CN1161973A - Two-step purification method of recombinant human tissue plasminogen activator - Google Patents

Abstract

A two-step decontamination method for recombining human tissue type plasminogen activating agent (tr-PA) is to utilize the epculiarity of micro-porous high-Si glass beads (MPG) in the MPG adsorptive chromatographic column capable of non-specifically combining tr-PA in large quantity, MPG is used for decontaminating tr-PA-contained serum cell culture supernatant as the first step; based on the peculiarity that lysine position points present in tr-PA molecules can combine the specificity of lysine on Lys.-Sepharose 4B-philic chromatographic analysis column, the Lys-Sepharose-philic chromatographic analysis is used for decontaminating tr-PA as the second step.

Description

Tr-PA's two-step purifying method

Invention relates to a kind of purification of Recombinant human histiotype plasminogen activator, and (recombinant tissue-typeplasminogen activator, two-step purifying method rt-PA) belongs to biotechnology.

(tissue-type plasminogen activator t-PA) is the physiological activator of fibrinolytic system in the blood in tissue-type plasminogen activator.It and thrombus stroma fibrin have stronger special avidity, can be a kind of efficient special thrombolytics at the local efficient fibrinolytic system that activates of thrombus.Because the transformation period of t-PA is short, clinical consumption is big, cultivate the t-PA that produces by human tissue cell and far can not satisfy clinical needs, the rt-PA that utilizes gene recombination technology to produce in prokaryotic expression system can not carry out posttranslational modification effectively, be difficult to form the configuration consistent, thereby to produce rt-PA with eukaryotic expression system be the approach that generally adopts at present with natural rt-PA.The purifying process of product is an important step in mass production people rt-PA process.

At present external used t-PA purification technique as:

1. Rijken and Collen adopt: 1). the chelates of zinc affinity chromatography; 2). the canavalin(e) affinity chromatography; 3) t-PA in three-step approach purifying human melanoma cell (Bowes) the serum-free culture supernatant such as .Sephadex G-150 gel-filtration.

2. Einarsson is with three-step approaches such as immunoaffinity chromatography, arginine affinity chromatography and Sephadex G-150 gel-filtration, the purifying that reorganization human melanoma cell serum-free culture excretory rt-PA is carried out.

European patent (application number: 87308392) record, adopt antibody affinity chromatography, saltout and purifying that ion exchange chromatography carries out the rt-PA that produces the production of rt-PA recombinaant CHO cell etc.

Form by three step purification steps in view of above-mentioned all purification process, step is various, has comprised desalination or gel permeation chromatography in the purge process, and scale is difficult for amplifying, and the rate of recovery of t-PA and specific activity are all low, and (rate of recovery is lower than 75%, and specific activity is about 2.0 * 10 ⁵IU/mg albumen); Adopt antibody affinity chromatography, in purge process,, mix in the t-PA product, cause quality product restive because of heterogenetic antibody comes off from stationary phase; In addition, above purification process all is only limited to the purifying of t-PA in the serum-free cell culture, can not be used for having the purifying of the cells and supernatant t-PA of serum.

The technical essential of two-step purifying rt-PA method provided by the invention is to adopt micro pore high silicon granulated glass sphere (Micropore Glass, MPG) adsorption chromatography is as the first step of this purification process, use Methionin (Lys-Sepharose 4B) affinity chromatography to be further purified then, both are in conjunction with the two-step purifying method that forms.This method is applicable to that purifying contains the cultivation of calf serum training base and produces the rt-PA that rt-PA CHO engineering cell (N4B3) is produced, and can be used for the industrial amplification production of rt-PA, and its purpose is to overcome existing purification process and is difficult to adapt to the defective that technical scale is amplified production.

Following technical scheme can realize purpose of the present invention:

1., belong to micropore or claim porous high silica glass pearl, the rt-PA in chromatography column in a large amount of adherent cell culture supernatant in the non-specific ground of energy so the present invention's employing micropore glass pearl is to contain abundant silicon hydroxyl because of its surface.

2. owing to contain lysine-binding site in the rt-PA molecule, can combine specifically with the Methionin on the Methionin affinity column,, separable the Methionin affinity chromatography to having and natural rt-PA identical configuration and bioactive highly purified rt-PA as the step of second in this two-step approach purifying.

Embodiment:

Example 1.

(1) preparation of micro pore high silicon granulated glass sphere (MPG) chromatography column: selecting particle diameter for use is 75～125 μ m, and micropore diameter is the MPG of 35nm, dries after the rinsing repeatedly with distilled water.

(2) consumption of MPG is the consumption calculating according to the required MPG1 gram of every processing 100～120ml cells and supernatant.

(3) MPG soaks back filling chromatography column with PBS.

(4) N4B3 that contains rt-PA with 0.8 μ m filtering with microporous membrane has the serum culture supernatant.

(5) supernatant after the filtration passes through MPG adsorption chromatography post with the flow velocity of 200cm/h.

(6) use 0.02M PBS, pH7.4 (E respectively ₁) and 0.02M Tris, 0.5M NaCl, pH7.4 (E ₂) the wash-out foreign protein.

(7) with 0.02M Tris, 0.5M NaCl, 0.5M Arginine, pH7.4 (E ₃) rt-PA of elution of bound on MPG, flow velocity is 100cm/h.

(8) collect the E that is rich in rt-PA ₃Elution fraction.

Example 2:

(1) E of rt-PA will be rich in deionized water in the MPG adsorption chromatography ₃4～5 times of elution fraction dilutions.

(2) flow velocity with 100cm/h passes through Lys-Sepharose 4B affinity column.

(3) use with the MPG adsorption chromatography in identical E ₁And E ₂The wash-out foreign protein.

(4) with 0.02MTris, 0.15M NaCl, 0.5M Arginine, the rt-PA of pH7.4 elution of bound on Lys-Sepharose 4B affinity chromatography.

(5) keep sample and measure activity and protein concentration, identify the purity of rt-PA with SDS-PAGE in conjunction with photoabsorption scanning.

(6) whole process of MPG adsorption chromatography and Lys-Sepharose 4B affinitive layer purification rt-PA is carried out under 4～8 ℃.

(7) all contain 0.01%Tween80 in all elutriants, the elution process wavelength is that the UV-light of 280nm detects.

The good effect that MPG adsorption chromatography and Lys-Sepharose 4B affinity chromatography two-step purifying method produce in actual applications:

It is big that 1.MPG adsorption chromatography has a binding capacity to the purifying of rt-PA, flow velocity is fast, concentrate and the effect of purifying aobvious The advantage of work.

2. the rate of recovery height of rt-PA (surpass 100%), the purification efficiency height, the purity of rt-PA improves 200～300 times, and the rt-PA behind the purifying accounts for 98% of total protein concentration, and specific activity reaches 4.2 * 10⁵IU/mg albumen. Through two-step purifying The concentration of rear rt-PA improves about 200 times.

3. operate simple and easyly, good reproducibility can be used for industrial amplification production.

Claims (9)

1. the two-step purifying method of a tr-PA (rt-PA) is made up of micro pore high silicon granulated glass sphere (MPG) adsorption chromatography and Methionin (Lys-Sepharose 4B) affinity chromatography, it is characterized in that MPG adsorption chromatography wherein,

2. the two-step purifying method of a kind of rt-PA according to claim 1 is characterized in that abundant silicon hydroxyl is contained on the surface of (1) MPG; (2) particle diameter of MPG is 75～125 μ m; (3) its micropore diameter is 35nm,

3. the two-step purifying method of a kind of rt-PA according to claim 1 is characterized in that MPG calculates with 100～120ml/g MPG the binding capacity of rt-PA,

4. the two-step purifying method of a kind of rt-PA according to claim 1, the serum cells and supernatant that has that it is characterized in that containing rt-PA is 200cm/h by the flow velocity of MPG adsorption chromatography post,

5. the two-step purifying method of a kind of rt-PA according to claim 1 is characterized in that unconjugated albumen washs with 0.02MPBS, 0.01%Tween80, pH7.4 in the MPG chromatography column,

6. the two-step purifying method of a kind of rt-PA according to claim 1 is characterized in that the foreign protein that part is incorporated on the MPG carries out wash-out with 0.02MTris, 0.5M NaCl., 0.01%Tween80, pH7.4,

7. the two-step purifying method of a kind of rt-PA according to claim 1, the rt-PA that it is characterized in that being incorporated on the MPG carries out wash-out with 0.02MTris, 0.5M NaCl, 0.5M Arginine, 0.01%Tween80, pH7.4,

8. the two-step purifying method of a kind of rt-PA according to claim 1,0.02MTris, 0.5M NaCl, 0.5M Arginine, 0.01%Twwen80, the pH7.4 elution fraction that it is characterized in that containing rt-PA with 4～5 times of deionized water dilutions after, flow velocity with 100cm/h passes through Lys-Sepharose 4B affinity column

9. the two-step purifying method of a kind of rt-PA according to claim 1 is characterized in that the whole process of MPG adsorption chromatography and Lys-Sepharose 4B affinitive layer purification is all carried out under 4～8 ℃.