CN101585864A - Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product - Google Patents

Abstract

The invention discloses a method for column chromatography granulocyte colony-stimulating factor nitrogen terminal fixed-point coupling polyethylene glycol, wherein in the coupling method, 1) the chromatographic medium is preferably a cation exchange medium; 2) the chromatographic column bed The upper limit of the recommended column height is highly preferred; 3) The sample volume of rhG-CSF protein is preferably 30-50% of the maximum load capacity measured in the medium; 4) With a lower sample flow rate and a longer sample time (3~ 5 hours), the pH value of the higher reaction rate was loaded with activated mPEG; 5) the amount of activated mPEG was loaded according to the reaction volume ratio of activated mPEG and rhG-CSF was 1 ~ 50mol: 1mol; 6) with 0.5 M ~ 1M sodium chloride salt solution gradient elution. The invention also discloses the coupling compound prepared by the method, that is, pegylated recombinant human granulocyte colony-stimulating factor, and its structural formula is: CH ₃ -(CH ₂ CH ₂ -O)n-(CH ₂ )r -NH-rhG-CSF or CH ₃ -(CH ₂ -CH ₂ -O)n-(CH ₂ )r-NH-rhG-CSFm, wherein n is 570-2200, r is 1-3; and the Application of the coupling compound as preparation for preventing and treating granulocytopenia.

Description

Column Chromatography Granulocyte Colony Stimulating Factor Nitrogen-terminal Site-specific Coupling Method and Its Product

technical field

In a broad sense, the present invention belongs to the field of protein modification and water-soluble multimer coupling protein, polypeptide and its mutants or analogues; more specifically, the present invention relates to a column chromatography polyethylene glycol protein nitrogen terminal A new method for point-specific coupling, and a class of pegylated recombinant human granulocyte colony-stimulating factor conjugated compound (PEG-rhG-CSF) obtained by this method and its long-acting, long-acting, Application of long-acting preparations of protein compounds with high efficiency and low immunogenicity characteristics.

Background technique

Recombinant human granulocyte colony stimulating factor (rhG-CSF) can promote the proliferation and differentiation of bone marrow granulocyte progenitor cells, increase the number and activity of mature neutrophils in peripheral blood, and large doses of rhG-CSF can still mobilize The progenitor cells of various bone marrow lines are released into the blood. Therefore, since it was approved for marketing in 1992, rhG-CSF has been mainly used in the clinical treatment of granulocytopenia caused by tumor or acute leukemia chemotherapy, bone marrow and peripheral blood stem cell transplantation, and chronic and primary granulocytopenia, etc. And achieved good curative effect and safety. However, the plasma half-life of rhG-CSF is short, only 3-4 hours, and it needs to be applied every day for 7-14 consecutive days, which brings a lot of inconvenience to the treatment and patients.

Royer GI was equal to 1977 (USP 4002531) announced the method of polyethylene glycol aldehyde derivatives (aldehydederivative of a monoalkyl polyethyleneglycol) coupling protein lysine and nitrogen terminal (N-terminal, N-terminal) amino, and for the first time It is proved that this PEGylated bacterial asparaginase (Asparaginase) and urokinase (Uricase) can significantly reduce the immunogenicity of the enzyme, increase its stability and activity in vivo, and can prolong the life of the plasma, so it has important pharmacological and clinical value. The patent (USP 4,179,337) applied by Davis FF in 1977 described in more detail the molecular characteristics of various PEGs that can be coupled with proteins or enzymes, and the above pharmacological properties. Since a protein or polypeptide sequence often contains multiple lysines, the method described by Royer and Davis often forms a mixture of PEG proteins, such as one protein molecule linked with 1 to 4 PEG molecules. Obviously, it is not easy to achieve the quality controllability requirements for the production of new drug molecules. Therefore, people have been devoting themselves to the research and development of site-specific pegylation (Site-specific Pegylation) technology.

As early as 1989, the work of Kuga et al. has confirmed that knocking out the 11 amino acids at the N-terminal of rhG-CSF does not affect the in vitro activity of rhG-CSF, indicating that the N-terminal of rhG-CSF is far away from the active site of the molecule. Therefore, rhG-CSF The N-terminus of CSF is one of the ideal sites for site-specific coupling of PEG molecules. There are a variety of strategies to achieve point-specific coupling of PEG to the rhG-CSF N-terminal, including: 1) Wetzel R et al. [1] proposed in 1990 to achieve high selection of alfa amino groups at the N-terminal of the polypeptide by controlling the pH value Sexual coupling; 2) Gaertner HF et al. [2] described various PEG active groups that can selectively couple the N-terminal amino groups of proteins in 1996, and applied them to molecules such as interleukin-8 and G-CSF; 3) ChangTK et al. [3] found in 1994 that subtilase (subtiligase) had the effect of enzymatic PEG selective coupling; 4) recently, some scholars also adopted recombinant DNA technology [4], through the expression of transglutaminase (transglutaminase) mediated In vivo PEG point specific N-terminal coupling target protein.

On the basis of the highly selective coupling of the N-terminal alfa amino group of the polypeptide by controlling the pH value proposed by Wetzel R et al. in 1990, Kinstler OB and Gabrial of the Amgen Company in the United States further described in detail that the single The method and the product of methylpolyethylene glycol aldehyde group (PEG-ALD) specific coupling rhG-CSF and compound interferon and other N-terminal amino groups, see USP 5,824,784/USP 5,985,265/USP 7,090,835B2 and US 2006/ Patents such as 0233746A1 and CN 1896103A; Kinstler et al. call this reaction by reducing the pH value to realize the N-terminal reaction of PEG-ALD selective coupling protein as reductive alkylation reaction (reductivealkylation). In fact, Royer GI is equal to 1977 U.S. Patent USP 4,002,531 and Shaw et al. have already mentioned this in 1990 U.S. Patent No. 4,904,584, which means that the amino group reaction between PEG-ALD and protein is divided into two steps To carry out, that is, the Shiff base is formed first, and then the imine is reduced to form an irreversible PEG-ALD and protein alkylation conjugate, and the selectivity of the coupling occurs in the first step. The principle of N-terminal selectivity in the reductive alkylation reaction is: under different pH conditions, the dissociation constants (pKa ) are different, so the coupling reaction rates of epsilon amino group and alfa amino group and PEG aldehyde group (PEG-ALD) are different, and selectivity appears; The coupling reaction probability of PEG-ALD is more than 50 times that of the epsilon amino group on lysine; therefore, when the coupling reaction lasts for a long time, most of the PEG-ALD will be coupled to the rhG-CSF molecule N- On the terminal alfa amino group, a single, site-directed PEG-rhG-CSF compound is formed; thus, conditions for formation of a single, highly selective protein N-terminus include low pH and long reaction times, in addition to requiring relatively low PEG - ALD input amount. In fact, the coupling of PEG-ALD to amino groups of protein molecules can be accelerated under near-neutral, slightly alkaline or alkaline conditions, but at this time the selectivity of PEG-ALD to the N-terminus of the protein will be lost, and a multi-modified PEG will be formed. - a mixture of proteins.

In the patent USP 5,824,784 mainly related to PEGylation coupling rhG-CSF, Kinstler OB specially protected PEG molecules with a molecular weight of 6kDa～25kDa (6,000-25,000 Daltons) and rhG-CSF molecules for pharmaceutical PEG-rhG-CSF molecules. The coupling product of a single, N-terminal amino group. At the same time, Amgen Corporation of the United States has also developed a new PEGylated rhG-CSF drug (PEG-rhG-CSF), namely Neulasta (pegfilgrastim), which is a compound that is coupled to the N-terminus of the rhG-CSF protein with 20kDa molecular weight PEG; The curative effect of one injection in one chemotherapy cycle, and the safety is also comparable to common rhG-CSF (filgrastim), but its single dose is as high as 6 mg (6 mg), which is the total dose of high-dose common rhG-CSF for ten consecutive days Twice of that (ie 300ug/dose/day×10×2=6mg), so Neulasta has defects in pharmaceutical economics. However, the molecular weight of PEG of a class of long-acting preparation products described by the inventors of the present application is just outside the scope of this patent claim, and it is surprising that a class of PEG-rhG-CSF compound molecules published in the patent application of the present invention , as a long-acting preparation, compared with the above-mentioned new drug Neulasta (pegfilgrastim), it has the characteristics of pharmaceutically obvious long-acting, high efficiency and low immunogenicity, so its clinical application dose is expected to be significantly lower than Neulasta.

Fee CJ (USP 2006/0128945A1) published a technology for protein PEG modification using gel chromatography, and proposed that due to the speed difference between protein and PEGylated protein in the mobile phase of gel chromatography, PEGylation using a chromatography column The coupling reaction may be easier to produce a single coupling product, but Fee CJ's method [6] does not involve the PEGylation coupling reaction that requires the addition of a catalyst, nor is it a specific coupling technology for the N-terminus of the protein. Recently, Lee BK et al. [5] described a method for point-specific modification of recombinant human interferon alfa-2a (IFN α-2a) using a solid-phase PEGylation technique, and obtained a method for coupling with the N-terminus of the IFN α-2a protein. However, this method obtains highly selective coupling products at the N-terminus of the protein by using catalytic reaction conditions very similar to those of Kinstler OB et al. Therefore, in essence, this method is still based on the above-mentioned The principle of reductive alkylation reaction, by controlling the pH value of the reaction system, PEG-ALD can be highly selectively coupled to the N-terminal of the protein, and due to the limitation of the reaction time, the coupling reaction rate is reduced, and more free IFN appears a-2a; at the same time, this method does not involve the PEG coupling of rhG-CSF; it is not as effective as the application of the present invention, that is, the use of extended strong cation exchange chromatography to simultaneously achieve PEG-ALD-specific rhG-CSF N -End coupling and one-step purification to obtain a single, site-specific N-terminal coupling PEG-rhG-CSF product; in addition, experiments have shown that factors such as different media, column bed heights, and chromatographic conditions can significantly affect the specificity of the chromatography column Coupling results, including PEG coupling reaction rate, coupling site, coupling product and its type, and separation effect of the final product, etc.

In summary, there are many kinds of PEG protein N-terminal point-specific coupling reactions in vitro, and the main strategy is to improve the selectivity of the PEG activating group to the N-terminal alfa amino group. The lysine epsilon amino group on the rhG-CSF molecule is "blocked" by exchange chromatography to realize the specific coupling of PEG-ALD and the rhG-CSF N-terminal methionine alfa amino group, and simultaneously realize the PEG-rhG-CSF coupling product of purification. There are many kinds of PEG-rhG-CSF new molecules formed by coupling rhG-CSF with PEG, and their properties may vary greatly. Even the specific coupling products of PEG and rhG-CSF N-terminal amino groups may have completely different properties. Different; as far as PEG molecules are concerned, different molecular weights, different molecular structures (single-chain, double-chain, multi-chain), different active groups, or even different numbers of carbon atoms in the same active group (such as propionaldehyde, pentamethylene Aldehydes, etc.) will significantly affect some properties of the coupling compound molecule; as far as the rhG-CSF molecule is concerned, whether it is accompanied by other coupling sites or whether the coupling product contains a certain lysine amino group may be obvious. Change the physicochemical, pharmaceutical and pharmacological properties of PEG-rhG-CSF, these changes include, but not limited to, protein stability, in vivo and in vitro activities, plasma half-life, distribution in vivo and its immunogenicity or antigenicity. Therefore, many previous patents on PEG-coupling proteins or polypeptides, including Kinstler et al. The actual application value and significance of these patents; let alone the use of column chromatography involved in the application of the present invention to directly affect the pharmaceutical and pharmacological properties of rhG-CSF in a specific and controllable manner, and Compared with Neulasta (pegfilgrastim), a new long-acting preparation with long-acting, high-efficiency and low immunogenicity characteristics has been developed.

references:

〔1〕Wetzel R, Halualani R, Stults JT, Quan C.A general method for highlyselective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal a-amino groups. Bioconjugate Chemistry1990;1:114-122.

[2] Gaertner HF and Offord RE. Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus proteins. Bioconjugate Chemistry 1996; 7: 38-44.

[3] Chang TK, Jackson DY, Burnier JP, Wells JA. Subtiligase: a tool for semisynthesis of proteins. Proc National Acad Science 1994; 91: 12544-12548.

[4] Sato H, Yamamoto K, Hayashi E, Takahara Y. Transglutaminase-mediated dual and site-specific incorporation of poly(ethylene glycol) derivatives into chimeric interleukin-2. Bioconjugate Chem 2000;11:502-509.

[5] Lee BK, Kwon JS, Kim HJ, Yamamoto S and Lee EK. Solid-phase PEGylation of recombinant interferon a-2a for site-specific modification: process performance, characterization, and in vitro bioactivity. Bioconjugate Chem. 2007; 18: 1728 -1734.

[6] Fee CJ. Size-exclusion reaction chromatography (SERC): A New Technique for protein PEGylation. Biotechnology and bioengineering 2003; 82(2): 200-206.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a new method for column chromatography polyethylene glycol protein nitrogen terminal point-specific coupling, that is, column chromatography granulocyte colony-stimulating factor nitrogen terminal point-specific coupling polyethylene glycol The method of diol, and a class of PEGylated recombinant human granulocyte colony-stimulating factor conjugated compound (PEG-rhG-CSF) obtained by the method and its pharmaceutically long-acting, high-efficiency, low-immunity compound Application of long-acting preparations of protein compounds with original characteristics.

Summary of The Invention

The present invention discloses for the first time a new method for specific coupling of PEG to the N-terminal point of rhG-CSF protein, that is, to realize the specific coupling and synchronization of PEG and rhG-CSF protein N-terminal point at the same time through ion exchange chromatography Enables purification of single, site-directed PEG-rhG-CSF compounds.

The selection conditions for PEG in the coupling method are: various activated monomethyl PEG (mPEG) molecules that can be coupled with protein amino groups, mainly including mPEG-ALD, PEG-succinimide α-methylbutyl Various mPEG-N-hydroxysuccinimide activated esters (mPEG-Succinimidyl α-methylbutanoate, mPEG-SMB), mPEG-Succinimidyl propionate (mPEG-SPA), etc. -N-Hydroxy succinimide active esters, mPEG-NHS ester), preferably mPEG-ALD; the molecular weight of mPEG is greater than 25kDa, preferably 26-40kDa, most preferably 30kDa; mPEG molecules can be single-chain, double-chain or multi-chain, preferably single-chain ; mPEG-ALD, its molecular formula is mPEG-(CH ₂ )r-CHO, wherein r is 1-3, and the more suitable range of r is 2-3, that is, mPEG-propionaldehyde and mPEG-butyraldehyde.

The selection conditions for rhG-CSF in the coupling method are: the amino acid sequence of rhG-CSF or rhG-CSF mutant (rhG-CSFm) should meet the amino acid sequence formula shown in SEQ ID NO.1; wherein the rhG - CSFm is the rhG-CSF whose N-terminal amino acid is knocked out and the N-terminal amino acid is still methionine; the recombinant human granulocyte colony-stimulating factor (rhG-CSF) of the present invention is preferentially expressed in a prokaryotic cell expression system, namely the large intestine Bacillus high-efficiency expression; including artificial synthesis and various other prokaryotic cells, eukaryotic cells, insects, plants or animal expression systems; at the same time, rhG-CSF/rhG-CSFm can be a protein composed of the entire amino acid sequence, or it can be mutated RhG-CSF variants composed of rhG-CSF sequences, or even rhG-CSF analogs composed of partial amino acid sequences, but their only characteristic is: the in vitro activity is equivalent to that of rhG-CSF or not lower than 3×10E7IU/mg protein ; The various rhG-CSF proteins mentioned above are within the scope of the present invention.

The selection conditions of chromatographic medium, column and condition in the coupling method are: 1) chromatographic medium can be selected various chromatographic mediums such as ion exchange, hydrophobicity, gel and affinity, wherein preferred cation exchange medium, such as MacroCap SP, Sepharose Fast Flow, etc.; 2) The height of the chromatographic column bed is preferably the upper limit of the recommended column height; 3) The loading of rhG-CSF protein is 30-50% of the maximum capacity of the medium measured; 4) A lower The sample loading flow rate, longer sample loading time (3-5 hours), higher reaction rate pH value loading activated mPEG, to obtain more suitable PEGylation reaction conditions; 5) loading amount of activated mPEG The reaction volume ratio of activated mPEG and rhG-CSF is 1-50mol: 1mol, preferably 2-20mol: 1mol, so as to facilitate the generation of single-modified PEG-rhG-CSF; 6) with 0.5M-1M sodium chloride Gradient elution of salt solution was used to facilitate the effective separation of the reaction products. The results are shown in Figure 1.

The single-modified PEG-rhG-CSF elution peak sample obtained by the above-mentioned method was analyzed by liquid chromatography-mass chromatography with trypsin digestion peptide map, and it was important and unexpected to find that the coupling site of the activated PEG is rh- The N-terminal of CSF, such as PEG-ALD with a molecular weight of 30kDa, 40kDa or 20kDa, can form a single, site-specific specific coupling product with the N-terminal of rhG-CSF protein, namely PG-N-30, PG-N-40 , PG-N-20 (see Example 3 for details); and the purity of the single-modified PEG-rhG-CSF elution peak sample confirms that its purity is greater than 95 through high-performance gel chromatography (SEC) and high-performance liquid chromatography (HPLC). %, as shown in Figure 3 and Figure 4.

The present invention also found that by selecting mPEG-ALD and only lowering the pH value of the above reaction system to 4.0, the elution peak area of single-modified PEG-rhG-CSF can be significantly reduced to 62% of the total elution peak area, and the unreacted rhG - The CSF elution peak accounted for a significant increase of 38% in the total elution peak area, and the elution peak of multi-modified PEG-rhG-CSF disappeared; Peptide map analysis confirmed that the coupling site of PEG-ALD was still the N-terminal of rh-CSF; it suggested that under the above ion-exchange chromatography conditions, acidic pH conditions were not the selective coupling of PEG-ALD to rh-CSF N- main factor at the end;

In the same way, select PEG-ALD and PEG-NHS ester (polyethylene glycol N-hydroxysuccinimide activated ester) modifiers that can react with protein amino acid, and adopt the above-mentioned cation exchange chromatography, and the pH values of the reaction system are respectively At 5.0-6.0 and 6.0-7.0, the N-terminal single and fixed-point coupling products of PEG and rh-CSF with a peak area greater than 90% can be obtained (as shown in Figure 1 and Figure 5);

In the liquid phase, using the same ratio of reactants, conditions and duration as the PEGylation reaction of the above-mentioned chromatography column, the coupling reaction products of mPEG-ALD and mPEG-SPA and rhG-CSF were determined by high-efficiency gel, usually Only about 23%-45% are single-modified PEG-rhG-CSF, and the remaining 36%-47% are multi-modified PEG-rhG-CSF compounds; 19-30% unreacted rhG-CSF, with the column The product ratio of PEGylation coupling reaction is obviously different;

The present invention surprisingly found through the above experiments: under certain conditions, the cation exchange medium adsorbs rhG-CSF protein, and can "block" the lysine epsilon amino group located on the surface of rhG-CSF molecules, so that PEG-ALD or PEG -NHS is specifically coupled to the alfa amino group of the N-terminal methionine of rhG-CSF molecules to form a single, N-terminal-directed PEG-rhG-CSF, such as PG-N-30 (30kDa PEG-ALD and rhG-CSF protein N- single-terminal, site-specific coupling product). At the same time, the purity of the collected single, N-terminal site-directed PEG-rhG-CSF compound is greater than 95% (as shown in Figure 2 and Figure 3); it shows that the PEGylation reaction of the chromatography column can simultaneously realize the specific rhG -CSF N-terminal single, site-specific coupling, and purification of PEG-rhG-CSF coupling compounds.

Detailed Description of The Invention

The method for column chromatography of the present invention to couple the nitrogen terminal of granulocyte colony-stimulating factor with fixed-point coupling to polyethylene glycol, the steps include:

Select the cation exchange medium, load it in the chromatographic column, the height of the column bed is the upper limit of the conventional recommended height, and connect it to the liquid chromatography system; 20mM sodium acetate buffer solution and 0.005% polysorbate 80 by mass percentage are fully balanced chromatographic column; use a feed pump to add rhG-CSF or rhG-CSFm stock solution into the chromatographic column at a flow rate of 8-10ml/min, The amount of sample loaded is 10% to 70% of the maximum capacity of the medium measured; then at a flow rate of 13 to 15ml/min, wash the column with 5 to 6 times the column volume of 20mM sodium acetate buffer solution with a pH of 3.5 to 6.0 to remove excess Substances that undergo electrostatic adsorption with the chromatographic medium; according to the reaction volume ratio of mPEG:rhG-CSF or rhG-CSFm at a ratio of 1 to 50mol:1mol, add a sample with a molecular weight range of 26 to 40kDa at a flow rate of 0.5 to 3ml/min Monomethyl PEG, the mPEG input total amount X of described different molecular weights is calculated by formula (1):

(1) X＝5～50×m×MW _PEG /MW _G-CSF

In the formula: X is the total input amount of mPEG with different molecular weights, m is the protein content of rhG-CSF added, MW _PEG and MW _G-CSF are the molecular weights of mPEG and G-CSF respectively;

Then calculate the add-on of reducing agent sodium cyanoborohydride by formula (2):

(2) NaBH ₃ CN addition amount = 10×MW _{reducing agent} ×X/MW _PEG

In the formula: MW _{reducing agent} is the molecular weight of the reducing agent sodium cyanoborohydride, X is the total amount of mPEG input with different molecular weights, and MW _PEG is the molecular weight of mPEG;

Load the sample continuously for 240±5 minutes; the pH of the reaction system is 3.5-6.0; then wash the column with 5-6 column volumes of 20mM sodium acetate buffer solution with a pH of 3.5-6.0 at a flow rate of 13-15ml/min to remove excess Substances that undergo electrostatic adsorption with chromatographic media; then use a mixture of 20mM sodium acetate buffer with pH 3.5 to 6.0 and 0.5M to 1M sodium chloride in the elution mode of 0 to 8%, 8% to 18% and 18% ~100% elution lasted for 150±2 minutes, and three elution peaks were obtained, namely P1, P2 and P3, which were multi-modified PEG-rhG-CSF, single-modified PEG-rhG-CSF and unreacted rhG-CSF respectively. Peak off, wherein the peak area of P2 accounts for 90±1% of the total peak area, which is a single modified PEG-rhG-CSF or PEG-rhG-CSFm, that is, polyethylene glycol and rhG-CSF or rhG-CSFm protein N- Single-terminal, site-specific coupling compounds;

in:

The amino acid sequence of the above rhG-CSF or rhG-CSFm should conform to the amino acid sequence formula shown in SEQ ID NO.1.

The amino acid sequence formula of said SEQ ID NO.1 is as follows:

-1 -2 -3 -4 -5 -6 -7 -8 -9 -10

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu

5 10 15

Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu

20 25 30

Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu

35 40 45

Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser

50 55 60

Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His

65 70 75 80

Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile

85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala

100 105 110

Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala

115 120 125

Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala

130 135 140

Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser

145 150 155 160

Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro ***

165 170 175

Wherein the rhG-CSFm is the rhG-CSF whose N-terminal amino acids 1-10 are deleted and the N-terminal amino acid is still methionine.

rhG-CSF consists of 175 amino acids, and the rhG-CSF mutant (rhG-CSFm) refers to rhG-CSF with 1-10 amino acid deletions at the N-terminal and methionine as the N-terminal amino acid. In the above amino acid sequence formula, -10Xaa is the deletion of 10 amino acids at the N-terminal, namely rhG-CSF-165; -9Xaa is the deletion of 9 amino acids at the N-terminal, namely rhG-CSF-166; -8Xaa is the deletion of 8 amino acids at the N-terminal Amino acid deletion, that is rhG-CSF-167; -7Xaa is N-terminal 7 amino acid deletion, that is rhG-CSF-168; -6Xaa is N-terminal 6 amino acid deletion, that is rhG-CSF-169; -5Xaa is N -Deletion of 5 amino acids at the end, i.e. rhG-CSF-170; -4Xaa, deletion of 4 amino acids at the N-terminus, i.e. rhG-CSF-171; -3Xaa, deletion of 3 amino acids at the N-terminus, i.e. rhG-CSF-172; -2Xaa is the deletion of 2 amino acids at the N-terminal, namely rhG-CSF-173; -1Xaa is the deletion of 1 amino acid at the N-terminal, namely rhG-CSF-174.

The above monomethyl PEG refers to various activated monomethyl PEG molecules that can be coupled to protein amino groups, preferably PEG-succinimide α-methyl in mPEG-ALD or mPEG-N-hydroxysuccinimide activated ester butyrate or mPEG-succinimidyl propionate.

Further, in the above-mentioned method of column chromatography granulocyte colony-stimulating factor nitrogen-terminal-specific coupling of polyethylene glycol:

The cation exchange medium is preferably MacroCap SP or Sepharose Fast Flow.

The rhG-CSF or rhG-CSFm is preferably prepared by expression in Escherichia coli in a prokaryotic cell expression system.

The loading amount of the rhG-CSF or rhG-CSFm is preferably 30-50% of the measured maximum load of the medium.

The molecular weight of the monomethyl PEG is preferably 30kDa; the mPEG molecule is preferably a single chain; the loading amount of the monomethyl PEG is preferably a ratio of 2 to 20 mol: 1 mol according to the reaction volume ratio of mPEG: rhG-CSF or rhG-CSFm .

The monomethyl PEG is preferably mPEG-ALD; its molecular formula is mPEG-(CH ₂ )r-CHO, wherein r is selected from 2 to 3, that is, mPEG-propionaldehyde or mPEG-butyraldehyde.

Wherein the molecular structural formula of mPEG-propionaldehyde is as follows;

Wherein the molecular structural formula of mPEG-butyraldehyde is as follows;

The pH of the reaction system of column chromatography rhG-CSF or rhG-CSFm nitrogen-terminally coupled monomethyl PEG is preferably 5.0-6.0.

A kind of PEGylated recombinant human granulocyte colony-stimulating factor prepared by column chromatography granulocyte colony-stimulating factor nitrogen terminal fixed-point coupling polyethylene glycol method, characterized in that: the PEGylated The molecular structural formula of recombinant human granulocyte colony-stimulating factor is:

CH ₃ -(CH ₂ CH ₂ -O)n-(CH ₂ )r-NH-rhG-CSF or CH ₃ -(CH ₂ -CH ₂ -O)n-(CH ₂ )r-NH-rhG-CSFm ,

In the formula, n is 570-2200, r is 1-3; at the same time, the pegylated recombinant human granulocyte colony-stimulating factor should also have the following characteristics:

1) The molecular ratio of rhG-CSF or rhG-CSFm to mPEG coupling is 1:1, and the coupling site is the N-terminal amino group of rhG-CSF or rhG-CSFm molecule;

2) In the in vitro anti-enzyme test: incubate trypsin with PEG-rhG-CSF or PEG-rhG-CSFm at 37°C for 4 hours, take samples and determine by SDS-PAGE method, PEG-rhG-CSF or PEG-rhG - The percentage of enzymatic hydrolysis of CSFm should be less than 50%;

3) In the determination of specific activity, the Lowry method is used to measure the protein concentration of PEG-rhG-CSF or PEG-rhG-CSFm to be tested, the NFS60 cell/MTT method is used to measure the in vitro activity, and the activity per mg of protein is calculated to obtain the specific activity. The specific activity of PEG-rhG-CSF or PEG-rhG-CSFm should be greater than or equal to 2.0X10E7U/mg protein;

4) The plasma half-life of PEG-rhG-CSF or PEG-rhG-CSFm should be more than 50% longer than the plasma elimination half-life of Pegfilgrastim (20kDa PEG-ALD and rhG-CSF N-terminal coupling product, namely PG-N-20);

5) In the assay of immunogenicity, the positive rate of PEG-rhG-CSF or PEG-rhG-CSFm at week 12 should be less than 20%.

Wherein: n in the molecular formula of the above pegylated recombinant human granulocyte colony-stimulating factor is 570-1000, the more suitable range of n is 700±100; the more suitable range of r is 2-3.

A class of PEGylated recombinant human granulocyte colony-stimulating factor prepared by the method of the present invention has both molecular weight and molecular structure uniformity, and is a single-chain, single macromolecule PEG and rhG-CSF or PEG-rhG-CSFmN -Terminal site-specific coupling product; the preferred molecular weight 30kDa PEG-ALD and rhG-CSF N-terminal site-specific coupling product of the PEGylated recombinant human granulocyte colony-stimulating factor is PG-N-30 molecule.

A class of pegylated recombinant human granulocyte colony-stimulating factor prepared by the method of the present invention is significantly better than ordinary rhG-CSF in in vitro solubility and resistance to enzymolysis, and its in vitro cell activity is greater than or equal to 2.0X10E7U /mg protein; when used in vivo, compared with the above-mentioned Neulasta (Pegfilgrastim, 20kDa PEG-ALD and rhG-CSF N-terminal coupling product, namely PG-N-20), it has significantly prolonged plasma half-life and significantly increased in vivo activity Pharmacokinetics and pharmacodynamics characteristics of long-acting preparations such as reduced immunogenicity and reduced immunogenicity; in animal models of granulocytopenia, the dosage of PG-N-30 to achieve equivalent levels is low by adopting the mode of daily continuous administration. 10 times the amount of common rhG-CSF; using the administration mode of injecting one injection in one chemotherapy cycle, the curative effect of PG-N-30 is significantly better than that of PG-N-20, achieving the same effectiveness as the administration of PG-N-30 The dosage can be significantly lower than 2 times of the dosage of PG-N-20. Based on this, a class of pegylated recombinant human granulocyte colony-stimulating factor of the present invention, such as PG-N-30 and PGm-N-30 (30kDa PEG-ALD and rhG-CSFm protein N-terminal site-specific coupling product) , has the characteristics of long-acting, high efficiency and low immunogenicity at the same time, and can show a good comparative advantage in the treatment mode of daily continuous application or once in a chemotherapy cycle, but from the perspective of pharmaceutical economics, each Daily continuous administration or administration with shorter dosing intervals may be a better treatment mode.

The PEGylated recombinant human granulocyte colony-stimulating factor of the present invention is used in the preparation and treatment of granulocytopenia caused by tumor or leukemia chemotherapy, bone marrow and peripheral blood stem cell transplantation, and primary chronic granulocytopenia. Application in parenteral preparations.

A further realized parenteral preparation for preventing and treating neutropenia caused by tumor or leukemia chemotherapy, bone marrow and peripheral blood stem cell transplantation and primary chronic neutropenia, which contains a therapeutically effective amount of the present invention Pegylated recombinant human granulocyte colony-stimulating factor and a pharmaceutically acceptable carrier.

The parenteral preparation made of PEG-rhG-CSF or PEG-rhG-CSFm molecules of the present invention obviously has the characteristics of long-acting, high efficiency and low immunogenicity, and can be injected once every 1-4 weeks, or can be Realize the clinical application mode of taking medicine once in a treatment cycle (course of treatment), and perform the functions of long-acting preparations as described in general; (such as intervals of 1 day, 2 days, etc.), to achieve the same curative effect as continuous application of rhG-CSF, the dosage of PG-N-30 needs to be doubled, and to realize the administration mode of one injection per chemotherapy cycle, The dosage of PG-N-30 may even reach 5 times of the total amount of continuous application of rhG-CSF (see Table 6-2, Table 7 for details). This result is quite unexpected, which shows that there is still a strong PG -N-30 clearance mechanism, a single or longer dosing interval in a chemotherapy cycle is far from economical or optimal, so continuous daily dosing or shorter dosing intervals may is a better treatment modality.

Experiments have confirmed that: with the treatment mode of continuous daily injection, the dose of PEG-rhG-CSF or PEG-rhG-CSFm continuously applied is significantly lower than that of ordinary rhG-CSF; according to the different application indications and therapeutic purposes, it can be used once as a long-acting preparation Or the dose of PEG-rhG-CSF or PEG-rhG-CSFm treated by daily continuous injection is 0.1ug～300ug/kg body weight/1 day～4 weeks.

The above rhG-CSF or rhG-CSFm is prepared by expression of prokaryotic cell expression system (Escherichia coli):

Cloning and high-level expression of rhG-CSF and rhG-CSFm genes

Human fresh bone marrow cells were cultured and activated with pokeweed mitogen (PWN mitogen). Human bone marrow cell mRNA was extracted, and hG-CSF gene was amplified by RT-PCR method. Apply multi-dimensional and multi-parameter nucleotide chain analysis technology to analyze and process the human G-CSF mRNA original sequence in detail, find the gene repression fragment in the G-CSF mRNA original sequence, and use PCR and site-directed mutagenesis to modify human natural G-CSF The gene sequence (without changing its coded amino acid sequence) makes it suitable for expression in bacteria. The modified gene is constructed on the pMD18-T vector, and the vector is transformed into the Top10 strain for preservation.

Using the modified rhG-CSF gene as a template, the rhG-CSF gene was amplified in vitro by PCR (polymerase chain reaction). The gene was digested with Nde I and EcoRI, cloned into the expression vector of Escherichia coli, and the correctness of the plasmid was verified. The recombinant plasmid was named vector-rhG-CSF.

The recombinant plasmid vector-rhG-CSF was transformed into Escherichia coli BL21(DE3)pLysS. Screen the strains that highly express the target protein rhG-CSF.

Preparation of rhG-CSF and rhG-CSFm

The rhG-CSF Escherichia coli engineering strain was subjected to high-density fermentation culture, post-fermentation treatment, rhG-CSF protein inclusion body washing, renaturation, separation and purification (see Example 1 for details), to obtain high-purity rhG-CSF or rhG- CSFm protein.

Under strict aseptic operation, inoculate rhG-CSF Escherichia coli engineered bacteria on LB/Amp plates and culture them upside down in a bacterial incubator at 37°C for 12-15 hours; select a single colony and transfer it to a 12ml LB/Amp culture test tube medium, 37°C and 250rpm shaking culture for 12 hours; take 10ml of fermentation broth and transfer it to 1000ml LB/Amp fermentation broth for shaking flask culture (37°C, 250rpm shaking), when the concentration of the bacterial solution reaches OD600 1.5-2.5; then inoculate at 15 Cultivate in a 1-liter fermenter (each liter of fermentation broth contains 12 grams of tryptone, 24 grams of yeast extract, 3.8 grams of potassium dihydrogen phosphate, 12.5 grams of dipotassium hydrogen phosphate, 0.5 grams of magnesium sulfate, 6.3 grams of glycerin, 0.9 liters of pure water, 20 grams of glucose and 0.1 gram of ampicillin); the fermentation parameters were set as temperature 37° C., pH 7.0, dissolved oxygen 35%, and the linkage between stirring speed and dissolved oxygen. When the OD ₆₀₀ value reaches 20, it is the middle stage of the logarithmic growth of the engineered bacteria, and IPTG is added to induce it, and the fermentation is terminated after 2 hours. Cool the fermented liquid to bring it down to 4-8°C. Centrifuge the bacterial liquid at 4000rpm with a large-capacity low-temperature refrigerated low-speed centrifuge for 30 minutes, and collect the bacterial cells.

Add engineering bacteria breaking buffer (50mM Tris, 1mM EDTA, pH7.5 buffer) to the collected engineering bacteria thalline precipitate. The bacteria were crushed twice under the pressure of 10000 psi by a high-pressure homogenizer. After breaking the bacteria, the liquid was centrifuged in a high-speed refrigerated centrifuge at 4°C and 10,000g for 20 minutes to obtain the crude engineering bacteria inclusion body (IB). Repeatedly wash three times with inclusion body washing solution (50mM Tris, 1mM EDTA, pH7.5, 2.5% Triton × 100, 0.15M NaCl), then fully mix the inclusion body washing solution and inclusion body with a high-speed liquid mixer, and then in 4 The inclusion bodies were collected by high-speed centrifugation at 10,000 g for 50 minutes, and this was repeated three times to complete the washing and purification of the inclusion bodies. The purified inclusion body was dissolved in inclusion body dissolving and denaturing solution (50mM Tris, 1mM EDTA, 8M guanidine hydrochloride, 1mM dimercaptoerythritol) according to the ratio of 10g wet weight to 500ml, and placed at 4°C for 12 hours, Obtain inclusion body dissolving and denaturing solution, centrifuge at 10000g high speed at 4°C for 40 minutes, and collect the supernatant.

Slowly add inclusion body dissolving and denaturing solution into 20mM sodium acetate buffer (pH 6.0), 150mM sodium chloride, 3M urea, 0.005% polysorbate 80 (polysorbate 80) refolding solution at a volume ratio of 1:100, 4 ℃ for 24 hours, after filtering through a 0.45 μm pore size filter membrane, the protein was purified.

The rhG-CSF protein refolding solution was subjected to Sepharose Fast Flow ion exchange chromatography, and the equilibrium buffer was 20mM sodium acetate buffer (pH 4.5), 150mM sodium chloride, 0.005% polysorbate 80; 60ml/min loading, using 0 ~ 1M sodium chloride gradient elution, collect A280d protein elution peak liquid. Then use buffer 10mM sodium acetate buffer (pH4.5), 0.005% polysorbate 80 buffer to balance the Superdex 75prep grade gel chromatography column, load the sample at a flow rate of 30ml/min, and collect the sample after 120 minutes. The A280 protein absorption peak that appears is the rhG-CSF stock solution.

The rhG-CSF mutant (rhG-CSFm), i.e. rhG-CSF-165-174, was obtained by knocking out 1-10 amino acid residues at the N-terminal of rhG-CSF and retaining the N-terminal amino acid as methionine. The rhG-CSFm stock solution can be obtained by the same method. The data provided in Example 4 show that knocking out 1-10 amino acid residues at the N-terminal of rhG-CSF hardly affects the in vitro activity of rhG-CSF, see Table 3 for details.

Taking PEG-propionaldehyde and rhG-CSF N-terminal specific site-specific coupling method as a preferred example, PEGylated rhG-CSF and its preparation process are described:

Select MacroCap SP or Sepharose FF cation exchange medium, load the chromatographic column, the height of the column bed reaches the upper limit of the recommended height, and connect it to the AKTA Explorer 100 liquid chromatography system (Amersham Bioscience, Sweden), using the equivalent of 5 times Column volume of 20mM sodium acetate buffer solution pH 3.5～6.0 (buffer solution A) and 0.005% polysorbate 80 to fully equilibrate the chromatographic column; use a feed pump to add the rhG-CSF stock solution to the chromatographic column at a flow rate of 10ml/min In this method, the sample load is 10%-70% of the maximum load capacity measured in the medium, and the more suitable range is 30-50%. Then, at a flow rate of 15ml/min, wash the column with about 5 times the column volume of buffer A to remove excess Substances that have electrostatic adsorption with the chromatographic medium; at a flow rate of 0.5-3ml/min, add 0.22mM-2.2mM 30kDa PEG-propionaldehyde (PEG-propionaldehyde) and 2.2mM-22mM NaBH ₃ CN solution 250ml-1500ml, continuously The sample is about 240 minutes; the pH of the reaction system is 3.5-6.0, more preferably pH 5.0-6.0; then at a flow rate of 15ml/min, wash the column with about 5 times the column volume of buffer A to remove the static electricity that does not occur with the chromatographic medium. Adsorbed substances; then use buffer B (i.e. buffer A + 1mol sodium chloride) to elute in the elution mode of 0-8%, 8%-18% and 18%-100% for about 150 minutes, namely Can record the ion-exchange chromatogram as shown in Figure 1, see from the figure, can obtain peak-1 altogether, three elution peaks of peak-2 and peak-3, i.e. P1, P2 and P3, wherein The peak area of P2 accounts for 90% of the total peak area; P2 is analyzed as single modified PEG-rhG-CSF through reduced SDS-PAGE, high performance gel chromatography (SEC) and high performance liquid chromatography (HPLC), with a purity greater than 95% (As shown in Figures 2, 3, and 4), the product was determined to be the site-specific coupling of PEG-ALD with a molecular weight of 30kDa and the rh-CSF N-terminus by liquid chromatography-mass chromatography with trypsin digestion peptide map analysis, that is, PG- N-30 (see Example 3 for details).

In the same way, PG-N-10, PG-N-20, PG-N-40, PGm-N-10, PGm-N-20, PGm-N-30, PGm-N-40, etc. can be obtained.

Similarly, taking PEG-NHS ester (polyethylene glycol N-hydroxysuccinimide activated ester) and rhG-CSF N-terminal specific site-specific coupling method as an example, describe PEGylated rhG-CSF and its preparation process:

Select MacroCap SP cation exchange medium, load the chromatographic column, the height of the column bed reaches the upper limit of the recommended height, and connect it to the AKTA Explorer 100 liquid chromatography system (Amersham Bioscience, Sweden), using the equivalent of 5 times the column volume 20mM sodium acetate buffer solution pH 3.5～6.0 (buffer solution A) and 0.005% polysorbate 80 fully equilibrate the chromatographic column; With the feed pump, with the flow rate of 10ml/min, rhG-CSF stock solution is added in the chromatographic column, The amount of sample loaded is 10% to 70% of the maximum capacity of the medium measured, and the more suitable range is 30-50%; then at a flow rate of 15ml/min, wash the column with about 5 times the column volume of buffer A to remove the Substances that are electrostatically adsorbed by the chromatographic medium; at a flow rate of 0.5-3ml/min, add 0.22mM-2.2mM 30kDa PEG-NHS ester (polyethylene glycol N-hydroxysuccinimide activated ester) solution 250ml-1500ml, continuously Load the sample for about 240 minutes; the pH of the reaction system is 4.5-7.0, more preferably pH 6.0-7.0; then wash the column with about 5 times the column volume of buffer A at a flow rate of 15ml/min to remove the Electrostatically adsorbed substances; then use buffer B (i.e. buffer A+1mol sodium chloride) to elute in the elution mode of 0-8%, 8%-18% and 18%-100% for about 150 minutes, The ion-exchange chromatogram as shown in Figure 5 can be recorded, and it can be seen from Figure 5 that the peak area of the single-modified PEG-rhG-CSF is greater than 90% of the total peak area, and the liquid-mass spectrometry trypsin enzyme Peptide mapping analysis determined that it was the N-terminal single, site-specific coupling product of 30kDa molecular weight PEG-NHS and rh-CSF (see Example 3 for details).

The method and results of the amino-specific coupling of PEG-ALD and rhG-CSF in a liquid phase reaction system carried out in an existing conventional manner:

Get the above rhG-CSF stock solution and concentrate it through ultrafiltration to make the protein concentration reach 2mg/ml, and dissolve PEG propionaldehyde (PEG-propionalaldehyde) with a molecular weight of 30kDa in the rhG-CSF stock solution of 2mg/ml according to the ratio of 8～40mol: 1mol; The appropriate ratio is that the molar ratio of 30kDa PEG-ALD and rhG-CSF stock solution is 8-20mol: 1mol; the reaction solution is 20mM-50mM sodium acetate buffer solution, pH 5.0-6.0; ) by adding sodium cyanoborohydride (NaBH ₃ CN) with a concentration of 7.5mg/ml, and the reaction solution was left to stand for 4 hours at 4°C to 25°C, and 1mM dilute hydrochloric acid was added in a ratio of 1:100 (v/v) solution (pH 3.5) to terminate the reaction.

Use 20mM～50mM sodium acetate buffer solution, pH 3.5～9.0, 150mM～500mM sodium chloride and 0.005% polysorbate 80 solution to equilibrate the Sepharose Fast Flow ion exchange chromatography column, and continuously load samples at a flow rate of 50ml/min The PEG-rhG-CSF reaction solution was eluted with a gradient of 0-1M sodium chloride solution, and the elution peak of the A280 protein was collected; three elution peaks were obtained, which were multi-modified PEG-rhG-CSF and single-modified PEG-rhG - Elution peaks of CSF and rhG-CSF. It is calculated that only about 45% of PEG-rhG-CSF is single-modified PEG-rhG-CSF, and the other 36% is multi-modified PEG-rhG-CSF compounds; the remaining 19% is unreacted rhG-CSF, the results are as follows Figure 6 shows.

The method and results of the amino-specific coupling of PEG-NHS and rhG-CSF in a liquid phase reaction system carried out in an existing conventional manner:

Take the above rhG-CSF stock solution and concentrate it by ultrafiltration so that the protein concentration reaches 1-2 mg/ml, and dissolve PEG-NHS with a molecular weight of 30 kDa in the rhG-CSF stock solution of 2 mg/ml according to the ratio of 5-50 mol:1 mol; the reaction solution is 20 mM ～50mM PBS buffer solution, pH 6.0～9.0, 150mM sodium chloride; the reaction solution was kept at 4℃～25℃ for 15～180 minutes.

Use 20mM～50mM PBS buffer solution, pH 4.5～7.0, 150mM sodium chloride and 0.005% polysorbate 80 solution to balance the Sepharose Fast Flow ion exchange chromatography column, and continuously load PEG-rhG at a flow rate of 50ml/min -CSF reaction solution, through gradient elution of 0 ~ 1M sodium chloride solution, the elution peak of A280 protein was collected; three elution peaks were obtained, which were multi-modified PEG-rhG-CSF, single-modified PEG-rhG-CSF and Elution peak of rhG-CSF. It is calculated that only about 23% of PEG-rhG-CSF is single-modified PEG-rhG-CSF, and the other 47% is multi-modified PEG-rhG-CSF compounds; the remaining 30% is unreacted rhG-CSF, the results are as follows Figure 7 shows.

Physicochemical properties and biological activities of PEG-rhG-CSF and PEG-rhG-CSFm

The present invention first discloses the in vitro physical and chemical properties and biological activity data of PG-N-30, PG-N-40, PG-N-20, PG-N-10, etc., see Example 4 for details.

The present invention found that the coupling of macromolecular PEG with a molecular weight of 30kDa to the N-terminal of rhG-CSF did not significantly affect the secondary structure and isoelectric point of rhG-CSF, while the solubility and stability against enzymolysis of rhG-CSF However, it has been significantly improved, which is one of the bases that such PEG-rhG-CSF and PEG-rhG-CSFm molecules have biological activities in vivo and in vitro (the physical and chemical properties and biological activity data in vitro are shown in Table 1).

The experimental data of the present invention also shows (see Table 3 for details), adopt NFS60 cell/MTT method to measure the in vitro activity of PEG-rhG-CSF and rhEPO simultaneously, find that the PG- The in vitro activity of N-10, PG-N-20, PG-N-30 and PG-N-40 has a tendency to decrease with the increase of the molecular weight of coupled PEG; while the in vitro activity of PG-N-10 is obvious higher than normal rhG-CSF, usually up to 50%, this result is greatly surprising, because it is generally believed that the conjugation of PEG macromolecules will significantly reduce the activity of rhG-CSF, obviously, this finding has potential and important clinical Application value; In addition, the in vitro activity of PG-N-20 and PG-N-30 differs by about 30%, but compared with PG-N-30, the molecular weight of PG-N-40 increases by about 30%, while its in vitro However, the activity was reduced by more than 2 times, and an obvious activity "inflection point" appeared. This also suggests that double-chain PEG (such as 40kDa PEG-ALD is a double-chain structure) has a greater impact on in vitro activity than single-chain PEG (such as 30kDa PEG-ALD is a single-chain structure). Therefore, the present invention confirms that single-chain PEG-ALD is preferred for coupling to the N-terminal amino group of rhG-CSF.

Pharmacokinetic and Pharmacodynamic Tests of PEG-rhG-CSF and PEG-rhG-CSFm

The present invention first discloses the systemic and standardized pharmacokinetic and pharmacodynamic test results of PG-N-30 and PG-N-20 in healthy Beagle dogs (as shown in Tables 4 and 5, Figures 19 and 20) , after subcutaneous injection of the same dose of PG-N-30 and PG-N-20, the half-life of the elimination phase of PG-N-30 was significantly longer than that of PG-N-20, which were 33.5±4.0 and 21.7±6.7 hours respectively; the AUC was significantly Greater than PG-N-20, the C _max of PG-N-30 is 120.5% of PG-N-20, the relative bioavailability is 158.7%, and the plasma clearance rate is significantly lower than PG-N-20, indicating that PG-N After subcutaneous injection, the residence time of -30 in the body is significantly longer than that of PG-N-20; therefore, the long-acting effect of PG-N-30 is better than that of PG-N-20. At the same time, it was shown that the change of this pharmacokinetic parameter also brought about the corresponding response of pharmacodynamics, that is, the half-life of the elimination phase of the effect (t _1/2(E) ) was significantly prolonged, the peak concentration of the effect (ANCC _max ) and the baseline Significant increase in the area of the effect curve (AOBEC _(0-192h) ); therefore PG-N-30 is more efficient than PG-N-20. This result is unexpected and unusual, because PG-N-20 is exactly Neulasta that has been on the market at present and achieved excellent curative effect, safety and high sales volume; Not the "best" long-acting formulation of rhG-CSF; obviously, PG-N-30 is better than Neulasta in terms of long-acting and high-efficiency.

Pharmacodynamics and immunogenicity of PEG-rhG-CSF and PEG-rhG-CSFm

The experimental results published by the present invention show (see Table 6-1, Table 7 for details), on the model of mouse leukopenia caused by ¹³⁷ Cs-r irradiation and chemotherapy-induced rat leukopenia, the pattern of continuous administration every day The results showed that the two test drugs could lead to a dose-dependent increase in white blood cells (WBC) and neutrophils (ANC), but beyond Unexpectedly, when the dose of rhG-CSF injected is ten times that of PG-N-30, the increase of WBC and ANC at the equivalent level can be achieved; or the increase of WBC and ANC caused by the same injected dose of PG-N-30 The value is three times that of rhG-CSF; this shows that in the treatment mode of daily continuous administration, PG-N-30 has obvious high-efficiency characteristics, and its dosage may even be lower than that of ordinary rhG-CSF; obviously, this One discovery has potential and important clinical therapeutic implications. The present invention also found that, on the above two animal models, with the gradual increase of the PG-N-30 administration interval (such as interval 1 day, 2 days, etc.), to achieve a curative effect equivalent to that of continuous application of rhG-CSF, The dosage of PG-N-30 needs to be doubled. To realize the administration mode of injecting one injection in one chemotherapy cycle, the dosage of PG-N-30 may even reach 5 times of the total amount of rhG-CSF used continuously ( See Table 6-2, Table 7 for details), this result is greatly unexpected, which shows that there is still a strong PG-N-30 clearance mechanism in the body, and the treatment mode of applying once or longer dosing intervals in a chemotherapy cycle is far from Economical or optimal mode of administration.

The present invention found that on the model of fluorouracil-induced leukopenia in mice, the administration method of one chemotherapy cycle was adopted, and the results showed that the single-dose application of 1200ug/kg of PG-N-30 and the application of 2400ug/kg of PG-N -20 equivalent (as shown in Figure 21); Adopt the same mode of administration, on the rat leukopenia model caused by cyclophosphamide, confirm that the PG-N-30 of single agent application 600ug/kg and the PG-N-30 of application 1200ug/kg PG-N-20 is equivalent (as shown in Figure 22); therefore, in the above two animal models, it is confirmed that the curative effect of PG-N-30 is also significantly better than that of PG in the treatment mode of one chemotherapy cycle application -N-20; therefore, the clinical use of PG-N-30 is expected to be significantly lower than that of Neulasta.

The present invention proves for the first time that PG-N-30 may be a better or more effective preparation, and it can show a good comparative advantage in the treatment mode of daily continuous application and once-in-one chemotherapy cycle, but from From the perspective of pharmaceutical economics, daily continuous dosing or shorter dosing intervals may be a better treatment mode.

The present invention also discloses the experimental data on the immunogenicity of the above-mentioned PEG-rhG-CSF and PEG-rhG-CSFm. On normal rats, the administration method of 1 time/week for 12 consecutive weeks shows that PG- The immunogenicity of N-20, PG-N-30 and PG-N-40 was significantly lower than that of rhG-CSF, and the immunogenicity of PG-N-30 and PG-N-40 was significantly lower than that of PG-N -20.

The PEG-rhG-CSF or PEG-rhG-CSFm coupling compound involved in the present invention is an active ingredient of a clinical pharmaceutical preparation, which may also include: diluents, stabilizers, preservatives ( Preservatives), dissolving agents (solubilizers), emulsifiers (emulsifiers), adjuvants (adjuvants) and carriers (carriers) etc. Specifically, such as: 1) diluent: Tris-HCL, phosphoric acid, acetic acid and other buffers; 2) pH value and ionic strength; 3) detergent and dissolving agent: polysorbate 20, polysorbate 80; 4 ) Filling agents (bulking substances): lactose, mannitol. See Remington's Pharmaceutical Science, ^18th Ed (1990, Mack Publishing Co., Easton, Pa. 18042) Pages 1435:1712 for details. The effective dose of the active ingredient refers to the effective therapeutic or preventive dose considering factors such as body weight and age. The PEG-rhG-CSF or PEG-rhG-CSFm involved in the present invention has an effective dosage range of 0.1ug-300ug/kg body weight/dose, and a more suitable effective dosage range is 0.5ug-200ug/kg body weight/dose.

The PEG-rhG-CSF or PEG-rhG-CSFm clinical pharmaceutical preparations involved in the present invention are long-acting, high-efficiency, and low-immunogenic preparations, usually injected once every 1-2 weeks, or once in a course of treatment; or The long-acting effect can reach 3-4 weeks; the continuous administration method of injecting once a day or the administration method at an interval of 1 to 3 days can also be used; the application of PEG-rhG-CSF or PEG-rhG-CSFm clinical pharmaceutical preparations The interval design is also related to the indications of the application and the actual condition of the patient. The PEG-rhG-CSF or PEG-rhG-CSFm clinical pharmaceutical preparation can be injected subcutaneously, intramuscularly, intravenously and intraperitoneally; it can also be administered by inhalation, sublingually or transdermally.

The pegylated recombinant human granulocyte colony-stimulating factor of the present invention can be applied to prevent and treat various granulocytopenias, common indications include: granulocytopenia caused by tumor or leukemia chemotherapy or AIDS chemotherapy drugs such as AZT , bone marrow and peripheral blood stem cell transplantation, and primary chronic neutropenia.

The data and methods provided by the present invention are also applicable to the development of other cytokines and antibody fragments Fab', scFv and Fc PEGylation and long-acting, high-efficiency, and low-immunogenicity preparations, such as recombinant human interferon alfas, interferon betas, tumor necrosis factor receptor protein and so on.

Description of drawings

Figure 1 PEG-ALD and rhG-CSF N-terminal specific site-specific coupling ion exchange chromatogram

Among them: peak 1 is double-modified or multi-modified PEG-rhG-CSF, peak 2 is single-modified PEG-rhG-CSF, and peak 3 is unreacted rhG-CSF.

Figure 2 SDS-PAGE PG-N-30 molecular weight determination (silver staining).

It shows that the molecular weight of PG-N-30 is 66.2kDa, which is a single modified PEG-rhG-CSF.

Fig. 3 High Performance Gel Chromatography (SEC) Determination of PG-N-30 Purity

Among them, the area of the main peak is greater than 95%, which is identified as single-modified PEG-rhG-CSF, and the small peak before it is multi-modified PEG-rhG-CSF.

Fig. 4 high performance liquid chromatography (RT-HPLC) measures PG-N-30 purity

Among them, the area of the main peak is greater than 98%, and it is identified as single-modified PEG-rhG-CSF.

Figure 5 PEG-NHS ester and rhG-CSF N-terminal specific site-specific coupling ion-exchange chromatogram

Display: the peak area of single modified PEG-rhG-CSF is greater than 90% of the total peak area, and it is determined by liquid chromatography-mass spectrometry that the N-terminus of PEG-NHS and rh-CSF with a molecular weight of 30kDa is single and fixed. Coupling product (see Example 3 for details).

Figure 6 High performance gel chromatography analysis of PEG-ALD and rhG-CSF coupling reaction products

Among them: peak-1 is multi-modified PEG-rhG-CSF, accounting for 33.12% of the total peak area, peak-2 is two-modified PEG-rhG-CSF, accounting for 2.68% of the total peak area, peak-3 is single-modified PEG- rhG-CSF accounted for 45.60% of the total peak area, and peak-4 was unreacted rhG-CSF, accounting for 18.60% of the total peak area.

Figure 7 High performance gel chromatography analysis of PEG-NHS and rhG-CSF coupling reaction products

Among them, peak-1 is multi-modified PEG-rhG-CSF, accounting for 44.6% of the total peak area, peak-2 is di-modified PEG-rhG-CSF, accounting for 2.7% of the total peak area, peak-3 is single-modified PEG- rhG-CSF accounted for 22.78% of the total peak area, and peak-4 was unreacted rhG-CSF, accounting for 29.92% of the total peak area.

Fig. 8 Construction of recombinant plasmid expressing rh-CSF.

Figure 9 SDS-PAGE expression analysis of rhG-CSF

Wherein, the arrow points to the expression band of the target protein rhG-CSF.

Figure 10 MALDI-TOF determination of rhG-CSF molecular weight.

Fig. 11 Determination of the purity of rhG-CSF stock solution by high performance liquid chromatography (HPLC).

Fig. 12 Determination of the purity of rhG-CSF stock solution by high performance gel chromatography (SEC).

Figure 13-1 is the analysis and detection results of M ¹ TPLGPASSLPQSFLLK ¹⁷ fragment after rhG-CSF proteolysis

It shows that after the primary mass spectrometry scan and the accurate mass number scan, it can be concluded that the peptide segment m/z=894.6 or so.

Figure 13-2 is the first-order mass spectrum, accurate mass scanning and second-order mass spectrometry analysis results of the M ¹ TPLGPASSLPQSFLLK ¹⁷ peptide of the PEG-ALD single modification product PEG-rhG-CSF at pH 8.0

The fragment showing m/z=894.3 still exists very clearly, and its content can be seen to be relatively high according to its abundance. This shows that compared with the rhG-CSF original protein, a large part of the site of the modified product under the condition of pH8.0 is not at the N-terminus.

Figure 13-3 is the primary mass spectrum and accurate mass scan of the M ¹ TPLGPASSLPQSFLLK ¹⁷ peptide of the PEG-ALD single modification product PEG-rhG-CSF, namely PG-N-30, at pH 4.5, after trypsin digestion And the results of the secondary mass spectrometry

The fragment showing m/z=894.3 hardly exists, and its content is very low. According to its abundance, it can be seen that its content should be less than 5-10%. This shows that compared with the modified product at pH 8.0, only a very small part of the modified product at pH 4.5 is not at the N-terminal, that is, the sites of the modified product are mainly concentrated at the N-terminal.

Figure 14-1 MALDI-TOF-MS molecular weight measurement results of PEG-ALD (30kDa).

Figure 14-2 MALDI-TOF-MS molecular weight determination results of peak 2 in Example 2.

Fig. 15 isoelectric focusing measurement results.

Figure 16 Circular dichroism spectrum determination of rhG-CSF and PEG-rhG-CSF secondary structure

Among them, the three lines show rhG-CSF, PG-N-20 and PG-N-30.

Figure 17-1 PG-N-30 (3mg/mL/bottle) after standing at 4°C for 24 months, the results of high-efficiency gel detection

Wherein: peak 1 is PG-N-30 multimer accounting for 0.5%, peak 2 is PG-N-30 dimer 1.1%, peak 3 is PG-N-30 accounting for 98.4%.

Figure 17-2rhG-CSF (3mg/mL/vial) after standing at 4°C for 30 days, the results of high-efficiency gel detection

Among them: peak 1 is rhG-CSF accounting for 41.2%, peak 2 is rhG-CSF aggregates accounting for 58.8%.

Fig. 18 In vitro enzymatic hydrolysis reaction diagram of rhG-CSF and PG-N-30.

Fig. 19 The drug-time curves of Beagle dogs after subcutaneous injection of the same dose of PG-N-30 and PG-N-20 (200 μg·kg ^-1 ).

Figure 20-1 Changes in ANC counts at different times after subcutaneous injection of PG-N-30 and PG-N-20 in Beagle dogs.

Figure 20-2 Comparison of changes in drug concentration and ANC counts after Beagle dogs were injected with 200 μg·kg ^-1 of PG-N-30.

Fig. 21 Effect of PG-N-30 on neutrophil level in mouse leukopenia model animals induced by fluorouracil.

Fig. 22 Effect of PG-N-30 on neutrophil level in rat leukopenia model animals induced by cyclophosphamide.

Detailed ways

The following examples help to further illustrate the present invention, but the content and scope of the present invention are not limited to these examples.

Example 1 Expression and preparation of rhG-CSF and rhG-CSFm

Expression of rhG-CSF and rhG-CSFm:

Fresh human bone marrow cells were cultured and activated with pokeweed kinogen (Sigma, USA). Human bone marrow cell mRNA was extracted, and RT-PCR method was used to amplify hG-CSF gene, which was confirmed to be hG-CSF gene through sequencing (completed by Beijing Boshang Biotechnology Co., Ltd.), and its cDNA sequence is shown in SEQ ID NO.2. The amino acid sequence is shown in SEQID NO.1. DNA Strider software was used to analyze hG-CSF gene DNA sequence analysis; PCR (polymerase chain reaction)) and site-directed mutagenesis were used to modify the human natural G-CSF gene sequence to obtain a new modified human granulocyte colony-stimulating factor ( rhG-CSF) gene coding sequence, its nucleotide sequence is shown in SEQID NO.3, this gene is constructed on the pMD18-T vector, and the vector is transformed into the Top10 bacterial strain and preserved.

The primer rhG-CSF_F (5-GATCCTAGCATATGACTCCATTAGGTCCT-3) was used as the front primer, the primer rhG-CSF_R (5-GATCCTAGAATTCTCAGGGCTGCGCAAG-3) was used as the back primer, and pMD18-T/rhG-CSF was used as the template to amplify rhG-CSF in vitro by PCR Sequence (PCR reaction conditions: primer concentration 20 μmol/L, 5 μl 10× buffer; 25 mmol/L MgCl ₂ 4 μl; 10 mmol/L four kinds of dNTP mixture 1 μl; upstream and downstream primers 1 μl; TaqDNA polymerase 0.5 μl; template DNA 1 μl , add water to make up to 50 μl; pre-denaturation at 97°C for 10 minutes, denaturation at 94°C for 60 seconds, annealing at 55°C for 60 seconds, extension at 72°C for 60 seconds, extension at 72°C for 10 minutes after 30 cycles, and storage at 4°C). The molecular weight of the fragments was detected by electrophoresis after the reaction (the concentration of the agarose gel was 0.8%).

The fragment obtained by PCR and the pGL1 plasmid were digested with Nde I and EcoR I endonucleases. Then, the PCR product recovery kit (purchased from OMEGA) was used to recover and purify the digested fragments. Use T4 ligase (purchased from MBI company) for ligation, and the reaction composition system (rhG-CSF gene fragment: 6 μl; pGL1 fragment 2 μl; ligation buffer (10X) 1 μl; T4 ligase 1 μl) for ligation reaction at 22°C , the time is 4 hours, and the recombinant environment-inducible expression plasmid pGL1-rhG-CSF is obtained after ligation.

With the above-mentioned method, use the following pre-primer and back primer rhG-CSF_R (5-GATCCTAGAATTCTCAGGGCTGCGCAAG-3) respectively to carry out PCR amplification in the above-mentioned manner and conditions, and clone the amplified product into Nde I and EcoR I of the pGL1 plasmid. Between the restriction sites, construct the expression plasmid of rhG-CSF mutant.

rhG-CSF-174_F: 5-GATCCTAGCAT ATGCCATTAGGTCCTGCA-3

rhG-CSF-173_F: 5-GATCCTAGCAT ATGTTAGGTCCTGCATCT-3

rhG-CSF-172_F: 5-GATCCTAGCAT ATGGGTCCTGCATCTTCT-3

rhG-CSF-171_F: 5-GATCCTAGCAT ATGCCTGCATCTTCTTTA-3

rhG-CSF-170_F: 5-GATCCTAGCAT ATGGCATCTTCTTTACCA-3

rhG-CSF-169_F: 5-GATCCTAGCAT ATGTCTTTCTTTACCACAA-3

rhG-CSF-168_F: 5-GATCCTAGCAT ATGTCTTTACCACAAGC-3

rhG-CSF-167_F: 5-GATCCTAGCAT ATGTTACCACAAAAGCTTC-3

rhG-CSF-166_F: 5-GATCCTAGCAT ATGCCACAAAGCTTCCTG-3

rhG-CSF-165_F: 5-GATCCTAGCATATGCAAAGCTTCCTGCTC-3.

The recombinant plasmid pGL1-rhG-CSF was transformed into Escherichia coli DH5α. Pick and transfer a single colony into LB liquid medium containing 100 μg/ml ampicillin, and culture overnight at 37° C. and 225 rpm. The plasmid kit was used to extract the recombinant plasmid pGL1-rhG-CSF, and the size of the plasmid was verified. And use Nde I, EcoRI endonuclease double digestion analysis, screen the recombinant plasmid transformant containing rhG-CSF gene fragment; The recombinant plasmid map is shown in Figure 8.

The verified recombinant plasmid pGL1-rhG-CSF was transformed into Escherichia coli BL21(DE3)pLysS. Pick the positive clones after screening on the LB plate, and inoculate 1-2 loops under sterile conditions into 5ml of fermentation medium with ampicillin at a final concentration of 50-150 μg/ml, 30-40 Under the condition of ℃, 150-300 rev/min shaker shake culture for 8-16 hours to obtain seed solution; transfer 1%-5% inoculum amount into a 300ml Erlenmeyer flask with 50ml medium. Incubate vigorously at 37°C for 3 hours, so that the bacteria are in the mid-logarithmic growth phase, and the OD ₆₀₀ value is about 0.8. Take 1mL from 50ml logarithmic growth phase bacterial liquid as a control, add 1mol/L IPTG solution to the remaining bacterial liquid to make the final concentration 1mmol/L. After adding IPTG, continue to culture the bacterial solution for 1-4 hours, centrifuge at 12,000 rpm for 10 minutes, take the supernatant and precipitate and store them separately for later use, and finally harvest the bacteria by centrifugation to detect the expression of the target protein. The results of SDS-PAGE are shown in Figure 2, and the rhG-CSF content accounts for about 25% of the total bacterial protein, as shown in Figure 9 .

With the above method, the constructed rhG-CSF mutant expression plasmid can also be screened to obtain engineering strains of 10 rhG-CSF mutants that can efficiently express the target protein, that is, 10 rhG-CSF mutants that are deleted one by one from the 2nd to the 11th amino acid at the N-terminal and Its rhG-CSF mutants, namely rhG-CSF-174, rhG-CSF-173, rhG-CSF-172, rhG-CSF-171, rhG-CSF-170, rhG-CSF-169, rhG-CSF-168, rhG-CSF-167, rhG-CSF-166, rhG-CSF-165; its amino acid sequence is shown in the amino acid sequence formula of SEQ ID NO.1.

Preparation of rhG-CSF or rhG-CSFm in E. coli expression system:

Take out the above rhG-CSF strains from the liquid nitrogen tank, and melt them quickly in a water bath at 37°C; under strict aseptic operation, inoculate the rhG-CSF E. , 0.5 g of yeast extract, 0.5 g of sodium chloride, 4 mg of sodium hydroxide, 1.5 g of agar powder, and 5 mg of ampicillin), cultivated in a 37°C incubator (SANYO MCO-17AIC) for 12-15 hours, and selected a single colony , transferred in a 12ml LB/Amp (100ml formula contains 1 gram of tryptone, 0.5 grams of yeast extract, 0.5 grams of sodium chloride, 4 mg of sodium hydroxide and 5 mg of ampicillin) culture tube, 37 ° C 250 rpm Shaking culture (HWY 111 constant temperature shaker) for 12 hours; transfer 10ml of fermentation broth to 1000ml LB/Amp fermentation broth for shaking flask culture (37°C, 250rpm shaking) for 8 hours; re-inoculate in a 15-liter fermenter (B.Braun Cultivated in BIOSTAT C), each liter of fermentation broth contains 12 grams of tryptone, 24 grams of yeast extract, 3.8 grams of potassium dihydrogen phosphate, 12.5 grams of dipotassium hydrogen phosphate, 0.5 grams of magnesium sulfate, 6.3 grams of glycerin, 0.9 liters of pure water, 20 grams of glucose and 0.1 grams of ampicillin; the fermentation parameters were set as temperature 37°C, pH 7.0, dissolved oxygen 35%, and the linkage between stirring speed and dissolved oxygen. When the OD ₆₀₀ value reaches 20, it is the middle stage of the logarithmic growth of the engineered bacteria, and IPTG is added to induce it, and the fermentation is terminated after 2 hours. Cool the fermented liquid to bring it down to 4-8°C. Bacteria were collected by centrifugation (4000 rpm, 30 minutes) in a Sorvall large-capacity low-temperature refrigerated low-speed centrifuge (Sorvall RC3B).

Add engineering bacteria breaking buffer (50mM Tris, 1mM EDTA, pH7.5 buffer) to the collected engineering bacteria thalline precipitate. The bacteria were crushed under the pressure of 10000psi by APV high-pressure homogenizer (APV 2000). After breaking the bacteria, the liquid was centrifuged at a speed of 11000 rpm in a high-speed refrigerated centrifuge (Sorvall RC5B) SLA-3000 (4°C, 20 minutes) to obtain crude inclusion bodies (IB). Use inclusion body washing solution (50mM Tris, 1mM EDTA, pH7.5, 2.5% Triton × 100, 0.15M NaCl) three times, mix well with inclusion body, and then centrifuge at low temperature (4°C) at high speed (Sorvall RC5B, 11000rpm) , 50 minutes) to collect the inclusion bodies, and complete the washing and purification of the inclusion bodies. Dissolve the purified inclusion body in inclusion body dissolving and denaturing solution (50mM Tris, 1mM EDTA, 8M guanidine hydrochloride, 1mM dimercaptoerythritol) at a ratio of 10 grams of wet weight to 500 ml, at 4°C for 12 hours to obtain Inclusion bodies were dissolved in denaturing solution, centrifuged for 40 minutes (Sorvall RC5B, 11,000 rpm), and the supernatant was collected.

Slowly add inclusion body dissolving and denaturing solution into 20mM sodium acetate buffer (pH 6.0), 150mM sodium chloride, 3M urea, 0.005% polysorbate 80 (polysorbate 80) refolding solution at a volume ratio of 1:100, 4 ℃ for 24 hours, after filtering through a 0.45 μm pore size filter membrane, the protein was purified.

The rhG-CSF protein refolding solution was subjected to ion exchange chromatography (chromatography equipment AKTA purifier, chromatography column INdEX200/500, chromatography medium Sepharose big bead), and the equilibrium buffer was 20mM sodium acetate buffer (pH 4.5), 150mM chloride Sodium chloride, 0.005% polysorbate 80; gradient elution with 0-1M sodium chloride, the separation result is shown in Figure 3, and the A280 protein elution peak solution was collected. Equilibrate the gel chromatography column (chromatographic column BPG100, chromatography medium Superdex 75 prep grade) with the above-mentioned equilibration buffer, load the sample at a flow rate of 30ml/min, and each loading lasts for 5 minutes (the loading volume is about 150ml), After 120 minutes of sample loading, the A280 protein absorption peak that appeared was collected, which was the rhG-CSF stock solution.

MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) measures the molecular weight of rhG-CSF (as shown in Figure 10), and high-performance liquid chromatography (HPLC) and high-performance gel chromatography (SEC) measure rhG-CSF stoste, the result As shown in Figure 11 and Figure 12, the method is the same as that described in Embodiment 3. The results showed that the molecular weight of rhG-CSF was about 18.688kDa; and the purity of rhG-CSF stock solution by HPLC and SEC was greater than 98%.

Example 2 Preparation of PEG-rhG-CSF by site-specific coupling of PEG-propionaldehyde and rhG-CSF N-terminus

Load MacroCap SP strong cation exchange chromatographic column (2.6cmX20cm), and connect it to AKTAExplorer 100 liquid chromatography system (Amersham Bioscience, Sweden), use 20mM sodium acetate buffer pH 5.5 (buffer solution A) and 0.005% poly Sorbitol ester 80 500ml fully equilibrates the chromatographic column; using a feed pump, add 0.5mg/ml 1500ml of rhG-CSF stock solution to the chromatographic column at a flow rate of 10ml/min; Wash the column with liquid A to remove substances that do not electrostatically adsorb with the chromatographic medium; add 5mg/ml 30kDa PEG-propionaldehyde (PEG-propionaldehyde) and 0.1mg/mlNaBH ₃ CN solution 1500ml at a flow rate of 3ml/min, and continuously add The sample is about 240 minutes; the pH of the reaction system is 5.5; then at a flow rate of 15ml/min, wash the column with about 500ml buffer A to remove substances that do not electrostatically adsorb with the chromatographic medium; then use buffer B (i.e. buffer A +1mol sodium chloride), with the elution mode of 0-8%, 8%-18% and 18%-100% elution, for about 150 minutes, the ion-exchange chromatogram as shown in Figure 1 can be recorded Fig. 1 sees, peak 1 can be obtained altogether, and three elution peaks of peak 2 and peak 3, namely P1, P2 and P3, wherein the peak area of P2 accounts for about 90% of total peak area; P1 and P3 respectively Accounted for 4% and 6% of the total peak area. P2 was analyzed as a single modified PEG-rhG-CSF by reducing SDS-PAGE, high performance gel chromatography (SEC) and high performance liquid chromatography (HPLC), with a purity greater than 95% (as shown in Figures 2, 3, and 4) . The product was analyzed by LC-MS using tryptic peptide map to determine that the coupling site of PEG-propionaldehyde with a molecular weight of 30 kDa was the N-terminal of rh-CSF, namely PG-N-30 (see Example 3 for details).

In the above-mentioned chromatographic column PEG protein N-terminal fixed-point coupling method, if only the pH value of the reaction system is changed, such as at pH4.0 and pH6.0 (see Table 2 for details), the composition and ratio of the elution peak will be It will change, but the single-modified PEG-rhG-CSF is still the fixed-point coupling between 30kDa molecular weight PEG-propionaldehyde and rh-CSF N-terminus, that is, PG-N- 30.

Table 2 The influence of the change of the pH value of the reaction system on the N-terminal coupling of the PEG protein on the chromatography column

Example 3 Analysis of the Coupling Site of PEG-ALD and rh-CSF Using Trypsin Digestion Peptide Mapping by Liquid-MS

1) Measurement method

Select liquid chromatograph Agilent 1100 and mass spectrometer LCQ Deca XP MS to analyze, and specific method is as follows:

Liquid chromatograph operating conditions:

The mobile phase is: A: 0.1% A: 0.1% TFA in water, B: 0.1% TFA in ACN;

Chromatographic conditions: the chromatographic column is Zorbax SBC18 (2.1 × 150mm, Agilent), the flow rate is 0.2ml/ml, the gradient is 0-60min, 5%B-60B, 60-120min, 60%B-90%B, and the detection wavelength is 214nm;

Mass spectrometer operating conditions:

Sheath gas: 60arb; Aux gas: 0; Spray voltage: 4.5kV; Detection method: Triple play; Primary mass spectrometry: Mass range: m/z300-2000; Zoom scan: Data dependent mode; Data processing software: Bioworks3.1; mass spectrometry data analysis software Turbosequest3.1.

The enzymolysis sample is injected into the chromatographic system through the liquid chromatography automatic sampling system for vigilance analysis.

Sample handling operating conditions:

Sample processing of unmodified protein to be tested:

①Take 200μl (protein concentration 1mg/mL) of the sample to be tested, add 10uL 8M urea, disperse the sample by ultrasonication for 10min, and adjust the pH to pH8.0 with 0.2M Tris-HCl;

② Add trypsin solution at a molar ratio of 50:1 (protein:trypsin), and incubate at a constant temperature of 37°C for 8 hours; trypsin was purchased from Promega, USA;

③Reduction of disulfide bonds: Add 10 μl of 1.4 mol/l dithiothreitol aqueous solution to the enzymatic hydrolysis product, shake and mix, and react at 37°C for 10 minutes;

The treatment method of the modified protein to be tested is the same as that of the unmodified protein, but the digestion time is extended to 12 hours.

2) Analysis of the amino acid sequence of rhG-CSF and its possible restriction sites

M ¹ TPLGPASSSL ¹⁰ PQSFLLK ¹⁷ CLE ²⁰ QVRK ²⁴ IQGDGA ³⁰ ALQEK ³⁵ LCATYK ⁴¹ LCHPEELVLLGHSLGIPWAPLSSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASLAFQRRAGGVLEVASHLQ.

Theoretically, the enzyme cleavage site of trypsin is the carboxyl terminal of lysine and arginine (Lys and Arg), and the peptide segment of rhG-CSF obtained by digestion with trypsin is:

①M ¹ TPLGPASSLPQSFLLK ¹⁷

②CLEQVRK ²⁴

③IQGDGAALQEK ³⁵

④LCATYK ⁴¹

⑤LCHPEELVLLGHSLGIPWAPLSSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQR ¹⁴⁷

⑥AGGVLVASHLQSFLEVSYR ¹⁶⁷

⑦HLAQP ¹⁷⁵

Since the modification sites of PEG-ALD are also Lys and Met at the N-terminal, we can focus on the analysis of those enzyme-cleaved polypeptide fragments before Lys41. Theoretically, trypsin digestion before Lys41 of rhG-CSF can obtain the following 4 polypeptide fragments:

①M ¹ TPLGPASSLPQSFLLK ¹⁷ ; ②CLEQVRK ²⁴ ; ③IQGDGAALQEK ³⁵ ; ④LCATYK ⁴¹

In the actual detection and analysis of restriction enzyme fragments, we obtained results that are in full agreement with the theoretical values, that is, the theoretical restriction fragments have been found one by one. Figure 13-1 shows the restriction fragments ①M ¹ TPLGPASSLPQSFLLK ¹⁷ , that is, at m /z=894.6 or so; because there are too many original pictures, the rest of the clips are not included.

3) Coupling site analysis of PEG-ALD single modified rhG-CSF

Analysis of the product of PEG-ALD modified rhG-CSF under different pH conditions, namely PEG-rhG-CSF, found that under the condition of pH=8.0, the m/z= The fragment of 894.3 exists very clearly, according to its abundance, it can be seen that the content is relatively high, as shown in Figure 13-2, while other ②CLEQVRK ²⁴ ; ③IQGDGAALQEK ³⁵ ; ④LCATYK ⁴¹ , the abundance decreases or the corresponding fragment disappears. However, under the condition of pH=4.5, on the contrary, the above-mentioned fragment ①M ¹ TPLGPASSLPQSFLLK ¹⁷ m/z=894.3 almost disappeared (as shown in Figure 13-3), and the remaining three enzyme-digested fragments were clearly present, and were compatible with the same amount of The abundance of the corresponding fragments of rhG-CSF was consistent; indeed. Both theory and experiments have confirmed that under the condition of pH=4.5, most of the monomodified PEG-rhG-CSF is PEG-rhG-CSF modified at the N-terminus of the protein; On the premise, whether the PEG coupling site is located at the N-terminus can be judged by judging the existence and abundance of the restriction fragment ① M ¹ TPLGPASSLPQSFLLK ¹⁷ (m/z=894.3).

4) Sample determination and analysis

The samples collected from peak-2 in Figure 2 and the sample collected from peak-2 in Figure 5 were measured. The results showed that the enzyme-cut fragment ①M ¹ TPLGPASSLPQSFLLK ¹⁷ (m/z=894.3) was not detected, while the other three enzyme-cut fragments were ②CLEQVRK ²⁴ ; ③ IQGDGAALQEK ³⁵ ; ④ LCATYK ⁴¹ are clearly identifiable, and the common rhG-CSF restriction pattern of the same amount of abundance shows that the single modified PEG-rhG-CSF of the above two samples is PEG-ALD or PEG-NHS and rhG - The product of site-directed coupling to the N-terminus of CSF protein.

Physicochemical properties and in vitro activity of embodiment 4 PEG-rhG-CSF or PEG-rhG-CSFm

1) MALDI-TOF-MS molecular weight determination

On the matrix-assisted laser desorption-ionization time-of-flight mass spectrometer Bruker Daltonics Autoflex 3 (Bruker Daltonics Billerica Company, the United States), the MALDI target was cleaned and dried according to the operating procedures, and the Zip Tip C4 (Millipore Company, the United States) was used for desalting, Concentrate the sample to be tested; draw 10ul of the sample to be tested and the standard target; set the operating program conditions of the instrument as follows: Delayed extraction time (Delayed extraction) 190ns, detection mode is Linear ion mode/positive ion mode, laser frequency (Laser frequency) 50Hz , Accelerating Voltage (Accelerating Voltage) 20KV, graphic overlay (sum shots) 400 times, calibration mode is External calibration; through the calibration of standard samples, sample determination is carried out, and the MALDI-TOF-MS molecular weight determination spectrum can be obtained by data processing; Figure 10, Figure 14-1 and Figure 14-2. Figure 14-1 is the MALDI-TOF-MS molecular weight measurement result of the modifier PEG-ALD (30kDa) used in Example 2, which is 32439.3 Daltons. The molecular weight is close to its marked molecular weight; Figure 14-2 shows the measurement results of peak 2 obtained in Example 2 above. The molecular weight of PG-N-30 is 50667.491 Daltons, indicating that it is a single modified PEG-rhG-CSF.

2) High Performance Liquid Chromatography (HPLC) protein determination

On the Agilent 1100 chromatographic instrument (produced by Agilent Corporation of the United States), select the carbon 4Vydac C4 protein chromatographic column (company), use A phase trifluoroacetic acid aqueous solution (take 1.0ml trifluoroacetic acid and add water to 1000ml) and B phase trifluoroacetic acid acetonitrile solution (Get 1.0ml trifluoroacetic acid and add chromatographically pure acetonitrile to 1000ml) as mobile phase; at room temperature, set flow rate as 1ml/min, pressure as 76bar, carry out gradient elution (0～70% B phase); The sample volume is 20ul, the sample volume is not less than 30ug, and the detection is performed at a wavelength of 280nm. The area of each peak was calculated by the area normalization method. The results are shown in Figure 4 and Figure 11.

3) Determination of protein purity by high performance gel chromatography (Size Exclusion Chromatography, SEC)

On the Agilent 1100 chromatograph (produced by U.S. Agilent), select gel filtration chromatography column Superdex ^TM 200, HR10/30 (GE Healthcare Biosciences company), mobile phase is 50mM sodium phosphate buffer (containing 0.1M sodium sulfate ), pH6.7; set the flow rate at 0.5ml/min, the pressure at 16bar, and the temperature at 25°C; the detection wavelength is 280nm, and the wave width is 4nm; the reference wavelength is 360nm, and the wave width is 100nm; the amount of sample to be tested is not less than 20ug ; Use the area normalization method to calculate the area of each peak. The results are shown in Figure 3, Figure 6, Figure 7, Figure 8, Figure 12, Figure 17-1, and Figure 17-2.

4) Determination of isoelectric point

On the Phastsystem electrophoresis instrument (Pharmacia Biotech, Sweden), select prefabricated gel PhastGel IEF 5-8 for isoelectric focusing (GE Healthcare Biosciences, USA); prepare fixative 20% (W:V) trichloroacetic acid, decolorization solution 30 % methanol + 10% acetic acid mixed solution and staining solution 0.02% PhastGel Blue R solution; according to the "Pharmacia Phastsystem Use and Maintenance Manual" to set the electrophoresis program, staining/decolorization steps and washing procedures, etc., the sample volume is 1ul (including 1~2ug protein), calculate and judge the isoelectric point of the sample according to the standard protein provided by the precast gel PhastGel IEF 5~8. As shown in Fig. 15, the isoelectric points of rhG-CSF, PG-N-20 and PG-N-30 are consistent, indicating that the coupling of PEG to the N-terminus of rhG-CSF does not change its isoelectric point.

5) Determination of secondary structure by circular dichroism spectroscopy

The samples to be tested were desalted and replaced with 20 mM acetic acid-sodium acetate buffer (pH 4.5) by G-25 gel chromatography column (GE Healthcare, USA), and the protein concentration was adjusted to 0.2 mg/ml. Operate the garden dichroic spectrometer (Jasco-810Spectropolarimeter, Jasco, Tokyo, Japan) according to the regulations, set parameters: sensitivity: 100mdeg, bandwidth: 1.0nm, rosolution: 0.5nm, response: 0.5sec, accumulation: 4, scanspeed: 500nm/min . The measurement results are shown in FIG. 16 . The results show that the circular dichroism patterns of PG-N-30, PG-N-20 and rhG-CSF are almost in perfect agreement, suggesting that the nitrogen end of the rhG-CSF protein molecule coupled with PEG of 20kDa and 30kDa molecular weight can well maintain its secondary structure.

6) In vitro activity

The in vitro activity of rhG-CSF and various PEG-rhG-CSF was measured and compared by NFS60 cell/MTT method in vitro, and the results are shown in Table 3.

NFS cell/MTT colorimetric method: For detailed methods, see Appendix X E of Part Three of the Pharmacopoeia of the People's Republic of China in 2005. NFS60 cells were purchased from China National Institute for the Control of Pharmaceutical and Biological Products. NFS60 cells were cultured in RPMI 1640 medium (containing penicillin 10E5IU/L and streptomycin 10E5IU/L) containing 10% newborn bovine serum and 20ng/ml rhG-CSF at 37°C for 5 % carbon dioxide culture, control the cell concentration to contain 1.0×10E5～4.0×10E5 cells per 1ml, subculture for 24～36 hours for activity determination; collect NFS60 cells by centrifugation, wash the cells 3 times with RPMI 1640 medium; prepare 1ml containing 2. Cell suspension of 0×10E5 cells; add 50ul of cell suspension to each well of a 96-well cell culture medium plate with standard solution and test solution, and incubate at 37°C and 5% carbon dioxide for 40-48 hours; Add 20uL thiazolyl blue reagent (MTT reagent, Sigma, USA) to each well, and continue to incubate for 5 hours; add 100uL lysate (made from 14ml hydrochloric acid, 50mL Triton X-100 solution and 500mL isopropanol) to each well, and mix well Then, put it into a microplate reader (Bio-Tek Company, USA), take 630nm as a reference wavelength, and measure the absorbance value at a wavelength of 570nm.

Table 3 rhG-CSF activity assay results in vitro

It can be seen from Table 3 that the in vitro activity of PG-N-10 is significantly higher than that of rhG-CSF; PG-N-10, PG-N-20 obtained by coupling PEG propionaldehyde to the nitrogen end of rhG-CSF protein, The in vitro activity of PG-N-30 and PG-N-40 has a tendency to decrease with the increase of the molecular weight of coupled PEG, and the decrease in in vitro activity of PG-N-40 is particularly significant, compared with PG-N-30 The in vitro activity decreased by more than 2 times, and an obvious activity "inflection point" appeared, while the in vitro activity of PE-N-10, PE-N-20 and PE-N-30 differed within 30%. The in vitro activity of di-PG-40 coupled with two 20kDa PEGs was significantly lower than that of PG-N-40 with the same molecular weight; while the in vitro activities of PG-165N-30 and PG-174N-30 were not different from PG-N-30 big.

7) Stability of PG-N-30 preparation

In a solution of 10mM sodium acetate buffer (pH 4.5), 0.005% polysorbate 80, 5% sorbitol, the protein concentration was 3mg/ml PE-N-30, kept at 4°C for 24 months, and tested by SEC No protein aggregation or degradation was found in the measurement (as shown in Figure 17-1), while a small amount of protein aggregation appeared in rhG-CSF stored under these conditions for 1 month, which was soluble aggregates (as shown in Figure 17-2); explanation PEGylated rhG-CSF significantly increased protein solubility or stability in water.

8) PE-N-30 in vitro anti-enzymatic stability

Dialyze the test sample with ultrapure water for 24 hours, freeze-dry (freeze dryer, Alphal-2, Matin Christ, USA) after the test sample, dissolve it with 1% ammonium bicarbonate aqueous solution to 1.5 mg/ml, press 1:25 (w/w) Add TPCK treated TRYPSIN (TPCK treated TRYPSIN, Sigma, USA) and incubate with the test samples PG-N-30 and rhg-CSF at a constant temperature of 37°C, respectively, at 15, 30, 60, Samples were taken at 120 and 240 minutes, and the hydrolysis percentage of the original protein was measured by SDS-PAGE. The results are shown in Figure 18. The results showed that after 4 hours of enzymolysis, 90% of PG-N-30 had not been enzymatically hydrolyzed, while rhG-CSF About 50% of the enzyme was digested in 1 hour. It shows that the anti-enzyme stability of PEG30-rhG-CSF is much higher than that of common rhG-CSF.

Embodiment 5 PEG-rhG-CSF pharmacokinetics and pharmacodynamics test

Choose healthy Beagle dogs to carry out pharmacokinetic and pharmacodynamic tests, through a single subcutaneous injection of PG-N-20 and PG-N-30 at the same dose of 200ug/kg, after administration of 0, 1, 3, 6 , 8, 10, 12, 24, 48, 72, 96, 120, 144, 168 and 192 hours later, blood was collected from the cephalic vein of the forelimb, and 6 animals were taken at the same time at each blood collection time point, and serum was prepared according to the conventional method, -20 Preserve at ℃, adopt ELISA method (American R & D company, Jingmei company sub-package) to measure the blood drug concentration; And measure the neutrophil count (ANC) of the whole blood of grid blood collection point, the results are shown in Table 4, Table 5 and Figure 19 , as shown in Figure 20-1.

Table 4. Comparison of main PK parameters after subcutaneous injection of different molecular weight PEG-rhG-CSF (200μg·kg ^-1 ) in Beagle dogs (X±s, n=6)

Table 5. Comparison of main parameters of PD after subcutaneous injection of different molecular weight PEG-rhG-CSF (200μg·kg ^-1 ) in Beagle dogs (X±s, n=6)

The above test results were quite unexpected, and confirmed for the first time that compared with PG-N-20 (that is, Neulasta, which is already on the market), the plasma elimination half-life of PG-N-30 is significantly prolonged, and the plasma drug clearance rate (CL/F) is significantly higher than that of PG-N-20. decrease, blood drug concentration (C _max ) and average area under the drug time curve (AUC _(0-192h) ) all increased significantly; at the same time, this change in pharmacokinetic parameters also brought about corresponding pharmacodynamic responses, That is, the half-life of the elimination phase of the effect (t _1/2(E) ) was significantly prolonged, the peak concentration of the effect (ANCC _max ) and the area of the effect curve on the baseline (AOBEC _(0-192h) ) were significantly increased; this shows that although Neulasta has achieved It has a good curative effect and sales volume, but it is not the "best" rhG-CSF long-acting preparation; obviously, the long-acting effect of PG-N-30 is better than Neulasta.

In the exploration of the changing rule of blood drug concentration and blood ANC, it was found that the two showed a negative correlation trend (as shown in Figure 20-2), indicating that rhG-CSF receptor has obvious clearing effect on blood PG-N-30. This has also been confirmed in the clinical application of Neulasta.

The pharmacodynamics test of embodiment 6 PEG-rhG-CSF

In the model of leukopenia induced by radiation irradiation ( ¹³⁷ Cs-r once, the dose is 3.78Gy, and the irradiation time is 4.7 minutes) BALB/C mice, the same dose of 12, 48 and 120ug/kg of PG-N- 30 and rhG-CSF, for 15 consecutive days, blood was taken on the 5th, 10th and 15th day after administration to measure the neutrophil count (ANC), etc., the results showed (shown in Table 6-1), since the 5th day The ANC of PG-N-30 and rhG-CSF groups was significantly higher than that of the model control group; and the increase of ANC between each dose group had a dose-dependent trend; surprisingly, it was found that the injection of 12ug/kg of PG-N-30 The increase of ANC caused by the injection of 120ug/kg rhG-CSF is equivalent to the result; while the increase of ANC caused by the injection of PG-N-30 is more than 3 times higher than the increase of ANC caused by the injection of the same dose of rhG-CSF. However, if PG-N-30 is administered every other day, every two days, or once in a chemotherapy cycle, as the administration interval prolongs, the dosage must be significantly increased by 2 to 5 times to achieve the same level as continuous chemotherapy. Applying 12μg/kg rhG-CSF for 15 days is equivalent, as shown in Table 6-2, which shows that in the case of the receptor clearance mechanism for PG-N-30 in the body, one chemotherapy cycle should be administered once or longer. A treatment pattern of drug intervals is far from being an economical or optimal dosing pattern.

On the animal model of cyclophosphamide-induced leukopenia in rats (intraperitoneal injection), PG-N-30 and rhG-CSF of the same dose of 10, 30 and 100ug/kg were subcutaneously applied every day for 10 consecutive days. 4, 7 and 10 days, blood test neutrophil count (ANC) etc. are taken, and the results are shown in Table 7. The conclusions obtained are completely consistent with the conclusions on the animal model of mouse leukopenia caused by previous radiation, that is, injection of 10ug The increase of ANC caused by PG-N-30/kg is equivalent to the result of injection of 100ug/kg rhG-CSF; and the increase of ANC caused by injection of PG-N-30 is higher than the increase of ANC caused by injection of the same dose of rhG-CSF more than 3 times higher. This shows that in the treatment mode of continuous daily administration, PG-N-30 has obvious high-efficiency characteristics, and its dosage may even be lower than that of common rhG-CSF. However, as with the model application results of radiation-induced leukopenia in mice, if PG-N-30 is administered with one injection per chemotherapy cycle, 500ug/kg/cycle (chemotherapy cycle) needs to be injected to obtain continuous application rhG-CSF 10g/kg/d curative effect equivalent results (see Table 7).

Table 6-1 The impact of continuous subcutaneous injection of PG-N-30 on the ANC of radiation-induced leukopenia mice X±SD (n=10)

Note: *P<0.01 compared with the value of the model group, ○P<0.01 compared with the value of the positive control group with the same dose

Table 6-2 The impact of continuous subcutaneous injection of PG-N-30 on the ANC of radiation-induced leukopenia mice X±SD (n=10)

Note: Compared with the value of the model group *P<0.01; compared with the value of the 12μg/kg/d positive control group ○P<0.01

Table 7 The impact of subcutaneous injection of PG-N-30 on the ANC of chemotherapy-induced leukopenia rats X±SD (n=10)

Note: *P<0.01 compared with the value of the model group, ○P<0.01 compared with the value of the same dose positive control group, ★P<0.05 compared with the value of the 10μg/kg/d positive control group.

On the animal model of 5-fluorouracil (5-Fu)-induced leukopenia in mice (tail injection of 120mg/kg of 5-Fu), on the 3rd day after chemotherapy drug injection, a subcutaneous injection of PG-N-30600ug/kg ( low dose group), 1200ug/kg (middle dose group) and 2400ug/kg (high dose group), and PG-N-202400ug/kg and rhG-CSF 60ug/kg/day for 9 consecutive days; 0, 3, 6, and 9 days blood sampling to measure ANC etc., the results are shown in Figure 21; On the second day after chemotherapy drug injection, a subcutaneous injection of PG-N-30 300ug/kg (low dose group), 600ug/kg (medium dose group) and 1200ug/kg (high dose group), and PG-N-20 1200ug /kg and rhG-CSF 30ug/kg/day for 10 consecutive days; blood was collected on days 0, 3, 5, 7, and 10 after administration to measure ANC, etc., and the results are shown in Figure 22; the above two tests all showed that: Subcutaneous application of high, medium and low doses of PG-N-30 and high doses of PG-N-20 can effectively correct neutropenia on the 3rd day after administration, while medium doses of PG-N-30 and high doses The dose of PG-N-20 caused the response of granulocytes to be equivalent to the pattern, and the difference was doubled. Therefore, in the above two animal models, it was confirmed that in the treatment pattern of one chemotherapy cycle, PG-N The curative effect of -30 is also significantly better than that of PG-N-20; therefore, the clinical dosage of PG-N-30 is expected to be significantly lower than that of Neulasta.

The immunogenicity test of embodiment 7 PEG-rhG-CSF

On the healthy SD rats, the test compound (15 rats in each group) was injected once a week, each 10ug/kg administration mode, administered continuously, observed for 12 weeks, in the 1st week and the 8th, At 10 and 12 weeks, blood was drawn to measure ANC and anti-PEG-rhG-CSF antibody; PEG-rhG-CSF antibody was determined by indirect ELISA (American R&D Company, PEG-rhG-CSF (antigen) was treated with Na ₂ CO at pH 9.6 _3- NaHCO ₃ solution was diluted to a concentration of 10 μg/ml, coated on a 96-well ELISA plate, 100 μl per well, overnight at 4°C. After blocking with 2% BSA blocking solution at 37°C for 2 hours, add 100 μl of sample diluent to each well For the diluted serum samples to be tested, set positive and negative control wells at the same time, and incubate at 37°C for 1 hour. After washing, add 100 μl horseradish peroxidase-labeled goat anti-rat IgG (1:60,000) to each well, and incubate again at 37°C 1h. After washing, add 100μl enzyme reaction substrate TMB to each well, and react at 37°C for 20min. Stop the reaction with 50μl stop solution, and read the OD value of each well at 450nm on a microplate reader.

Judgment of the positive rate of antibodies: 2.1 times the OD value measured in the serum samples of animals in the excipient control group during the same period is used as the threshold value for antibody production, and those whose OD value measured in the serum samples after administration is greater than or equal to the threshold value are judged as positive . The results are shown in Table 8. The results showed that: PG-N-20, PG-N-30, and PG-N-40 had lower immunogenicity than rhG-CSF, while PG-N-30 and PG-N-40 The immunogenicity is lower than that of PG-N-20.

Table 8 The comparison of the immunogenicity of continuous application of various PEG-rhG-CSF in rats

Note: *significantly different from rhG-CSF at P<0.05; △significantly different from PG-N-20 at P<0.05.

sequence listing

<110> Tianjin Paige Biotechnology Co., Ltd., Institute of Process Engineering, Chinese Academy of Sciences

<120>Column chromatography granulocyte colony-stimulating factor nitrogen-terminal site-specific coupling method and its product

<141>2008-12-1

<160>3

<210>1

<211>175

<212>PRT

<213> Recombinant human granulocyte colony-stimulating factor

<221> amino acid sequence formula of rhG-CSF or rhG-CSFm

<222>(1)...(175)

<400>1

-1 -2 -3 -4 -5 -6 -7 -8 -9 -10

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu

5 10 15

Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu

20 25 30

Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu

35 40 45

Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser

50 55 60

Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His

65 70 75 80

Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile

85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala

100 105 110

Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala

115 120 125

Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala

130 135 140

Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser

145 150 155 160

Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro ***

165 170 175

<210>2

<211>528

<212>DNA

<213> Recombinant human granulocyte colony-stimulating factor

<221> Naturally encoded human granulocyte colony-stimulating factor (rhG-CSF) gene

<222>(1)...(528)

<400>2

atgacccccc tgggccctgc cagctccctg ccccagagct tcctgctcaa gtgcttagag 60

caagtgagga acatccaggg cgatggcgca gcgctccagg agaagctgtg tgccacctac 120

aagctgtgcc accccgagga gctggtgctg ctcggacact ctctgggcat cccctgggct 180

cccctgagct cctgccccag ccaggccctg cagctggcag gctgcttgag ccaactccat 240

agcggccttt tcctctacca ggggctcctg caggccctgg aagggatatc ccccgagttg 300

ggtcccacct tggacacact gcagctggac gtcgccgact ttgccaccac catctggcag 360

cagatggaag aactgggaat ggcccctgcc ctgcagccca cccagggtgc catgccggcc 420

ttcgcctctg ctttccagcg ccgggcagga ggggtcctgg ttgctagcca tctgcagagc 480

ttcctggagg tgtcgtaccg cgttctacgc caccttgcgc agccctga

<210>3

<211>528

<212>DNA

<213> Recombinant human granulocyte colony-stimulating factor

<221> Modified gene encoding human granulocyte colony-stimulating factor (rhG-CSF)

<222>(1)...(528)

<400>3

atgactccat taggtcctgc atcttcttta ccacaaagct tcctgctcaa gtgcttagag 60

caagtgagga agatccaggg cgatggcgca gcgctccagg agaagctgtg tgccacctac 120

aagctgtgcc accccgagga gctggtgctg ctcggacact ctctgggcat cccctgggct 180

cccctgagct cctgccccag ccaggccctg cagctggcag gctgcttgag ccaactccat 240

agcggccttt tcctctacca ggggctcctg caggccctgg aagggatatc ccccgagttg 300

ggtcccacct tggacacact gcagctggac gtcgccgact ttgccaccac catctggcag 360

cagatggaag aactgggaat ggcccctgcc ctgcagccca cccagggtgc catgccggcc 420

ttcgcctctg ctttccagcg ccgggcagga ggggtcctgg ttgctagcca tctgcagagc 480

ttcctggagg tgtcgtaccg cgttctacgc caccttgcgc agccctga

Claims (10)

1. A method for column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol, the steps comprising: Select the cation exchange medium, load it in the chromatographic column, the height of the column bed is the upper limit of the conventional recommended height, and connect it to the liquid chromatography system; 20mM sodium acetate buffer solution and 0.005% polysorbate 80 by mass percentage are fully balanced chromatographic column; use a feed pump to add rhG-CSF or rhG-CSFm stock solution into the chromatographic column at a flow rate of 8-10ml/min, The amount of sample loaded is 10% to 70% of the maximum capacity of the medium measured; then at a flow rate of 13 to 15ml/min, wash the column with 5 to 6 times the column volume of 20mM sodium acetate buffer solution with a pH of 3.5 to 6.0 to remove excess Substances that undergo electrostatic adsorption with the chromatographic medium; according to the reaction volume ratio of mPEG:rhG-CSF or rhG-CSFm at a ratio of 1 to 50mol:1mol, add a sample with a molecular weight range of 26 to 40kDa at a flow rate of 0.5 to 3ml/min Monomethyl PEG, the mPEG input total amount X of described different molecular weights is calculated by formula (1): (1) X＝5～50×m×MW _PEG /MW _G-CSF In the formula: X is the total input amount of mPEG with different molecular weights, m is the protein content of rhG-CSF added, MW _PEG and MW _G-CSF are the molecular weights of mPEG and G-CSF respectively; Then calculate the add-on of reducing agent sodium cyanoborohydride by formula (2): (2) NaBH ₃ CN addition amount = 10×MW reducing agent×X/MW _PEG In the formula: MW reducing agent is the molecular weight of the reducing agent sodium cyanoborohydride, X is the total amount of mPEG input with different molecular weights, and MW _PEG is the molecular weight of mPEG; Load the sample continuously for 240±5 minutes; the pH of the reaction system is 3.5-6.0; then wash the column with 5-6 column volumes of 20mM sodium acetate buffer solution with a pH of 3.5-6.0 at a flow rate of 13-15ml/min to remove excess Substances that undergo electrostatic adsorption with chromatographic media; then use a mixture of 20mM sodium acetate buffer with pH 3.5 to 6.0 and 0.5M to 1M sodium chloride in the elution mode of 0 to 8%, 8% to 18% and 18% ~100% elution, lasting 150±2 minutes, three elution peaks were obtained, namely P1, P2 and P3, among which the peak area of P2 accounted for 90±1% of the total peak area, which was mono-modified PEG-rhG-CSF Or PEG-rhG-CSFm, that is, polyethylene glycol and rhG-CSF or rhG-CSFm protein N-terminal single, site-specific coupling compound; in: The amino acid sequence of the above rhG-CSF or rhG-CSFm should conform to the amino acid sequence formula shown in SEQ ID NO.1, wherein the rhG-CSFm is the N-terminal knockout of 1-10 amino acids and the N-terminal amino acid is still methionine rhG-CSF; The above monomethyl PEG refers to various activated monomethyl PEG molecules that can be coupled with protein amino groups, which are PEG-succinimide α-methyl in mPEG-ALD or mPEG-N-hydroxysuccinimide activated ester butyrate or mPEG-succinimidyl propionate. 2. The method for column chromatography of granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1, characterized in that: the cation exchange medium is MacroCap SP or Sepharose Fast Flow. 3. The method of column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1, characterized in that: the rhG-CSF or rhG-CSFm adopts Escherichia coli in the prokaryotic cell expression system Expression preparation. 4. The method of column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1, characterized in that: the loading amount of the rhG-CSF or rhG-CSFm is the maximum load of the medium actually measured 30-50% of the volume. 5. the method for column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1, is characterized in that: the molecular weight of described monomethyl PEG is selected 30kDa; mPEG molecule is selected single chain; The loading amount of monomethyl PEG is in the ratio of 2-20 mol:1 mol according to the reaction amount ratio of mPEG:rhG-CSE or rhG-CSFm. 6. The method for column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1 or 5, characterized in that: the monomethyl PEG is selected from mPEG-ALD; its molecular formula is mPEG- (CH ₂ )r-CHO, wherein r is selected from 2 to 3, that is, mPEG-propionaldehyde or mPEG-butyraldehyde. 7. The method for column chromatography granulocyte colony-stimulating factor nitrogen-terminal fixed-point coupling polyethylene glycol as claimed in claim 1, characterized in that: the column chromatography rhG-CSF or rhG-CSFm nitrogen-terminal fixed-point coupling single The pH of the reaction system of methyl PEG is 5.0-6.0. 8. A class of PEGylated recombinant human granulocyte colony-stimulating factor prepared by column chromatography granulocyte colony-stimulating factor nitrogen terminal fixed-point coupling polyethylene glycol method according to claim 1, characterized in that: the polyethylene glycol The molecular structural formula of the glycolated recombinant human granulocyte colony-stimulating factor is: CH ₃ -(CH ₂ CH ₂ -O)n-(CH ₂ )r-NH-rhG-CSF or CH ₃ -(CH ₂ -CH ₂ -O)n-(CH ₂ )r-NH-rhG-CSFm , In the formula, n is 570-2200, r is 1-3; at the same time, the pegylated recombinant human granulocyte colony-stimulating factor should also have the following characteristics: 1) The molecular ratio of rhG-CSF or rhG-CSFm to mPEG coupling is 1:1, and the coupling site is the N-terminal amino group of rhG-CSF or rhG-CSFm molecule; 2) In the in vitro anti-enzyme test: incubate trypsin with PEG-rhG-CSF or PEG-rhG-CSFm at 37°C for 4 hours, take samples and determine by SDS-PAGE method, PEG-rhG-CSF or PEG-rhG - The percentage of enzymatic hydrolysis of CSFm should be less than 50%; 3) In the determination of specific activity, the Lowry method is used to measure the protein concentration of PEG-rhG-CSF or PEG rhG-CSFm to be tested, the NFS60 cell/MTT method is used to measure the in vitro activity, and the activity per mg of protein is calculated to obtain the specific activity, PEG - The specific activity of rhG-CSF or PEG-rhG-CSFm should be greater than or equal to 2.0X10E7U/mg protein; 4) The plasma half-life of PEG-rhG-CSF or PEG-rhG-CSFm should be more than 50% longer than the plasma elimination half-life of Pegfilgrastim, namely PG-N-20; 5) In the assay of immunogenicity, the positive rate of PEG-rhG-CSF or PEG-rhG-CSFm at week 12 should be less than 20%. 9. The pegylated recombinant human granulocyte colony-stimulating factor as claimed in claim 8, characterized in that: n in the molecular structural formula of the pegylated recombinant human granulocyte colony-stimulating factor is 700 ± 100; r is 2~3. 10. The PEGylated recombinant human granulocyte colony-stimulating factor according to claim 8 is used for preventing and treating granulocytopenia, bone marrow and peripheral blood stem cell transplantation and primary chronic granulocytopenia caused by tumor or leukemia chemotherapy. Application in parenteral preparations of drugs.

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