Note: Descriptions are shown in the official language in which they were submitted.
e s ~
~ - 2071954
A matrix with cells adherently bound thereto, as well
as a method of producing virus/virus antigen
The invention relates to a matrix with human or
animal cells adherently bound thereto, as well as to a
method of producing virus/virus antigen, in particular
tick-borne enceph~litis (TBE) virus antigen.
Infections with the virus of TBE have been
observed in Europe since World War II. In Austria, in
Southern Germany and in ~7-echoslovakia, several hundred
patients are stationarily treated each year because of
a TBE infection.
The TBE vlrus is assigned to the group of
flaviviruses of the earlier serological group B of the
arboviruses, which constitutes a genus of the
,o~aviridae virus group.
Inactivated v~cc~nes against one of the most
important and most frequent ence~h~litis pathogens in
man, the JAPA~eSe encerhAl itis B virus, have been
available for some time. These inactivated v~cc~nes are
recovered from the brains of infected mice, purified,
and are a~h,l~.tledged to be safe and effective (Hoke et
al., N. Engl. J. Med., 319, 608 (1988)).
Slnce 1976 a vacclne against TBE has been
avallable and admitted by the health authorities. For
pro~llc~ng this vA~ ne, the virus is grown in the
- '_ 2071954
brains of infected baby mice, propagated in chick
embryo cells, inactivated with formalin and
subsequently subjected to an efflcient purification
procedure (Heinz et al., J. Med. Vlrol., 6, 103
(1980)).
In the literature a number of possibllities for
propagating arboviruses with a view to the possible
production of a vaccine have been described. The method
mostly used today is the inocculation of chick embryo
fibroblasts with a TBE seed virus recovered from a
mouse brain, and the cultivation of the inocculated
cells. This method reguires a complicated purification
of the antigen so as to remove complex, heterological
biological material and so as to avoid a sensitizing
effect in the persons to be v~ccln~ted when repeatedly
administering v~cç~ne doses obtA~ne~ therefrom.
For providing chick embryo cells, one has to
depart from SPF (= specific pathogen free) eggs. These
SPF eggs must be subjected to a great number of time-
consuming examinations to maintain their SPF status
prior to each use.
Furthermore, chick embryo cell cultures exhibit
only low generatlon numbers in cont~ culturlng,
thereby llmltlng the size of batches, the primary
culture was difficult to keep sterile and the ~uality
of the primary cells with regard to virus propagation
and antigen production was not constant.
These disadvantages exist not only with the
-- 2 --
207 1 954
methods of produelng TBE vlrus antlgen, but exlst qulte
generally ln the produetlon of antlgens.
The lnventlon has as lts ob~eet to lmprove the
produetlon of vlrus/vlrus antlgen, ln partleular of TBB
vlrus/vlrus antlgen sueh that the above-mentloned dlsad-
vantages are ellmlnated, and to provlde a method of growlng
vlrus/vlrus antlgen ln cell eultures whleh partleularly
enables productlon on a large scale, whereln slmultaneously
the eulture can be malntalned sterlle ln a simple manner.
Furthermore, the dellvery of undeslred eellular protelns lnto
the eulture supernatant 18 to be mlnlmlzed.
For obtalnlng the above lndleated ob~eets, a matrlx
18 provlded, l.e. a earrler materlal, havlng human or anlmal
eells adherently bound thereto, the eells belng lnfeeted wlth
vlrus. The lnventlon 18 based on the flndlng that surfaee-
dependent eells sultable for vlrus propagatlon remaln
adherently bound to a matrlx even ln the vlrus-lnfeeted state,
eontlnuously produee vlrus antlgen over relatlvely long
perlods of tlme and dellver lt lnto the eulture medlum.
Aeeordlngly, ln one aspeet the present lnventlon
provldes a serum-free eell eulture eontalnlng a matrlx wlth
human or anlmal eells adherently bound thereto for the
produetlon of flavlvlrus/vlrus antlgen or arena vlrus/vlrus
antlgen, whlch cells are lnfected wlth TBB vlrus or arena
vlrus.
In another aspeet the lnventlon provldes a method of
produelng tlek-borne eneephalltls (TBE) vlrus antlgen uslng a
matrlx as deflned above, eharaeterlzed ln that surfaee-
~.,
~ L 24242-492
2071 954
dependent permanent cells lnoculated wlth the TBE vlrus are
kept adherently bound to the matrlx ln a serum-free medium
whlle thelr vlablllty 18 malntalned, 80 that there occurs
antlgen formatlon ln the cells and antlgen dellvery lnto the
medlum, and antlgen-contalnlng medlum 18 separated from the
carrler-bound cells.
It 18 posslble to store the matrlx accordlng to the
lnventlon loaded wlth lnfected cells for several days at a
temperature of between 0C and 8C, l.e. under condltlons
under whlch the cell metabollsm and thus the vlrus productlon
are stopped. A matrlx thus stored may later on be used for
produclng vlrus
- 3a -
- 24242-492
207195~
antigen without any problems, by introducing it lnto a
culture medium and ad~usting the respectlve growth
conditions. The matrix according to the invention thus
constitutes a starting culture capable of being
produced in stock at constant quality and activity,
whose sterile condition is easy to check and which may
be used for virus antigen production at any time.
The binding of the antlgen-producing cells to the
carrier furthermore allows for an extremely slmple
handling of the virus infected cells that are ready for
productlon. Thus it is, e.g., possible to carry out the
virus/virus antigen production continuously in a
perfusion reactor. Separation of the cells from the
antigen-containing medium is substantially facilitated
by their being bound to the matrix, whereby the matrix
according to the invention simplifies the commercial-
scale production of virus/virus antigen.
A preferred embodiment of the matrix according to
the invention consists in that Vero cells ATCC CCL 81
are provided as the adherently bound cells, which
preferably are provided for the production of TBE virus
antigen and thus are infected wlth TBE vlrus.
The cells adherently bound to the matrix may,
however, also be infected with flavlvirus or with arena
virus.
Glass, cross-llnked dextran, gelatine or synthetic
materlal has proved to be well suited as the materlal
2Q71954
for the matrix, it being best if the matrix is formed
as a microcarrier whose particle diameter preferably is
in the range between lOO~m and 3000~m. These
microcarriers may have a smooth surface or a porous
structure.
A further suitable embodiment of the matrix
accordlng to the invention is characterised in that
between lx105 and 4x105 cells are adherently bound to
its surface per cm thereof.
The invention also relates to a method of
producing TBE virus antigen by using the matrix
according to the invention, which is charaterised in
that the surface-dependent permanent cells, preferably
the Vero cells ATCC CCL 81, are inoculated with the TBE
virus, and the cells are kept adherently bound to a
matrix in a serum-free medium while maintA1 n1 ng their
viability so as to maintain an antigen formation and an
antigen delivery into the medium, whereupon the
antigen-contAi n~ ng medium is separated from the
carrier-bound cells and is pr~ss~ to a galen1cAlly
acceptable preparation in a known manner by
~-o~ce~tration, inactivation and purlficatlon.
The Vero cell llne A~CC CCL 81 i8 obt~1ne~ from
the kidney t1~?ue of the green monkey (Cercopithec
aethiops) and may be kept metAhol1c~11y actlve ln
serum-free medium. For such a permanent cell line, a
mother seed cell bank and a working seed cell bank are
20719S4
started and all the tests for contaminating substances
are carried out. This permanent cell line thus can be
prec1sely characterised not only with regard to its
freedom from contaminating microorganisms, but also
with regard to is growth behaviour, starting culture,
propagation behaviour, and, once optimized, may be
considered as constant.
With the method according to the invention,
preferably Vero cells bound to microcarriers are used.
Thereby a high cell density can be obt~ne~ which, with
the primary cell cultures hitherto used, could be
obtA~ne~ neither in Roux flasks nor in suspension, and
which enables a considerable increase in virus and
virus antigen yields per fermentation volume.
An advantageous embodiment of the method according
to the invention consists in that virus propagation and
antigen formation are carried out in a continuously
operated perfusion reactor for a period of at least 5
days, at a temperature of between 34 and 37C, wherein
perfusion may be effected at a perfusion rate of from
0.3 to lO v/v/day. Furthermore, in the perfusion
reactor there may be provided a cell density of from 2
x 109 to 2 8 101 cells per liter of fermentation
volume, the latter in a fl~ 7e~ bed fermenter.
The virus propagation of the lnvention ln a
perfusion culture enables a sub~ ial reduction of
the - perfusion-rate dep~nAent - dwell time of the
- ~ 2071954
virus and of the virus antigen in the medium as
compared to that of the cultivatlon in batches. The
shorter dwell time causes a much slighter thermal
inactivation and thus results in a higher productivity
of the method according to the invention. Thus, an
antigen conce~tration of from 1 to 10 ~g/ml may be
reached and maintA~ned in the perfusion medium.
With the method accordlng to the invention,
optimum conditions for cultivation may be ad~usted in a
simple manner. Furthermore, substantially less
manipulations are required for its execution than with
all the known methods, thus making handling of the
infectious material safer and enabling continuous and
quick working up of the virus and of the virus antigens
from the culture medium.
In the following, the production of the virus
inoculum, the culture of the cells for the virus and
virus antigen production, and the virus and virus
antigen production as such will be described in more
detail.
1. Virus ~no~ um
Cells (e.g. Vero ATCC CCL 81) are cultivated in
Roller flasks at 37-C up to confluence snd infected
with 1 ml of a seed virus su~rens~on. Starting from the
2nd day after infection, half the medlum i8 replaced
with serum-free medlum each day. The medlum
supernatants from the 4th to the 8th day contain
~ 2071954
2-5 x 107 p.f.u. per ml and are stored at -20C untll
they are used as virus ~oc~lum.
2. Culturing the cells for the virus/virus antigen
production
Starting from the ATCC CCL 81 working seed cells
stored in liquid nitrogen, these cells are propagated
in tissue culture flasks until an amount of cells is
obt~ne~ which allows ~nocl)lation of a fermenter.
Further culturing of the cells is effected in
fermentation vessels at 37C, and as much surface as
possible should be provided for adherence of the
adheringly growing working seed cells. Such large
surfaces are obt~ne~ by using Roller flasks of glass
or of pol~s~y~ene of by using microcarriers (MC). MC of
cross-linked dextran having a size of between 170~m and
250~m are best suited.
The MC loaded with seed cells are cultured at 37C
until a cell density of from lx105 - 4x105 cells per
cm2 has been reAch~. Generally, this cell density is
rç~c~e~ after six days. During culturing, the
microcarrier is completely overgrown with cells, and
finally individual microcarriers may grow together to
groups via the cell sheet adhering to their sur~aces.
3. Virus/virus antigen production
When the cell density indicated has been re~-h~,
the cells bound to MCs are infected with the virus
1 ~oC~l um (1-0.01 pfu/cell, preferably 0.1 pfu/cell) to
207195~
produce the matrix accordlng to the invention. The
matrix according to the invention may be stored for
several days at a temperature of between 0C and 8C or
it may immediately be used for the production of virus
antigen.
For the production of antigen, the MCs loaded with
lnfected cells are introduced into a perfusion reactor.
From this time of virus infection onwards, only serum-
free medium will be used in the culture, which is
pumped continuously through the perfusion reactor,
while the cells cultured on the microcarriers are
retained in the reactor by a retention arrangement.
Starting from the 2nd day post infection, virus antigen
in a high concentration is present and may be
continuously recovered therefrom for at least lO days.
The method according to the invention will be
explained in more detail by way of the following
Examples. Determination of the virus antigen was
effected in all the Examples with an antigen-ELISA.
Example 1
Vero cells ATCC CCL 81 were cultured in a 6-l-
fermenter on microcarriers (Cytodex 3 of Pharmacia) at
370C up to a cell n~ ~r of 2X106 per ml of culture
medium (DMEM - D~llhPc~s Eagle Medium), and
a) was infected with TBE virus (0.1 pfu/cell), and
virus propagation was effected in batches.
_ g _
'-- 2071~54
Table 1
Days post infection virus/virus antigen
~g/ml
2 0.20
3 0.70
4 1.60
2.70
6 4.00
7 3.80
8 2.90
The productivity amounted to 4 mg of virus/virus
antigen per 1 of fermentation volume.
b) was infected with TBE virus (0.1 pfu/cell) and
culture medium was continuously perfused at O.S
volumes/fermenter volume/day.
Table 2
Days post infection virus/virus antigen
~g/ml
2 0.30
3 1.60
4 4.50
4.50
6 2.50
7 3.20 E
8 2.90
9 2.50
2.30
-- 10 --
~ 20719S4
The productivity amounted to 13.7 mg of
virus/virus antigen per 1 of fermentation volume.
c) was infected with TBE virus (0.1 pfu/cell) and
culture medium (DMEM) was continuously perfused at 1
volume/fermenter volume/day.
Table 3
Days post infection virus/virus antigen
~g/ml
2 0.45
3 1.40
4 2.00
2.00
6 1.70
7 1.60
8 1.10
9 1.10
- 90
The productivity amounted to 12.4 mg of
virus/virus antigen per 1 of fermentation volume.
Example 2
Vero cells (ATCC CCL 81) were cultured in as 40-1-
fermenter on microcarriers (Cytodex 3 of Pharmacla) at
37C up to a cell number of 2X108 cells/ml and, after
infection with TBE virus (0.1 pfu/cell), contl n~o.-~l y
perfused with medlum (DMEM) (0.33 vol/fermenter
volume/day).
~ 20719~4
Table 4
Days post infectlon virus/virus antigen
~g/ml
2 1.60
3 3.50
4 5.00
4.30
6 4.00
7 2.90
8 2.70
9 2.10
2.00
The productivity amounted to 10.7 mg of
virus/virus antigen per 1 of fermentation volume.
Example 3
Vero cells (ATCC CCL 81) were cultured in a 40-1-
fermenter on microcarriers (Cytodex 3 of Pharmacla) at
37C up to a cell number of 3X106 cells /ml and
continuously perfused with medium (DMEM) (1
vol/fermenter volume/day) after infection with TBE
virus (0.1 pfu/cell).
20719~4
Table 5
Days post infection Virus/virus antigen
~g/ml
2 1.10
3 3.80
4 3.90
3.00
6 2.30
7 2.20
8 2.00
9 3.15**)
2.30
**) The perfusion rate was reduced to
0.5 v/fermenter volume/day.
The productivity amounted to 21.7 mg of
virus/virus antigen per l of fermentation volume.
Example 4
Vero cells (ATCC CCL 81) were cultured in a 150-l-
fermenter on microcarriers (Cytodex 3 of Pharmacia) at
37C up to 2X106 cells/ml and continuously perfused
with medium (DMEM) (O.33 vol/fermenter volume/day)
after $nfection with TBE virus (0.1 pfu/cell).
` ~ 20719~4
Table 6
Days post infection Virus/Virus antigen
~g/ml
2 0.20
3 1.90
4 2.40
4.80
6 5.40
7 4.10
8 4.40
9 3.20
4.50
The productivity amounted to 14.7 mg of
virus/virus antigen per 1 of fermentation volume.
- 14 -
