LT3062B - Method for preparing recombinant human tumour necrolysis factor-alpha - Google Patents

Abstract

The point of this invention, which applies to biotechnology, is a method of homogeneous tumour necrosis α factor albumen purification obtaining from microbiological autotrophic organisms in order to produce medicinal preparations. This purification method may be used in the medical and biochemistry industries producing tumour α factor intended for treatment of oncology diseases. The invention aims to increase the output amount of the preparation and create such a control scheme that enables it to manage without a high pressure chromatography in the Phenyl-TSK column. This is achieved by cleaning tumour necrosis α factor from the Eschericnia coli SG200 50 microbiological autotrophic organism, that has a recombinant pTNF311Δ plasmid, and using the following processes: the breaking of cells and further albumen cleaning by chromatography on hydroxyapatite, DE-52 celluloses, Red Sepharose CL-6B and desalination on the Sephadex G-25.

Description

This is a biotechnological invention and relates to the production of 'homogeneous human tumor necrosis factor-α for medical purposes using a bacterial' cell with a cloned plasmid containing the human tumor necrosis factor-α gene. This method is for the production of human tumor necrosis factor-α protein for use in the medical and microbiological industry.

To a limited extent, homogeneous human tumor necrosis factor-10 is derived from cells of human hematopoietic origin (see Williamson B. 'D. · et al., Proc. Natl.

Acad. Sci., USA, 1983-, v. 80, pp. 5397-5401; EP 0218868;

C12P 21/02., C12N 15/00, 1985) 1

In recent years, human tumor necrosis factor-α is derived from 'microorganisms capable of synthesizing this protein by recombinant deoxyribonucleic acid technology (E'p 0220966, CO7K 3/20, 1985; US 4677063, C12P 21/00, C12N: 15/00 ; EP 0263518, CO7K 3/18).

The use of microbiological producers enables to solve the problem of large-scale production of human tumor necrosis factor-α protein and to meet medical needs.

The closest technological approach to obtaining human tumor necrosis factor-α from Escherichia coli K-12 DG95 λ cells containing the recombinant plasmid pAW711 is as follows: suspending the microorganism cells in 10 mM Tris buffer (pH 7.0) and sonication; pre-cleaning,

- using DEAE-agarose with an ionic force gradient ··· to 1 M NaCl in 10 mM Tris buffer (pH 8, -2); further cleaning. by high-performance chromatography, P'applies on a preparative phenyl-TSK column equilibrated in 0.1N phosphate buffer (pH 7.0)

In this manner, only 0.6 mg protein (6X1Q ⁶ V) is obtained per cycle. In addition, this technique limits the industrial production of human tumor necrosis factor-α from bacterial producer biomass, since only small amounts of cells can be disrupted by ultrasound. The process is not technological and due to the use of high pressure chromatography.

The object of the present invention is to increase the yield and purity of human tumor necrosis factor-α preparation and to provide a more economical technological purification scheme.

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The objective is achieved by the following bacterial-producer purification of human tumor necrosis factor-α

Scheme for Escherichia coli SG200 50, in which the tumor necrosis factor-α gene has been cloned into the plasmid pTNF31lA obtained by known means (see USSR Authorization No. Γ445193): -;

1. Cell disruption under high pressure (MantonGaulin or equivalent apparatus), temperature 3-5 ° C;

2. Removal of Nucleic Acids Using DE-52 Cellulose (Whatman, United Kingdom) at 3-5 ° C;

'3. Primary' purification stage using hydroxylapatite (KPaCHbJkl XHMHK, CIS);

4. Chromatography on DE-52: Cellulose (Whatman, UK);

5. Affinity chromatography using Red-Sepharose CL-6B (Pharmacia, Sweden);

6. Desalting using Sephadex G-25 5 (Pharmacia, Sweden).

All purification operations, starting with chromatography on hydroxylapatite, are carried out under sterile conditions using. apyrogenic sorbents and buffers.

The presented method of producing human tumor necrosis factor-α differs in that bacterial cells were disrupted by high pressure (Manton-Gaulin

15. apparatus), which enables unlimited hassle of the process. Substitution of hydrophobic high pressure chromatography on a phenyl-TSK column for affinity (Ręd-Sepharose CL-6B) e

enables up to 2.2χ10 ^1θ V preparation per cycle at 6x10 ⁶ V (prototype). Substantial changes allow unlimited process reduction, while significantly increasing yields and purity, which is important in industrial production.

Examples of implementation of the presented method

Example I.

1.1. Cell disruption of strain producer by Manton-Gaulin apparatus

120 g of Escherichia coli SG200 50 (pTNF31lA) biomass, frozen at -70 ° C, are pulverized into 0.5-1 cm ³ pieces, placed in a dish and 2400 ml of 20 mM Tris containing 200 mM NaCl, pH 7.5, stir until homogeneous suspension at 3-5 ° C. The resulting suspension was allowed to flow 2-3 times through a Manton-Gaulin apparatus at 500-600 kg / cm ^{2 for} 1 hour in a Beckman

Extract J2-21 centrifuge

1400 rpm 2350 ml of protein extract is obtained (see table) R '

1.2. Nucleic Acid Removal 'r'R- ^? ''-r;'.'11'R'Xr'<R; -R

Add 500 ml of DE-52 to the resulting protein solution. cellulose washed with 20 mM Tris containing 200 mM: R NaCl, pH 7.5 and stirred for 15-20 min at 3-5 ° C. By decantation, the currant is separated from the protein solution and rinsed with 50.0 ml of 20 mM Tris, 200 mM NaCl, pH 7.5. The protein solutions are combined. 2900 ml of protein solution is obtained. .

1.3. Chromatography on hydroxylapatite ¹⁵ ~? ^>'RR': UrR - / 1 / .R.7 'RR

• ... o .... R '' R '. R .'RR 'R; ·. '. - RR ' ¹ '

The resulting protein solution was diluted 3 times with 20 mM Tris. pH 7.0 and passed through a K45 / 100 column ('' Pharmacia, Sweden) filled with 1000 ml of hydroxylapatite at 3-5 ° C, after which it was washed with 50 mM Na ₂ HPO ₄ , pH 7.0. The sorbent is then rinsed with the same buffer having a volume of 4-5 sorbent.- Human Tumor Necrosis Factor-oy • Removed with an ionic force linear gradient to 220 mM Na ₂ HPO ₄ , RpH 7.0. Elution rate 500 ml / h. Fractions containing: · human tumor necrosis factor-a are harvested (see Table 3). This and all subsequent steps are performed under sterile conditions.

1.4. - Ultrafiltration R

R.r'r ' _: ' t? Y ''; toR <^;'k'R^;'' ^: R '' ⁵ R ^; · R’R ·. Combined. The fractions are titrated under hydroxylapatite fluorescence using a Minitan apparatus (Millipore, USA). Five cycles are performed. The membrane pore size is' 10,000 Daltons. 20 rnM Tris pH 7.5 buffer was used. .

RR. ^? '-RrR''R,

1.5. Chromatography using DE-52 cellulose

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After dialysis protein solution passed through a column of 'K45 / 100 (Pharmacia, Sweden) packed with 2000 ml of DE-52 celuilioze, "against tai'serbentas washed with 20 mM Tris / pH 7 .5, the temperature 3-5 C. ^c

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Continue flushing through the sorbent until a baseline is reached, fixed in protein detector; ^{: their} control equipment UV-I type (Pharmacia, Sweden). The elution is performed by a linear gradient of the ionic force to

140 mM NaCl, 20 mM Tris, pH 7.5, in buffer. Speed 2000 ml / h. Fractions containing human tumor necrosis factor-α are pooled. Total volume 3000 ml (see table). ^; 3 ^{: /} '' to '/ ·''·' ·

1.6. Affinity chromatography using. Red- to.

SepharoseCL-6B.

The sorbent was washed with 25 mM Na ₂ HPO , _; , pH 7.2 buffer ... The pooled fractions were passed through a K50./40 column (Pharmacia, Sweden) loaded with 300 ml of Red-Sepharose CL-6B sorbents. A flushing buffer up to the baseline is passed through the sorbent. Elution 'is performed with an ionic force gradient up to 1.5 M NaCl,' 25 · mM Na ₂ HPO ₄ ,. pH 7.2,. The sorbent was then further washed with buffer containing 1.5 m NaCl, pH 7.2. Fractions containing human tumor necrosis factor-α are pooled. Total volume / 1600 ml (see table).

1.7. Ultrafiltration

3 ^ // 333 // y. / '.,. // - 3' .// 'ΐ · 3 / ^

The pooled fractions are ultrafiltrated under a Red-Sepharose CL-6B apparatus in a Minitan apparatus (Millipore, USA). The membrane pore size is 10,000 / dalton. Ultrafiltration / seeding at a protein concentration of not more than 4 mg / ml.

Γ.8. Desalting column Sephadex G-25

T he Column Κ10Ό / 100 Pharma e i a ”Liquid column (a) Filled with 7500 ml Sephadex G-25 sorbent, washed with 100 mM phosphate buffer, 200 mM NaCl, pH 7.2. Concentrated protein. the solution is passed through a sorbent at a rate of '400 ml / h. Fractions containing human tumor necrosis factor-ct are pooled. Total volume 800 ml (see table ·).

Example II.

2.1. The destruction of biomass cells is carried out analogously to 1.1.

The pressure of the bacterial producer is 700-800 kg / cm ² using the Manton-Gaulin apparatus.

2.2. Nucleic acids are removed analogously 1.2. to

2.3. Chromatography using hydroxylapatite. Elution of human tumor necrosis factor-α is performed using a linear gradient of ionic strength to 300 mM Na ₂ HPO ₄ , PH 7.0.

2.4. Ultrafiltration.

Perform by analogy 1.4.

2.5. Chromatography on DE-52 cellulose. Elution is performed using a linear gradient of ionic strength to 200 mM NaCl, 20 mM Tris, pH 8.0.

2.6. Affinity chromatography using Red-Sepharose

CL-6B. '' -

Elution is performed by ionic force linear up to 2 M NaCl, 25.'MM Na ₂ HPO ₄ , pH 7.2.

2: 7.

-; ; . . : i '

Performing the same procedure 1.7. ·

2.8. Desalting column Sephadex G-25. ../ ::

Performed by analogy 1.8.

Example III.

3.1., The destruction of the biomass cells is carried out analogously

1.1. Pressures of 300-400 kg / cm ^{2 are} used to destroy bacterial product cells using 10 Manton-Gaulin apparatus.

3.2. Nucleic acids, acids removed by analogy 1.2.

3.3. Chromatography on hydroxylapatite is carried out analogously to 1.3.

3.4. Ultrafiltration is carried out analogously to 1.4.

3.5. Chromatography on DE-52 cellulose, 20 ·. performed analogously 1.5.

3.6. A-final chromatography using Red-Sepharose CL-6B is performed using a linear gradient of ionic strength ^u to 1.5 M NaCl, 25 mM Na ₂ HPO ₄ , pH 7.2.

3.7. Ultrafiltration is carried out analogously to 1.7.

3.8. Sephadex G-25 column desalting is performed analogously to 1.8.

The resulting preparation is pyrogen-free, sterile, homogeneous in electrophoretic and immunochemical manner, and has a biological activity of not less than 3x10 ^?

35.; ///// '/' .. y .- / '/, ////; ./ /./-y// /

The homogeneity of the resulting preparation of human tumor necrosis factor-α was confirmed by electrophoretic polyacry

in a laminated gel in the presence of sodium dodecyl sulfate.

• Automated sequencing modified by Edman method

Charigo showed that the resulting preparations of tumor necrosis factor-a have a single polypeptide chain called Ser5 'Aris-Thr-Pro-.7. amino acid sequence at the N-terminus.

The latter sequence of tumor necrosis factor-α differs from the natural type in that the N-terminus does not contain the four amino acids Val-Arg-Ser-Ser-...

Effectiveness of the method of obtaining human tumor necrosis factor-α ·

Cleaning stage Example I Example II Example III Exact- -Ύ; '· / / 77 / nis protein mg Yield % Target protein mg Yield % Target protein mg Yield % 1. Surpematant after breaking up ultrasound 2300 100. 2300 100 2250 100 2. Chromatogram, utilizing hydroxylapatite 1150 50 1150 50 1100 48.9 3. Chranatogram, using EE-52 cellulose 966 42 828 36 918 40, 8 4. Chranatogram, using Red- Sepharose-CL-6B 782 34 414 17.7 595 26, 4 5. Salinisation, utilizing Sephadex G-25 736 32 300 13.5 538 23.9 Bio Preparation logical active imam prescribed s-pauc io j an t

murine fibroblastic cells L929 equivalent to 3x10 ⁷ U / mg protein accustomed to protein 7 7 e.s / The use of the presented technological scheme enables to increase the yield of human necrosis factor-α up to 32%, in the prototype 30%, and. per cycle 2,2xl0 ^10V preparation prototype V. 6x10 ⁶ '''' 77B7 / 7 7 / 7.77: /:/::7^7/7:/77:7./ / 7: 777 /</.::7: _{s:.: -:;} ,, _;;

The replacement of high pressure chromatography allows for a more technological, hassle-free purification scheme. 7 .7 / ':' 7'7 / '

The resulting preparation can be used as a biologically active substance for cell cultures as well as for the preparation of a pharmaceutical form. e: '7 7ą: //...-- p · pą-. / p · .'- ::::

Claims (3)

DEFINITION OF INVENTION A method of producing recombinant human tumor necrosis factor from Eschęrichia coli cells. having 5 recombinant plasmid, using anion exchange sorbent and desalting in chromatographic processes, characterized in that it destroys Escherichia coli. SG200- 50 recombinant plasmid pTNF31lA under high pressure for initial purification. 10 hydroxylapatite and elution with a gradient of 220-300 mM Na ₂ HPO ₄ , pH 7.0, further purified by DE-52 cellulose, elution with 20 mM Tris buffer containing 140-200 mM NaCl, pH -7.5-8.0, then uses, affinity chromatography Red-. Sepharose CL-6B, elution using 25 mM Na ₂ RPO ₄ , • .i57: - + -. + Turfritis + '' l>: 5.72 Sodium phosphate buffer, pH 7.2. performs desalting on a Sephadex G-25 column. SL 1520. Tir. 52 copies Ordering 33 State Patent Bureau of the Republic of Lithuania, Algirdo 31,2600 Vilnius, Lithuania. Printed by the printing house of the Lithuanian Information Institute, Totorių 27,2000 Vilnius.