12_ANIMAL CELL TECHNOLOGY_707

435

binding activity is presented as the anti-D titre determined by haemagglutination performed

according to Whitson (1985). The percentage of active antibody was calculated by

subtracting the mean antibody content of supernatant contacted with from the mean

content of supernatant contacted with and dividing this figure by the latter and

multiplying by 100. The antibody content was determined by IgG ELISA after purified

anti-D was mixed with either washed cells and incubated at 37°C for 3 hr. The

suspension was centrifuged at 2000 g for 10 min to pellet red cells. Functional (Fc-
dependent) binding activity was determined by measuring monocyte-mediated antibody

dependent cellular cytotoxicity (ADCC) performed according to Kirkwood et al., (1993).

In addition resetting with U937 monocyte-like cells was determined as described by
Krumpel and Hadley (1990). The analysis of monosaccharide and sialic acid content was

performed as described by Cant et al., (1994).

Results and discussion
The effects of the different purification schemes on antibody yield and purity are shown

in Table 1. The antibody yield was clearly highest using the protein G purification scheme
but highest purity was achieved using protein A. The Fab and Fc activities of antibodies

generated by different purification schemes are shown in Table 2. It was noted that the
purification scheme did affect the amount of antibody in the final purified product which
was bound to antigen. The HiC (2 stage) and combined methods produced the highest Fab-
dependent activity and the anti-human IgG-agarose the lowest activity. In terms of Fc-
dependent activity rosetting activity was highest in the anti-human IgG-agarose
preparation. The increase in functional activity compared to the raw superantant pool

implies that a preferential separation was occurring and this most likely reduced the

proportion ofaglycosyl (and hence functionally inactive) molecules in the purified product.
The effect may have been more subtle however, with more active species being

preferentially separated from less active ones. The monosaccharide composition of the
purified products was then analysed to determine which ofthese mechanisms was the most
likely. It was concluded that the amount of aglycosyl anti-D present was the main
determinant of the overall sugar content and was the most likely explanation for the
increase sugar content of the combined HiC/lon X/GF-purified anti-D. This scheme also

separated certain sugar species over others generating a product with a lower sialic acid

content and appeared the most applicable for production scale applications although its cost
would be a disadvantage compared to either protein A or G. In conclusion the purification
system did affect the Fab and Fc activities and is clearly an issue which must be addressed
in designing a manufacturing process.

References
Cant, D., et al. (1994) Cytotechnology, 14, 223-228.
Kirkwood, J., et al. (1993) Trans. Med. 3, 269-273.

Krumpel, B.M. and Hadley, A.G. (1990) Mol. Immunol. 27 (3) 247-256.
Whitson, K.J. (1985) Aust. J. Med. Lab. Sci., 6, 106-109.

INTERFACING OF PROTEIN PRODUCT RECOVERY WITH AN INSECT
CELL BACULOVIRUS PRODUCTION SYSTEM

I.A. CARMICHAEL, M. AL-RUBEAI AND A. LYDDIATT
Biorecovery Group, Centre for Bioprocess Engineering, School of
Chemical Engineering, University of Birmingham, Edgbaston,
Birmingham B15 2TT, U.K.

1.Abstract

Conventional protein recovery requires several unit operations to clarify, concentrate
and isolate the product, resulting in high processing costs, extended separation times
and reduction in molecular yield. Any foreshortening in this purification process will
therefore be beneficial. Interfacing of fluidised bed adsorption with production systems
achieves this goal, negating the need for problematic centrifugation and filtration
steps. Direct processing of an unclarified insect cell-Baculovirus -galactosidase
feedstock with a custom made, fluidisable adsorbent has facilitated successful
clarification, 10-fold concentration and purification, and greater than 30%
improvement in capture efficiency over conventional purification operations.

Keywords: Fluidised Bed, Product Recovery, -galactosidase.

2. Introduction

Animal cell culture is commonly used for the production of complex, high-value
protein therapeutics, often characterised by extensive post-translational modification.
Regulatory approval requires stringent levels of reproducible purity and defined
molecular integrity. To achieve such demands, product purification conventionally
uses many unit operations to clarify, concentrate and isolate the product. However, this
is disadvantaged by high capital, space and time investments1, reduction in yield (due
to multistep operations) and concomitant product degradation. Rapid purification
would therefore confer benefits of reduced costs and increased product quality.
Realisation of this goal has been achieved by replacing centrifugation and
microfiltration steps in the purification process with fluidised bed adsorption2. The
fluidisable adsorbent employed herein facilitates capture, concentration and
purification of product, without the need for centrifugation or filtration of feedstock,
which is mandatory for conventional chromatography and contribute to product losses
(see Figure 1).

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

438

3. Materials and Methods

SF9 cells were grown in suspension at using TC-100 + 5% FCS. Infection was

achieved by resuspension of cells, at late log phase, in fresh media and addition of

recombinant Baculovirus lacZ-ACMPV at a multiplicity of infection (m.o.i.) of 10.
Protein concentrations were determined by the method of Bradford3, -galactosidase
assays were performed using a modification of the method of Miller4. The fluidisable

affinity matrix was constructed by CDI activation of Macrosorb-K4AX (Phase

Separations Ltd) and sequential derivatisation with 3,3’-iminobis-propylamine

(DADPA), glycidol and the affinity ligand p-aminobenzyl-1-thio -D-
galactopyranoside (ABTG)5. The matrix was designated Macrosorb-ABTG. Fixed bed

operation involved loading and elution at a linear flow rate of 0.22cm/hr. Desorption
was achieved by a step of 0.1 M borate buffer, pH 10, followed by immediate desalting
in Sephadex G50. Fluidised bed operation was achieved by recirculation of unclarified

feed upwards through a custom-built fluidised contactor (dimensions 20x2.2cm),

containing 7mls of Macrosorb K4AX-ABTG, at 600cm/hr. This yielded a bed

expansion of 100%. Bound protein was desorbed as in conventional fixed bed mode.

4. Results

Macrosorb-ABTG was constructed with the final chemistries of 10 mole/ml DADPA,
13 mole/ml glycidol modified DADPA and1 mole/ml ligand.

As can be seen in Figure 1, lab scale
purification involved losses of 27% in
steps which serve only to clarify the
feedstock for further processing. Fixed
bed affinity chromatography achieved a

final concentration factor of 20-fold and
purification factor of 10-fold. The final
specific activity of the product was
~400units/mg protein (data not shown).

Figures 2 and 3 show the loading and elution data for interfaced recovery of product

from unclarified culture. 180mls of unclarified insect cell culture was contacted with
Macrosorb-ABTG in a fluidised bed. The matrix removed 85% of enzyme activity,
conferring a capacity of 2.5 mg enzyme/ml matrix. Figure 3 shows fixed bed
desorption of the saturated matrix, yielding a recovery of >90% of total protein, with
an overall concentration of 4.5-fold, of which 30% was in a 0.1 M borate pool (pH10)
that retained activity upon desalting (specific activity ~700units/mg protein). Elution
of Macrosorb-ABTG, in 3M potassium thiocyanate, recovered the remaining protein.

439

5. Conclusions
The opportunity for enhancement of the conventional purification process by
circumvention of clarification steps is clearly shown in Figure 1. Judicious sizing of
the matrix volume allows removal of at least 85% of enzyme activity from unclarified
culture. Greater than 90% of total protein can then be recovered in a two stage
desorption strategy. The first stage recovers -galactosidase with a purity 40% higher
than conventional recovery, achieving an overall purification factor and volume

reduction both of ~10 fold, together with clarification of the product stream. The

remaining bound protein can then be desorbed with 3M KSCN. Although overall
recovery of enzyme activity from this system is only ~30%, this is clearly a system
specific problem (compare with previous work with yeast and bacteria2,6) concerned
with heterogeneous interaction of -galactosidase with the surface chemistries of the
Macrosorb-ABTG. In order to increase recovery of activity, (i) manipulation of contact
time, (ii) matrix construction and (iii) ligand chemistries are currently under
investigation. A logical extension of this work is the use of direct product
sequestration (DPS) during fermentation. This will offer the advantages outlined
above, coupled with potential to decrease negative feedback of protein synthesis,
stabilise product structure and steepen secretion gradients2.
6. References
1) Datar, R., (1986) Process Biochemistry 21 19-26.
2) Morton, P.M. and Lyddiatt, A., (1994) In: Separations for Biotechnology 3. Ed;
Pyle, D.L., Elsevier Applied Science pp. 329-325.
3) Bradford, M., (1972) Analytical Biochemistry 72 248-254.
4) Miller, I.M., (1972) In: Experiments in Molecular Genetics, Ed; L. Cold Spring
New York, Cold Spring Laboratories pp. 352-355.
5) Hermanson, G.T., Krishna Mallia, A. and Smith, P., (1992) Immobilised Affinity
Ligand Techniques. Academic Press Inc, New York.
6) Burns, M.T. and Lyddiatt, A., (1996), Proceedings of the 5th World Congress Of
Chemical Engineering 2 633-639.
Acknowledgements : IC was funded by a BBSRC quota studentship.

PREPARATION OF PLASMID DNA USING DISPLACEMENT CHRO-
MATOGRAPHY

R. FREITAG and S. VOGT
Laboratoire de biotechnologie cellulaire
Institut de Génie Chimique IV
ETH Lausanne
Switzerland

1. Introduction

Large amounts of pure plasmid DNA are increasingly in demand. Possible areas of applica-

tion include, of course, gene therapy but also the transient transfection of mammalian cells.

The plasmids are routinely produced in E. coli and their recovery from the cell lysates may
involve precipitation and extraction operations, -density gradient centrifugation or

various adsorption techniques. By these operations numerous E coli proteins, other polynu-
cleotides, the toxic lipopolysaccharides (LPS) stemming from the cell membrane, etc. are

removed.

While extraction and precipitation are easily scaled,
the resulting product is often of insufficient quality.

The -density gradient centrifugation yields a
highly purified product, but is difficult to use at
large scale. Adsorption and especially chromatogra-
phic techniques combine scalability with high reso-
lution and thus present a possibility for large scale
applications. Concentrated DNA-solutions are vis-
cous, however, thus only diluted fractions may be
collected from ordinary gradient elution chromatog-
raphy, Figure 1.

Displacement chromatography (DC) is a little-
known alternative to preparative elution chromatog-
raphy [1]. In DC the substances are focused into

consecutive zones of constant concentration by an

advancing displacer front. The displacer may be any

substance which shows a superior binding to the
stationary phase under operating conditions. The

concentration in the zones can be controlled via the
displacer concentration and thus be kept in its en-
tirety just below the critical value, Figure 2. The
overall concentration in the pooled product fractions

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442

may thus be considerably higher than in the elution mode, while smaller columns can be
used to give the same amount of product due to a more effective use of the column’s capac-
ity [2]. The results are higher throughputs and easier scale up [3].

2. Experimental

Plasmid preparation:: Double-stranded plasmid DNA (DH5 , C2007-1, Lot 56682, Clon-
tech Laboratories Palo Alto, USA) was produced in E. coli. Qiagen kits were used for isola-
tion according to the manufacturer’s instructions. The final preparations were free of protein
and non-plasmid DNA, but some of the large scale preparations contained residual LPS [4].
Sample preparation for DC: Samples were prepared by spiking pure plasmid DNA prepara-
tions with equal amounts of protein (holo-transferrin, Sigma) and/or E. coli LPS (scrotype
026:B6, Lot 46H4024, Sigma).
Displacement chromatography: Investigated were a 2 ml Bio-Scale Q2 strong anion ex-
changer column (7 x 52 mm, porous 10 m beads, Bio-Rad), a 1.3 ml UNO™ Ql continu-
ous bed strong anion exchanger column (7 x 35 mm, Bio-Rad), and two columns (250 x 4
mm) filled with porous, ceramic hydroxyapatit beads (10 m, type I and type II, Bio-Rad).
The carrier was a 0.02 M TRIS/HC1 buffer, pH 8.0. A flow rate of 0.1 ml/min was used
throughout. Column regeneration was by a high salt step (2 M NaCl). Fractions were col-
lected twice per minute.
Analytical methods: Protein concentrations in solution were measured either by the BCA
assay (Pierce) according to the manufacturers instruction or by reversed phase chromatogra-
phy (RPC) as described previously [2]. Plasmid DNA in the DC fractions was quantified as
described by Sten et al. [5]. LPS concentrations were measured either by a kinetic LAL
assay or by capillary electrophoresis as described previously [4].

3. Results

The purification of plasmid DNA from cell lysates involves the removal of proteins,
polynucleotides, and lipopolysaccharides. Residual LPS does present a major problem to
both gene therapy and transient transfection. Its maximum residual dosis in parenteralia is
0.5 ng/kg of body mass and LPS has also been known to influence the transfection effi-
ciency in eukaryotic cells [6]. The possibility of separating all three substance classes by
displacement chromatography was investigated by us.

Mass transfer tends to present a problem in plasmid preparation with columns packed from
conventional i.e. porous stationary phase particles. The major part of the adsorptive surface
in the pores can only be reached by diffusion, a time consuming task for large biopolymers.
Evidence has been presented that within the normal time frame of a chromatographic separa-
tion the inner pore surface is not at all accessible to the plasmid molecules and adsorption is
restricted to the - small - outer surface of the particles [7].

3.1 CONVENTIONAL STATIONARY PHASES (POROUS PARTICLES)

Displacement chromatography is based on the displacer enforced competition of the feed
components for the stationary phase binding sites. If this is not possible, e.g. because the
inner article surface is accessible to the proteins but not to the larger DNA molecules or the
equally large LPS aggregates, no separation takes place. This was indeed observed by us in
the case of the BioScale Q 2 column. Plasmid DNA and LPS aggregates are of equal size

443

and do compete for binding. Their separation is possible, but the zone overlap (shock layer)
between the zones is broad, presumably also due to pronounced mass transfer limitations.

3.2 HYDROXYAPATIT

Hydroxyapatit (HA) is a crystalline calciumphosphate modification with known affinity to
molecules containing phosphate groups. For HPLC application two types of ceramic porous
particles are available. The type II material is advertised for its DNA binding ability. While
the DNA binding capacity of the type I material was indeed unsatisfactory, and the corre-
sponding column had to be abandoned, columns packed with the type II HA showed similar
problems as the particulate anion exchanger as far as the DNA / protein separation were
concerned. A separation of LPS and DNA was not possible at all with the type II material,
both substances were found in all fractions at roughly the same concentration ratio.

3.3 CONTINUOUS BED COLUMNS (UNO™ Q COLUMN)

The mass transfer poses a problem to all types of biopolymer chromatography, not only to
displacement. Recently, special stationary phases have been introduced that hope to circum-
vent the problem. The continuous bed UNO™ column by Bio-Rad is investigated here. The
UNO column is a polymer rod prepared in situ by radical polymerisation. It consists of a
continuous network of polymer nodules with interconnecting channels. Thus the surface is
high, while there is no « intraparticle » surface. Band broadening is small even at high flow
rates. In the displacement mode the UNO™ Q column behaves as the comparable BioScale
Q anion exchanger from the same manufacturer. Displacers designed for the BioScale Q
column [8,9] could also be used with the UNO™ Q. The separation between plasmid DNA
and LPS aggregates became sharper, although it was still not possible to collect the pure
substances.

4. Conclusions

The isolation of plasmid DNA at larger scale continuous to present a problem to preparative
biotechnologists. Given both its scalabilty and its high resolution, chromatographic opera-
tions and especially displacement chromatography may well contribute to the final answer to
this problem.

5. References

[1] Freitag R.: In: Analytical and Preparative Separation Methods for Biomolecules.
H.Y. Aboul-Enein (Ed.) Marcel Dekker, 1998

[2] Kasper C., Vogt S., Breier J., Freitag R. Bioseparation 6 (1996) 247-262
[3] Gerstner J.A. BioPharm 9 (1) (1996), 30-35
[4] Freitag R., Fix M., Brüggemann O.: Analysis of Endotoxins by Capillary Electro-

phoresis. Electrophoresis (1997), in press
[5] Sten R.G., MacDonald C.G., Weaver A.L., Pitt A.M. Bio Techniques 15(5) (1993)

932-933
[6] Weber M., Möller K., Welzeck M., Schorr J. BioTechniques 19(6) (1995) 930-

940
[7] Schwarz A., Data presented at the PREP´97, Washington DC, June 1 to 4, 1997
[8] Vogt S., Freitag R. J. Chromatogr., 760 (1996) 125-137
[9] Freitag R., Vogt S., Mödler M.: Customised in affinity and solubility - new pro-

tein displacers for ion exchange protein displacement chromatography. (submitted)

NOVEL METHODS FOR LARGE-SCALE PREPARATION OF NUTRIENT MEDIA AND
BUFFERED SALT SOLUTIONS.

David W. Jayme
Life Technologies, Inc.
3175 Staley Road, Grand Island, NY 14072 USA

1. ABSTRACT

Large-scale manufacture of solutions used to feed bioreactors and purify biologicals
creates logistical challenges which impact facility footprint, personnel utilization and
overall manufacturing cost. In collaboration with large volume users, we have identified
novel formulation configurations and reconstitution options for preparation of nutrient
media and buffered salt solutions. Some options exploit continuous reconstitution from
concentrated intermediates using an automated mixing device. Other options enhance
traditional batch reconstitution processes by automatically adjusting pH and osmolality
(conductivity) to target ranges in a closed formulation tank or bioreactor. We have also
identified hybrid options for campaigned manufacture of similar formulations from
common intermediates. These alternatives produce solutions of comparable quality,
reduce capital investment in facility and equipment, improve space/time utilization
within kitchens, optimize personnel utilization, focus on user core competencies, and
reduce the overall cost of biological fluid production.

2. INTRODUCTION

Traditional approaches to formulate nutrient medium and buffered salt solutions from
biochemical constituents or homogeneous powders have been adequate for production
requirements of a few thousand liters. Biotechnology industry maturation has escalated
required volumes substantially and justified exploration of alternative approaches [1].

2.1. The Challenge

Beyond the daunting volumes of batch-scale mammalian cell fermenters ( 10,000 liters)
perfusion bioreactor feeding during an extended production campaigns (30-200 days),
requiring 1-2 volume exchanges per day, can consume vast quantities of nutrient fluids.
A pilot-scale bioreactors (100L) operating over a 30 day campaign will consume 3000-
6000 liters of nutrient medium. By comparison, a production scale bioreactor (1000L)

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446

operating over a 200 day campaign may consume over 400,000 liters. Protein-free
supplemental nutrient concentrates, designed to replenish exhausted nutrients in fed-
batch mode, have been implemented to reduce medium cost and harvest volume [1-2].

Beyond logistical concerns relating to nutrient medium quantities, downstream
product purification typically requires 1-3 times that volume in buffered salt solutions for
column equilibration, elution, and regeneration and final product formulation [3].

2.2. The Issues

Escalating volumetric demands for nutrient medium or buffer to meet production
projections stresses the kitchen facility, equipment and personnel. Balancing capital
investment for facility space, formulation and holding tanks, and water systems against
risks for a biological undergoing clinical investigation is a challenging task. Various
additional factors contribute to the complexity of determining the “preferred” method for
preparing large-scale biological fluids:
• Technical factors: solubilization time, constituent degradation, endotoxin generation,

number and diversity of formulations;
• Quality assurance factors: batch-to-batch precision, status of existing facility and

equipment, batch-driven testing requirements, project regulatory status;
• Human resource factors: recruitment and training, utilization efficiency;
• Business factors: storage space, geographic location, distribution logistics, current

and projected volumetric scale, accuracy of planning and market projections

3. THE OPTIONS

Alternatives used or under evaluation by global biotechnology and biopharmaceutical
manufacturers for large-scale production of nutrient media and buffers include:

3.1. Continuous preparation from intermediate concentrates using a mixing device.

Liquid medium or buffer concentrates are purchased in bulk packaging compatible with
mixing device connections. A mixing device [4], custom-engineered to user
specifications, is resident at the user facility. High precision I X fluid is reconstituted at
the user site either in large batches or as a continuous feedstream. Advantages include
increased batch size or potential for continuous bioreactor feeding, and reduction in
facility footprint, technical labor and overall manufacturing and qualification cost.
Considerations include the design and capital cost of the mixing device, requirement for
supplemental validation, and maximal benefit derived by commitment to mixing device
and concentrate utilization at the design phase.

3.2. Campaigned production of similar formulations

Campaigning [1] permits manufacture of multiple, similar formulations from a minimal
number of common concentrated intermediates, with reconstitution performed either in

447

batch or continuous mode. Examples include growth and perfusion media or column
equilibration, elution and regeneration buffer combinations. Advantages include
increased manufacturing flexibility, facilitated inventory management, and more efficient
use of kitchen space and formulation/holding tanks. Considerations include the initial
design complexity of concentrate sub-groups and formulation-specific limitations.

3.3. Hybrid of powdered base and liquid concentrated additive

A common example is a complete medium which combines a powdered component with
a concentrated liquid supplement of “difficult to solubilize” components. Another option
is a “salt-free” concentrated supplement designed to replenish key nutrients which have
been metabolically consumed or degraded [1-2]. Advantages include the ability to
produce a complex nutrient formulation which may be challenging to solubilize with full
potency and biological performance from a single component powder. Bulk powders
may be solubilized in a pre-existing kitchen facility. Nutrient replenishment improves
bioreactor productivity in terms of space/time utilization and specific product yield.
Nutrient supplement composition may vary with bioreactor type and feeding regimen.

3.4. Batch formulation using either single concentrated component or multiple
concentrated sub-groups

The multiple component option formulates IX fluid from 2-5 concentrated sub-groups
[1]. Single component concentrates may be provided in bulk containers with integral
filter and recirculation tubing for user dilution [3]. Concentrate formulation accelerates
mixing time, personnel utilization, and batch consistency, particularly as reconstitution
may be performed in closed vessels without manual adjustment of pH or osmolality
(conductivity). Diluting a single component concentrate within a disposable container
with an integral filter reduces capital investment in facility and equipment and may be a
useful transition strategy pending regulatory agency approval and volumetric scale-up.

3.5. Internal production vs. out-sourcing

Ready-to-use IX fluids are manufactured per user specification in large batches (10-
50,000 liters), aseptically dispensed into bulk containers, and released for manufacturing
based on vendor certification. Advantages include forestalling of capital investment in
facility, equipment and utilities. Considerations include the aseptic connections to the
bioreactor and inventory management based upon consumption rate, batch size and
stability and refrigerated storage space. Cost effectiveness will depend upon volumetric
requirement and regulatory status and capacity of existing kitchen facility [4].

4. SUMMARY

Escalating volume requirements for nutrient media and buffered salt solutions demand
novel formulation options to improve quality and consistency, increase batch size,

448

decrease fully-burdened cost and retain manufacturing flexibility. Stable concentrated
intermediates have been successfully implemented at pilot and production scales to
accelerate media and buffer production time, increase batch size and decrease batch-
driven costs, combine with powdered components to improve solubility and performance
of complex formulations, replenish consumed nutrients in fed-batch and perfusion
bioreactor feeding modes, and facilitate inventory management flexibility.

Coupling bulk concentrates with an automated, high precision mixing device
provides additional options. Large-volume batches (10-50,000 liters) of liquid medium
have been commercially produced from concentrated intermediates using this validated
process. Customized mixing devices for continuous reconstitution of concentrated fluids
are under active design and qualification for various biotechnology applications.

5. REFERENCES

1. Jayme DW, Fike RM, Kubiak JM, Nash CR and Price PJ (1993), "Use of
Liquid Medium Concentrates to Enhance Biological Productivity", In Animal
Cell Technology: Basic & Applied Aspects. Volume 5, eds. S. Kaminogawa,
A. Ametani and S. Hachimura (Kluwer) pp. 215-222.

2. Jayme D, Kubiak J, Fike R, Rashbaum S, and Smith S (in press), “Cost-
saving Design and Operational Options for Large-Scale Production of
Nutrient Medium and Buffers” (JAACT ‘96 meeting in Yokohama, Japan,
September 1996)

3. Fike R, Kubiak J, Price P and Jayme D, BioPharm (1993) 6(8): 49-54,
"Feeding Strategies for Enhanced Hybridoma Productivity: Automated
Concentrate Supplementation".

4. Jayme DW, Kubiak JM, Battistoni, and Cady DJ Cytotechnology (1996) 22:
255-261, “Continuous, High Capacity Reconstitution of Nutrient Media from
Concentrated Intermediates.”

LARGE SCALE APPLICATION OF THE SEMLIKI FOREST VIRUS SYSTEM:
5-HT3 RECEPTOR PRODUCTION

H.D. BLASEY, B. BRETHON , R. HOVIUS ,
K. LUNDSTRÖM , L. REY, H. VOGEL , A.-P.TAIR1 AND
A.R. BERNARD

Geneva Biomedical Research Institute, 14 Chemin des Aulx, CH 1228 Plan les
Ouates, Switzerland

EPFL, Lausanne, Switzerland, Institut National des Sciences Appliquées,
Toulouse, France, Hoffmann-La Roche, Basel, Switzerland

Abstract

After we had applied the Semliki Forest Virus (SFV) expression system to produce
large amounts of hCOX-2 in spinners, we succeeded in the first reactor runs with the
SFV used and produced the serotonin receptor 5-HT3 in large quantities. We
successfully scaled up the SFV technology allowing rapid gene expression at 11.5 litre
scale. BHK cells were chosen as host cells for expression. They were grown in a
bioreactor, equipped with a Vibromix agitation system providing for mixing and
preventing cell aggregation. Medium exchange and subsequent viral infection was
followed by a one day expression phase yielding 3 million of serotonin receptors per
cell. After the harvest the, receptor protein could be purified in a single step at high yield
(50%).

Introduction

The Semliki Forest Virus expression system (Liljeström and Garoff, 199la) is a recent
system for the expression of heterologous genes in mammalian cells cultures offering
distinct advantages over other mammalian systems with respect to expression levels and
speed. Co-transfection of in vitro transcribed recombinant and helper RNA into BHK
cells leads to the in vivo packaging of only recombinant RNA and the production of
infectious, but non replicative, recombinant virus particles at high titer (approximately

This virus can infect most mammalian, insect and amphibian
cells.. On the other hand, the ability of the virus to infect almost any eukaryotic cell
raises a biological safety issue. These concerns however have been met by the
production of recombinant virus particles which are only conditionally infectious due to
three mutations in the pSFV-Helper2 plasmid (Berglund et al, 1993).

Various recombinant proteins (enzymes, receptors, viral proteins and others) have been
expressed with the SFV system (reviewed by Bernard and Blasey, in press). Table 1

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450
summarises one specific class of proteins, the receptors, which in addition to the 5-HT3
receptor were expressed with SFV.

Recently we have applied the SFV technology to the expression of human
cyclooxygenase-2 in spinners and produced this enzyme at the one litre scale (Blasey et
al., 1997). Already at this scale we could satisfy the demand for large amounts of active
cytosolic recombinant protein preparations to run high-throughput compound screens.
The 5-HT3 receptor has become of great interest as an object for functional and
structural studies due to its important role as a drug target. The serotonin receptor is a
ligand-gated cation channel and belongs to the group of nicotinic acetylcholine receptors
which are involved in several CNS disorders and for which drugs are on the market (e.g.
against emesis induced by chemotherapy). In order to enable structural studies of the 5-
HT3 receptor we scaled up receptor expression. To achieve the goal of producing
between 10-20mg receptor protein, we had to demonstrate satisfactory protein
production at the scale of 11.5 litres using a mechanically mixed bioreactor, and thus
showing for the first time the feasibility of using the SFV expression system at large
scale.

Materials and methods
Medium, cell maintenance and glucose/ lactate analysis were reported earlier (Blasey et
al., 1997). The plasmids pSFV3-LacZ, pSFVHelper-2 have been described elsewhere
(Liljestrom and Garoff, 1991; Berglund et al., 1993). The pSFVl-5HT3 plasmid has
been described earlier by Werner et al. (1994). However, to facilitate purification, an in-
frame hexa-histidine tag was introduced at the C-terminus of the 5-HT3 receptor by
PCR technique. The amplified 5-HT3 receptor gene was then subcloned into the BAM
HI site of the pSFVl vector.
Generation of recombinant 5-HT3 SFV. In-vitro RNA transcripts were made from
pSFVl-5-HT3 and pSFV Helper plasmids (Lundström et al., 1994) and the RNA
products were co-electroporated into BHK cells according to Liljeström and Garoff
(1991b). The in-vivo packaging of virus particles was completed 24 hours after

451
clectroporation, virus stocks collected and stored at -80° C after eliminating cell debris
by centrifugation (5 min at 200g).
Activation of virus stocks generated with pSFV-Helper2 was done with Chymotrypsin
followed by enzyme inactivation with Aprotinin. The virus titre estimation was
described earlier (Lundström et al., 1994).
5-HT3 receptor characterisation: The and total binding were determined by binding
of [3H]-GR65630 in absence or presence of the 5-HT3 antagonist quipazine. Assays
were done in duplicates.

Results and Discussion
After we had found that 5-HT3 receptor expression was highest in BHK (amongst a
variety of 10 cell lines, Fig. 1) we had selected BHK cells as host for the production of
5-HT3 receptors. Also, BHK cells are widely used to express a large variety of different
recombinant proteins.

Large scale production was carried out in a mechanical mixed reactor from MBR
(Switzerland) with a working volume of 11.5 litres. Oxygen was supplied via a silicone
tubing (length 28m, 1.2mm, thickness: 0.2mm). Our objective for the scale up of
BHK cell culture was to obtain a single cell suspension culture i.e. with no aggregation
of cells (cell aggregates prevent optimal virus infection therefore potentially reduce
recombinant protein yield, nor shown). Therefore, mixing was carried out with a
VIBROMIX (Gerber+ Pfenninger, Switzerland): 2 stainless steel plates of 5.5 cm
diameter, fixed on a vibrating shaft (vibration: 50Hz, amplitude 1.5 mm). The plates
carry 20 conical holes each. The oscillation of the plates also induces shear forces which
provide conditions for non aggregate, single cell growth of BHK.

452

The culture was grown to Since the medium is widely used up at this stage

and because the infected cells have a significantly increased metabolic rate (Blasey et
al., 1997), we exchanged about 90% of the medium, using external crossflow filtration

device (CellFlow, MICROGON, USA,

The culture was then infected by addition of 330 ml activated virus, containing an
estimated virus per ml to yield MOI of 30. The pH at infection was 7.3, but not
controlled. The cells were harvested 20 hours post infection by pelleting (250 ml
centrifuge tubes, Corning, USA, spun for 5 minutes at 420 g in a RC3B centrifuge
Sorvall, USA). Aliquots were frozen at -

As shown in Table 2, the two reactor runs yielded very high level receptor expression,
significantly higher than has been obtained from stable, amplified cell lines.

453

Optimisation pH: a glass spinner (Bellco, USA) was equipped with a pH probe
(Ingold, Switzerland) connected to a pH controller (Bioengineering) controlling two
peristaltic pumps (Chemap). The pH was adjusted by addition of 5% (w/v) NaOH
and 10 % (w/v) acetic acid. BHK cells from T225 flasks cultures were transferred
with 270 ml medium into the spinner (at and infected at an MOI of
10. We compared 5-HT3 receptor expression for cultures run at pH 6.9 (optimal for
infection) and pH 7.3 (identified to be optimal for SPAP expression by BHK cells
after SFV infection, data not shown). Results (from two independent experiments
each) showed an advantage by a factor two for pH 7.3 (Table 3) versus pH 6.9.

Purification of the 5-HT3 receptor was performed by exploiting the C-terminally
engineered His-tag by immobilized metal-ion chromatography. The final preparation
was judged pure by SDS-PAGE: one band was observed at 65 kD corresponding to

the glycosylated receptor. After deglycosylation with
peptide N-glycosidase F, a band at 49 kD appeared. The
specific ligand binding activity was found to be
pmol [3H]-GR65630 binding per mg protein.
The molecular size of the receptor complex was
determined by gel filtration chromatography to be
approximately 280 kD, agreeing well with the calculated
molecular mass of 270 kD for a pentameric structure. The
secondary structure of the purified receptor protein was
probed by circular dichroism and appeared to be
dominated by helical elements (48 %) and poor in non-
regular structure (9 %).

454

Summary

This is the first report of the SFV expression system being applied successfully for the
expression of recombinant proteins at reactor scale. Previously reported experience with
the expression of human Cyclooxygenase-2 using the SFV at the 1 litre spinners allowed
to take the step towards large scale application of the virus. BHK cells were grown in
suspension using a specially designed reactor system at a scale of 11.5 litres. Medium
exchange before infection then allowed the culture to express more than 3 million
receptors per cell before harvesting at 24 hours p.i. Studies on pH optimisation revealed
the potential for increasing the receptor yield to 8 million receptors per cell when
stabilising the pH at 7.3.

Acknowledgement

We thank Dr. J. Delamarter (Geneva Biomedical Research Institute) for his continuing
support of this project.

References:

Berglund P, Sjödberg M, Garoff H, Atkins J.G, Sheahan B.J. and Liljeström P. (1993) Semliki Forest virus
expression system: production of conditionally infectious recombinant particles. Bio/Technology 11, 916-
920.

Bernard A.R. and Blasey H.D. (in press) Transient expression systems, in M.C. Flickinger and S.W. Drews
(eds.), The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis & Bioseparation, John
Wiley & Sons.

Blasey, H.D., Lundström, K., Tate S. and Bernard, A.R. (1997) Recombinant protein production using the
Semliki Forest virus expression system Cytotechnology 24:65-72.

Liljeström P and Garoff H. (1991a) A new generation of animal cell expression vectors based on the Semliki
Forest virus system Bio/Technology 9, 1356-1361.

Liljeström P and Garoff H. (1991b) Current Protocols. Mol. Biol. 2, 16-22.
Lundström K, Mills A, Buell G, Allet E. Adami N and Liljeström P. (1994) High-level expression of the

human neurokine-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur.
J. Bioehem. 244, 917-921.
Lundström K, Vargas A. and Allet B. (1995) Functional activity of a biotinylated human neurokinin 1
receptor fusion expressed in the Semliki Forest virus system. Biochem. and Biophys. Res. Comm. 208, 1,
260-266.
Lundström, K., Michel, A. Blasey, H., Bernard, A.R. Hovius, R., Vogel, H. and Suprenant, A. (1997)
Expression of ligand-gated ion channels with the Semliki Forest virus expression system , J. of Rec. &
Signal Transductuction Research 17(1-3), 115-126.
Lundström, K., Mills, A. Allet, E., Ceszkowski, K., Agudo, G. Chollet, A. and Liljeström P. (1995) High-
level expression of G-protein-coupled receptors with the aid of the Semliki Forest virus expression system,
J. of Receptor & Signal Transduction Research 15(1 -4), 23-32.
Werner P. , Kawashima, E., Reid, J. Hussy, N., Lundström, K., Buell, G., Humbert, Y and Jones, K.A. (1994)
Organisation of the mouse 5-HT3 receptor gene and functional expression of two splice variants. Molecular
Brain Research 26, 233-241.

Discussion 455
Grammatikos:
A slide comparing CHO and BHK cells showed that when the cells
Blasey: go into suspension, the BHK cells produce more than when
attached. However, the CHO cells are producing twice as much in
suspension. Why did you conclude, therefore, that BHK cells are
more appropriate?
I did the comparisons in spinner culture as well and the expression
level in BHK cells is always several factors higher than with CHO.
We have found this for other proteins as well.

FROM APPLIED RESEARCH TO INDUSTRIAL APPLICATION:
THE SUCCESS STORY OF MONITORING INTRACELLULAR
RIBONUCLEOTIDE POOLS

S. I. GRAMMATIKOS, K. TOBIEN, W. NOÉ and R.G. WERNER
Department of Biotech Production
Dr. Karl Thomae GmbH (Boehringer Ingelheim)
Birkendorfer Str. 65, D-88397 Biberach an der Riss, Germany

Extended abstract of the oral presentation1

The development of industrial cell culture processes for the production of
commercially-relevant recombinant proteins is governed by constraints which pertain to
issues such as costs, competitiveness and the meeting of project timelines. Usually, the
time allowed for proper process development is rather short and in this time culture
conditions and scale-up protocols have to be defined in such a way as to maximize cell
productivity and final titers and to minimize process length and overall costs. The
developer tries to satisfy these demands by working on various development areas
(rapid cell growth, high cell densities, no lags during scale-up, long periods of high cell
viability, homogeneous and good product quality, defined culture conditions,
straightforward process etc.). The whole excercise is characterized by a constantly
urgent need for better and better tools.

Central to this situation are cell growth and viability and the ability to obtain
information on changes in cell physiology as early as possible. In this regard, it is
important to realize that cell growth and viability are consequences of physiological
processes and represent a balance of metabolic, growth-promoting and growth-
inhibiting events that occur inside the cell. Because of this, information gained from
monitoring only cell concentration and viability and a number of other on-line and off-
line extracellular data (glucose, lactate, ammonia, oxygen uptake rates etc.), however
useful, suffers from inherent limitations. First and foremost, it comes too late and
allows no predictions. This is immediately evident in the case of cell number and
viability monitoring. When a sample from a bioreactor is taken and it is determined
that, since the previous sample, cell concentration has doubled and viability is at 95%,
nothing can be deduced about the state of the cells at the time of sampling, let alone
predicted what the cell number and viability will be 24 hours later. Secondly, all of the
conventional on-line and off-line parameters are no help in pinpointing the exact time
of physiological change inside the cell. For example, is a nutrient limiting at a
concentration which correlates with entry into a stationary phase or with drop in

viability from 95 to 75%, or is its limiting concentration truly higher, affecting cell
physiology and growth potential already long before reduced growth and viability are
noted? One could most certainly not tell by looking at the usual on-line and off-line
parameters. Some on-line parameters like the oxygen uptake rate correlate well with

cell concentration and viability and are very useful in the monitoring of already
established production processes. Even these parameters, however, are insensitive at

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

458

low cell concentrations. Furthermore, it should be considered that oxygen consumption
is only one aspect of cell metabolism and as such it can only be regarded as a rough
indicator of cell physiology. Based on these considerations, then, it is logical to expect
that the most useful information to complement all of the above-discussed extracellular
measurements will be obtained from intracellular physiologic data.

Some years ago, Roland Wagner and Thomas Ryll at the GBF in
Braunschweig, published a paper in which they described the sensitivity of certain
ratios of nucleotides in responding to physiological changes of BHK and hybridoma
cells and speculated that these parameters could prove useful in the monitoring of
production processes2. These investigators formulated three ratios which they used to
describe the physiological condition of cells: The so-called NTP ratio of purine to
pyrimidine nucleotide triphosphates increases as cell condition and growth potential
worsen, mainly because UTP and CTP decrease as cells enter the reduced exponential
and eventually the stationary phase (Fig. 1). The U ratio of UTP to its derivative UDP-
N-acetylhexosamines is a measure of growth potential versus the accumulation of toxic
metabolic by-products such as ammonia which lead to increases of UDP-activated
sugars3. The U ratio decreases as cell condition worsens, first because of decreased

UTP and further because of possible increases in UDP-activated sugars (Fig. 1). The
NTP/U ratio combines the NTP and U ratios in one very sensitive parameter which
increases as cell condition, growth potential and eventually viability become worse.

459

At THOMAE the sensitivity of the NTP/U ratio and its ability to report on cell
status and growth potential were tested. A large number of fermentations with CHO and
hybridoma cells producing a variety of products, at scales from 4 to 10000 liters and
under various culture modes were analyzed. In this presentation several examples were
shown from CHO cells under development and in large-scale production processes, in
which it was confirmed that the NTP/U ratio is a very sensitive parameter which allows
prediction of the behavior of cells in culture up to two days before any changes are
noted by classical cell number and viability measurements (Fig. 2).

This information is used to advantage in the timely development of efficient
cell culture processes. An example of the applicability of the method in process
development was shown in which nucleotide information was used to select the optimal
perfusion starting point of a perfusion culture. Further applications of this method in

460

process development include the optimization of feed start and the selection of optimal
feeding strategies for fed-batch processes, the optimization of perfusion rates in
perfusion processes and the selection of optimal scale-up schedules and inoculum
conditions. Finally, an additional advantage of the method, is the insights one obtains
on cell metabolism and clues about improving the culture conditions.

References:
1 This work will be published in detail elsewhere.
2 Ryll, T., Wagner, R. Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro

cultivated mammalian cells during production processes. Biotechnol. Bioeng. 40, 934-946, 1992.
3 Ryll, T., Valley, U., Wagner, R. Biochemistry of growth inhibition by ammonium ions in mammalian cells.

Biotechnol. Bioeng. 44, 184-193, 1994.

Discussion 461
Aunins:
Grammatikos: When you showed a slide on scale-up to 10,000 1, you had
dislocations on your NTPU ration whenever you had a split. You
Barteling: indicated that the high starting level limited your growth. Do you
Grammatikos: have an explanation for those dislocations?
Ozturk: The NTPU ratio is variable dependent on cell type, medium
Grammatikos: conditions, and culture mode. In this 10,000 1 reactor, 3 changes
Guan: occurred: lower initial cell densities, the medium exchange to go
into production conditions, and the new production medium. In
this case we reached a new basal level and reflected a slower
growth rate of the cells.
When you scale-up you see this change all the time. Do you take
care to have the medium as well conditioned as possible, in the
sense that the temperature is right or that you start at a low volume
and then add the medium slowly, as that can make a big difference?
The cells entered the 10,000 1 reactor in a good physiological state,
so the changes were not there. With tPA these changes are
according to the protocol, and there is nothing that can be done to
change the process. The only thing is to monitor the process by
nucleotides.
If you control the environment, then you are maybe controlling the
cells. This ratio might be controlled by the metabolite
concentrations in the medium. If you measure the nutrients such as
glucose and glutamine available to the cell, can you predict the
nucleotide ratio?
We could try to correlate this ratio with certain nutrients in the
medium, or certain conditions in the reactor, and then perhaps
monitor these instead of the nucleotide ratio. It is possible that in
the later stages of this project, when we rely on this parameter, we
could make such correlations.
You have only correlated this NTPU ratio with cell concentration.
Have you correlated it to any other ratios such as productivity or
product quality?

462 It is not true that we correlate this with cell number and viability.
Grammatikos: We use this as a mirror of cell physiology, and it so happens that
we can predict changes in viability and cell growth with this NTPU
Konstantinov: ratio before they happen. We could correlate the ratio with other
Grammatikos: parameters but I think this ratio should be looked at on its own.

Ryll: There is value in defining indices representing physiology.
However, I would give higher priority to indices that measure on-
line as you do not need to sample. How does your index correlate
with oxygen uptake rate, which is easy to measure and immediate?

There could be on-line parameters that could be monitored, and
oxygen is a good one, which may be more suitable for the
production setting. In development you need to have information
as early as possible on physiological changes and the OUR may still
be a late indicator of this change.

On-line parameters, which you can correlate and then define, are

much better to control fermentation processes. The power of this
ratio technique is that it is very universal for animal cells, and you
can use it early on before on-line parameters have been developed.
These physiological parameters depend a lot on cell cycle
distribution, for example. So it is a direct measurement for the
growth rate, etc, so you can use these data for things other than for
just controlling fermentation processes.

NEW TECHNOLOGY OF PRODUCTION RECOMBINANT HUMAN
ERYTHROPOIETIN FOR PERORAL ADMINISTRATION

T.D. KOLOKOLTSOVA, N.E. KOSTINA, E.A. NECHAEVA,
N.D. YURCHENKO, O.V. SHUMAKOVA, A.B. RIZIKOV,
N.A. VARAKSIN, T.G. RYABICHEVA
Research Institute of cell cultures, State Research Centre of Virology
and Biotechnology Vector, Koltsovo, Novosibirsk region, 633159,
RUSSIA

1. Introduction
Erythropoietin (EPO) is a glycoprotein hormone that stimulates erythrocyte for-
mation in human blood. At present, EPO is regarded as an effective medicament
for treating patients with anemia caused by renal insufficiency, being a side-effect
of treatment for AIDS with azidothymidine (AZT) or other medicines (1,2). Such
patients need frequent blood transfusions or EPO hormone injections. Since trans-
fusion and injections entail the possibility of transfer of infecting agents, peroral
form of EPO would make treatment more safe and effective.
The present work was aimed at the development of a new technology for produc-
tion of recombinant human erythropoietin (rhEPO) for peroral administration
and testing the new preparation in experiments involving animals.

2. Materials and methods
rhEPO substance has been produced in the new strain of cell culture CHOpE . Cell
culture CHOpE using methods of genetic and cellular engineering has been devel-
oped (3). Cultural media containing rhEPO was centrifugated and concentrated
(1:50-1:100). Isolation, concentration, purification and certification of rhEPO sub-
stance were performed in accordance with the modern requirements .
The tabletted form of rhEPO was produced under laboratory conditions by mixing
erythropoietin substance with stabilizers followed by lyophilization, addition of
admixtures and tabletting.
Specific activity of the new tabletted form of rhEPO was studied by the method of
enzymoimmune assay (EIA method) (4) and in experiments involving animals. In-
tensity of erythropoesis in mice C57BL strain was registered by measuring reticu-

463
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 463-465.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

464

locyte concentration in the animals’ blood. Placebo tablets and EPO for injections
(St. Petersburg, Russia) were used as controls.
Control of tablets harmless and toxicity was carried out on laboratory animals.
Tablets was dissolved and 5 ml of rhEPO solution (1 tablet) was injected subcuta-
neously in each uinea pig (weight 300+ 50g) and on 0.2 ml in each mice (weight 17-
20g.). In each experiment were involved not less than 5 animals.

3. Results and Discussion
Erythropoietin is known to be of great importance for practical medicine. The de-
mand for EPO for treatment of patients is rather high. At the same time produc-
tion of EPO from physiological materials (urine and plasma) is a labour-
consuming and expensive procedure. Establishment of rEPO producers by genetic
and cell engineering methods and production of recombinant preparation turned
out to be most promising.
A strain of CHO cells producing rhEPO using methods of genetic and cellular en-
gineering has been developed in State Research Center of Virology and Biotech-
nology “Vector” ; the strain was patented and certified in accordance with con-
temporary requirements; saving and working banks of the cell culture have been
created and certified in the State Institute of Control and Standardization of Im-
munological preparations.
As a result of the performed studies conditions on cultivation, isolation, concen-
tration, purification and certification of rhEPO substance have been developed,
the effects of conditions of drying the substance are being studied. The results of
this work was represented in the previous publication. The selection of admixtures
for stabilization showed that lactose, gelatose and sacharose were the best to be
added. The more best mixture of tablets for maintaining of specific activity stabil-
ity of rhEPO consists of 12-18% gelatose, 12-18% saharose, 25-70% lactose, not
more than 1% calcium stearat and 0,2 % vanilin.
The tablets produced in this way preserved a stable double-convex form, had the
taste and the smell of vanilin and contained EPO in the dose of 100-2000 U. The
tablets does not contein patogenic microorganisns was harmless and does not call
the toxicity or pyrogenic effect on enimals. The microscopic studies of animal tis-
sue samples does not show necrosis or pathologic process in the places of injec-
tions.
Control of specific activity of rhEPO in tablet’s form by the method of enzymo-
immune assay demonstrated stability of properties for 10-12 months. Taking into
account the obtained data of experimental series, the tabletted form of the prepa-
ration supplemented with admixtures for stabilization has been obtained.
Specific activity of the tabletted form of rhEPO was studied in experiments on
registration of intensity of reticulocyte formation involving mice strain C57BL.
Tablets were pressed in powder and added in the food of mice. Injection form
rhEPO was injected intramuscular. Fig. l presents the results of measuring the
hormone activity in vivo.

465

Experimental results showed that EPO injected directly into blood promoted
erythropoiesis faster than the tabletted form of rhEPO administered per os. How-
ever, on day 3 an increase in reticulocyte concentration was observed in murine
blood at the level which does not significantly differed from that after EPO injec-
tion. According to the date of another autors the effect of EPO significantly de-
pend on the dose and on the way of introduction (5).

CONCLUSION
Thus, new technology has been developed for production of human recombinant

erythropoietin in the tabletted form for peroral administartion.
The more stable tablet’s form of rhEPO consists of 12-18% gelatose, 12-18% sa-
harose, 25-70% lactose, not more than 1% calcium stearat and 0,2 % vanilin.
New form rhEPo for peroral administration demonstrated the specific effect on
erythropoiesis in white mice.

REFERENCES

1. Besarab, A.., Ross, R.P., Nasca, T.J. (1995) The use of recombinant human erythropoietin in pre-
dialysis patients, Curr. Opin. Nephrol. Hypertens 4(2), 155-61,

2. Muirhead, N., Bargman, J., Burgess, E., Jindal, K.K., Levin, A., Nolin, L., Parfrey, P. (1995) Evi-
dence-based recommendations for the clinical use of recombinant human erythropoietin, Am. J. Kid-
ney. Dis., 26(2), 1-24.

3. Patent U S S R - N 1 8 0 1 1 1 8 - 1994.
4. Storring, P.L., Tiplady, R.J., Gaines Das, R.E., Rafferty, B., Mistry, Y.G. (1996) Lectin-binding as-

says for the isoforms of human erythropoietin: comparison of urinary and four recombinant
erythropoietins, J. Endocrinol 150(3), 401-12.
5. Breymann, C., Bauer, C., Major, A., Zimmermann, R., Gautschi, K., Huch, A., Huch, R. (1996)
Optimal timing of repeated rh-erythropoietin administration improves its effectiveness in stimulating
erythropoiesis in healthy volunteers, Br. J. Haematol 92(2), 295-301.

GENERAL SAFETY ASPECTS

SIGNIFICANCE OF MULTIPLE TESTING ON
MURINE LEUKEMIA VIRUS OF MOUSE HYBRIDOMAS

E.J.M. AL, T. JORRITSMA, A. BLOK, H.M.G. SILLEKENS AND P.C. VAN MOURIK.

CLB Biotechnology Services
PO Box 9190, 1006 AD Amsterdam, The Netherlands.

1. Introduction
Monoclonal antibodies derived from mice are known to produce Murine Leukemia
Viruses (MuLV; Ref.l) and if intended for human in vivo use they should be tested for
production of these viruses according current guidelines (Refs.2-4). Detection of MuLV
can be done in the following ways:

- XC-Plaque Assay for infectious ecotropic (i.e. mouse-tropic) MuLV;
- S + L-Focus Assay for infectious xenotropic (i.e. not mouse-tropic) MuLV
- Electron Microscopy (EM) studies on retroviral particles.

- Reverse Transcriptase enzyme activity (RT) assay.
The objective of this study was to reveal correlation and redundancy of multiple MuLV
testing of 5 mouse hybridomas. Furthermore, both cell banks and crude harvests of these
hybridomas were studied for determination of the optimal test strategy for MuLV-testing.

2. Test Procedures

The XC-Plaque-Assay was performed by inoculation of subconfluent mouse SC-1
fibroblasts and, at confluency, UV-irradiation and subsequent overlay with rat XC-cells
(Ref.5). Plaques were detected macroscopically after staining of the cell layers. The
Extended XC-Plaque Assay was done by performing 5 passages on SC-1 cells prior to the
direct assay. The S+L-Focus Assay was performed by inoculation of subconfluent
(S + L-)- mink lung cells and microscopic detection of foci after reaching confluency up
to 10 days (Ref.5). The Extended S+L-Focus Assay was done by performing 5 passages
on prior to the direct assay. Reverse Transcriptase (RT) Assay was
performed on polyethylene glycol precipitated culture supernatants, using Poly rA/dT-
template and either as divalent cation (Ref.6). Positive test results are
subjected to confirmatory testing by using Poly dA/dT template for detection of

contaminating DNA-polymerase activity. Electron Microscopy studies (EM) were
subcontracted. Either transmission EM (TEM) on cultured cells or negative staining (NS-
EM) on viral pellets obtained after ultracentrifugation of crude harvests were performed
(Ref.7).

469

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 469-472.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

470

3. Results

Table 1 presents the results of the two types infectivity assays, both direct as well as
extended. The results show that four out of the five hybridomas tested were positive in
either of the assays; three hybridomas produced ecotropic MuLV and two hybridomas
produced xenotropic MuLV. Hybridoma #2 was positive in both infectivity assays
indicating both the presence of ecotropic and xenotropic MuLV. This was confirmed by
performing XC-Plaque assays with culture supernatants after 5 passages on cells,
which assay yielded > 2000 focus-forming units in the S + L-Assay but only 1 plaque in the
XC-Assay. In the reciprocal test, culture supernatants after 5 passages on SC-1 cells
yielded >200 plaque forming units in the XC-Assay but very low number infectious units
in the S + L-Focus Assay. Therefore, we concluded that this hybridoma produced both
types of MuLV.

All tests on infectious MuLV on the bulk harvests derived from the 5 hybridomas yielded
negative results (not shown).

Table 2 presents the results of the RT-Assays performed with both cell bank
culture supernatants of the cell banks and bulk harvest material obtained after large-scale
fermentation.

Table 2 shows that RT-activity was detected in cell banks of three hybridomas

(#l-#3) using the Poly rA/dT template; however, only in one case (Hybridoma #2) this

RT-activity was confirmed by a low DNA-polymerase activity using the poly dA/dT
template. When testing bulk harvest preparations, none of the samples were clearly

positive for RT-activity.

471

Table 3 presents the results of EM-studies on both cell banks (by TEM) and
crude harvests (by NS-EM).

Table 3 shows that intracellular Type A Retroviral particles were detected in all five cell
banks; the percentage of positive cells ranged from 40 to 100%. The production of
retroviral particles (Type C) by the Cell Banks, however, was only clearly positive in 4 out
of five cell lines. Viral-like particles were also detected in 3 of the 5 Bulk Harvest
samples. However, there is no clear correlation of the bulk harvest EM-results with the
EM results of the cell banks tested.

472

4. Discussion

European guidelines (Note for Guidance) differ from US Points to Consider by their

description of MuLV testing: whereas the EC guidelines stress the tests for infectious

MuLV in cell banks, the US prescribes EM-studies especially in Bulk Harvests.

Moreover, since mouse hybridomas are a priori considered to be able to produce MuLV,
the US guideline indicate that MuLV testing was not required for murine hybridoma

seedlots. The recent international guideline from the ICH includes both infectivity tests,
EM and RT testing.
Our results show that the most informative results were obtained by infectivity

tests on seedlots: 4 out of 5 hybridomas tested were positive in either XC or S + L-Assay.

These results were confirmed by EM-testing on Type C Retroviral particles whereas RT-
testing yielded only positive results with the hybridoma producing the highest amounts of

infectious virus (see Table 1, ). Since EM-studies require specialized equipment and
procedures, we promote the application of infectivity tests on cell banks.

Analysis of bulk harvests may be compromised by long storage time and probably

unfavourable storage conditions: in contrast to seedlot testing, all infectivity tests and RT-

test were negative with this material. By EM-studies viral-like particles (VLPs) were
detected in bulk harvests by negative stain EM, however, these results did correlate with

neither the infectivity tests nor the RT-tests. The hybridoma producing thew highest
number of infectious units did not produce detectable levels of VLPs. The absence
of a clear relation between MuLV detected in cell banks and the VLPs detected in bulk

harvests, questions the relevance of bulk harvest testing for MuLV.

References

1. Weiss RA (1982) Retroviruses produced by hybridomas. N Eng J Med 307:1587.
2. European Commission Note for guidance: Production and quality control of monoclonal antibodies.

Ad hoc Working party on Biotechnology/Pharmacy. III/5271/94.
3. Points to consider in the manufacture and testing of monoclonal antibody products for human use

(1994). Center for Biologics Evaluation and Research (CBER).
4. Note for Guidance on quality of Biotechnological Products: Viral safety evaluation of biotechnology

products derived from cell lines of human or animal origin. CPMP/ICH/295/95.
5. Purification and Assay of Murine Leukemia Viruses. Sherr CJ and Todaro GF (1979). Methods in

Enzymology, Vol. LVIII, pages 412-424.
6. Assay of C-type infectivity by measurement of RNA-dependent DNA polymerase activity. Kellof GJ,

Hatanaka M and Gilden RV (1972). Virology, Vol.48, pages 266-269.
7. Detection and Identification of Viruses by Electron Microscopy. Miller SE (1986) Journal of

Electron Microscopy Technique, Vol. 4, pages 265-301.

MURINE RETROVIRUS DETECTION USING MUS DUNNI CELLS AND
CELLS

P Seechurn, B Mortimer, P Newton, C Martin
Covance Laboratories,
Otley Road,
Harrogate,
North Yorkshire
HG3 1PY,
United Kingdom

1. Introduction

Regulatory authorities require that murine cells used for the production of
biopharmaceuticals and gene therapy vector-banks are evaluated for the presence of
murine leukaemia viruses (MuLV).

The classification of the four classes of MuLV is based on the ability of the virus to
infect and replicate in different cell types. Ecotropic MuLV have a limited host
range, only replicating in cells of mouse or rat origin. Xenotropic MuLV infect
xenogeneic cells but are not infectious for murine cells. Polytropic retroviruses,
which are formed by a recombination between exogenous ecotropic retrovirus and
non-ecotropic endogenous proviral DNA sequences, are characterised by their ability
to induce focus formation on cultured mink lung cells; hence their designation as
mink cell focus forming (MCF) viruses. These viruses can infect murine cells but
have limited infectivity to non-murine cells. Amphotropic MuLV differ from MCF in
their wider host range for non-rodent cell lines.

Lander MR and Chattopadhyay SK (1984) have shown that, with the exception of
ecotropic Moloney MuLV, the Mus dunni cell line will detect all four major classes of
MuLV when directly inoculated with MuLV. The "Points To Consider in
Manufacturing and Testing of Monoclonal Antibody Products for Human Use (1997)"
suggests that infectivity assay should be comprised of an amplification step on Mus
dunni cells coupled with detection of infectious virus on a suitable cell line. We
have evaluated the level of extracellular virus following passage of Mus dunni cells
infected with the four classes of MuLV. In addition, we have evaluated the retroviral
host range of various S+L- cells.

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 473-480.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

474

2. Method

2.1 MCF AND AMPHOTROPIC VIRAL ASSAYS

The method used was essentially as described by Peebles (1975). Briefly S+L- cells
were seeded and polybrene treated. Following polybrene treatment, cells were
inoculated and refed as necessary with growth medium until foci appeared.

2.2 ECOTROPIC VIRAL ASSAY

Two methods were used to detect ecotropic MuLV. Supernatant samples were
assayed on murine S+L- cells (FG-10) essentially as described above. Ecotropic viral
evaluation was also carried out using the SC-l/XC method as described by Rowe et al
(1970).

2.3 INFECTION OF MUS DUNNI CELLS

Cells were inoculated as described by Lander & Chattopadhyay (1984). Briefly, the
cells were seeded and polybrene treated prior to inoculation with 200 infectious units
of each type of MuLV. Once confluent, the supernatant was collected (PO) and the
cells passaged twice. The supernatant samples (P1 & P2) were collected each time
the cells reached confluence.

3. Results

Subconfluent cultures of Mus dunni cells were separately inoculated with xenotropic,
ecotropic, polytropic and amphotropic MuLV. When the cells reached confluence,
culture supernatant samples were collected. The cells were passaged on two occasions
and culture supernatant samples collected each time confluence was reached. The
supernatant samples were assayed for infectious MuLV. During the course of the
experiment cells were observed for cytopathic effect (CPE).

No CPE was observed when Mus dunni cells were inoculated with xenotropic and
amphotropic MuLV either following direct inoculation or after passage. Similarly no
morphological effects were observed on Mus dunni cells following direct inoculation
with ecotropic and polytropic MuLV. After passage, however, CPE was observed with
these viruses. The CPE was more pronounced by the second passage (Figures 1-3).

475

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The results in Table 1 show that for each type of MuLV, there was an increase in viral
titre following passage of infected Mus dunni cells. By passage 2 there was a
substantial increase in titre values. The results from Table 1 are also expressed
graphically in Figure 4.

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The sensitivity of various indicator cells to the four classes of MuLV was evaluated
and the results presented in Tables 2a and 2b also shown graphically in Figures 5 and
6. These results show that feline cells were the most sensitive cell line for the
detection of amphotropic MuLV. Mink cells were more suitable for the
detection of xenotropic and polytropic MuLV. The SC-l/XC assay and murine
cells were the most sensitive indicator system for the detection of ecotropic MuLV.
Table 2b also shows that comparable titre values were obtained when ecotropic MuLV
was assayed on SC-l/XC cells compared to murine cells.

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4. Conclusion
• Mus dunni cells amplified the four types of MuLV used; the litre increased

with passage number
• Feline cells provided the most sensitive infectious assay system for

amphotropic virus
• Xenotropic and polytropic MuLV were more readily detected on mink

cells compared to feline cells.

479
• Murine cells were the most suitable indicator cell line for the

detection of ecotropic MuLV.

5. Discussion

The data presented here are in disagreement with the findings of Hughes et al (1996)
where the authors show that feline cells were more sensitive for the detection of
xenotropic MuLV and polytropic MuLV compared to mink cells. In their study
the level of detection of xenotropic MuLV on feline was variable when using the
same stock, ranging from 2 to 55 times higher titres on feline compared to mink

Our findings indicate that the most sensitive cell line for the detection of
xenotropic and polytropic MuLV was mink cells. Our observation supports the
findings (Hughes et al 1996) that feline cells are the most sensitive indicator cell
line for the detection of amphotropic MuLV. This observation is in agreement with
those of Dr Janet Hartley (NIH), personal communication.

The reason for the differences in observation between the work presented here and
that of Hughes et al (1996) is unclear. However, differences in strains of MuLV used
may account for these observations. It would appear that in the method employed by
Hughes et al (1996) indicator cells were not treated with polybrene. Differences in
methodology between these two studies may be a contributing factor.

The effect of passage of retrovirally infected Mus dunni cells on supernatant viral titre
was evaluated. The viral titre for the four types of retroviruses increased with passage
number, suggesting that the maximum level of virus may not have been achieved by
the second passage. The findings presented here do not support the observation made
by Hughes et al 1996; these authors reported that there were no differences in the titre
of supernatant samples from amphotropic infected Mus dunni cells between passage 1
and passage 2. We would recommend that when using Mus dunni to amplify MuLV,
the cells should be passaged twice as a minimum.

6. References

Hughes, J.V., Messner, K., Burnham, M., Patel, D., White, E.M. (1996) Validation of Retroviral Detection for
Rodent Cell-Derived Products and Gene Therapy Applications. Developments in Biological
Standardisation 88. 297-304

Lander, M.R., Chattopadhyay, S.K. (1984) A Mus dunni Cell Line that Lacks Sequences Closely Related to
Endogenous Murine Leukemia Viruses and Can Be Infected by Ecotropic, Amphotropic,
Xenotropic, and Mink Cell Focus-Forming Viruses. Journal of Virology 52 695-698

Peebles, P.T., (1975) An In Vitro Focus-Induction Assay for Xenotropic Murine Leukemia Virus, Feline
Leukemia Virus C, and The Feline-Primate Viruses Rd-114/CCC/M-7. Virology 67 288-291

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Rowe, W.P., Pugh, W.E. and Hartley, J.W. (1970) Plaque Assay Technique for Murine Leukaemia Viruses.
Virology 42 1136-1139.

Acknowledgements
Thanks to Karen Heelan and Kay Beddoe for secretarial support.

Covance Laboratories Ltd
Otley Road, Harrogate, North Yorkshire, HG3 1PY
United Kingdom
Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595
Website: http:\\www.covance.com

THE REMOVAL OF MODEL VIRUSES DURING THE PURIFICATION OF
HUMAN ALBUMIN USING CHROMATOGRAPHIC PROCEDURES

R. CAMERON1, C. HARBOUR1, Y. COSSART1 and J.P. BARFORD2
Departments of Infectious Diseases1 and Chemical Engineering2,
University of Sydney, NSW 2006, Australia

Abstract

The capacity of an ion exchange chromatography system, designed to purify human
albumin from plasma, to remove two model viruses, i.e. polio virus type 1 and canine
parvovirus, has been evaluated. The viral removal studies were performed using a scaled-
down model of the production scale process which was shown to perform within the limits
established for the production process with regard to product purity. An aliquot of either
high titre polio virus or canine parvovirus was added to human plasma (supernatant 11 +
111) in a 1:10 ratio and then applied to a DEAE column. This column was then washed
and eluted and the eluant applied to a CM column. The latter was also washed and then
product eluted. Cell culture assays were then used to establish the levels of both viruses

in the different chromatography fractions. Following the two-stage purification
procedure which generated high purity albumin (>99%) it was shown that polio virus levels
were reduced by 5.8 logs and canine parvovirus 2 logs thus demonstrating that
chromatographic procedures can play a role in increasing the safety of biopharmaceuticals
with respect to viral transmission.

Introduction

Chromatographic procedures are now widely used at some stage during the downstream
processing or purification of the majority of recently developed biopharmaceuticals, such
as monoclonal immunoglobulins produced by hybridoma technology and recombinant
proteins derived from microbes or cell culture systems. There is also an increasing trend
towards the incorporation of some chromatographic steps in the purification of biologicals
which have not traditionally employed these techniques for large-scale production
including, for example, the manufacture of plasma-derived products such as the
coagulation factors, immunoglobulins and albumin. A chromatographic procedure for the
purification of human plasma albumin was first developed almost twenty years ago
(Curling et al., 1977) but did not gain widespread approval among plasma product
manufacturers for a number of reasons but most significantly the excellent safety record
of heat-treated albumin produced by the traditional Cohn-ethanol fractionation procedure.
There are, however, advantages claimed for the production of plasma proteins such as

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 481-483.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

482

albumin by using chromatographic procedures including increased purity of product and
the capacity to recover more proteins and, as a result, some manufacturers have begun to
adopt this approach while being mindful of the need to validate their processes for viral
elimination. As yet there have only been a few reports which have investigated the
capacity of chromatographic processes to reduce viral loads and this project was designed
to collect data in this important area. A new process for manufacturing albumin which uses
a combination of traditional Cohn fractionation and chromatographic methods, has been
investigated for its capacity to remove viruses.

Materials and methods

The source material used in this study, supernatant 11 + 111, was generated from human

plasma by Cohn fractionation as described previously (Yap et al., 1993). Ion-exchange

chromatography was performed using Pharmacia XK/20 columns containing DEAE

Sepharose Fast Flow and CM Sepharose Fast Flow resins connected in series. Albumin

was eluted from the DEAE column and the resulting eluate applied to the CM column

which was equilibrated and washed and then proteins eluted. Protein levels of samples
were determined by capillary electrophoresis (Applied Biosystems). Viral clearance during

the process was assessed following spiking of supernatant 11 + 111 with virus in a 10:1

ratio of supernatant : virus. Polio type 1 (Mahoney vaccine strain) was assayed for

cytopathic effect in HeLa cells and canine parvovirus was assayed by haemagglutination

of porcine red blood cells following growth in and lysis of feline kidney cells. Titres of

both viruses are expressed as log and calculated using the Spearman and

Karber method. Viral clearance was calculated as follows

Clearance = total virus input spike (calculated from real value)

total virus detected in product

Results and discussion

The results shown in table 1 demonstrate the viral clearance factors for three consecutive
process runs or cycles. The polio clearance factor was 6.1, 5.5 and 5.9 for cycles 1,2 and
3 respectively while that for canine parvovirus was 3.3, 1.3 and 1.3 respectively. There is
an indication that the clearance of polio virus is slightly more efficient in the first cycle
than in cycles 2 and 3 but the results are probably within experimental error. A similar
trend was observed for canine parvovirus where the clearance factor in the first cycle is
clearly greater than that in either cycles 2 and 3. In an attempt to determine whether or not
this effect could be reversed by cleaning of the gels between cycles a sanitization regime
was introduced after each cycle and these results are reported elsewhere (Cameron et al.,
Biologicals, in press).

This study has established the capacity of an ion-exchange process used in the production
of a human biopharmaceutical to reduce viral load. Non-enveloped viruses were selected

483
for analysis because, unlike enveloped viruses such as hepatitis B and C and HIV, they are
not effectively inactivated using solvent/detergent treatments. In addition polio virus
serves as a useful model for hepatitis A virus and canine parvovirus for human parvovirus
B19 both of which have been transmitted to patients during treatment with coagulation
factors derived from human plasma. The results demonstrate that the ion-exchange process
studied can remove reproducibly large amounts of non-enveloped viruses. The levels of
clearance which were found using chromatographic processes therefore add to the overall
safety of therapeutic proteins and augment other removal/inactivation procedures. The
results also show however that viruses can behave quite differently in the same system
which emphasises the need to carry out further research in this area so that the complex
interactions between viruses and matrices in the presence of different biological materials
can be more clearly understood and hence predicted.

References
Curling, J.M., Berglof, J., et al. (1977) A chromatographic procedure for the purification
of human albumin, Vox Sang 33,97-107.
Yap, M.B. et al. (1993) Development of a process for the preparation of human serum
albumin using chromatographic methods, Biotechnology of bloodproducts 227, 143-149.

Acknowledgements
The authors would like to thank the Australian Research Council for funding for this
project and a scholarship to RC and Arthur Webster Pty. Ltd. for supplies of canine
parvovirus and CSL Pty. Ltd. for helpful advice and super II + III.

INACTIVATION OF HEPATITIS A VIRUS DURING THE PRODUCTION OF
NORDIMMUN

D W BIRCH* P KAERSGAARD C MARTIN*

*Covance Laboratories Ltd HemaSure A/S
Harrogate Gentofte
North Yorkshire, Denmark
HG3 1PY
UK

Introduction
One of the process steps in the manufacture of Nordimmun is a Pasteurisation step
(60°C heat treatment for 10 hours). Parenterally administered plasma products should
be free of any infectious agents and historically, Pasteurisation has commonly been
used to inactivate both enveloped and non enveloped viruses in plasma products.
Thermal stabilizers such as sucrose are added to protect the plasma product against
thermal inactivation. These stabilizers however, also protect any contaminating viruses
present in the material. Additionally, the presence of virus neutralising antibodies in
the product may also mask or elevate any virus clearance demonstrated by the process.

Concerns regarding the transmission of hepatitis A virus (HAV) in blood products was
reported in 1992 with haemophiliacs in Italy contracting the disease after treatment
with purified factor VIII [1]. The factor VIII preparation in question had been
prepared using a solvent/detergent virus inactivation step, which although effective
against lipid enveloped viruses (eg HIV), is ineffective against non-enveloped viruses
such as HAV.

Poliovirus has previously been used as a virus with some degree of thermal stability. It
has been reported that HAV is significantly more heat stable than poliovirus and the
use of poliovirus in heat inactivation validations may have overestimated the degree of
virus inactivation caused by the process. The strain of HAV we have used is a lytic
strain and has been shown to be more thermostable than poliovirus[2].

In a recent paper, Niessen et al [3] demonstrated that the addition of sucrose and BSA
stabilisers influenced the stability of infectious viruses under Pasteurising conditions
and that HAV showed greater thermostability under such conditions than poliovirus.

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 485-490.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

486

Methods

Aliquots from three different batches of purified and concentrated IgG solution
(Nordimmun), each with different levels of HAV antibodies were separately spiked
with HAV. In addition a placebo solution was also spiked with virus.

An appropriate volume of 0.5M HC1 was added to each spiked test solution such that
pH values of between 5.4 - 5.5 were obtained. Sucrose was added to each solution.
Each spiked sample was then dispensed into 8 appropriately labelled vials.

Six vials from each solution were completely immersed in a water bath maintained at
60°C. At 0.5, 1, 2, 4, 7 and 10 hours after incubation, a vial of each sample was
removed and diluted 1 in 4 with ice-cold diluent. This was further diluted 1 in 2.5
with diluent. The samples were serially diluted 10 fold with diluent and assayed.

The contents of one of the remaining vials was diluted and assayed immediately. The
other remaining vial from each batch was incubated under ambient conditions for 10

hours, whereupon a sample was removed, diluted and assayed.

To assess the effect of the heat treatment alone, HAV was spiked into a portion of
virus diluent and incubated at 60°C along with the spiked test solutions. Samples were
withdrawn at the same sampling points as for the spiked test material, diluted 1 in 4
with ice cold virus diluent, followed by a 1 in 2.5 dilution and then further serially
diluted and assayed.

Virus diluent was spiked with HAV and samples withdrawn and assayed immediately
(assay positive control) and after 10 hours incubation under ambient conditions.

Virus diluent was used as the assay negative control.

Results

In the treatment control, which did not contain IgG or sucrose, infectious virus was not
detected beyond 4 hours incubation at 60°C.

After 10 hours incubation at 60°C, the test solution A, containing (w/w) sucrose, IgG
but no anti HAV antibodies contained infectious virus.

Test solution B, contained antibodies against HAV and (w/w) sucrose. This solution
contained the higher amount of antibody and was detected at the 2 hour sampling
point but not at the 4 hour sampling point.

487

In the other HAV antibody containing test solution (test solution C), which contained
a lower level of anti HAV antibody, virus was present at the 4 hour sampling point but
was not detected at the 7 hour sampling point.

In test solution D, which contained sucrose but not IgG, virus was detected in the 7
hour sample but not in the 10 hour sample.

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489

Summary of Sample Titres

Conclusion

The Pasteurisation step was effective in removing HAV over a 10 hour incubation
period in the presence of sucrose and anti HAV antibodies.

It appear's that anti HAV antibodies alone do not appreciably reduce viral titre during
incubation under ambient conditions. Heating of HAV to 60°C in test solution D,
which did not contain IgG but did contain sucrose, lead to no detectable virus being
present at 10 hours incubation.

A combination of heating to 60°C in the presence of anti HAV antibodies, lead to an
accelerated viral clearance, with no detectable virus present 2 - 4 hours after spiking
depending on the level of antibodies present. It is not possible to explain the
mechanism by which this phenomenon occurs.

Incubation of HAV at 60°C over a 10 hour incubation period, in an IgG solution
which did not contain anti HAV antibodies but did contain sucrose (test solution A),
resulted in an approximate 4.5 log reduction in viral titre over 10 hours but not total
clearance of virus.

From a comparison between a treatment control which contained no sucrose or IgG
and a placebo solution (test solution D, which contained sucrose but no IgG), it can be

490
inferred that the presence of sucrose imparts a protective effect on HAV during
incubation at 60°C.
References

1. Mannucci PM. (1992) Outbreak of Hepatitis A among Italian Patients with Haemophilia. The Lancet,
339, 819

2. Seechurn, L.P., Birch, D.W., Mortimer, B, Devlia, A., Martin, C. and Loudon, P.T. (1995)
Inactivation of Hepatitis A Virus. Animal Cell Technology: Developments Towards the 21st Century,
(1995), 625-629

3. Niessen, E., König, P., Feinstone, S. M. and Pauli, G. (1996) Inactivation of Hepatitis A and other
Enteroviruses During Heat Treatment (Pasteurisation). Biologicals, (1996), 24, 339-341

Acknowledgements
Thanks to Kay Beddoe and Karen Heelan for secretarial support.

Covance Laboratories Ltd.
Otley Road, Harrogate, North Yorkshire, HG3 1PY United Kingdom
Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595
Website: http:\\www.covance.com

SESSION ON :

VIRAL VECTOR PRODUCTION FOR GENE THERAPY

The application of gene therapy is moving from fundamental research to clinics and
questions concerning new production technologies, biodistribution of the transgene and
safety issues become more important. This increasing orientation towards clinical
application is reflected by the fact that the ESACT-Meeting was the third one, after
the ESACT-Meeting held in Vilamoura in 1996 and the preconference symposium of
the ESACT-Meeting in Veldhoven in 1994, during which a gene therapy session was
organized.
Among the different viral vectors which have been used for gene transfer, three had
expanded from research and developmental assessment to gene therapy clinical trials:
retroviral, adenoviral, and adeno-associated viral (AAV) vectors. Whereas the two first
ones are already used since several years, the interest in AAV vectors is constantly
increasing, due to the fact that they are nonpathogen for humans and that they can
transfect dividing and non-dividing cells of almost any tissue.

Besides the search for gene transfer efficacy, probably one of the major challenge gene

therapy had to face is linked to the safety and genetic stability of viral vectors.
Considerable efforts are spent in the development of new optimal packaging cell lines
preferable of human or old world monkey origine, for the production of different viral
vectors.
Aspects of the scale-up and the optimisation of the production and purification of
retroviral and adenoviral vectors were presented. As elsewhere in animal cell technology,
the trends in the production are going to <<large-scale>> reactor processes (for
retroviruses as well as for adenoviruses) by using serum-free media where feasible. These
developments are all in vain, when there is not a validated safety precedure for verifying
not only the absence of mycoplasmas and other microorganisms (classical) but more
important, proving the absence of replication-compentent viruses.
Other aspects of this session were the use of macrophages as potential shuttles for
widespread/systemic targeting in neuromuscular disorders, like Duchenne Muscular
Dystrophy, for which also a dog model has been described.
Finally, the analysis of biodistribution is of utmost importance in order to verify which
percentage of cells of which tissue of treated animals had been transfected. This analysis
plays a key role for the evaluation of efficacy and safety of any viral vector.
In conclusion, this chapter gives an overview about the actual problems and
developments in gene therapy and the production and use of viral vectors for gene
therapy.

J.-M. Guillaume, A. Crespo
Chairpersons

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.