C5 subclones decreased significantly. Accordingly, the changes in intracellular antibody contents
of subclones correlate with those in suggesting the potential of flow cytometric measurement of

intracellular antibody as a useful tool determining production stability during long-term culture.

83

CONCLUSIONS
1. CHO cells become quite heterogeneous in regard to antibody production stability in the course of

dhfr-mediated gene amplification, though they are derived from a single parental clone
2. Clonal analysis enables one to screen the stable clone.
3. The antibody production stability of each clone cannot be predicted based on itsinitial andIg

gene copy numbers.
REFERENCES
1. Kaufman. R.J., Wasley, L.C., Spiliotes, A.J., Gossels, S.D. Latt, S.A., Larsen, G.R., Kay, R.M.

1985. Coamplification and coexpression of human tissue-type plasminogen activator and murine
dihydrofolate reductase sequences in Chinese hamster ovary cells. J. Mol. Biol. 5:1750-1759.
2. Pendse, G.J., Karkare, S., Bailey, J.E. 1992. Effect of cloned gene dosage on cell growth and
hepatitis B surface antigen synthesis and secretion in recombinant CHO Cells. Biotechnol. Bioeng.
40: 119-129.
3. Page, M.J., Sydenham, M.A. 1991. High level expression of the humanized monoclonal antibody
Campath-lH Chinese hamster ovary cells. Bio/Technology 9: 64-68.
4. Sinacore, M.S., Brodeur, S., Brennan, S., Cohen, D., Fallon, M., Adamson, S.R. 1995. Population
analysis of a recombinant Chinese hamster ovary cell line expressing recombinant human protein
cultured in the presence and absence of methotrexate selective pressure, pp 63-67. In: E.C.
Beuvery, J.B. Griffiths, and W.P. Zeijlemaker (eds.), Animal cell technology, Kluwer Academic
Publishers, Dordrech.
5. Weidle, U.A., Bucke, P., Wienberg, J. 1988. Amplified expression constructs for human tissue-type
plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective
pressure. Gene 66:193-203.
6. Zettlmeissl, G., Ragg, H., Karges, H. 1987. Expression of biologically active human antithrombin
I I I in Chinese hamster ovary cells. Bio/Technology 5: 720-725.

IMMUNOFLUORESCENCE DETECTION OF RECOMBINANT MONO-
CLONAL ANTIBODY AS A TOOL IN THE CHARACTERIZATION OF
CHINESE HAMSTER OVARY CELL LINES

CHERYL ANN S. KINNEY, ROBIN E. TRALA, LINDA C.
HENDRICKS, and TIMOTHY D. HILL

SmithKline Beecham Pharmaceuticals, Biological Process Sciences, PO
Box 1539, King of Prussia, PA 19406, USA

1. Abstract

A highly sensitive and specific method was developed for direct immunofluorescence
analysis of recombinant Chinese Hamster Ovary (rCHO) cells expressing IgG
monoclonal antibody (mAb). Fluorescence signal-to-noise ratio using
labeled goat anti-human IgG was improved through optimization of fixation, staining
and mounting protocols. These improvements enabled measurement of relative cell to
cell differences and the detection of non-producing cells when they represented only
2.5% of a mixed cell population. Heterogeneity of fluorescence signal with both intense
and low-staining cells was noted for some rCHO cell lines. Low mAb-specific signal
was unrelated to cell viability since low-staining cells excluded the viability indicator
ethidium monoazide bromide. Furthermore, positive staining of the soluble ER resident
protein BiP in dual labeling studies with anti-mAb reagents indicated that low level
staining was not an assay artifact related to poor access of fluorescent antibody into
secretory compartments. Correlations of signal heterogeneity to long-term culture
stability and process performance is under investigation.

2. Introduction

CHO cells are a convenient expression system for the generation of protein
biopharmaceutical therapeutic agents. Productivity levels for monoclonal antibodies
(mAb) achieved in rCHO effectively compete with traditional hybridoma/myeloma cell
systems, thus enabling rCHO to serve as an economical antibody production system. In
addition, although rCHO contain non-infectious retroviral-like particles, they have been
extensively characterized and found free of adventitious viruses, making them a safe
substrate for production of biopharmaceuticals.

Cell lines developed for industrial processes often vary in productivity and clonal
stability. Current methods for evaluating rCHO cells rely on whole population

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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measurements, such as supernatant titer by HPLC or ELISA, and typically require
optimization of growth conditions and qualified assays, while providing no information
about homogeneity of cell populations. Immunofluorescence microscopy methods were
developed to rapidly profile individual cells within a population, regardless of culture
conditions. Through use of immunofluorescence, information critical to selection of the
lead process cell line may be available earlier in the development process, before costly
development resources are committed.

3. Methods

3.1 CELL LINE

Suspension-adapted host CHO cells (DG-44 clone as described by Urlaub et. al., 19831)
and genetically modified recombinant CHO (rCHO) were maintained in SB proprietary
medium at a minimum culture viability of 85%. The gene-minus control cell line was
generated by transfection with plasmids lacking antibody genes.

3.2. CELL PREPARATION, FIXATION AND PERMEABILIZATION

3.2.1. Slide Preparation
HTC™ coated slides (Cel-Line Assoc., New Field, NJ, 4mm wells) were pre-treated for 2
min at room temperature with 2% (v/v) 3-amino-propyl silane (Sigma Chemical , St.
Louis, MO) diluted in acetone, washed 3X in water for injection (WFI), then dried 2hr at
37°C.

3.2.2. Cell Attachment
Cells were washed 1X with Dulbecco's phosphate buffered saline (PBS), then
cells were added to slide wells and incubated in a humidified environment for 30
minutes at 37°C. PBS was carefully aspirated prior to fixation.

3.2.3. Fixation/Permeabilization
Cells were fixed for 15 minutes at room temperature in 1% buffered

paraformaldehyde (ultrapure, Polysciences,Inc.,Warrington, PA), washed twice
in PBS, then permeabilized by 2 minute treatment with 0.2% Triton X-100 in
PBS, followed by a 5 minute wash in PBS. To reduce background signal
associated with free aldehydes, cells were treated for 5 minutes in 0.1M glycine
(J.T. Baker, Phillipsburg, NJ) in PBS, followed by two washes in PBS. To
enhance reagent uptake, cells were dehydrated stepwise in ethanol (50%, 70%,
95%, and 100%) at room temperature. Slides were air dried prior to addition of
probe.

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3.3. IMMUNOFLUORESCENCE STAINING

3.3.1. Antibodies and Reagents
Cy3-conjugated goat anti-human IgG (Fc specific) was obtained from Sigma
ImmunoChemical, St. Louis, MO. Cy2-conjugated goat anti-human IgG (Fc specific)
and Cy3-conjugated goat anti-mouse IgG (H&L) were obtained from Jackson

ImmunoResearch, West Chester, PA. Mouse anti-rat Grp78(BiP) was obtained from
StressGen Biotechnologies, Victoria, BC. Ethidium monoazide bromide (EMA), a
fluorescent photoaffinity label used as a viability indicator, was purchased from
Molecular Probes, Eugene, OR. Cy3 and Cy2 are registered trademarks of Amersham

International, Inc.

3.3.2. Direct Detection of Intracellular Antibody
Cy2- or Cy3-conjugated anti-IgG probes were diluted in PBS containing 1% heat-

inactivated (65°C, 1hr) fetal bovine serum (FBS). Probe was applied to permeabilized
samples on slides and incubated 30 minutes at 4°C in the dark, followed by wash in 1%

FBS at 4°C, wash in cold PBS, and final wash at room temperature in WFI.

3.3.3. DNA staining with Ethidium Monoazide Bromide(EMA)
EMA is a fluorescent photoaffinity label that after photolysis, binds covalently to nucleic
acids in cells with compromised membranes.2 Viability assessment with EMA was
performed prior to cell attachment and fixation. Cells were transferred to polypropylene
plates (Costar) on ice and incubated 15 min with 4ug/ml EMA dissolved in PBS, then
exposed at 15 cm under a 13 watt fluorescent light source to activate EMA. Following

exposure, cells were washed 2X in PBS, and processed for attachment, fixation,
permeabilization and immunofluorescent staining.

3.3.4. Indirect Detection and Dual Labeling
Permeabilized cell samples were incubated 30 minutes at 4°C with anti-BiP antibody

diluted in PBS containing 1% heat-inactivated FBS, then washed 2X in 1% FBS/PBS.

Samples were incubated an additional 30 minutes at 4°C with secondary Cy3-anti-mouse
IgG antibody, washed 2X in cold PBS and 1X in room temperature WFI.

3.3.5. Mounting of coverslips

Residual WFI was removed from processed slides, then samples were mounted under

coverslips using AntiFade reagent (Molecular Probes, Eugene, OR) according

to manufacturers instructions. Slides were stored in the dark under low-humidity.

3.4. IMAGE ANALYSIS

Images were acquired using a Nikon Optiphot epifluorescence microscope fitted with

fluorophore-specific filter sets from Omega Optical (Brattleboro, VT), and captured at

1000X magnification using an IS-2000 CCD camera system and software
(Alpha Innotech, CA).

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4. Results

Immunofluorescence methods were developed to generate high resolution images of
rCHO cell intracellular targets. Fluorescent-labeled antibodies specific for the Fc portion
of human IgG were used to detect intracellular mAb in the secretory pathway of CHO
cells. Figure 1A and C illustrate the high signal specificity of anti-mAb probes within
secretory pathway compartments exemplified by fluorescent staining pattern typical of
endoplasmic reticulum and Golgi elements. Non-transfected parental CHO or gene-
minus rCHO generated by transfection with plasmids lacking mAb genes were tested as
negative controls and appeared as faint opaque spheres, lacking any definition of
intracellular membranes and requiring extended exposure times for visualization (Fig
1B and D).

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The limit of detection was determined by mixing up to 50% gene-minus negative control
cells with CHO cells expressing mAb (Fig.2). Data demonstrated that the method is
highly sensitive, capable of detecting a low level of negative cells (2-3 per 100) within a
population, and that fluorescence intensity differences are readily distinguished across a
population.

Clonally selected rCHO cells derived from lines genetically engineered to secrete mAb
were characterized at the single cell level using anti-mAb immunofluorescence.
Subpopulations of cells weak in fluorescence intensity, ranging from 5-50% were

observed in various rCHO cell lines (Fig. 3). Low staining cells show structural patterns
consistent with higher intensity cells(Fig. 3B). Detection of subpopulations varying in
mAb expression suggests that lines are unstable and /or no longer clonal. Thus, this
technique may be useful, particularly early in development, to support cell line selection.