CN101711876A - Polyethylene glycol modified recombinant human granulocyte colony-stimulating factor and preparation method thereof - Google Patents

Abstract

The invention discloses a polyethylene glycol modified recombinant human granulocyte colony-stimulating factor coupling substance and a preparation method thereof, and particularly relates to a coupling substance of N-end proline of a polyethylene glycol site-directed modified recombinant human granulocyte colony-stimulating factor. The substance is absent in the technical field; and compared with the marketed like products abroad, the substance has lower immunogenicity, less activity loss and higher preparation yield.

Description

A kind of polyethyleneglycol modified recombinant human granulocyte colony stimulating factor and preparation method thereof

Technical field

The present invention relates to a kind of proteinic polyethyleneglycol modified technology, relate to a kind of modified by polyethyleneglycol recombinant human granulocyte colony stimulating factor and preparation method thereof and its application in field of medicaments particularly.

Background of invention

Granulocyte colony-stimulating factor (G-CSF) is a kind of hemopoietic growth factor, act on the bone marrow neutrophil series and promote its proliferation and differentiation, and promotion neutrophils maturation and release, strengthen the function of ripe neutrophilic granulocyte, its early stage clinical indication is that the neutrophilic granulocyte after cancer chemotherapy and the bone marrow transplantation reduces, and progressively expands the neutrophilic granulocytopenia that the various causes of disease cause later on to.But G-CSF has only 1.3-4.2 hour in people's body-internal-circulation half-life, needs administration every day, can cause side effect such as drug eruption, heating, myalgia, osteodynia and continue medication, and causes actual amount far below theoretical expense, directly influences clinical therapeutic efficacy.

At prolonging the protein medicaments half-life, reducing its Studies on Immunogenicity has launched for many years, a kind of method wherein is that protein drug and water-soluble polymer covalent bond are formed conjugate, using commonplace water-soluble polymer is exactly Polyethylene Glycol, because Polyethylene Glycol has more outstanding clinical applicability-good heat stability, resistance to enzymolysis, increase water solublity, prolong half-life, reduction clearance rate, reduce immunogenicity and toxicity.

In the prior art field of PEG-G-CSF, the difference of various PEG-G-CSF is the different of Polyethylene Glycol and G-CSF method of attachment and site, thereby the connection site of conjugate, stability and biological activity difference, the method of attachment of having reported has: the active propionic ester of PEG is modified G-CSF, the PEG-propionic aldehyde modify the G-CSF mutant that N-terminal added methionine (total length 175aa, PEG-Met-G-CSF).Some European patents have also been reported other method of modifying about G-CSF, as reaching the immunogenic purpose of attenuating etc. by removing without one or more t cell epitopes in the modified protein molecule.

Conjugate of the present invention is the initial proline of modified by polyethyleneglycol N end, its structure with reported all different, its activity is higher than PEG-propionic aldehyde modification N-terminal (PEG-Met-G-CSF), the product structure homogeneous.The theoretical basis of this inventive concept is: the N of natural G-CSF end can contain threonine or not be with threonine in the human body, and latter's abundance is about 30%.Naturally occurring molecular forms is the optimum selection as curative drug in the body.And in the research of bibliographical information at present, also the natural molecule form is not carried out polyethyleneglycol modified research.On the proline of natural molecule N-terminal, modify and have certain technical difficulty, also do not have the correlational study report at present.Add a methionine at the medicine Neulasta of U.S.'s listing at the natural molecule N-terminal and realize long-actingization as modifying point, this molecule sudden change can be brought out human body and produce drug resistance antibody, influences therapeutic effect.

In order to modify on first proline that is implemented in natural molecule, we carry out some trials, and have obtained gratifying modification effect.

Summary of the invention

An object of the present invention is to provide a kind of new modified by polyethyleneglycol G-CSF conjugate, the higher and structure of its activity is homogeneous more.Another object of the present invention provides a kind of preparation method of modified by polyethyleneglycol G-CSF conjugate.

G-CSF in a kind of polyethyleneglycol modified G-CSF conjugate of the present invention is the active peptide section of naturally occurring G-CSF (173aa) in the human body.Its polymeric chain length range of the used Polyethylene Glycol of modification reaction is 5KDa to 100KDa, preferred 10KDa to 50KDa, and more preferably 20KDa, used activated polyglycol is a methoxy poly (ethylene glycol) aldehyde.Adopt the mono-modified N end with the initial G-CSF of proline of 20KDa Polyethylene Glycol propionic aldehyde, relatively it is higher than living with the product of modifying N end methionine, and the product homogeneity is better.

Conjugate of the present invention is to be formed by covalent bond by G-CSF and Polyethylene Glycol, and general reaction condition is: pH5-10, temperature 4-25 ℃, the response time is 30 minutes-24 hours, reactant molar ratio example (PEG: G-CSF=1: 1 to 100: 1).Low temperature, low pH value, short reaction time are more conducive to generate mono-modified product generally speaking, the preparation method of a kind of polyethyleneglycol modified G-CSF conjugate of the present invention is characterized in that controlling pH value of reaction system, reaction temperature, Polyethylene Glycol and the G-CSF concentration of catalyst SODIUM CYANO BOROHYDRIDE when that feeds intake.Optimum condition is: the G-CSF original liquid concentration is 2-5mg/ml, pH is 4.0-5.0, and the final concentration that adds SODIUM CYANO BOROHYDRIDE is 20mM, and the amount that adds activated polyethylene glycol is 3-5 a times of G-CSF, preferred 1-2 of response time hour, add the glycine cessation reaction that is equivalent to twice activated polyethylene glycol amount.

Modify for guaranteeing efficient, the Polyethylene Glycol in the most literature report and the rate of charge of protein molecular be up to 1: 50, but the rate of charge more than 10 can make the product component more complicated-single, double, modify conjugate more and deposit.In general, if reactant liquor pH is lower than pK, the dressing agent ratio should substantially exceed protein molecular, if pH is higher than pK, then the ratio of dressing agent and protein molecular needn't differ too big, the difference of reaction system pH can cause the difference of the binding site of dressing agent and protein molecular, the extent of reaction in time prolongation and increase, and relevant with reaction temperature.We are by the factors such as the reaction temperature that feeds intake when of pH, dressing agent and the G-CSF of control reaction system, the control activated polyethylene glycol holds the amino covalence of initial proline to combine with N, under the prerequisite that guarantees certain modification efficient, the amount of used activated polyethylene glycol significantly reduces.

Mixture after PEG modifies, its molecular weight difference is more obvious, suits to select for use the gel chromatography medium to separate.Early stage gel exclusion filler such as cross-linking dextran, polyacrylamide biogel etc., chemical inertness better and the fillers of different big small-bores are arranged, adapting to the proteinic fractionated of different molecular weight, but the bad mechanical strength of these gels is difficult to the separation under the extensive condition.In recent ten years, the gel commercialization preferably of several mechanical strengths comprises Sepharcryl, Superdex etc.The initial gross separation experiment is carried out on the Sephacryl of 5.6cm * 100cm S-200 chromatographic column, it is NaNC-HAC (pH4.0) that level pad is selected the weakly acidic solution of rhG-CSF preference for use, because Sephacryl S-200 chromatographic column is alkalescence, easy adhesion protein during meta-alkalescence, add 0.1M NaCl to increase ionic strength, reduce proteic non-specific adsorption.Can remove through this step operation that molecular weight makes PEG-rhG-CSF be able to preliminary purification greater than the most of molecule and the unreacted rhG-CSF of destination protein in the reactant mixture, purity can reach about 90%.

Another effect of this step effectively separates with Reducing agent the PEG that has neither part nor lot in reaction in the reactant mixture exactly.Used PEG molecular weight is the neutral molecule of 20KD, and Reducing agent is the inorganic molecules material, and they do not produce adsorption with filler; And the molecular weight of destination protein matter trim is 39KD, and it is bigger in the eluting behavior of S-200 column chromatography and PEG, Reducing agent difference, can reach effective removal of PEG and Reducing agent a step.

There is some difference on surface charge effect for the many trims of PEG and mono-modified thing, utilizes this point will can not effectively be removed by complete isolating many trims of PEG composition in the S-200 sample, reaches making with extra care sample.According to rhG-CSF more stable characteristics under mild acid conditions, purification PEG-rhG-CSF divide the period of the day from 11 p.m. to 1 a.m, and we select to be applicable to that the CM-Sepharose Fast Flow of mild acid conditions is as the proteic solid-phase media of binding purpose; The effect of DEAE post is in conjunction with the endotoxin in the sample, behind the protein solution upper prop, destination protein hangs on the CM post after passing the DEAE post, after pH4.0,20mMNaCN-HAC balance, with 0.03MNaCl eluting foreign protein, 0.3M NaCl eluting destination protein can reach more than 95% through this step sample purity.If still have small amount of residual PEG and Reducing agent component through the S-200 step, further to remove through DEAE-CMSepharose FF chromatography, it is minimum that its component is dropped to.

Based on characteristics such as the administering mode of PEG-rhG-CSF and injected dose are big, require more strict to the endotoxin of PEG-rhG-CSF stock solution.For guaranteeing the endotoxin conformance with standard, the dosing water is to meet the water for injection that pharmacopeia requires, and part reagent and used glass drying oven are done roasting 2hrs for 180 ℃ in advance.The ion-exchange chromatography flow velocity is fast, and the carrying capacity height only needs to increase the column jecket size, strengthens the chromatography media volume, just can carry out the transition to big production scale.

Above-mentioned stock solution adds the acceptable pharmaceutic adjuvant, is prepared into preparation.

The preparation of embodiment 1 polyethyleneglycol modified granulocyte colony-stimulating factor

Get granulocyte colony-stimulating factor stock solution 100ml (buffer system is pH4.0,50mM acetate buffer, and protein concentration is 5mg/ml).Take by weighing the 0.126g SODIUM CYANO BOROHYDRIDE, add in the granulocyte colony-stimulating factor stock solution, stir and make it rapid dissolving.Take by weighing 2g activated polyglycol (20KDa), stir and make it rapid dissolving, stirred 1.5 hours under the room temperature.Add 4g glycine cessation reaction.

With S-200 post on the reactant mixture, level pad is selected NaNC-HAC (pH4.0) for use, adds 0.1MNaCl to increase ionic strength.Applied sample amount is the 2-5% column volume, when flow velocity is 30ml/min, both can obtain separating effect preferably, has saved the operating time again.For determining that further experimental result shows, can reach better separating effect equally when applied sample amount is the 4-5% column volume at the maximum sample size that obtains under the satisfied separating effect prerequisite.

Ion exchange.DEAE on the above-mentioned molecular sieve purpose peak, destination protein directly go up the CM post after passing the DEAE post, and after pH4.0,20mMNaCN-HAC balance, with 0.03MNaCl eluting foreign protein, 0.3M NaCl eluting destination protein can reach more than 95% through this step sample purity.

The determination of activity of embodiment 2 polyethyleneglycol modified granulocyte colony-stimulating factors

The PEG-G-CSF Determination of biological activity adopts the MTT colorimetry.The specific activity that embodiment 1 sample records is 8 * 10 ⁷IU/mg.Concrete grammar is as follows:

Reagent

The RPMI1640 culture fluid: every liter is added 2.0g sodium bicarbonate, 2.383gHEPES, and the 0.2923L-glutamine is through 0.22 μ m membrane filtration degerming.4 ℃ of preservations.

Basic culture solution: get 100ml hyclone (FBS), add in the 900ml RPMI1640 culture fluid.4 ℃ of preservations.

Complete culture solution: basic culture solution add the reorganization Filgrastim to final concentration be 20ng/ml or 3000IU/ml.

Phosphate buffer (PBS): take by weighing 8g sodium chloride, 0.2g potassium chloride, 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, add water and make and be dissolved into 1000ml, through 121 ℃, 15 minutes autoclavings.

Tetrazolium bromide (MTT) solution: get MTT powder 0.25g, add among the 50ml PBS, be mixed with the solution of 5.0mg/ml, through 0.22 μ m membrane filtration degerming.4 ℃ keep in Dark Place.

Lysate: draw hydrochloric acid 14ml, Triton X-100 solution 50ml adds the solution that isopropyl alcohol is mixed with 500ml.

The preparation of standard solution: after getting 1 recombinant human granulocyte colony stimulating factor titration standard substance by specification dissolving, be diluted to 50～100IU/ml with basic culture solution.In 96 porocyte culture plates, continue to do dilution with 2 times of dilution factors, totally 8 dilution factors, each dilution factor is done two holes.More than operate under the aseptic condition and carry out.

The preparation of need testing solution: with test sample by after the labelled amount dissolving, with the basic culture solution dilution into about 50～100IU/ml.In 96 porocyte culture plates, continue to do dilution with 2 times of dilution factors, totally 8 dilution factors, each dilution factor is done two holes.More than operate under the aseptic condition and carry out.

Algoscopy (a～e step is carried out under aseptic condition)

The aNFS-60 cell strain uses complete culture solution in 37 ℃, 5%CO ₂Cultivate, the control cell concentration is 1.0 * 10 ⁵～4.0 * 10 ⁵/ ml, the back of going down to posterity was used for titration in 24～36 hours.

B will test the pre-temperature of used solution to 37 ℃.

C gets capacity NFS-60 cell culture, and centrifugal collection NFS-60 cell with basic culture solution washing 3 times, is resuspended in basic culture solution then and is made into 2.0 * 10 ⁵The cell suspension of/ml, put 37 ℃ standby.

D every hole in being added with 96 porocyte culture plates of standard solution and need testing solution adds 50 μ l cell suspension, 37 ℃, 5%CO ₂Cultivated 40～48 hours.

The every hole of e adds 20 μ l MTT solution, and 37 ℃, 5%CO2 cultivated 5 hours.

The every hole of f adds 200 μ l lysates, behind the mixing on microplate reader colorimetric, measure wavelength 540nm, reference wavelength 630nm, record measurement result.

The result calculates

Test data adopts computer program or rectilinear regression computing method to handle.And be calculated as follows the result:

Pr is that standard substance are tired in the formula, IU/ml,

Ds is the pre-extension rate of test sample;

Dr is the pre-extension rates of standard substance;

Es is that test sample is partly imitated extension rate;

Er is that standard substance are partly imitated extension rate.

Claims (8)

1. a polyethyleneglycol modified granulocyte colony-stimulating factor (G-CSF) conjugate.

2. the described conjugate of claim 1, wherein said G-CSF are the Filgrastims of N end disappearance threonine, 173 aminoacid of total length.

3. the described conjugate of claim 1, the molecular weight of wherein said Polyethylene Glycol is that 5KDa is to 100KDa.

4. the molecular weight of Polyethylene Glycol described in the claim 3 is that 10KDa is to 50KDa.

5. Polyethylene Glycol described in the claim 3 to 4 is a methoxy poly (ethylene glycol) aldehyde.

6. Polyethylene Glycol described in the claim 5 is that molecular weight is the methoxy poly (ethylene glycol) aldehyde of 20KDa.

7. preparation method for preparing Polyethylene Glycol-granular leukocyte colony-stimulating factors coupling agent.It is characterized in that:

(1) adjusts G-CSF original liquid concentration and pH value, add SODIUM CYANO BOROHYDRIDE and activated polyglycol,, add the glycine cessation reaction in 4 ℃ of reactions.

(2) adopt ion exchange and sieve chromatography that reactant mixture is given separation and purification.

8. be used for the application of the medicine of promoting leucocytes in preparation as Polyethylene Glycol-granular leukocyte colony-stimulating factors coupling agent as described in the claim 1-7.