Note: Descriptions are shown in the official language in which they were submitted.
WO 91tO0347 PCr/DK89/00168
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METHOD OF PRODUCING PROTEINS WITH FVIII A~ vl~lY AND/OR
FVIII DERIVATIVES
The present invention relates to a method of producing
5 proteins with FVIII activity and/or FVIII derivatives by
in vitro culturing of mammalian cells.
The bleeding disorder Hemophilia A is caused by the absence
of Factor YIII (FVIII). FVIII is a glycoprotein which can
10 be isolated from blood plasma (US no. 4,650,858). Purified
FVIII is used in the treatment of Hemophilia A. By the use
of gene technology it is possible to synthesize ~VIII (EPO
160,457, Wo 85/01961, US 570,062). The amount of FVIII,
which can be produced in mammalian cells is rather low
15 compared with other human proteins. The amount of protein
can be increased if truncated variants of FVIII are
biosynthesized (WO 86/06101, DK 3442/87), or if the two
subunits (FVIII heavy chain and FVIII light chain) are co-
produced tUS 822,989). It is also possible in vitro to
20 assemble active FVIII from separately produced subunits
(DK 2957/86). The above-mentioned forms of FVIII all have
the characteristics of human FVIII: activity in bioassays,
activatable by thrombin, and biological activity in
hemophilia dogs.
Mammals are in part characterized by the ability to keep a
constant ~ody temperature near 37-C. Therefore mammalian
cells in general are grown in vitro at 37 C. FVIII
circulates in the body at that temperature, and by
30 culturing in serum cont~ining medium (which mimic body
fluid) one may expect that FVIII has optimal stability.
FVIII produced in serum free medium is rather unstable,
but nevertheless it is attractive to on~t serum from the
medium, and Wo 81~04187 shows that FVIII c~n be stabilized
35 in serum free medium by addition of the carrier protein
von Willebrand Factor (vWF). DK 3594/87 shows that FVIII
~;~
WO 91/00347 PCI~DK89~00168 .
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can also be stabilized in serum free medium by addition of
lipoproteins. These stabilizing agents exhibit no
pronounced effect in serum containing medium.
5 It has surprisingly been found that by culturing ma~malian
cells at a temperature below 37-C, more precisely below
33-C, the yields of truncated FVIII variants and of FVIII
derived subunits (especialy FVIII heavy chain) are
increased drastically, both in serum containing and in
10 serum free medium.
It is preferred to carry out the culturing at a temperature
from 10 to 32 C, more preferred at a temperature from 2
to 30 C, and most preferred at 27-C.
Furthermore it has surprisingly ~ppeared that the yields
are raised by ~hortening the medium residence time below
the usual 24-72 hours, which normally gives the optimal
yields from mammalian cells.
It is preferred to use a medium residence time of 30 hours
or below, more preferred 2~ hours or below, even more
preferred 10 hours or below, and most preferred below 4
hours.
The combined effect of low growth temperature and short
medium residence time is especially pronounced in the case
of FVIII heavy chain for which the yield can be raised 2S
times.
The increased yield of FVIII heavy chain (HC) at low
temperature and short medium residence time can be related
to unstability of the product at normal growth conditons.
Table 1 shows that FVIII heavy chain at 37-C loses the
3~ a~ility to combine ~ith ~ Ylll light chain. Fig. 1 shows
that FV~II heavy chain form aggregates at 37-C. These
Wosl/~ ~7 PCT/DK89t~l~
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aggregates can be dissolved upon reduction. Truncated
variants of FVIII may behave like FVIII-HC and form
aggregates at high temperature. However, the increased
yield at low temperature may also be caused by other
5 factors than a decrease in ayy-eyation. For example a
greater resistance against proteolytic degradation might
be important.
The preferred host cells will include mammalian cells such
10 as CH0 cells, COS-7 monkey cells, melanoma cell lines such
as Bowes cells, mouse L-929 cells 3T3 lines, Balb-c or NIH
mice, BHK or HAR hamster cell lines and the like.
The examples show that the yields of truncated FVIII
15 variants, FVIII heavy chain, FVIII light chain and FVIII
obtained from cells cotransfected with plasmids encoding
each of the two subunits are increased at low temperature.
Table 1
Lane Incubation of Treatment for Combination
FVIII-HC sample SDS-PAGE capacity for
(Fig. 1) diluted sample,
FVIII:C mU/ml
0 h, 37 C reduced
6 4 h, 37 C
7 26 h, 37 C - -
8 0 h, 37-C unreduced 10.4
9 4 h, 37-C - 4.0
26 h, 37 C - <o.s
11 26 h, 22-C unreduced 8.3
35 12 4 h, 22-C - 8.6
13 0 h, 22-C - 10.6
14 26 h, 22 C reduced
4 h, 22 C
40 16 0 h, 22 C
19 MW markers
WO 91/00347 PCI/DK89/00168
20~878S
Plasma derived FVIII-HC (300 U/ml) was diluted 4 fold in
O.05% BSA, 50 mM tris, 0.1 M Nacl, 0.02% NaN3, 150 ~M
2-ME, pH 7.4 and incubated at 22-C and 37-C. At t=0, 4, 24
h ~amples were freezed at -80-C. The samples were thawn and
5 analysed (unboiled) reduced and unreduced in Western blot
Furthermore, the ~amples were diluted 300 fold ~nd tested
for combination with FVIII-LC (W0 88/00210).
The invention is further explained with reference to the
10 drawings in which Fig. 1 shows Western blot of FVIII-HC
incubated at 22-C and 37-C (samples are the same as in
Table 1).
Descri~tion of ~lasmids
15 Plasmid Description
pPR49 cDNA ~ncoAing a Factor VIII variant, in which
880 amino acids are deleted in the ~-region
(the variant is identical to the one encoded
by the pLA-2 plasmid in PCT Patent Application,
Publication no. WO 86/06101), has been inserted
into the expression vector pSV7d (Truett et
al., 1985, DNA 4; 333-349).
pPR60 cDNA encoding a Factor VIII variant, in which
Arg-740 has been fused directly to Ser-1690,
has been inserted into pSV7d.
pSVF8-92E cDNA encoAing the Factor VIII derived 92 kD
peptide (heavy chain) has been inserted into
pSV7d.
pSVF8-80K cDNA enCoA;ng the Factor VIII derived 80 kD
peptide (light chain) has been inserted into
pSV7d.
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~YamDle 1
The effect of growth tem~erature on the vield of Droteins
with Factor VITI activity
5 ~he expression plasmids pPR49 and pPR60 were transfected
to COS-7 monkey cells (Gluzman, 1981, Cell ~; 17S-182) by
usage of the calcium phosphat- technique (Graham and van
der Eb, 1973, Virology ~; 4S6-467) with th- modifications
described in DNA Cloning, a Practical Approach, Vol
10 I+II/IRL Press (Each plasmid was totally transfected to
eight S cm's ~i~hes 2 tim-s 2 dish- determined for
expres-ion at 37 C and 27 C, respectively, in serum
containing medium (10%), and the same number of dishes in
serum fre- m-dium) 16 hours post transfection the ~edia
lS wsr- ch~nged; h~lf th~ w-r- ~hifted to serum free
medium 40 hours post transf-ction th- media were changed
and half the ~h~g w-r- tran-f-rr-d to a 27 C incubator
After additional 24 hours the m-dia wer~ harvested and the
Factor VIII activity wa~ d-termined by usag~ of the Kabi
20 coat-~t chromog-nic assay method The results ar- listed
in Table 2
T~hle 7
Plasmid Temp ( C) Chromogenic activity (mU/ml/day)
+ serum - s-rum
pPR49 37 1023 277
_n_ 37 1028 248
30 -~- 27 1862 1051
_n_ 27 1695 977
pPR60 37 89 >138
-"- 37 104 ~138
35 _n_ 27 336 387
-~- 27 322 401
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ExamDle 2
The combined effect of low arowth temDerature and short
medium residence time on the Yield of Factor VIII heavy
chain from a CHO cell line
s
The CHO (Chin~e Hamster Ovary) cell line DUKX-811 Urlaub
and Chasin, 1980, PNAS 77; 4216-4220), which is mutated in
the dihydrofolate reductase gene, was co-transfected with
the plasmids pSVF8-92E and pSVF8-80K plus a plasmid
10 encodinq the dihvdrofolate reductase.
Hereby a clone (lOC2D2) was isolated, characterized by
15 producing 10 fold more heavy chain (HC) than light chain
(~C) when it is grown at 37-C. When lOC2D2 is grown at
27 C the yield of HC is raised dramatically seen in
relation to the yield of LC (see Table 3).
20 Table 3
Temp. Culturing Vol.med./T-80 flask HC:Ag* LC:Ag*
( C~ time (ml)(U/ml) (U/ml)
(hours)
37 24 10 3.2 0.36
27 24 10 18.4 0.28
* HC:Ag and BC:Ag were measured in specific immuno assays
30 (Nordfang et al., 1988, Br.J.Haematol. 68; 307-312;
Nordfang et al., 1985, Thromb.Haemostas. 53:346-350).
By shortening the medium residence time to only 2 hours a
greater yield of HC per day is achieved (Table 4):
A
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Table 4
Temp. Culturing Vol.med./T-80 flask HC:Ag HC:Ag
( C) time (ml) (U/ml) (U/ml/
(hours) day)
37 24 10 3.2 3.2
27 24 10 18.4 18.4
27 2 10 7.5 go
27 2 3 24.0 288
~YamDle 3
15 Production of FVITI:HC in Q~ticell Bioreactor at 28-30-C
bY 10C2D2
*
In the opticell bioreactor the cells are cultured on a
ceramic matrix, the opticore*, and the culture media is
20 circulated through the Opticore. The oxygen content, pH,
medium feed and harvest ~re all measured and controlled by
the system.
The average medium circulation time was 5 hours tl50 ml per
25 hour with a volume in the reservoir + Opticore of 750 ml).
The harvest was collected at 5-C and frozen every 24 h at
-80-C.
The cells were cultured at 37-C until near confluent
30 (measured by the oxygen comsumption rate) and the
temperature was lowered to 28-30-C for production of
FVIII:HC.
The cells were kept at the production temperature for 1000
35 hours. The oxygen comsu~ption rate decreased in a couple
of hours when the temperature was lowered from 37-C to
30 C and from 30-C to 28-C, but each time the oxygen
comsumption rate gradually increased again. Thus the cells
*~.~ rks
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WO 91/00347 PCr/DlC89/00168
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could be maintained at the lower temperature even for
longer than the 1000 hours.
In Table 5 the medium composition and feed/harvest volumes
S as well as the FVIII:HC levels for some of the harvest
samples are shown.
Table 5
Hours after Medium Feed/Har- FVIII:HC FVIII:HC
temperature vest U/ml U/day
shift to ml/h
28-30 C
DMEH +
1% ITS
408 ~ 2% FCS 100 15.1 36240
432 1 2% FCS 150 10.9 39240
456 1 2% NCS 150 11.0 39600
Exam~le 4
The effect of low ~rowth temDerature on yield of Factor
VIII li~ht chain from COS-7 monkev cells
30 An expression plasmid designated pPR77 encoding Factor
VIII light chain was transfected to COS-7 cells in the
same manner as described in Example 1. The plasmid was
transfected to four 5 cm dishes: two times two dishes
determined for expression at 37-C and 27-C, respectively.
35 The media (DNEM I 10% FCS) were changed 16 and 40 hours
post transfection (at 40 hours half the ~icheC were
transferred to a 27 C inCllhAtor). After additional 24
hours the media were harvested. The content of Factor VIII
light chain was determined as described in Example 2. The
40 results are given in Table 6.
.... . .. . . . ..
WO 91/00347 PCr/DK89/00168
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Table 6
Temp. (-C) LC:Ag (U/ml/day)
37 0.33
37 0.30
27 1.25
27 0.85
Exam~le 5
The effect of low ~rowth temDerature on the Yield of
protein-comDlex with Factor VTII activitY from CH0 cells
transfected with Dlasmids encodina each of the two subunits
20 of Factor VTII: the heaw chain and the liqht chain
The DHFR(-) CH0 cell line DG44 (cf. G. Urlaub et al.,
Proc.Natl.Sci., USA 77; 4216-4220, 1980) was first
transfected with a plasmid enGo~ing the light chain of
25 Factor VIII and the dhfr gene. By selection of DHFR(+)
cells a stable light-chain producer was isolated. This new
cell line was co-transfected with a plasmid encoding the
heavy chain of Factor YIII (and the dhfr gene) and a
plasmid encoding the neo gene (pSV2neo; P.J. Southern and
30 P. Berg, Journal of Molecular and Applied Genetics 1; 327-
341, 1982). Transfectants were isolated in medium
containing 700 ~g Geneticin (G418 Sulphate, Gibco) per ml.
Cells from the primary pool were propagated directly into
medium containing 0.1 ~M MTX. Cells isolated in this way
35 were seeded into two T-80 flasks, called A and B. At
confluence the media (DMEM + 10% DFCS + 700 ~g Geneticin/ml
+ O.1 ~M MTX) were changed tlO ml) and the flasks were
incubated 24 hours at 37 C whereafter media samples were
collected. The media were renewed and the B flask was
,
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205878~
transferred to a 27 C incubator. Again the flasks were
incubated for 24 hours followed by collection of media
samples and renewing of media. This procedure was repeated
for another two days (the A flask still at 37 C and the B
5 flask still at 27 C). The ~actor VIII activity was
determined by the Kabi coatest chromogenic assay method.
The results are given in Table 7.
Table 7
Day Temp. ( C) Chromogenic activity
(U/ml/day)
Flask A: l 37 0.42
2 37 0.77
3 37 0.84
4 37 1.0
Flask B: 1 37 0.50
2 27 1.20 -~
3 27 1.72
4 27 3.2
