CN103275210B - Chromatographic purification method of fatty acid mono-acylation insulin - Google Patents
Chromatographic purification method of fatty acid mono-acylation insulin Download PDFInfo
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Abstract
The invention discloses a chromatographic purification method of fatty acid mono-acylation insulin. The chromatographic purification method comprises the steps of separating the fatty acid mono-acylation insulin by employing a medium- and low-pressure chromatographic system, and using an organic polymer reversion phase chromatographic material as a stationary phase, and taking at least one organic solvent mixable with water and at least one buffering agent substance as an elution moving phase with the pH (Potential of Hydrogen) value of 2-4. The chromatographic purification method has simple steps, is easy to amplify, and low in economic cost, and can increase the purity of a sample from original 50-60% to 98% by one step.
Description
Technical field
The present invention relates to a kind of chromatographic purification method, particularly a kind of chromatographic purification method of lipid acid list acylated insulin.
Background technology
Aminoacylates is common protein modified method, and the universal method of acidylate is common in " Enzymology method " published by Elsevier of C.H.W.Hirs chief editor, 25:494-499(1972).
Namely the insulin detemir that Novo Nordisk Co., Ltd of current Denmark produces is the common insulin derivates utilizing this principle to obtain after the epsilon-amino acidylate of B29 position Lys residue is modified.And in the aminoacylation of Regular Insulin; generally use active ester, carboxylic acid halides or acid anhydrides in the industry at present; react with proinsulin or analogue and prepare single acylated insulin; but due in proinsulin or analogue containing the free epsilon-amino (epsilon-amino of such as Lys) be positioned on the amino acid whose free alpha-amino group of A1, B1 position and other amino acid side chains; these three kinds of free amine groups all can form covalent bonds with active ester, carboxylic acid halides or acid anhydrides, thus define the monosubstituted Regular Insulin of above common different loci modification or two replacement insulin protein product.Therefore, how selective modification Regular Insulin and subsequent purification thereof just important problem can be become preferably.At biology magazine Biochem.J121:737-745(1971) Lindsay etc. provides the experimental data of Regular Insulin and the reaction of acetic acid N-succinimide ester in The acylation of insulin mono-literary composition published, mainly generates Phe
b1the monosubstituted Regular Insulin of-ethanoyl, Gly
a1the monosubstituted Regular Insulin of-ethanoyl, Lys
b29-monosubstituted ethanoyl Regular Insulin and a small amount of two replacement Regular Insulin, it adopts DEAE ion-exchange packing to study purifying research of product afterwards.Find use the method to have certain purification effect but be also not so good as people's will, although because the display of purity result can reach more than 90% on its document, but due on purity result decision method, Lindsay the electric gel electrophoresis method such as to be only the use of as detection method, and the method is compared with HPLC method conventional in current industry, sensitivity is low.At protein & peptide letters, Synthesis 13:135-142(2006) published, in Characterization and Biological Activity of Chemically Modified Insulin DerivativeWith Alpha Lipoic Acid mono-literary composition, Tao Huang etc. adopt lipoylbenzotriazole and insulin human to react in the basic conditions, generate with ε-Lys
b29-lipoyl-insulin is main single acidylate modified outcome.Afterwards, the RP-HPLC that it has mainly used with C18 reverse phase silica gel is stationary phase carries out purifying, obtain purity higher than 95% single acylated insulin.Chinese patent CN1171742 provides the method for the epsilon-amino of a kind of selective reaction acidylate modified human proinsulin and analogue thereof; in the disclosure in this patent; which employs with C4 reverse phase silica gel be stationary phase RP-HPLC or semipreparative column; moving phase is the method for carrying out gradient elution containing the solvent systems of 0.1%TFA, acetonitrile and water, and when 53% acetonitrile concentration, obtain single acylated insulin (purity data does not disclose).
As can be seen here, the Regular Insulin after modifying for acidylate is at present more adopt RP-HPLC or high pressure to prepare liquid phase to carry out liquid phase separation purifying on reverse phase silica gel solid substrate.Term RP-HPLC refers to the method adopting anti-phase stationary phase separation and purification on high performance liquid phase instrument, and high pressure is prepared liquid phase and referred under high pressure the liquid chromatography that (the general pressure that uses is at 5-15Mpa) runs.Reverse phase silica gel is then interpreted as hydrophobic matrix and has applied superincumbent silica material, wherein the alkane of hydrophobic matrix example to be chain length be C3-C20, particularly C4-C18.
Although employing is prepared in liquid phase at HPLC or high pressure can reach higher separation purity with reverse phase silica gel material as stationary phase, the method has some obvious shortcomings.Such as reverse phase silica gel is only stable within the scope of pH2-10; and easily produce a large amount of hydrophobicity very strong impurity during fatty-acylation modification Regular Insulin; the dimer of such as free fatty acids or fatty acid modifying or polymer; it is very firm that they easily adsorb on silica gel; and neither easily by the regenerative elution of routine; along with the prolongation of time, impurity is easily concentrated on filler.Alkali washing method can only be adopted to clean, but alkali lye easily destroy the chemical property of reverse phase silica gel and then affect the separating effect of filler.Meanwhile, if adopt RP-HPLC or high pressure half preparation liquid phase instrument as separation and purification instrument, although it has accurately feature efficiently, its cost high maintenance bothers, and especially cost at least exceeds 50% than mesolow chromatographic system.
Summary of the invention
For overcoming above-mentioned shortcoming and defect, the object of the present invention is to provide with a kind of chromatographic purification method of lipid acid list acylated insulin.This purification process uses a kind of than the more convenient use of reverse phase silica gel, cheap chromatographic material, and chromatogram purification acidylate modifies Regular Insulin thereon.Use this method can reach use one step chromatogram purification and can obtain highly purified acidylate modification Regular Insulin.
Object of the present invention is achieved through the following technical solutions: a kind of chromatographic purification method of lipid acid list acylated insulin, comprise following steps: utilize mesolow chromatographic system to be separated lipid acid list acylated insulin, use organic polymer reversion phase chromatographic material to be stationary phase, the moving phase of wash-out is can the organic solvent miscible with water and at least one buffer material, pH value be 2 ~ 4 containing at least one;
Described lipid acid list acylated insulin refers to the naturally occurring longer chain fatty acid or its analogue that are connected to side chain at the alpha-amino group place of parent insu B chain-terminal amino acid residue or the epsilon-amino place of Lys residue that exists on B chain, and this side chain has following general general formula:
-X-Y；
Wherein X be and parent insu B chain-terminal amino acid residue alpha-amino group or and the epsilon-amino of parent insu B chain Lys residue between covalent linkage;
X is :-CO-;
Y is :-(CH
2) m-, wherein m is the integer of 6 ~ 32;
And wherein parent insu is selected from insulin human, des(B1) insulin human, des(B30) insulin human, Gly
a21insulin human, Gly
a21des(B30) insulin human, Asp
b28insulin human, pork insulin, Lys
b28pro
b29insulin human, Gly
a21arg
b31arg
b32insulin human or Lys
b3glu
b29insulin human.
Described lipid acid list acylated insulin is preferably (N
ε B29-myristoyl) Lys
b29des(B30) insulin human, (N
ε B29-myristoyl) Lys
b29insulin human or (N
ε B29-hexadecanoyl) Lys
b29des(B30) insulin human; Be more preferably (N
ε B29-myristoyl) Lys
b29des(B30) insulin human;
Described mesolow chromatographic system refer to operating pressure be 2 ~ 25bar(bar) chromatographic system; Being preferably operating pressure is the chromatographic system of 2 ~ 20bar, is more preferably the chromatographic system that operating pressure is 5 ~ 10bar, most preferably is the chromatographic system that operating pressure is 5 ~ 8bar; Chromatographic system comprises analytical chromatographic, half preparing chromatography system, preparing chromatography system;
Described organic polymer reversion phase chromatographic material is the material for reversed phase chromatography separation purifying that polymethacrylate (PMA) or polystyrene (PS)-Vinylstyrene (DVB) are matrix, is preferably polystyrene (PS)-Vinylstyrene (DVB) material; Its mean particle size preferably 5 ~ 300 μm, is more preferably 10 ~ 50 μm; Granularity is less, and resolution is better, but the more short grained pressure stability of granularity is lower;
Described organic polymer reversion phase chromatographic material can select commercially available organic polymer reversion phase chromatographic material as shown in table 1:
Table 1
| Manufacturers | Trade(brand)name | Material | Minimum particle size | Aperture |
| Mitsubishi Chemical | MCI GEL CHP | PS/DVB、PMA | 4μm | 10nm |
| GE | Source15、30 | PS/DVB | 5μm | 10nm |
| AMBERCHROM | CG161、CG300、CG1000 | PS/DVB | 35μm | 15nm |
| Receive micro- | UniPS | PS/DVB | 3μm | 10nm |
Described organic polymer reversion phase chromatographic material is more preferably receives micro-Uni PS30-300 filler or GEsource30 filler;
Described wash-out can isocratic elution, and namely wherein have constant buffer substance concentration and constant organic solvent ratio, or can carry out by linear gradient elution, namely organic solvent ratio increases progressively in time, preferred isocratic elution;
Described organic solvent that can be miscible with water is preferably the alcohol that acetonitrile, ketone or carbonatoms are C1 ~ C4; Be preferably acetonitrile, Virahol or ethanol;
The described volumetric concentration of organic solvent in moving phase that can be miscible with water is 1 ~ 90%, is preferably 30 ~ 60%;
Described buffer material can be selected from phosphoric acid salt, acetate or Citrate trianion, is preferably SODIUM PHOSPHATE, MONOBASIC or sodium-acetate;
Described pH value can regulate by adding acetic acid, phosphoric acid, hydrochloric acid or sodium hydroxide;
Described pH value is preferably 3.0 ~ 3.5.
The present invention relative to the advantage of prior art and effect is:
1, operation steps is succinct: purification process of the present invention uses a kind of than the more convenient use of reverse phase silica gel, cheap organic polymer reversion phase chromatographic material, and chromatogram purification acidylate modifies Regular Insulin thereon.Use this method can reach use one step chromatogram purification can obtain high purity (purity reaches 98%-98.5%) acidylate modify Regular Insulin.
2, the purity requirement of purifying initial sample is low: the initial purity of sample is 50 ~ 60% can apply purification process of the present invention and carry out purifying, and the purity of the sample after purifying can reach 98%.
3, mesolow chromatographic system is adopted: adopt another benefit of the present invention to be that it can run in mesolow chromatographic system, and reasonable separation and purification effect can be obtained.
4, method of the present invention is applicable to various chromatographic system, analyzes chromatogram, half preparative chromatography, preparative chromatography, especially mesolow chromatogram.Use the method to carry out separation and purification, good cost advantage can be had.
In a word, purification process step provided by the invention is simple, a step sample purity can be brought up to 98% from 50 ~ 60%, and is easy to amplify, and Financial cost is low.
Accompanying drawing explanation
Fig. 1 is the RP-HPLC figure of the component 1 obtained when pH value is 4.0 in embodiment 1.
Fig. 2 is the RP-HPLC figure of the component 2 obtained when pH value is 4.0 in embodiment 1.
Fig. 3 is the RP-HPLC figure of the component 3 obtained when pH value is 4.0 in embodiment 1.
Fig. 4 is the RP-HPLC figure of the component 4 obtained when pH value is 4.0 in embodiment 1.
Fig. 5 is the RP-HPLC figure of the component 1 obtained when pH value is 4.3 in embodiment 1.
Fig. 6 is the RP-HPLC figure of the component 2 obtained when pH value is 4.3 in embodiment 1.
Fig. 7 is the RP-HPLC figure of the component 3 obtained when pH value is 4.3 in embodiment 1.
Fig. 8 is the RP-HPLC figure of the component 4 obtained when pH value is 4.3 in embodiment 1.
Fig. 9 is the RP-HPLC figure of the component 1 obtained when pH value is 7.0 in embodiment 1.
Figure 10 is the RP-HPLC figure of the component 2 obtained when pH value is 7.0 in embodiment 1.
Figure 11 is the RP-HPLC figure of the component 3 obtained when pH value is 7.0 in embodiment 1.
Figure 12 is the RP-HPLC figure of the component 4 obtained when pH value is 7.0 in embodiment 1.
Figure 13 is the RP-HPLC figure of the highest component of purity obtained when operating pressure is 2bar in embodiment 2.
Figure 14 is the RP-HPLC figure of the qualified component mixing obtained when operating pressure is 5bar in embodiment 2.
Figure 15 is the RP-HPLC figure of the qualified component mixing obtained when operating pressure is 8bar in embodiment 2.
Figure 16 is the RP-HPLC figure of the sample to be purified in embodiment 3.
Figure 17 is the RP-HPLC figure of the component 1 in embodiment 3.
Figure 18 is the RP-HPLC figure of the component 2 in embodiment 3.
Figure 19 is the RP-HPLC figure of the component 3 in embodiment 3.
Figure 20 is the RP-HPLC figure of the component 4 in embodiment 3.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1:
(1) preparation of sample to be purified:
With reference to the embodiment 7 disclosed in patent WO98/02460,0.50g Des B30 proinsulin human is dissolved in 7.0ml dimethyl sulfoxide (DMSO) (DMSO) with the mixing solutions of 3.5ml water, and regulate pH to be 10.2-10.5 toward wherein adding 0.4ml triethylamine, obtain mixing solutions.Afterwards 0.025g N-succinimido myristic acid is dissolved in 1.8ml N-Methyl pyrrolidone solution; and be added dropwise in aforementioned mixing solutions; stir reaction in 2 hours under finally the solution mixed being placed in 0 DEG C of environment, the insulin derivates modified containing acidylate can be obtained, i.e. (N
ε B29-myristoyl) Lys
b29des(B30) reaction solution of insulin human.Sample to be purified is obtained afterwards with its acidifying being diluted 10 times containing vinegar aqueous acid (concentration of acetic acid is volume percent 10%).The purity of this sample to be purified as shown in figure 16.
(2) QuickSep medium pressure chromatography system purifying is used:
The chromatography column of diameter 16mm and long 250mm is tested, in chromatography column load filler be 30ml receive micro-Uni PS30-300 filler (particle diameter 30 μm, aperture 300 dust), bed height is 150mm.Loading target protein amount is 100mg.
Chromatography buffer is: A phase, the 0.1M phosphate sodium dihydrogen buffer solution containing 50mM anhydrous sodium sulphate, regulates phosphoric acid or Sodium phosphate dibasic ratio in damping fluid, makes it pH value and be respectively 4.0,4.3 and 7.0; B phase, aqueous isopropanol.Pillar is first with 30%B(and B phase and the 3:7 mixing by volume of A phase) balance each other, regulate loading sample elution to adopt 38%B equality wash-out, elution flow rate 70ml/h, operating pressure 5bar, 280nmUV detector monitors out Fractional Collections behind peak, every section of 10ml.
(3) method for detecting purity:
Adopt RP-HPLC method detecting step (2) to collect component, instrument is Shimadzu LC-2010CHT, and pillar adopts Sepax GP-C4 reversed-phase column.With ammoniumsulphate soln (ammonium sulfate 40g, the 1900ml that adds water dissolve after, with 1mol/L sulfuric acid adjust pH to 2.3, add water to 2000ml): acetonitrile by volume the mixing solutions that obtains of 1800:200 as mobile phase A; With acetonitrile-water (800:225 mixing by volume), for Mobile phase B, flow velocity is 1.0ml/min; Column temperature is 50 DEG C, determined wavelength 214nm.30 ~ 68%B phase gradient wash-out 75min.Retention time 30 ~ 31min unimodal namely for the purpose of product.Purity calculates and adopts area normalization method.
Fig. 1 ~ 12 are the RP-HPLC collection of illustrative plates of each component.
List in table 2 and use polymer materials to receive the purity of micro-Uni PS30-300 each collection component of Virahol wash-out under different pH, can find out, the collection component that purity is greater than 98.0% can be obtained when pH4.0, then can not obtain at pH4.3 and pH7.0 the component that purity is greater than 98.0%.
Table 2
Embodiment 2:
(1) preparation of sample to be purified: with step (1) in embodiment 1.
(2) QuickSep medium pressure chromatography system purifying is used:
That in the chromatography column of diameter 11mm and long 250mm, loads 10ml receives micro-Uni PS30-300 filler (particle diameter 30 μm, aperture 300 dust), bed height 110mm.
Moving phase configuration proportion is with embodiment 1, and the pH regulating mobile phase A is 3.0.Regulate loading sample size, make filler target protein lifting capacity in pillar be 4.0g/L.Regulating course analysis system operating pressure, makes it to be respectively 2bar, 5bar and 8bar.Type of elution is with embodiment 1.280nm UV detector monitors out Fractional Collections behind peak, every section of 10ml, then after component mixing purity being greater than 98.0%, then detects purity and concentration, calculate the rate of recovery.
(3) method for detecting purity: with step (3) in embodiment 1.
Figure 13 is the RP-HPLC collection of illustrative plates of the highest purity using Uni PS30-300 polymer materials wash-out under 2bar pressure condition.
Figure 14 ~ 15 are for using Uni PS30-300 polymer materials mixed RP-HPLC collection of illustrative plates of the qualified component of wash-out under 5bar and 8bar pressure condition.
List experimental result in table 3, be analyzed as follows: under different chromatographic system operating pressure conditions, the purity of more than 98.0% can be reached under 5bar and 8bar pressure condition, and productive rate can reach more than 70%.And under the operating pressure of 2bar, the highest purity of component can only reach 96.24%, therefore its yield is 0.
Table 3
Embodiment 3:
(1) preparation of sample to be purified: with step (1) in embodiment 1.
(2) QuickSep medium pressure chromatography system purifying is used:
The GE source30 filler (particle diameter 30 μm) of 2.5L is loaded, the long 165mm of bed in the chromatography column of diameter 140mm and long 500mm.
Chromatography buffer is: A phase, 0.15M sodium-acetate buffer, pH3.0; B phase, containing TFA(trifluoroacetic acid) acetonitrile solution, the concentration of TFA is volume percent 0.1%.
Pillar first balances each other with 10%B, i.e. A phase and B phase 9:1 mixing by volume, after balance, loading makes filler target protein lifting capacity be 4.0g/L, 40%B equality wash-out, elution speed 5.7L/h, operating pressure is that 8bar, 280nmUV detector monitors out Fractional Collections behind peak, every section of 1.0L volume.
(3) method for detecting purity: with step (3) in embodiment 1.
Figure 16 is the RP-HPLC collection of illustrative plates of the sample to be purified before loading, and its purity area is 58.31%.
Figure 17 ~ 20 are the RP-HPLC collection of illustrative plates of each collection component.
The result listing each collection component in table 4 can be clear that, use the method also can have good separation and purification effect at the GE source30 polymer packing of 2.5L scale, and purity can bring up to 98% from 58.31% of sample to be purified, the component being greater than 98% with purity is qualified, then yield reaches 71.90%.
Table 4
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the chromatographic purification method of a lipid acid list acylated insulin, it is characterized in that comprising following steps: utilize mesolow chromatographic system to be separated lipid acid list acylated insulin, use organic polymer reversion phase chromatographic material to be stationary phase, the moving phase of wash-out is can the organic solvent miscible with water and at least one buffer material, pH value be 2 ~ 4 containing at least one;
Described lipid acid list acylated insulin is Methionin B29 (N
ε-mnyristoyl) go (B30) insulin human;
Described organic polymer reversion phase chromatographic material is polystyrene-divinylbenzene;
Described can the alcohol of the organic solvent miscible with water to be acetonitrile, ketone or carbonatoms be C1 ~ C4;
Described buffer material is phosphoric acid salt, acetate or Citrate trianion.
2. the chromatographic purification method of lipid acid list acylated insulin according to claim 1, is characterized in that: the chromatographic system of described mesolow chromatographic system to be operating pressure be 2 ~ 25 bar.
3. the chromatographic purification method of lipid acid list acylated insulin according to claim 1, is characterized in that: the mean particle size of described organic polymer reversion phase chromatographic material is 5 ~ 300 μm.
4. the chromatographic purification method of lipid acid list acylated insulin according to claim 3, is characterized in that: described organic polymer reversion phase chromatographic material is one in MCI GEL CHP, Source15, Source30, CG161, CG300, CG1000 and Uni PS or at least two kinds.
5. the chromatographic purification method of lipid acid list acylated insulin according to claim 1, is characterized in that: described pH value is 3.0 ~ 3.5.
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| CN109748948B (en) * | 2018-11-27 | 2020-04-21 | 珠海冀百康生物科技有限公司 | Purification method of palmitoyl tetrapeptide-7 |
| CN111518193B (en) * | 2020-06-11 | 2023-09-26 | 南京赛诺生物制药有限公司 | Purification method of fatty acid acylated insulin and GLP-1 polypeptide |
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| CN1313866A (en) * | 1998-08-24 | 2001-09-19 | 阿文蒂斯药物德国有限公司 | Method for chromatographically purifying insulins |
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| Title |
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| 邵承伟.高聚物型液相色谱柱在蛋白分离中的应用研究.《分析试验室》.2008,第27卷(第12期),摘要,第15页左栏第1-2段. * |
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