DK168713B1 - Process for preparing proteins which possess FVIII (factor VIII) activity, and/or FVIII derivatives - Google Patents

Description

The present invention relates to a particular method for producing proteins with FVIII activity and / or FVIII derivatives by culturing mammalian cells capable of producing the protein in vitro. which method is peculiar to the characterizing part of claim 1.

Bleeding the disease Hemophilia A is caused by a lack of coagulation factor VIII (FVIII). FVIII is a glycoprotein recoverable from blood plasma (US No. 4,650,858) and used to treat Haemophilia A. In addition, FVIII can be produced in mammalian cells by gene technology (EPO 160 457, WO 85/01961, EP 150 735) . The amount of FVIII that can be produced by genetic engineering is modest compared to other human proteins. However, the amount of protein can be increased if biosynthesized shortened variants of FVIII (WO 86/06101) or if different subunits (FVIII light chain and FVIII heavy chain) of the FVIII molecule (EP 232 112) are co-produced. In addition, it is possible in vitro to collect active FVIII from separate subunits (DK 2957/86). The above modified forms of FVIII have properties such as intact FVIII (~ full length FVIII): activity in 20 bioassays, thrombin activatability and biological activity in haemophilia dogs.

Mammals include characterized by maintaining a constant body temperature near 37 ° C and therefore mammalian cells are generally grown in vitro at 37 ° C. FVIII circulates in the body at this temperature, and when grown in serum-containing medium (similar to body fluid) one should expect FVIII to have optimal stability.

It also turns out that FVIII is unstable when cultivated without serum. However, it is desirable to be able to exclude serum from the culture medium and WO 87/04187 shows that FVIII can be stabilized in serum-free medium if the carrier protein vWF (von Willebrand Factor) is added. DK 3594/87 shows that FVIII can also be stabilized in serum-free medium if lipoproteins are added. These stabilizers have no significant effect when added to serum-containing medium. In the above references, the cultivation temperature is used at 37 ° C. X WO 88/05825 the produced FVIII is also stabilized by the addition of vWF, but since a temperature sensitive SV40 large T antigen allele (tsA58), which is 5 fully functional at 33 ° C, is used in the expression system used, this is used. value as culture temperature.

Surprisingly, by performing mammalian cell cultures at a temperature in the range of 10-32 ° C, the yields for shortened FVIII variants and for FVIII subunits (especially 10 FVIII heavy chain) are significantly increased in both serum-containing and serum-free medium.

Thus, the invention relates to a particular method for producing proteins with FVIII activity and / or FVIII derivatives by culturing mammalian cells capable of producing the protein, in vitro, and the method is peculiar in that the culture is carried out at a temperature in the range of 10. to 32 ° C.

It is preferred to carry out the cultivation in the range of 25 to 30 ° C and especially at 27 ° C.

In addition, it has surprisingly been found that the yield is increased by cultivation for a shorter time than the 24-72 hours, which is usually optimal for product formation from mammalian cells.

A cultivation time of 30 hours or less is used, the period of 24 hours or less being preferred. Particularly 25 are preferred for 10 hours or less and especially used for 4 hours.

The increased yield of FVIII at low temperature and short growing time is especially true for FVIII heavy chain (FVIII-HC), where the yield can be increased 25 times.

3 DK 168713 B1 The reason for the increased yield at low temperature and short growing time may be related to product instability under normal growing conditions. Table 1 shows that FVIII heavy chain loses the ability to connect with FVIII light chain (FVIII-LC).

5 FVIII heavy chain aggregates at 37 ° C.

These aggregates can be dissolved after reduction. Abbreviated variants of FVill can behave as FVIII heavy chain and form aggregates at high temperature. However, an elevated yield at low temperature may also have causes other than a decreased aggregation. For example, greater resistance to proteolytic degradation may be of importance.

The preferred host cells include mammalian cells such as CHO cells, COS-7 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cell 3T3 lines, Balb-c or NIH mice, BHK 15 or HAK hamster cell lines, and the like.

The examples show that the yields of abbreviated FVIII variants, FVIII heavy chain, FVIII light chain and FVIII obtained from cells co-transfected with plasmids encoding each of the two subunits are increased by low temperature culture.

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Table 1

Trace Incubation of Treatment Combination Capacitance FVIII heavy prior to SDS diluted chain electrophoresis samples FVIII: C mU / ml 5 0 hours, 37 ° C reduced 6 4 hours, 37 * C reduced 5 7 26 hours, 37 ° C reduced 8 0 hours, 37 "C unreduced 10.4 9 4 hours, 37" C unreduced 4.0 10 26 hours, 37 ° C unreduced <0.5 11 26 hours, 22 "C unreduced 8.3 10 12 4 hours, 22 ° C unreduced 8.6 13 0 hours , 22 ° C unreduced 10.6 14 26 hours, 22 ° C reduced 15 4 hours, 22 ° C reduced 16 0 hours, 22 ° C reduced 15 19 Molecular weight markers

Plasma derived FVIII-HC (300 U / ml) was diluted 4 times

0.05% BSA, 50 mM Tris, 0.1 M NaCl, 0.02% NaN3, 150 βΚ 2-ME, pH

7.4 and incubated at 22 ° C and 37 ° C, respectively. At time t = 0, 4 and 24 hours the samples were frozen to -5-80 ° C. The samples were thawed and analyzed without boiling, with and without reduction in Western blotting. Further, the samples were diluted 300 times and tested for combination with FVIII-LC (W088 / 00210).

5 DK 168713 B1

Overview and description of the plasmids used

PLASMID DESCRIPTION

pPR49 cDNA encoding Factor VIII variant in which 880 amino acids of the B mid region 5 have been subdivided (the deletion is identical to that described for the pLA-2 plasmid in PCT Patent Application, Publication No. W086 / 06101) is inserted on the expression vector pSV7d . (Truett et al. 1985 DNA4: 333-349).

10 pPR60 cDNA encoding Factor VIII variant (where Arg-740 is fused directly to Ser-1690) is inserted on pSV7d.

pSVF8-92E cDNA encoding the Factor VIII derived N-terminal 92 kD peptide (heavy chain) is inserted at 15 pSV7d.

PSVF8-80K cDNA encoding the Factor VIII derived c-terminal 80 kD peptide (light chain) is inserted on pSV7d.

EXAMPLE 1 The effect of the dwell temperature on the yield of proteins

Factor VIII activity

The expression plasmids pPR49 and pPR60 were transfected into COS-7 monkey cells (Gluzman, 1981, Cell 23: 175-182) by the calcium phosphate technique (Graham and van der Eb, 1973, Virology 52: 456-467) with the modifications , described in DK 168713 B1 ei: DNA Cloning, a Practical Approach, Vol. I + II / Glover, IRL Press. (In total, each plasmid was transfected into 8 x 5 cm dishes: 2 times 2 dishes determined for expression at 37 ° C and 27 ° C in serum-containing medium, plus the same 5 number of dishes in serum-free medium). 16 hours after transfection, medium was changed; half of the dishes were changed to serum-free medium. 40 hours after transfection, medium was changed on all dishes and half of the dishes were transferred to incubator set at 27 ° C. After a further 24 10 hours media was harvested for Factor VIII activity determination by the Kabi Coatest method. The results from the activity assays appear of Table 2:

Plasmid Temperature Chromogenic activity (mU / ml / day) + serum -5 serum pPR49 37 ° C 1023 277 pPR49 37 "C 1028 248 pPR49 27 ° C 1862 1051 pPR49 27 ° C 1695 977 pPR60 37 ° C 89> 138 pPR60 37 ° C 104> 138 20 pPR60 27 ° C 336 387 pPR60 27eC 322 401 EXAMPLE 2

The combined effect of low animal feeding temperature and short media residence time respectively on the yield of FVIII heavy chain from a 25 CHO cell line CHO (Chinese hamster ovary) cell line DUKX-B11 (Urlaub and Chasin, 1980, PNAS 77: 4216-4220) that is mutated in the dihydrofolate reductase (dhfE) gene, has been co-transfected with plasmids pSVF8 “92E and pSVF8-80K (see page 5) as well as a plasmid encoding dihydrofolate reductase (these co-transfections are described in EP 232 112 ). Hereby, among other things, isolated clone 10C2D2, characterized in that it produces 10 times as much heavy chain (HC) as light chain (LC) when grown under normal conditions at 37 ° C. When grown at 27 ° C, the yield of the FVIII heavy chain increases dramatically compared to the yield of the FVIII light chain, as shown in Table 3: 10 TABLE 3

Temp. Growing time Vol.med./T-80 fl. HC: Ag * LC: Ag * ("C) (hours) (ml) (U / ml) (U / ml) 37 ° C 24 10 3.2 0.36 27 ° C 24 10 18.4 0.28 15 * HC: Ag and * LC : Ag was measured in specific immunoassays (Nordfang et al., 1988, Br. J. Haematol. 68: 307-312; Nordfang et al., 1985, Thromb. Haemostas. 53: 346-350).

By shortening the media dwell time to just 2 hours, an even greater yield of FVIII heavy chain per hour is obtained. day (Table 4): 8 DK 168713 B1 TABLE 4

Temp. Growing time Vol.med./T-80 fl. HC: Ag HC: Ag CC) (hours) (ml) (U / ml) (U / ml / day) 37 ° C 24 10 3.2 3.2 5 27 ° C 24 10 18.4 18.4 27 ° C 2 10 7.5 90 27 ° C 2 3 24.0 288 EXAMPLE 3

Preparation of FVIII-HC in Ooticell Bioreactor at 28-30 ° C at 10 10 C2D2 In the Opticell bioreactor, cells are grown on a ceramic matrix, the Opticoren, and the nutrient medium is circulated through the Opticor. The oxygen content, pH, media additions and harvest are all measured and controlled by the system.

The average media circulation time was 5 hours (150 ml per hour with a volume in the reservoir + Opticor of 750 ml). The harvest was collected at 5 ° C and frozen to -4-80 ° C every 24 hours.

The cells were grown at 37 ° C until almost confluence (measured at 20 degrees of oxygen consumption) and then the temperature was lowered to 28-30 ° C during the production of FVIII-HC.

The cells were kept at the production temperature for 1000 hours. The rate of oxygen consumption decreased over a few hours as the temperature was lowered from 37 ° C to 30 ° C and from 30 ° C to 25 28 ° C, but each time the degree of oxygen consumption gradually increased again. In this way, the cells could be preserved even beyond the 1000 hours.

i e-iBr ^ 9 DK 168713 B1 Table 5 shows the composition of the medium and volumes for addition / harvest as well as the level of FVIII-HC in some of the harvested samples. Dulbecco's Modified Eagle Medium (DMEM) was used as a medium and as a medium supplement Insulin, Transferrin, 5 Selenium (ITS). Fetal Calf Serum (FCS) and New-Born Calf Serum (NCS).

Table 5

Hours after Medium Add- FVIII: HC FVIII: HC

temperature / harvest U / ml U / day 10 change to ML / h

28-30 ° C

DMEM + 1% ITS

408 + 2% FCS 100 15.1 36240 432 + 2% FCS 150 10.9 39240 15 456 + 2% NCS 150 11.0 39600 EXAMPLE · 4

Effect of low growth temperature on the yield of FVIII-LC from COS-7 monkey cells.

An expression plasmid designated pPR77 encoding FVIII-LC 20 was transfected into COS-7 cells in the same manner as described in Example 1. The plasmid was transfected into four 5 cm dishes; twice two dishes destined for expression at 37 ° C and 27aC, respectively. The medium (DMEM + 10% FCS) was changed at 16 and 40 hours after transfection (at 40 hours, half of the 25 dishes were transferred to a 27 ° C incubator). After another 24 hours, the medium was harvested and the content of FVIII-LC was determined as described in Example 2. The results are shown in Table 6.

10 DK 168713 B1

Table 6

Temp. (° C) LC: Ag (U / ml / day) 37 0.33 37 0.30 5 27 1.25 27 0.85 EXAMPLE 5

The effect of low growth temperature on yield, of a protein complex with FVIII activity from CHO cells transfected with plasmids encoding FVIII-HC and FVIII-LC, respectively.

The DHFR (-) CH0 cell line DG44 (G. Urlaub et al., Proc. Natl. Sci., USA 77? 4216-4220, 1980) was first transfected with a plasmid encoding the FVIII-LC and the dhfr gene. In selecting DHFR (+) cells, a stable FVIII-LC producer was isolated. This new cell line was co-transfected with a plasmid encoding the FVIII-HC (and dhfr gene) as well as a plasmid encoding the neo gene (pSV2neo? PJ Southern and P. Berg, Journal of Molecular 20 and Applied Genetics 1? 327 -341, 1982). The transfectants were isolated in a medium containing 700 µL of Geneticin (G418 sulphate, Gibco) per ml. ml. Cells from the primary pool were distributed directly into the medium supplemented with 0.1 μΜ MTX (Methotrexate). Cells isolated in this way were partitioned into two T-80 bottles 25 named A and B. At confluence, the medium (DMEM + 10% DFCS (Dialyzed Fetal Calf Serum) + 700 µg Geneticin / ml + 0.1 µΜ MTX) was changed (10 ml ) and the bottles were incubated for 24 hours at 37 ° C, after which media samples were harvested. The medium was renewed and the B-bottle was transferred to a 27 ° C incubator. Then, the 30 bottles were again incubated for 24 hours with subsequent harvesting of media samples and renewal of medium.

11 DK 168713 B1

This procedure was repeated for another two days (the A bottle still at 37 ° C and the B bottle still at 27 ° C). Factor VIII activity was determined by the Kabi Coatest method.

The results obtained are shown in Table 7.

Table 7

Day Temp. (C) Chromogenic activity (U / Ml / day)

Bottle A 1 37 0.42 2 37 0.77 3 37 0.84 4 37 1.0 10

Bottle B 1 37 0.50 2 27 1.20 3 27 1.72 4 27 3.2

Claims (7)

A process for producing proteins with FVIII (Factor VIII) activity and / or FVIII derivatives by culturing mammalian cells capable of producing the protein, in vitro characterized in that the culture is carried out at a temperature between 10 and 32 ° C. C. Process according to claim 1, characterized in that the culture temperature is between 25 and 30 ° c. Process according to claim 2, characterized in that the culture temperature is 27 ° C. A method according to any one of claims 1-3, characterized in that the FVIII derivative produced is the 92.5 kD heavy chain fragment (HC). A method according to any one of claims 1-3, characterized in that the manufactured FVIII derivative is the 80 kD light chain fragment (LC). A method according to any one of claims 1-3, characterized in that the resulting product is a protein complex with FVIII activity obtained from cells transfected with plasmids encoding each of the two FVIII "subunits": the heavy chain and the light chain. A method according to any one of claims 1-6 characterized in that the mammalian cells used are COS cells, CHO cells or BHK cells.