CN104558125A - Method for purifying polypeptide PDGF receptor inhibitor - Google Patents

Abstract

The invention relates to the field of polypeptide purification, especially to a purification method of a polypeptide PDGF receptor inhibitor prepared by a solid-phase synthesis method. According to the method, polypeptide undergoes crude extraction through ion exchange resin, and refining is carried out by high performance liquid chromatography after conversion in the form of salt. Finally, purity reaches more than 95%, and yield reaches 25%. The polypeptide purification method can be used for large-scale preparation of the polypeptide.

Description

A kind of method of purified polypeptide class pdgf receptor inhibitor

Technical field

The present invention relates to the purifying of the pdgf receptor inhibitor for the disease treatment such as tumour, organ fibrosis, this inhibitor is made up of 47 amino acid, adopt process for solid phase synthesis, what the present invention relates to is that the thick peptide obtained by solid phase synthesis is slightly carried, refining purification process.

Background technology

Tumour is the major disease of harm humans health, there is millions of patient infections every year, cause a large amount of death, for liver cancer, as one of the tumour of high malignancy, China dies from the patient of liver cancer and complication thereof every year up to 470,000 examples, and the expense for liver cancer treatment reaches 20,000,000,000 yuans, seriously occupying medical resource and funds, becoming for carrying out effective treatment the significant problem being badly in need of solving.

Existing achievement in research display, Thr6 PDGF BB (platelet derived growth factor, be called for short PDGF)---platelet derived growth factor receptor (platelet derived growth factor receptor, be called for short PDGFR) the active expression of signal transduction pathway (hereinafter referred to as PDGF-PDGFR signal transduction pathway) and liver cancer, cancer of the stomach, the generation of the solid tumor such as mammary cancer and lung cancer and development have certain dependency, prompting blocks PDGF-PDGFR signal transduction pathway will contribute to the treatment of tumour, describe the technology contents of this signal transduction pathway in the patent of patent No. ZL 2,008 1 0200937.8 in detail and applied for structure to by the polypeptide PDGFR inhibitor (hereinafter referred to as P47) be made up of 47 amino acid for the treatment of for tumour and fibrotic disease, sequence and use patent, the method of the thick peptide of method synthesis P47 adopting solid phase synthesis is described in detail in the patent of number of patent application 201310458634.7.

The method of solid phase synthesis is adopted to carry out the synthesis of polypeptide drugs, to suitable cutting liquid be used to make thick peptide and resin isolation and the protecting group removed on thick peptide side chain to polypeptide-resin cutting after end of synthesis, through washing, the thick peptide of desired polypeptides content at 20%-70% is obtained after dry, can be used for clinical medicinal polypeptide, its content should reach more than 95% usually, in order to improve the purity of polypeptide, the hollow peptide of effective removal, the impurity such as catalyzer, the most frequently used way in this area uses suitable solvent such as aqueous acetic acid, after the acetate acetonitrile aqueous solution etc. dissolves, reverse-phase HPLC technique is utilized to carry out purifying.Reversed-phase HPLC has very high resolving power, but have that Reusability easily makes that pressure increases, salt component to problems such as filler damage are larger, therefore need to introduce recycling reversed-phase HPLC after new method carries out pre-treatment to polypeptide and refine.

Ion-exchange is a kind of conventional isolation technique, along with the continuous effort of material produce enterprise, increasing medium is developed and is widely used in production practice, ion exchange resin have cheap, working range wide, to Organic Solvent Tolerant, can the advantage such as the incumbent firms of resistance to alkaline solution, therefore in the separation and purification of biotech drug especially reconstituted drug, become conventional means of purification, but the application in the purifying of chemically synthesized polypeptide medicine is less.

P47 is made up of 47 amino acid, there is certain protein characteristic, at solid phase synthesis and containing inorganic salt and organic solvents such as trifluoroacetic acids after cutting, direct employing reversed-phase HPLC can cause certain damage to medium, and the existence of impurity can affect the carrying capacity of reversed-phase column, therefore be necessary to carry out pre-treatment to it, the content of P47 brought up to about 80% after slightly carrying and remove inorganic salt and organic solvent to greatest extent, will follow-up refining be conducive to.

Summary of the invention

The invention provides a kind of method of purifying P47, the method, by using anion-exchange column slightly to carry P47 and adopting reversed-phase HPLC to refine again after changing salt, finally obtains the refining P47 of purity more than 95%, may be used for clinical.Simple and the yield had up to 25% of method provided by the present invention, is convenient to large-scale production, meets industry requirement.

The invention provides a kind of method of purifying P47; the method is applicable to adopt solid phase synthesis process synthesize and complete the purifying of the thick peptide of cutting; the starting material of present method is the polypeptide-resin adopting solid phase synthesis process to obtain; polypeptide is cut from resin through suitable cutting agent and excises the thick peptide of Side chain protective group; this thick peptide is also fully dry through washed with diethylether, and follow-up purifying comprises the steps.

Step 1: adopt anionite-exchange resin slightly to carry.Dissolve the thick peptide of P47, balance liquid A counterion exchange resin, loading is adsorbed, and balance liquid B balances and removes impurity, uses elution to obtain rough P47 solution.

Step 2: adopt reversed-phase HPLC to refine.Take 18 alkyl silica gel as stationary phase, balance liquid C balances, and by rough P47 solution loading, carries out wash-out using the acetate acetonitrile aqueous solution of finite concentration ratio as moving phase, collects main peak and both must refine P47 solution.

Step 3:: will refine the organic solvents such as P47 solution removal acetonitrile, is placed in freeze drier and carries out the P47 sterling that vacuum lyophilization obtains drying.

As preferably: the starting material of present method synthesizes via solid phase synthesis process, if the method described by employing this patent that the sample adopting liquid phase synthesis and/or biosynthetic method to obtain also can be part or all of carries out separation and purification.Part or all of technology contents in this method is can obtain and use for reference use by simple inference for professional and technical personnel.

As preferably: be the aqueous solution for dissolving the liquid of P47 in step 1, also the aqueous solution containing organic solvent can be adopted, with an organic solvent can increase the solubleness of P47, adoptable organic solvent includes but not limited to acetonitrile, methyl alcohol, ethanol etc., and wherein preferred organic solvent is acetonitrile.

As preferably: be 5%-40%(V/V for the organic solvent concentration dissolved in the solvent of crude product in step 1).

As preferably: in step 1 quality (mg) of P47 dissolving crude product with solvent volume (ml) than being 10:1-50:1(W/V).

 

As preferably: in step 1, balance liquid A used is basic solution.

As preferably: in step 1, balance liquid B used is saliferous basic solution.

As preferably: in step 1, elutriant used is acidic solution.

As preferably: with carrying out according to the following steps in step 1.

Step a: clean chromatography media to specifications, then balance liquid A carries out balancing to flowing liquid pH value consistent with balance liquid A.

Step b: with the aqueous solution or the thick peptide of aqueous dissolution P47 containing organic solvent, dissolving ratio is 10:1-50:1(W(mg)/V(ml)), loading under ultraviolet monitor function condition is until sample all enters chromatography column or sees that sample flows out through in peak.

Step c: rinse resin until flowing liquid pH value is constant with balance liquid A.

Steps d: with balance liquid B rinse resin until flowing liquid without ultraviolet radiation absorption.

Step e: with elution, collects elution peak according to ultraviolet monitor function, obtains P47 crude extract.

As preferably, the resin adopted is strong anion exchange resin.

As preferably: the carrying capacity of resin is not more than 200:1(mg:ml), preferred scheme is the carrying capacity of resin is 30:1-80:1.

As preferably: balance liquid A is basic solution, and pH value is 7.2-12, and preferred pH value is 9.5-11.5.

As preferably: balance liquid B is the basic solution of saliferous, pH value is 7.2-10.0, preferred pH value is 8.5-9.5, salt is wherein sodium-chlor or sodium carbonate or sodium-acetate or ammonium acetate or potassium primary phosphate, the ionic concn of above-mentioned salt is 20-1000mmol/L, and preferred ionic concn is 50-200mmol/L.

As preferably: elutriant is acidic solution, and pH value is 2.0-6.5, and preferred pH value is 3.5-4.5.

As preferably: acidic solution is acetum, and concentration (V/V) is 0.1%-1%.

As preferably: the moving phase of step 2 contains the acetum of acetonitrile, and preferred type of elution is gradient elution, is 5%-95% from the acetonitrile concentration (V/V) of 0-180min.The elution protocol with effect same also comprises stepwise elution, that is points three is three phases, and acetonitrile concentration (V/V) is respectively: 1%-10%, 11%-45%, 46%-90%, and time corresponding to different steps is respectively 5-50min, 5-50min, 10-100min.

As preferably: the silica filler diameter used is 5-15 μm, and preferred filler diameter is 5-10 μm.

As preferably: the mode that step 3 removes acetonitrile can adopt rotary evaporation, desalination chromatography to condense the modes such as filtration.

embodiment:

The invention discloses a kind of method of separation and purification P47, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter all realizes.Special needs to be pointed out is, all similar amendments or replacement will be readily apparent to persons skilled in the art, and they will be regarded as the content included by the present invention.Method of the present invention and application be described by preferred embodiment, those skilled in the art obviously can not depart from content of the present invention, principle and scope to methods and applications of the present invention carry out suitable amendment or suitable change combination realize and apply the present invention.

Embodiment 1:

Solid-phase synthesis obtains the thick peptide of P47

Sample preparation: dissolve the thick peptide of P47 according to the ratio of 30mg/ml with 1% ammonia soln and make the P47 solution that pH is 10.5, sample dissolves the membrane filtration of rear use 0.45 μm completely, and collect filtrate, 2-8 DEG C of placement is for subsequent use.

Ion exchange resin is slightly carried: use pH be 11 sodium hydroxide solution balance resin to PH detect constant.P47 solution is injected ion exchange column until sample all enters chromatography media, rinse until balance with the sodium hydroxide solution that pH is 11, then reequilibrate is carried out to remove impurity with the solution that the pH that sodium chloride concentration is 100mmol/L is 11, be the acetum wash-out of 4.0 with pH after balance, under ultraviolet monitor function condition, collect main peak, obtain P47 crude extract.

Reverse HPLC-purified: in P47 crude extract, add acetonitrile to final concentration is 5%(V/V), add with in the silicagel column of 5% acetonitrile solution balance, wash-out according to the following steps: 5-10%, 0-10min; 20-45%, 11-40min; 60-95%; 41-60min.Fraction collection main peak, merges the sample that P47 purity is greater than 95%, is P47 and refines peptide.

Revolve steaming and freeze-drying: revolve steam to remove acetonitrile, be placed in vacuum freeze drier and carry out freeze-drying, results be P47 sterling.

Utilize the purity of the P47 of the present embodiment separation and purification to be 98.2%, purification yield is 26.7%.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improve and modify and also should be considered as protection scope of the present invention.

Claims (8)

1. a method for purified polypeptide class pdgf receptor inhibitor, comprises the following steps:

1) polypeptide crude product solution anion chromatography medium adsorbs, carry out rinsing with balance liquid until remove trifluoroacetic acid root, wash-out also collects elution peak, and the principal constituent at this peak is acetate polypeptide, wherein balance liquid is the basic solution of basic solution and saliferous, and elutriant is for containing acetum;

2) elution peak that step 1) is collected take 18 alkyl silica gel as stationary phase, and with acetonitrile and aqueous acetic acid for moving phase carries out gradient elution, wherein in aqueous acetic acid, the content of acetic acid is 0.1%-2%, pH is 3-5;

3) step 2) elution peak collected removes freeze-drying after acetonitrile through underpressure distillation, obtains polypeptide raw material.

2. method according to claim 1, is characterized in that: step 2) described in moving phase pH of cushioning fluid be 3.6-4.5.

3. method according to claim 1, is characterized in that: described employing anion-exchange column method converts trifluoroacetate to acetate, and use acetum to carry out wash-out as elutriant, acetum concentration is 0.1-1.0%(V/V).

4. method according to claim 3, is characterized in that: described employing anion-exchange column method converts trifluoroacetate to acetate, and use acetum to carry out wash-out as elutriant, its concentration is 0.3%-0.5%.

5. the method according to claim 1-4 any one, it is characterized in that: described employing anion exchange method converts trifluoroacetate to acetate, use anionite-exchange resin, resin anion(R.A) is the anionite-exchange resin of the same or similar model of Cellufine MAX Q-h or Cellufine MAX CM or other producers.

6. the method according to claim 1-4 any one, is characterized in that: isocratic elution, and the concentration of acetonitrile aqueous acetic acid is 23%-45%(V/V).

7. method according to claim 5, is characterized in that: isocratic elution: use aqueous acetic acid to carry out wash-out.

8. method according to claim 1, is characterized in that: aqueous acetic acid concentration is 0.2%-0.4%(V/V).