CA2058785C - Method of producing proteins with fviii activity and/or fviii derivatives - Google Patents

Abstract

In a process for producing proteins with FVIII activity and FVIII derivatives by in vitro culturing of mammalian cells, the culturing is carried out at temperatures below 32°C and the culturing times used are below 24 hours.

Description

WO 91tO0347 PCr/DK89/00168 ~ ~ ~ 8 7 8 5 -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

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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~

3 20~785 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 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 * Tr~ rk ~ ' - WO9l/~ ~7 PCTtDK89/~1~

6 ~ ~ 5~7 ~ ~ ' 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):

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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 L~

WO 91/00347 PCr/DlC89/00168 8 ~587~

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 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.

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WO 91/00347 PCr/DK89/00168 9205878a 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 , W09l/~ ~7 PCT/DK89/~t68 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

Claims (6)

1. A method for producing proteins with Coagulation Factor No. VIII
(FVIII) activity and/or FVIII derivatives by in vitro culturing of mammalian cells, transfected with plasmids encoding for said proteins, said culturing being carried out at a temperature from 25°C to 30°C, whereafter the said proteins are expressed in the medium.

2. A method as claimed in Claim 1, wherein the temperature is 27°C.

3. A method as claimed in Claim 1 or Claim 2, wherein the FVIII
derivative produced is the 92.5 kD fragment or the "Heavy Chain".

4. A method as claimed in Claim 1 or Claim 2, wherein the FVIII
derivative produced is the 80 kD fragment or the "Light Chain".

5. A method as claimed in Claim 1 or Claim 2, wherein the resulting product is a protein-complex with FVIII activity obtained from cells transfected with plasmids encoding each of the two subunits of FVIII: the heavy chain and the light chain.

6. A method as claimed in Claims 1 through 5, wherein the mammalian cells used are COS cells, CHO cells or BHK cells.