Vlak J.M., de Gooijer C.D., Tramper J., Miltenburger H.G. (Eds.) Insect Cell Cultures: Fundamental and Applied Aspects

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252

should be addressed further. Additionally, the grad-

ual retention of recombinant product over the course

of hollow fibre perfusion or other cell separation pro-

tocols that involve membranes with low porosity has

also been observed. There is a clear need to carefully

monitor samples before and after each process to iden-

tify problem areas specific to each protein. Although

knowledge of the biochemical properties of the recom-

binant proteins is useful for the bioreactor production

process, minimisation of product loss during down-

stream processing requires thorough characterisation

of more basic physicochemical as well as biological

properties. Since prevention of cell lysis is not pos-

sible in this naturally lytic system a number of steps

should be taken to avoid excessive degradation of the

product once expressed. Immediate product separation

is optimal.

Optimisation, for downstream processing, of the

production bioreactor configuration and its mode of

operation has been previously overlooked but may

be an effective method to ensure maximal purifica-

tion yields. During batch cultivation, the recombinant

product is exposed to prolonged incubation periods

which may be associated with proteolytic degradation

as well as physicochemical stress including subopti-

mal temperature, pH and mechanical shear. In the

bioreactor, the product should be separated from the

cell suspension and the cell debris and then stored

at low temperature. Cell separation devices incorpo-

rated into the bioreactor design can be exploited to

allow direct processing of the extracellular compart-

ment. The possibility of on-line purification processes

should be considered in the future and those involving

secreted products may be possible now.

Although perfusion bioreactors have the added

advantage of built in cell separation and no addition-

al microfiltration step is necessary, the production of

unwanted filter cakes and molecular sieving have been

observed. Experiments involving back flushing cycles

and flow rate optimisation may have to be addressed

depending on the product involved and the density of

cells achieved in the reactor. Compared to mammalian

systems, the issue of molecular sieving and membrane

fouling may be a greater problem in insect cell culture

due to the high densities achieved and the release of

cellular components and debris due to virally induced

cell lysis.

If low MOI is employed in large scale batch cul-

tures then prior establishment of product stability at

small scale is essential to determine the potential loss of

product due to instability under prolonged culture con-

253

ditions. Low MOI protocols may only be suitable for

products that are maximally expressed intracellularly

prior to the onset of cell lysis. The effect of low MOI

on secreted products needs to be further addressed.

Another relevant disadvantage predicted to occur dur-

ing low MOI cultures is the increased extent of release

of intracellular proteins at the time of maximal product

expression that substantially complicates downstream

processing.

In conclusion the shortest possible culture time

appears to be optimal for product recovery in terms

of yield stability and decreased risk of contaminating

proteins. The time of harvest remains one of the most

important process parameters that will effect the effi-

ciency of downstream processing. However the opti-

mal time of harvest in terms of product expression may

not correlate exactly with the optimal time of harvest

in terms of product recovery.

Three main parameters must be monitored over the

production process to determine the optimal time of

harvest for downstream processing, i) Characterisa-

tion of cell lysis kinetics in parallel with ii) expres-

sion kinetics must be carried out before implementa-

tion of lenghthy and expessive downstream procedures.

iii) Additionally the biological activity kinetics must

also be monitored over the production period. This is

to ensure that host cell function is maintained through-

out the entire infection period allowing the continued

and correct post-translational modifications, particu-

larly around the onset of cell lysis. In most cases the

optimal time of harvest in terms of product recovery

does occur around the onset of cell lysis and although

biological activity may be necessary at all costs, it

may be possible to trade off between maximal product

expression and possible protein degradation and con-

tamination by release of host cell proteins to optimise

the yield recovered.

These areas have been neglected in the literature

and should be addressed as a starting point to change

approaches to downstream processing.

Feed back of downstream processes to molecular

biology level is also essential. Most of the develop-

ments and novel recent discoveries involving the bac-

ulovirus expression vector system have been made at

the molecular biology and cloning level.

Recovery, however, may contribute 50–90% to the

production costs. The need for feed back from down-

stream processing to genetic engineering is essential.

For example, the use of early viral promoters (Chapter

9, this issue) to direct expression of the recombinant

protein should be developed to solve many problems

associated with late harvest times. Interdisciplinary co-

ordination has been shown to facilitate product recov-

ery. Fusions proteins allow efficient affinity purifi-

cation with a ligand binding the fusion partner. The

same purification approach is then applicable to almost

any recombinant protein. Process engineering must be

implemented to translate the ideas and observations at

the molecular biology level into usable processes.

Scale-up of downstream processing may also be

an impending hurdle. The effects of scale up on each

downstream unit operation is relatively unknown, even

less well documented is the collective effects of each

unit operation which will inevitably complicate scale-

up. As oxygen and other nutrient limitation issues

have been successfully addressed at the bioreactor level

extremely high density batch and continuous fermenta-

tions become reality. Each downstream unit operation

will have to handle volumetrically large and concen-

trated process streams. The need for individual char-

acterisation and optimisation of downstream opera-

tions will be essential. The indications are that gen-

eralisations are difficult to make about the choice of

downstream processing protocols. Each step has to be

assessed not only individually but collectively in order

to achieve optimal recovery of the purified product.

Acknowledgment

We thank Drs HD Blasey, L Overton & T Kost for their

constructive comments.

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!"#$%&'()%#*+)*+#,*'--.%-)/+%0-'*1

PART IV

APPLICATIONS

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Cytotechnology

20:

261–270, 1996.

261

Parvovirus diagnostics and vaccine production in insect cells

José Ignacio Casal

Inmunologia y Genética Aplicada S.A. (INGENASA) Hnos, Garcia Noblejas 41–2, 28037, Madrid, Spain

Key words: insect cells, parvoviruses, virion, immune response, capsid proteins, VLPs

Introduction

Baculoviruses have been successfully used to express

large amounts of heterologous proteins for various

applications (Luckow & Summers, 1988; Luckow,

1991). Recombinant baculoviruses can be produced

on a large scale in insect cells. Insect cell cultures offer

an attractive alternative to traditional systems for man-

ufacturing of pharmaceuticals, biologicals and diag-

nostics products.

High level expression of heterologous genes makes

the baculovirus expression system specially suitable

for those cases where large amounts of protein are

needed, for instance, production of subunit vaccines

or diagnostic reagents. These new generation vaccines

will help to obviate the risks associated to the pro-

duction and use of live-modified viruses in classical

vaccines. The aim of this review is to summarize the

use of recombinant baculoviruses for the production of

vaccines and diagnostic reagents useful for parvovirus-

es (Table 1). Also, possible ways to improve the levels

and the quality of the expression are presented. Final-

ly, an study about the genetic stability of the system is

shown.

Parvoviruses

The family Parvoviridae is composed by a group of

small, non enveloped, linear single-stranded DNA

viruses (for a review, see Cotmore & Tattersall, 1987).

They depend on certain cellular helper functions.

Therefore, virus replication will require rapidly divid-

ing tissues, such as fetal tissues, intestinal epithelium

and the hemopoietic system.

Parvoviruses cause a number of diseases in man

and animals. They are endemic and widely prevalent

in populations of their natural hosts, between 70–90%

of world populations are seropositive for the respec-

tive parvoviruses. In this review, particular emphasis is

given to some of the most important parvoviruses, v.g,

the human parvovirus B19 and two economically rele-

vant veterinary viruses: porcine parvovirus (PPV) and

canine parvovirus (CPV). B19 is distantly related to

the other autonomous parvoviruses. In contrast, PPV

and CPV share many structural characteristics (Tsao et

al., 1991) and are classified in the rat parvovirus group.

B19 parvovirus has been associated with a vari-

ety of different human diseases including aplastic cri-

sis, erythema infectiosum, hydrops fetalis and pol-

yarthralgia syndrome in normal adults. PPV is one of

the major causes of reproductive failure in pigs. The

infection occurs without clinical symptoms in adults,

however the virus can cross the placental barrier dur-

ing the infection and cause the death of the foetuses

or embryos. CPV is responsible for acute enteritis in

young dogs, that may cause the death of the animal

in some specially serious cases. CPV is antigenically

and genetically very related (98% homology) with oth-

er relevant parvoviruses as feline panleukopenia virus

and mink enteritis virus. Therefore, vaccines used to

induce protection in dogs are predicted to have the

same effect in other hosts, as cats and minks.

Parvovirus infections are mainly controlled by the

antibody response. A number of classical vaccines

based on inactivated or attenuated viruses are avail-

able for controlling some of the most relevant diseases

induced by parvoviruses. In general, since the par-

voviruses do not replicate well in vitro

the possibility

of developing a recombinant vaccine was very inter-