Method for preparing and isolating protein eliciting activity of factor viii, strain of cells hkb expressing protein eliciting activity of factor viii (variants)

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

 

Applications relating to the same subject: Proposal Cho, marked MSB-7241 (U.S. Ser. No. 09/209,920) Hybrid cells-human hosts for the expression of mammalian genes and the application Cho and Chan, marked MSB-7254 (U.S. Ser. No. 09/209,915) Vectors with a sequence of terminal repeat of Epstein-Barr contain related material. Both applications registered 10 December 1998.

Prior art

Scope: This invention relates to an improved method of producing factor VIII and its derivatives. The method in General relates to the design of vectors, transfection and selection of cell lines with improved products in a protein-free conditions. In particular this invention relates to the process of obtaining protein with protagonizada activity of factor VIII in an industrial scale.

Prior art: the human Factor VIII is a trace of a plasma glycoprotein that participates as a cofactor in the activation of factors X and Ha. Inherited deficiency of factor VIII leads to X-linked disease hemophilia a, which can be successfully treated with purified factor VIII. Substitution therapy of hemophilia a has evolved from the use of factor VIII derived from plasma to recombinant factor VIII obtained by cloning and expression of cDNA of factor VIII in mammalian cells (Wood et al., 1984, Nature 312: 330).

Factor VIII has a domain organization A1-A2-B-A3-C1-C2 and synthesized as single-chain polypeptide of 2351 amino acids, from which you cut the signal peptide of 19 amino acids during translocation into the lumen of the endoplasmic reticulum. Due to the fact that factor VIII is bad glycosylases, it was difficult to achieve a high expression level (>0.2 PG/cell/day) factor VIII (Lind et al., 1995, Eur J Biochem. 232: 19-27; Kaufman et al., 1989, Mol Cell Biol. 9: 1233-1242). Expression of factor VIII in mammalian cells usually 2-3 orders of magnitude lower than the expression of other genes using similar vectors and techniques. The productivity of cell lines to obtain factor VIII is in the range of 0.5-1 MCED/KL/d (0.1-0.2 PG/CL/d).

It was shown that the B-domain of factor VIII is irrelevant to protagonizada activity. Different groups reported improvement in the expression of factor VIII in mammalian cells using truncated variants of factor VIII (Lind et al., 1995, Eur J Biochem 232: 19-27; Tajima et al., 1990, Proc 6thInt Symp H.T. p.51-63; US Patent 5,661,008 Almstedt, 1997). However, the level of expression of variants of factor VIII remained below 1 PG/CL/d for the stable cell clone.

Summary of invention

We opened (i) a method of obtaining cell lines with very high productivity proteins with protagonizada activity of factor VIII, and (ii) free from proteins PLA is we the process of obtaining protein, with protagonizada activity of factor VIII.

The process of obtaining proteins with protagonizada activity of factor VIII in an industrial scale. Using the newly created cells of the hosts were obtained cell clones with specific performance within 2 - 4 PG/CL/d (10-20 MCAD/KL/d). In serum-free conditions, one clone was supported daily productivity 2-4 PG/CL/A. Clones with such a high level of productivity can produce 3-4 million units per day in 15-liter perfusion fermenter. By definition one unit of activity of factor VIII is the activity contained in one milliliter of plasma. One PG of factor VIII in General equivalent to 5 MCAD activity of FVIII.

In this work, the protein having protagonizada activity of factor VIII is a protein that causes the activation of factor X in model systems in vivo and in vitro. As non-limiting examples, this definition includes a full-size recombinant human factor VIII and factor VIII deletionism In the domain, the sequence of which is shown in figure 1.

High level of expression of a protein having protagonizada activity of factor VIII, means at least about 2 MCED/KL/d or more, preferably 4 MCED/CL/l, most preferably 5 MCED/KL/d activity of factor VIII in the capacity in which, free of plasma proteins; or at least 4 MCED/CL/d, preferably 8 MCAD/CL/l, most preferably 10 MCAD/KL/d activity of factor VIII in the build environment add-derived plasma protein. If the expressed protein - BDD-FVIII, then the described method can be used to obtain cell lines with specific productivity up to 15 MCAD/CL/l, preferably up to 20 MCAD/CL/A.

When describing the origin of the cell lines, the term "obtained from" unlimited includes normal mitotic cell division and processes type of transfection, merge cells, or other genetic engineering techniques used to modify cells or obtain cells with new properties.

Brief description of drawings

Figure 1 - amino acid sequence of BDD-FVIII (SEQ ID NO:1).

Figure 2 - sequence of the terminal repeat (TR) of Epstein-Barr (SEQ ID NO:2).

Figure 3 - map of plasmid pCIS25DTR.

Figure 4(a) to obtain a clone OF.

Figure 4(b) - comparison of productivity of several clones in different environments.

Presents three pooled data of two-month stability studies of each clone.

5 is a volumetric productivity of clone OV.

The best ways of carrying out the invention

The study FVIII

Activity of derivatives of factor VIII obtained by expression of a recombinant gene in methotrexate (MTX)-Usto the sustainable populations of cells, was determined by chromogenic assay. Activity was measured by Coatest kit® factor VIII:C/4 (Chromogenix, Molndal, Sweden) according to manufacturer's instructions. As the standard of measurement in this study used anti-hemophiliac factor (factor VIII) U.S. standard, known as MEGA I (Office of Biologics Research and review, Bethesda, MD). Cm. Barrowcliffe, 1993, Thromb Haem 70: 876.

Construction of expression vectors for FVIII with deletionism In-domain

The sequence of FVIII with deletionism In-domain (BDD) is presented in figure 1. 90-kDa and 80 kDa-chains were connected by a linker consisting of 14 amino acids. Cm. Chan, S.-Y., "preparation of recombinant factor VIII in the presence of liposomally substances complex structure" ("Production of Recombinant Factor VIII in the presence of Liposome-like Substances of Mixed Composition"), application for U.S. Patent N 08/634,001, registered on April 16, 1996. The expression vector for BDD-FVIII was obtained using standard methods of recombinant DNA. The structure of the expression vector (pCIS25DTR) is presented in figure 3. The vector contains the transcription for BDD-FVIII and selective marker, dihydrofolate reductase (dhfr). In addition, to improve the efficiency of the integration vector was included sequence terminal repeat of Epstein-Barr (Figure 2), which has a high level of drug selection. A vector is essentially a design vector (the room is built in ATSC number 98879), which was created to enable transcription of the plot, corresponding to the sequence shown in figure 1. Additional sequence information terminal repeat can be found in the related patent application Cho and Chan, marked MSB-7254 included in this description by reference, Sequence terminal repeat of Epstein-Barr increases the level of drug selection", registered on the same day as this application.

Experts in the art may construct and use the same vectors to obtain cells expressing proteins with protagonizada activity of factor VIII. For example, coding sequences of known variants of factor VIII, preserving procoagulation activity, can be replaced by the coding sequence of BDD-FVIII. Also, instead of dhfr you can use other selective markers, such as glutamine synthetase (gs) or the gene of resistance to multiple drugs (mdr). The choice of agent selection should be carried out according to the facts known in the art, i.e. for dhfr preferred agent selection is methotrexate for gs - methionine sulfoximine, and for mdr - colchicine.

Examples

Obtain cell lines expressing BDD-FVIII: Transfection. selection for drug and amplification of the s.

Cells NCV (Depositary room in ATSS - CRL 12568 - hybrid 293S cells and human cell lymphoma, Burkitt's lymphoma, see the application for U.S. Patent Cho et al., filed on the same day as this application and identified MSB-7241 included in this description by reference) were transliterowany 30 micrograms DNA pCIS25DTR method of electroporation at 300 volts and 300 microfarad (WITH Electro cell Manipulator 600) when using a 2-mm cuvette (WITH #620). In a comparative experiment carried out in parallel with the cells NCV, CHO cells (Chinese ovary hamster) and 293S (kidney of a human embryo) was transfusional when using a cationic lipid reagent DMRIE-C (Life Technologies, Gaithersburg, MD) according to the Protocol offered by Life Technologies. Amplification of transfected cells was performed with increasing concentrations of methotrexate (MTX) (100 nm, 200 nm, 400 nm and 800 nm) with 1×106cells in 96-well plate in MTX-selective medium without gipoksantina and thymidine (Wednesday DME/F12 without thymidine and gipoksantina with the addition of 5% cialisbuynow fetal bovine serum (FBS), supplied by Hyclone, Logan, UT). Were considered grown MTX-resistant cells and approximately 2-3 weeks after transfection was verified secretion of BDD-FVIII using Coatest kit® factor VIII. The cultivation was carried out at 37°C in humidified incubator with 5% CO2.

Cloning end what swedanam

Clones of a single cell (SCC) were obtained by cloning with the final dilution (LDC) high producing populations in 96-well-plates in serum-free conditions. Cells were seeded in quantities of 1-10 cells per well in medium DME/F12 with the addition of 10 μg/ml of recombinant human insulin Humulin® (Lilly, Indianapolis, IN), 10X indispensable aminocell (Life Technology, Gaithersburg, MD) and protein fraction of human plasma Plasmanate® (Bayer, Clayton, NC). Protein fraction of human plasma (NRR) Plasmanate® contains human albumin (88%) and various globulins (12%). Clones were tested for the production of BDD-FVIII using sets Coatest® factor VIII. Clones with the highest productivity were selected to assess the stability in shaker bottles. For cells NKW the first stage of the cloning was performed using selective medium with addition of 5% cialisbuynow FBS. The second stage of the cloning was performed in medium without serum but with added protein fraction of human plasma Plasmanate® when the primary clones isolated cells adapted to serum-free medium with the addition of the protein fractions of human plasma Plasmanate®.

Obtaining a clone LCI OF

As shown in Figure 4(a), the primary population SO was obtained from cells LCI, transfected pCIS25DTR, after amplification with 400 nm MTX in selective medium with 5% FBS. One of the primary clones single who's cells (SCC), OA obtained from SO cloned with the final dilution in the use of selective medium with 5% FBS, was adapted in serum-free medium with the addition of the protein fractions of human plasma Plasmanate®. It was unexpectedly found that the clone OA has greatly increased level of rFVIII products at this stage (Fig.4b). Thus, it was held the second clone with the final dilution in the environment with the addition of the protein fractions of human plasma Plasmanate®. The productivity of clones SCC (for example, OV)obtained in the second cloning with the final dilution was similar productivity clone OA adapted when adding protein fraction of human plasma Plasmanate®. Clone OF had higher levels of BDD-FVIII than the original clone OA obtained with the first clone with the final dilution in medium containing serum. In conclusion, the clone OF was adapted to growth in an environment without plasma proteins (PPF). Samples of clone OV were placed in the American Collection of Type Cultures (ATSS) (Manassas,VA) (number in ADS: CRL-12582).

As shown in Table 1, clones LCI have overproductivity BDD-FVIII. In cells LCI was observed 10-20-fold increase in productivity compared to clones obtained after transfection cells SNO and 293S. Cells LCI, do not form large cell aggregates with the growth in susp nionoy culture, are preferred for the expression of proteins with protagonizada activity of factor VIII.

Table 1
Expression of FVIII and BDD-FVIII in human cell lines and rodent
 Specific productivity (MCED/KL/d)*
Derived FVIIIKSS293SSNONKW
Full-FVIII0.451.20.51.0
BDD-FVIIIThe concentration is2.51.020
*The average value for 5 wysokoprocentowych clones (in serum-free medium) Acting= not specified

Adaptation of clones in the absence of plasma proteins

Clones LCI, adapted to growth in serum-free suspension cultures, then was adapted to growth without additives plasma proteins. This process was performed in sterile polycarbonate shaker bottles (Cornig, Cornig, NY) at a density of cells approximately 0.5×106cells/ml when using environment free from plasma proteins. Environment, free from plasma proteins (PPF), was Wednesday DME/F12 with the addition pluronic F68 (0.1%), CuSO4(50 nm) and FeSO4/EDTA (50 m is M). Complete medium was replaced every 48 hours, and shaker bottles again were seeded at a density of 0.5×106cells/ml

Fermentation clone OF

Productivity clone OF was evaluated in a 15-liter perfusion fermenter. The fermenter was seeded by cells of the clone OV at a density of about 3×106cells/ml Fermenter was watered with a speed of 4 volume day in serum-free medium, as described in the previous paragraph. During the evaluation period (45 days) was supported by a final cell density of 2×107. As shown in Figure 5, during the first 4 weeks of fermentation cell clone OV was watered serum-free medium with the addition of the protein fractions of human plasma Plasmanate® and were able to maintain high productivity. With 28 days before the end of the fermentation the cells were watered the same serum-free medium, but without the protein fraction of human plasma Plasmanate®. As shown in Figure 5, the cells continued to produce FVIII at a high level in the environment without plasma proteins. "An environment free from plasma proteins" means any protein, secreted from the plasma to the environment was not added.

Discussion

Obtaining cells LCI provides a protein-free system to obtain not only the BDD-FVIII, and other therapeutic proteins. Proteins obtained in cells LCI, glycosylated human models that can increase the half-life of kotoryj glycoproteins in vivo. These cells can also be used to generate adenoviral and adeno-associated lines developed for the purposes of gene therapy.

The above examples are intended to illustrate the invention, and specialists in this field technicians can use their options. Accordingly, it is assumed that the volume of applications limited only by the following claims.

1. The method of preparation and the selection of a protein having the activity of factor VIII, which includes the cultivation of LCI cells, identified by the American type collection culture (ATS) as CRL-12568, or LCI A cells transfected pCIS25DTR, or LCI S cells transfected pCIS25DTR, or LCI V cells, designated as ATSS CRL-12582 that contain a sequence encoding a protein having the activity of factor VIII, operatively associated with the promoter, and the cultivation is carried out under conditions sufficient for expression of a protein having the activity of factor VIII, and the allocation of a protein having the activity of factor VIII.

2. The method according to claim 1, wherein the protein has the amino acid sequence designated as SEQ ID NO:1.

3. The method according to claim 1, in which the protein is expressed at a level at least 2 µa/CL/d, when cells are grown in an environment free from plasma proteins.

4. The method according to claim 3, in which the protein expre Seretse level, at least 4 µa/CL/A.

5. The method according to claim 4, in which the protein is expressed at a level at least 5 µa/CL/A.

6. Cell line LCI A, transfusiona pCIS25DTR, which expresses a protein having the activity of factor VIII.

7. Cell line LCI S, transfusiona pCIS25DTR, which expresses a protein having the activity of factor VIII.

8. Cell line LCI V, ATS CRL-12582, which expresses a protein having the activity of factor VIII.



 

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