RU2125093C1 - Method of preparing human recombinant erythropoietin, strain of cultured chinese hamster ovarian cells - a producer of erythropoietin - Google Patents

Abstract

FIELD: biotechnology, molecular biology. SUBSTANCE: method is carried out by culturing the strain of Chinese hamster ovarian cells (CHO) transformed with plasmid that contains human erythropoietin gene in growth and accumulative nutrient media. Accumulative medium has the following components (in 1 l): Fetaclone 11 (10 ml), glutamine 200 mg, HEPES 3.57 g and a mixture of media RPM1, 1640 and DMEM taken at equal parts. Strain is obtained by transformation of Chinese hamster ovarian cell (CHO) к 1DHFR with plasmid pBD carrying the complete human erythropoietin gene followed by selection on medium containing 1 mM methotrexate. Strain is deposited at number VSKK (P)-651 D. EFFECT: high yield of erythropoietin under industrial conditions. 5 cl, 6 dwg, 1 tbl, 7 ex

Description

 The invention relates to the field of biotechnology, and in particular to a technology for producing human erythropoietin by genetic engineering.

 Erythropoietin is a growth factor and terminal differentiation of red blood cell precursors in the bone marrow.

 The synthesis of erythropoietin in animals and humans is carried out mainly in the kidneys and liver, and the level of its secretion normally does not exceed several tens of milliunits. With a number of pathologies, especially with kidney diseases, the synthesis of endogenous erythropoietin is significantly reduced, which leads to the development of severe anemic conditions. The introduction of exogenous erythropoietin can effectively restore the formation of red blood cells, thereby normalizing the condition of patients. Due to the practical impossibility of obtaining erythropoietin in sufficient quantities for therapeutic use from natural sources, widespread technology for its production based on the use of recombinant animal cell lines that can secrete human erythropoietin (rEPO) when cultured on synthetic nutrient media in vitro (acc. PCT application NN 85-03079, class C 07 H 7/10, 1985; Heb. Pat. NN 0205564, class C 12 P 21/00, 1990; 0209539, class C 07 K 7/10, 1992; 0255231, C. C 12 P 21/02, 1995).

 As producers of rEPO, mammalian cell strains are mainly used, most often strains of cultured Chinese hamster cells. In particular, such producer strains as VSCK (P) -162D, VSCK (P) -626D are known (Pat. RF NN 1555359, class C 12 N 5/00, 1990, 2070931, class C 12 N 5/10, 1996). The level of production achieved during their cultivation and in stationary conditions on synthetic nutrient media is about 4 mg / l of recombinant erythropoietin.

 The disadvantages of the known methods are the relatively low yield of the target product and the complicated cultivation technology, as well as the need for periodic amplification of the erythropoietin gene to maintain the achieved level of production by selection on a nutrient medium containing hypoxanthine, aminopterin and thymidine (GAT), which increases the cost of production of erythropoietin.

 The prototype of the claimed group of inventions is a strain of HSC cells (P) -637D obtained by transforming the original cell line with the plasmid pSv-dEp-l-Mo carrying the full-sized gene of human erythropoietin. (Pat. RF N 2089611, class C 12 N 5/16, 1995). The strain is maintained on Eagle’s medium with 10% cattle serum (cattle) and is commercially cultivated on microcarriers with purification of the final product by chromatographic methods.

 When using this solution, the level of erythropoietin synthesis reached higher values and amounted to 1500-1800 U per ml.

 The main disadvantage of this strain and the method of its use are that the achieved level of erythropoietin biosynthesis in this case is still insufficient to create a cost-effective production.

 The challenge facing the authors was to obtain a highly productive strain of animal cells that constitutively produces human rEPO without additional periodic selection.

 The task is achieved as a result of the development of a technology that includes a new strain of cultured Chinese hamster ovary cells, which, upon registration, HSCC (P) -651D and the cultivation technology of this and similar strains.

 The strain was obtained by technology, which included the transformation of the Chinese hamster ovary cell line CHO K1 DHFR- (Urlaub, et al., 1980, PNAS. USA., 77, 4461) with the pBD plasmid (Glanville, Durham & Palmiter, 1981, Nature, 292, 267-269), which carries the MT-I promoter, the DHFR + marker, and a full-sized human erythropoietin gene, followed by one-step selection of target transfectants on a nutrient medium containing 1 mM metatrexate and subsequent cloning of the producers by limiting dilution with the control of the productivity of the colonies by solid-phase immuno-enzyme-linked immunosorbent assay.

The resulting cell strain (CHO-EPO / SPM was deposited in the Russian collection of cell cultures under the number VSCK (P) -651D
The strain is characterized by the following features:
1. Morphological features.

 Cell culture is represented at the initial stage of cultivation with fibroblast-like elements with large oval nuclei containing several nucleoli. As the monolayer consolidates, the cells acquire a rounded, epithelial-like shape, with the further growth of the culture, the formation of a multilayer cell structure is observed.

 2. Cultural traits.

 The strain CHO-EPO / SPM is maintained on a mixture (50% / 50%) of RPM11640 and DMEM culture media supplemented with 5% FETACLONEll serum substitute (HighClone, USA) and 40 μg / ml gentamicin.

Cells are substrate-dependent; when cultured in rich nutrient media (at a high fetal serum content of up to 10%), they are prone to partial detachment from the substrate and growth in suspension through the formation of multicellular clusters. Cells are repassed using a 0.25% trypsin solution, a 0.02% versene solution, or a mixture of 50% / 50%. The multiplicity of sieving 1/3 - 1/5, the sowing dose is 2-3 • 10 ⁴ cells per 4 square. see the surface of the culture bottle. The formation of the cell monolayer is completed 2-3 days after reseeding.

 3. Resistance to selective factors.

The strain shows resistance to metatrexate in a dose of up to 1 mm
4. Cryopreservation.

 Cryopreservation of the culture is carried out in RPMI1640 medium with the addition of 50% fetal serum and 7% dimethyl sulfoxide as a cryoprotectant at a cell concentration of 1.5-2.0 million cells / ml.

Cryoculture is stored in liquid nitrogen vapor or by immersion in liquid nitrogen. Thawing of the culture is carried out by rapid transfer of the ampoule with the culture to a water bath at 37 ^o C, followed by centrifugation and resuspension of cells in fresh growth medium. The viability of the culture after thawing is 85-95%.

 5. Control of species identity.

Compliance with the species is confirmed by sensitivity to proline
6. Contamination.

 When controlling the cells of the strain for the presence of bacterial, fungal and viral contamination in passages 10 and 40, no bacteria, mycoplasmas, fungi and viruses were detected.

 The main differences of the new strain was the possibility of producing rEPO in the absence of selective pressure. The strain retained the ability to constitutively produce repo for more than 40 passages without reducing the level of production and additional selection.

A feature of the proposed method of cultivation is that the cells are cultured in roller bottles on a nutrient medium containing 1 l of the following ingredients:
Fetaclone II - 80 - 20 ml
glutamine - 200 mg,
HEPES - 3.57 g,
a mixture of equal proportions of RPMI 1640 and DMEM media - up to 1 liter.

 In this case, the best results are achieved with the following cultivation parameters: the speed of rotation of the roller bottles -1.5 - 2.0 rpm, and the total cultivation time is at least 14 days with a duration of each accumulation cycle of 2 days.

As a rule, strains are grown before cultivation for at least 2 days in roller bottles at a rotation speed of 1.5-2.0 rpm on a nutrient medium containing 1 l:
fetal calf serum - FCS - 80-120 ml,
glutamine - 200 mg,
gentamicin - 0.04 mg
a mixture of equal proportions of RPMI 1640 and DMEM media - up to 1 liter.

 The best results are achieved when VSCK (P) -651D is used for culturing a strain of cell cultures, however, this method can also be used for other known strains.

 The usefulness and applicability of the obtained cell strain and a new method of producing rEPO for industrial use is illustrated by the following examples.

Example 1. To assess the productivity of cells of strain CHO-EPO / SPM, the pre-accumulated suspension of cells was sown at a concentration of 3-10 cells / ml in a volume of 100 ml of growth medium (50% / 50% mixture of RPMI 1640 media and DMEM containing 10 ml embryonic calf serum -FCS, 20 mg glutamine and 0.004 mg gentamicin) in each of 10 T175 culture bottles with a growth surface of 175 cm ² and a volume of 650 ml. The vials were incubated at 37 ^o C for 3 days under stationary conditions. At the end of the incubation, the growth medium in the bottles was replaced with an equivalent volume of the storage medium containing 1 l:
Fetaclone II - 10 ml
glutamine - 200 mg,
HEPES - 3.57 g,
a mixture of equal proportions of RPMI 1640 and DMEM media - up to 1 l,
and the incubation of the bottles at 37 ^o C continued for another 2 days. At the end of the incubation, the accumulation medium from the vials was collected in a collection container and placed after adding phenylmethyl sulfonyl fluoride (PMSF) for storage in a refrigerator at + 4 ^° C. A fresh portion of the accumulation medium was added to the culture bottles with cell culture and the entire erythropoietin accumulation cycle was repeated. The collected accumulative medium was saved as in the previous cycle. A total of 7 cycles of erythropoietin accumulation were carried out and 6.8 L of culture fluid containing erythropoietin was collected. At the end of the accumulation period, cells from each vial were collected after processing the cell monolayer with a trypsin / versene (50% / 50%) mixture by resuspension in 500 ml of accumulation medium and their concentration was determined using a hemocytometer.

The total number of cells collected from 10 vials was 22.75 • 10 ⁷ cells. Given that the total growth surface for 10 T175 bottles is 1750 cm ² , the average culture density per 1 cm ² was (1.3 ± 0.1) • 10 ⁵ cells. The concentration of erythropoietin was determined in the collected storage medium by enzyme-linked immunosorbent assay using the Amgen Clinigen reagent kit. The content of erythropoietin in the collected storage medium was 1160 ± 120 U / ml. Thus, the specific productivity of the obtained cell strain CHO-EPO / SPM was 5098 ± 527 E / 10 ⁶ cells / 48 h, which is several times higher than that achieved in the prototype.

 Example 2. Obtaining rEPO and its identification.

The collected storage medium was purified in order to isolate and characterize the synthesized erythropoietin and prove its identity to the natural analogue. For this, the culture fluid was clarified by microfiltration through membrane filters with a pore size of 0.8 μm (Millipore) and purified by immunoaffinity and ion exchange chromatography to a homogeneous state. The resulting preparation was evaluated according to the following criteria: purity, isoform composition, N-terminal chain identity, in vivo specific biological activity, immunological identity and the presence of oligomers. The obtained erythropoietin preparation according to the PAGE and HPLC electrophoresis was almost homogeneous and did not contain impurities of oligomeric forms of erythropoietin (Fig. 1a, b and 2). Its molecular weight was ≈35 kd, which is equivalent to the molecular weight of natural erythropoietin. The drug had microheterogeneity detected by isoelectric focusing (Fig. 3) and typical of erythropoietin due to differences in the degree and nature of glycosylation. During isoelectric focusing, 4–6 bands are detected in pl 3.5–4.5 zones, which is also characteristic of erythropoietin. The identity of the N-terminal sequence of the isolated erythropoietin of the previously described structure was demonstrated by solid-phase degradation of 16 N-terminal residues according to Edman (Fig. 4), which were identical to the natural hormone. The specific biological activity of the obtained erythropoietin was controlled in vivo in mice with hyperbaric-induced anemia by inclusion of ⁵⁹ Fe in EPO-stimulated erythroblasts. The 2nd international standard of erythropoietin was used as a standard. The specific activity of the obtained erythropoietin was (140 ± 14.5) • 10 ³ IU / mg protein, which corresponds to the activity of pure erythropoietin. The immunological identity of the obtained erythropoietin to its natural analogue was demonstrated in a competitive enzyme-linked immunosorbent assay, where natural erythropoietin isolated from the urine of polycemic patients was used as a competitor. With a 10-fold excess, cold natural erythropoietin inhibits almost 90% the binding of biotinylated recombinant erythropoietin to polyclonal rabbit antibodies to human erythropoietin immobilized on the cell surface of a polystyrene microplate (High bond. Costar). Thus, the totality of the criteria used allows us to state with certainty that the recombinant protein produced by the CHO-EPO / SPM cell strain is erythropoietin identical to the natural hormone.

Example 4. In order to demonstrate the stable productivity of the strain of cells SNO-EPO / SPM in the absence of selective pressure, the pre-prepared culture was seeded in 5 culture bottles T50 in 10 ml of growth medium growth composition (see example 1) at a concentration of 3 • 10 ⁴ cells / ml Vials were incubated at 37 ^° C. under steady-state conditions. Every 3 days, samples of the culture fluid were taken from each vial to determine the erythropoietin content in them, the remaining medium was removed and the cell monolayer was trypsinized as described in Example 1. 20% of the obtained cell suspension was plated in 10 ml of growth medium in a new T50 vial, and the remaining cells placed in cryopreservation medium and frozen for long-term storage in liquid nitrogen. Repassing of cultures and determination of erythropoietin content in the culture fluid at each passage was carried out for 4.5 months (45 passages). The results are presented in the table.

As follows from the data presented in the table, the level of erythropoietin production during passage of the CHO-EPO / SPM cell strain in the absence of selective pressure remained constant for at least 45 passages
Example 6. A variant of the method for producing rEPO cell strain SNO-EPO / SPM. A pre-prepared suspension of strain cells was plated in roller bottles (V = 2.6 L; S = 850 cm ² ) in a growth medium (see Example 1), except that significantly cheaper reconstructed Fetaclone serum was used instead of FCS 11 (HighClone, USA) in a final proportion of 1: 10 v / v ≈8 • 10 ⁶ cells were sown in each roller bottle in a volume of 200 ml. The vials were installed in a roller cultivator and incubated at +37 C at a rotation speed of 0.8 rpm for 3 days. At the end of this time, the growth medium in roller bottles was replaced by a storage medium consisting of a mixture of RPMI1640 and DMEM 50% / 50% culture media containing 200 mg of glutamine, 3.57 r HEPES (pH 7.4) and 10 ml Fetaclone II. 400 ml of storage medium were added to each vial. The incubation of cultures in a roller cultivator was continued for another 48 hours at a rotation speed of 1.5-2.0 rpm. At the end of the accumulation time, the culture medium from the roller bottles was collected in the storage tank, and a fresh 400 ml portion of the storage medium was introduced into the roller bottles and the accumulation procedure was completely repeated, collecting a new portion of the culture fluid at the end of the accumulation time in the storage tank. The described procedure for the accumulation of culture fluid with erythropoietin was repeated 7 times, stopping it due to the appearance of signs of a violation of the integrity of the cell monolayer due to its re-compaction. Erythropoietin was monitored in each portion of the collected culture fluid by enzyme-linked immunosorbent assay using an Amgen kit. The results are presented graphically in figure 5. In this example, the study of industrial potential was carried out on 30 roller bottles with a growth surface of 850 cm ² each with a storage medium volume of 400 ml and an accumulation time of 48 hours with a total number of accumulation cycles equal to 7. A total of 84 l of culture liquid was collected at a content erythropoietin 2611 ± 346 U / ml or about 1.5 g of erythropoietin. The result obtained, thus, demonstrates the high industrial potential of the proposed cell strain.

 Example 7. A variant of the method of obtaining rEPO. cell strain CHO-EPO-1. A pre-prepared suspension of strain cells was seeded in 5 roller vials (V = 2.6; S = 850 sq. Cm) and then cultivated in accordance with the conditions of Example 6. As a result of the experiments, 14 L of accumulating nutrient medium containing rEPO 360+ was collected 80 U / ml. Under standard conditions (Aut. St. USSR N 155359, class C 12 N 500, 1988), the level of production of rEPO for this strain was 12-15 U / ml.

Claims (4)

1. The method of producing recombinant human erythropoietin, comprising cultivating a strain of Chinese hamster ovary cells, transformed by pre-introducing a plasmid containing the human erythropoietin gene, sequentially in growth and accumulating nutrient media, followed by isolation of the target product sequentially by microfiltration and chromatographic methods, characterized in that the cultivation is carried out in roller bottles at a rotation speed of roller bottles of 1.5 - 2.0 rpm n, on the storage medium containing the following ingredients in 1 liter:
Fetaclone 11, ml - 10
Glutamine, mg - 200
HEPES, g - 3.57
A mixture of equal proportions of RPMI 1640 and DMEM media, l - Up to 1
and the total duration of cultivation in the growth medium is at least 2 days, and in the storage medium is at least 14 days with a duration of each storage cycle of 2 days.  2. The method according to claim 1, characterized in that as the cultured strain using a strain of cells of the ovary of a Chinese hamster N VSKK (P) N 651D. 3. The method according to claim 1 or 2, characterized in that as the growth medium is used growth medium containing 1 l:
Fetal calf serum - FCS, ml - 80 - 120
Glutamine, mg - 200
Gentamicin, mg - 0.04
A mixture of equal proportions of RPMI 1640 and DMEM media, l - Up to 1
4. The method according to claim 1 or 3, characterized in that the rotation speed of the roller bottles at the stage of growth cultivation is 1.5 to 2 rpm  5. The strain of cultured cells of the ovary of the Chinese hamster N VSCK (P) N 651D - producer of erythropoietin.

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