CN103275210A - 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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- CN103275210A CN103275210A CN2013102564216A CN201310256421A CN103275210A CN 103275210 A CN103275210 A CN 103275210A CN 2013102564216 A CN2013102564216 A CN 2013102564216A CN 201310256421 A CN201310256421 A CN 201310256421A CN 103275210 A CN103275210 A CN 103275210A
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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 chromatogram purification method, particularly a kind of chromatogram purification method of lipid acid list acylated insulin.
Background technology
Amino acidylate is common protein modified method, and the universal method of acidylate is common in C.H.W.Hirs chief editor's " Enzymology method " by the Elsevier publication, 25:494-499(1972).
The insulin detemir that present Denmark Novo Nordisk Co.,Ltd produces namely is the common insulin derivates that utilizes this principle to obtain after the epsilon-amino acidylate of B29 position Lys residue is modified.And in the amino acylation reaction of Regular Insulin; generally use active ester, carboxylic acid halides or acid anhydrides at present in the industry; with proinsulin or the single acylated insulin of analogue prepared in reaction; but owing to contain amino acid whose free alpha-amino group and the free epsilon-amino on other amino acid side chains (such as the epsilon-amino of Lys) that is positioned at A1, B1 position in proinsulin or the analogue; these three kinds of free amine groups all can form covalent bonds with active ester, carboxylic acid halides or acid anhydrides, thereby have formed single Regular Insulin or two replacement Regular Insulin protein product of replacing that above common different loci is modified.Therefore, how preferably selective modification Regular Insulin and subsequent purification thereof just become an important difficult problem.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 one literary composition of publication, has mainly generated Phe
B1-ethanoyl list replaces Regular Insulin, Gly
A1-ethanoyl list replaces Regular Insulin, Lys
B29-single ethanoyl Regular Insulin and a small amount of two Regular Insulin that replaces of replacing, it adopts the purifying research of DEAE ion-exchange packing research product afterwards.Discovery is used this method to have certain purification effect but also is not so good as people's will, because although purity result demonstration can reach more than 90% on its document, but because in purity as a result on the decision method, Lindsay such as only has been to use as detection method at electric gel electrophoresis method, and HPLC method commonly used is compared in this method and the present industry, and sensitivity is low.At protein﹠amp; Peptide letters, the Synthesis that 13:135-142(2006) publishes, in Characterization and Biological Activity of Chemically Modified Insulin DerivativeWith Alpha Lipoic Acid one literary composition, employing sulphur decoyl benzotriazoles such as Tao Huang and insulin human react under alkaline condition, have generated with ε-Lys
B29-lipoyl-insulin is main single acidylate modified outcome.Afterwards, it has mainly used with the C18 reverse phase silica gel is that the RP-HPLC of stationary phase carries out purifying, has obtained purity and has been higher than single acylated insulin of 95%.Chinese patent CN1171742 provides a kind of selective reaction acidylate to modify the method for the epsilon-amino of proinsulin human and analogue thereof; in this patent application; it has adopted with the C4 reverse phase silica gel is RP-HPLC or the semipreparative column of stationary phase; moving phase is that the solvent systems that contains 0.1%TFA, acetonitrile and water carries out the method for gradient elution, and obtains single acylated insulin (purity data does not disclose) when 53% acetonitrile concentration.
This shows that the Regular Insulin after modifying for acidylate at present more is to adopt RP-HPLC or high pressure to prepare liquid phase to carry out the liquid phase separation purifying at the reverse phase silica gel solid substrate.Term RP-HPLC refers to adopt at the high performance liquid phase instrument method of anti-phase stationary phase separation and purification, and high pressure prepares the liquid chromatography that liquid phase refers under high pressure (general working pressure is at 5-15Mpa) operation.Reverse phase silica gel then is interpreted as hydrophobic matrix and has applied superincumbent silica material, and wherein hydrophobic matrix example is that chain length is the alkane of C3-C20, particularly C4-C18.
Though adopt to prepare liquid phase at HPLC or high pressure and can reach higher separation purity with the reverse phase silica gel material as stationary phase, this method has some significant disadvantages.Only stable in the pH2-10 scope such as reverse phase silica gel; and the lipid acid acidylate is easy to generate the very strong impurity of a large amount of hydrophobicitys when modifying Regular Insulin; the for example dimer of free fatty acids or fatty acid modifying or polymer; it is very firm that they adsorb on silica gel easily; and it is neither easily by the regenerative elution of routine; along with the prolongation of time, impurity concentrates at filler easily.Can only adopt the alkali cleaning method to clean, but alkali lye destroy the chemical property of reverse phase silica gel easily and then influence the separating effect of filler.Simultaneously, if adopt RP-HPLC or high pressure partly to prepare the liquid phase instrument as the separation and purification instrument, though it has accurate characteristics of high efficiency, its cost high maintenance trouble, especially cost exceeds 50% than mesolow chromatographic system at least.
Summary of the invention
For overcoming above-mentioned shortcoming and defect, the object of the present invention is to provide the chromatogram purification method with a kind of lipid acid list acylated insulin.This purification process is to use a kind of chromatographic material than the more convenient use of reverse phase silica gel, cheapness, and the chromatogram purification acidylate is modified Regular Insulin thereon.Use this method to reach and use a step chromatogram purification can obtain highly purified acidylate modification Regular Insulin.
Purpose of the present invention is achieved through the following technical solutions: a kind of chromatogram purification method of lipid acid list acylated insulin, comprise following steps: utilize the mesolow chromatographic system that lipid acid list acylated insulin is separated, use organic polymer reverse-phase chromatography material to be stationary phase, the moving phase of wash-out is that containing at least a can be 2～4 with miscible organic solvent and at least a buffer material, the pH value of water;
Described lipid acid list acylated insulin refers at the alpha-amino group place of parent insulin B chain-terminal amino acid residue or the epsilon-amino place of the Lys residue that exists at the B chain has connected naturally occurring longer chain fatty acid or its analogue of side chain, and this side chain has following general general formula:
-X-Y；
Wherein X be and the alpha-amino group of parent insulin B chain-terminal amino acid residue or and the epsilon-amino of parent insulin B chain Lys residue between covalent linkage;
X is :-CO-;
Y is :-(CH
2) m-, wherein m is 6～32 integer;
And wherein parent Regular Insulin 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
B29The insulin 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; (N more preferably
ε B29-myristoyl) Lys
B29Des(B30) insulin human;
It is 2～25bar(bar that described mesolow chromatographic system refers to operating pressure) chromatographic system; Be preferably the chromatographic system that operating pressure is 2～20bar, more preferably operating pressure is the chromatographic system of 5～10bar, most preferably is the chromatographic system that operating pressure is 5～8bar; Chromatographic system comprises analytical chromatographic, half preparative chromatography system, preparative chromatography system;
Described organic polymer reverse-phase chromatography material is that polymethacrylate (PMA) or polystyrene (PS)-Vinylstyrene (DVB) are the material that is used for the reversed phase chromatography separation purifying of matrix, is preferably polystyrene (PS)-Vinylstyrene (DVB) material; Preferred 5～300 μ m of its mean particle size, more preferably 10～50 μ m; Granularity is more little, and resolution is more good, but the more short grained pressure stability of granularity is lower;
Described organic polymer reverse-phase chromatography material can be selected commercially available organic polymer reverse-phase chromatography material as shown in table 1:
Table 1
| Manufacturers | Trade(brand)name | Material | Minimum particle size | The 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 little | UniPS | PS/DVB | 3μm | 10nm |
Described organic polymer reverse-phase chromatography material is more preferably received little Uni PS30-300 filler or GEsource30 filler;
Described wash-out can isocratic elution, just wherein has constant buffer substance concentration and constant organic solvent ratio, perhaps can carry out by linear gradient elution, and namely the organic solvent ratio increases progressively in time, preferred isocratic elution;
Describedly can be preferably the alcohol that acetonitrile, ketone or carbonatoms are C1～C4 with the miscible organic solvent of water; Be preferably acetonitrile, Virahol or ethanol;
Described can be 1～90% with the miscible volumetric concentration of organic solvent in moving phase of water, 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 be regulated by adding acetic acid, phosphoric acid, hydrochloric acid or sodium hydroxide;
Described pH value is preferably 3.0～3.5.
The present invention with respect to advantage and the effect of prior art is:
1, operation steps is succinct: purification process of the present invention is to use a kind of organic polymer reverse-phase chromatography material than the more convenient use of reverse phase silica gel, cheapness, and the chromatogram purification acidylate is modified Regular Insulin thereon.Use this method to reach and use a step chromatogram purification can obtain the acidylate modification Regular Insulin of high purity (purity reaches 98%-98.5%).
2, the purity requirement of purifying initial sample is low: the initial purity of sample is 50～60% can use purification process of the present invention and carry out purifying, and the purity of the sample behind the purifying can reach 98%.
3, adopt the mesolow chromatographic system: adopting another benefit of the present invention is that it can move in the mesolow chromatographic system, and can access reasonable separation and purification effect.
4, method of the present invention is applicable to various chromatographic systems, analyzes chromatogram, half preparative chromatography, preparative chromatography, especially mesolow chromatogram.Use this method to carry out separation and purification, cost advantage preferably can be arranged.
In a word, purification process step provided by the invention is simple, can one the step sample purity is brought up to 98% from 50～60%, and be easy to amplify, Financial cost is low.
Description of drawings
Fig. 1 is that the pH value is the RP-HPLC figure of the component 1 that obtained in 4.0 o'clock among the embodiment 1.
Fig. 2 is that the pH value is the RP-HPLC figure of the component 2 that obtained in 4.0 o'clock among the embodiment 1.
Fig. 3 is that the pH value is the RP-HPLC figure of the component 3 that obtained in 4.0 o'clock among the embodiment 1.
Fig. 4 is that the pH value is the RP-HPLC figure of the component 4 that obtained in 4.0 o'clock among the embodiment 1.
Fig. 5 is that the pH value is the RP-HPLC figure of the component 1 that obtained in 4.3 o'clock among the embodiment 1.
Fig. 6 is that the pH value is the RP-HPLC figure of the component 2 that obtained in 4.3 o'clock among the embodiment 1.
Fig. 7 is that the pH value is the RP-HPLC figure of the component 3 that obtained in 4.3 o'clock among the embodiment 1.
Fig. 8 is that the pH value is the RP-HPLC figure of the component 4 that obtained in 4.3 o'clock among the embodiment 1.
Fig. 9 is that the pH value is the RP-HPLC figure of the component 1 that obtained in 7.0 o'clock among the embodiment 1.
Figure 10 is that the pH value is the RP-HPLC figure of the component 2 that obtained in 7.0 o'clock among the embodiment 1.
Figure 11 is that the pH value is the RP-HPLC figure of the component 3 that obtained in 7.0 o'clock among the embodiment 1.
Figure 12 is that the pH value is the RP-HPLC figure of the component 4 that obtained in 7.0 o'clock among the embodiment 1.
Figure 13 is the RP-HPLC figure of high component of the purity that obtains when operating pressure is for 2bar among the embodiment 2.
Figure 14 is the RP-HPLC figure that the qualified component that obtains when operating pressure is for 5bar among the embodiment 2 is mixed.
Figure 15 is the RP-HPLC figure that the qualified component that obtains when operating pressure is for 8bar among the embodiment 2 is mixed.
Figure 16 is the RP-HPLC figure of the sample to be purified among the embodiment 3.
Figure 17 is the RP-HPLC figure of the component 1 among the embodiment 3.
Figure 18 is the RP-HPLC figure of the component 2 among the embodiment 3.
Figure 19 is the RP-HPLC figure of the component 3 among the embodiment 3.
Figure 20 is the RP-HPLC figure of the component 4 among the embodiment 3.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, 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 that discloses among the patent WO98/02460,0.50g Des B30 proinsulin human is dissolved in the mixing solutions of 7.0ml dimethyl sulfoxide (DMSO) (DMSO) and 3.5ml water, and toward wherein adding the 0.4ml triethylamine to regulate pH be 10.2-10.5, obtain mixing solutions.Afterwards 0.025g N-succinimido myristic acid is dissolved in 1.8ml N-Methyl pyrrolidone solution; and it is added dropwise in the aforementioned mixing solutions; at last the solution that mixes is placed and stir reaction in 2 hours under 0 ℃ of environment, can obtain containing acidylate modified derivatives of insulin, i.e. (N
ε B29-myristoyl) Lys
B29Des(B30) insulin human's reaction solution.With the aqueous solution (concentration of acetic acid is volume percent 10%) that contains acetic acid its acidifying dilution is obtained sample to be purified for 10 times afterwards.The purity of this sample to be purified as shown in figure 16.
(2) use QuickSep medium pressure chromatography system purifying:
Chromatography column at diameter 16mm and long 250mm is tested, and the filler that loads is the little Uni PS30-300 of receiving of 30ml filler (particle diameter 30 μ m, aperture 300 dusts) in the chromatography column, and bed height is 150mm.Last sample target protein amount is 100mg.
The chromatography damping fluid is: the A phase, contain the 0.1M phosphate sodium dihydrogen buffer solution of 50mM anhydrous sodium sulphate, and regulate phosphoric acid or Sodium phosphate dibasic ratio in the damping fluid, make it the pH value and be respectively 4.0,4.3 and 7.0; The B phase, aqueous isopropanol.Pillar is B phase and A 3:7 mixing by volume mutually with 30%B(earlier) balance each other, all product wash-outs adopt 38%B equality wash-out in the adjusting, elution flow rate 70ml/h, operating pressure 5bar, the 280nmUV detector is monitored out Fractional Collections behind the peak, every section 10ml.
(3) method for detecting purity:
Adopt the RP-HPLC method to detect step (2) and collect component, instrument is Tianjin, island LC-2010CHT, and pillar adopts Sepax GP-C4 reversed-phase column.With ammoniumsulphate soln (ammonium sulfate 40g, add water 1900ml dissolving 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; Be Mobile phase B with acetonitrile-water (800:225 mix) by volume, flow velocity is 1.0ml/min; Column temperature is 50 ℃, detects wavelength 214nm.30～68%B phase gradient wash-out 75min.Retention time is at the unimodal purpose product that is of 30～31min.Purity is calculated and is adopted area normalization method.
Fig. 1～12 are the RP-HPLC collection of illustrative plates of each component.
Listed the purity of respectively collecting component of using polymer materials to receive little Uni PS30-300 Virahol wash-out under different pH in the table 2, as can be seen, when pH4.0, can obtain purity greater than 98.0% collection component, then can not obtain purity greater than 98.0% component at pH4.3 and pH7.0.
Table 2
Embodiment 2:
(1) preparation of sample to be purified: with step (1) among the embodiment 1.
(2) use QuickSep medium pressure chromatography system purifying:
That loads 10ml in the chromatography column of diameter 11mm and long 250mm receives little Uni PS30-300 filler (particle diameter 30 μ m, aperture 300 dusts), bed height 110mm.
The moving phase configuration proportion is with embodiment 1, and the pH that regulates mobile phase A is 3.0.Sample sample size in the adjusting makes that filler target protein lifting capacity is 4.0g/L in the pillar.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 is monitored out Fractional Collections behind the peak, and every section 10ml after purity being mixed greater than 98.0% component, detects purity and concentration, calculate recovery rate more again.
(3) method for detecting purity: with step (3) among the embodiment 1.
Figure 13 is the RP-HPLC collection of illustrative plates of the highest purity of use Uni PS30-300 polymer materials wash-out under the 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.
Listed experimental result in the table 3, be analyzed as follows: under different chromatographic system operating pressure conditions, can reach the purity more than 98.0% under 5bar and the 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%, so its yield is 0.
Table 3
Embodiment 3:
(1) preparation of sample to be purified: with step (1) among the embodiment 1.
(2) use QuickSep medium pressure chromatography system purifying:
The GE source30 filler (particle diameter 30 μ m) of filling 2.5L in the chromatography column of diameter 140mm and long 500mm, the long 165mm of bed.
The chromatography damping fluid is: A phase, 0.15M sodium-acetate buffer, pH3.0; The B phase contains the TFA(trifluoroacetic acid) acetonitrile solution, the concentration of TFA is volume percent 0.1%.
Pillar elder generation balances each other with 10%B, i.e. A phase and B 9:1 mixing by volume mutually is after the balance, it is 4.0g/L that last sample makes filler target protein lifting capacity, 40%B equality wash-out, elution speed 5.7L/h, operating pressure is 8bar, and the 280nmUV detector is monitored out Fractional Collections behind the peak, every section 1.0L volume.
(3) method for detecting purity: with step (3) among the embodiment 1.
Figure 16 is the RP-HPLC collection of illustrative plates of the sample to be purified before the last sample, and its purity area is 58.31%.
The RP-HPLC collection of illustrative plates of component is collected in Figure 17～20 for each.
Having listed the result who respectively collects component in the table 4 can be clear that, use this method at the GE of 2.5L scale source30 polymer packing good separation and purification effect can be arranged also, and purity can bring up to 98% from 58.31% of sample to be purified, be qualified with purity greater than 98% component, then yield reaches 71.90%.
Table 4
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. the chromatogram purification method of a lipid acid list acylated insulin; it is characterized in that comprising following steps: utilize the mesolow chromatographic system that lipid acid list acylated insulin is separated; use organic polymer reverse-phase chromatography material to be stationary phase, the moving phase of wash-out is that containing at least a can be 2～4 with miscible organic solvent and at least a buffer material, the pH value of water.
2. the chromatogram purification method of lipid acid list acylated insulin according to claim 1; it is characterized in that: described lipid acid list acylated insulin is naturally occurring longer chain fatty acid or its analogue that the epsilon-amino place of the Lys residue that exists at the alpha-amino group place of parent insulin B chain-terminal amino acid residue or at the B chain has connected side chain, and this side chain has following general general formula:
-X-Y；
Wherein X be and the alpha-amino group of parent insulin B chain-terminal amino acid residue or and the epsilon-amino of parent insulin B chain Lys residue between covalent linkage;
X is :-CO-;
Y is :-(CH
2) m-, wherein m is 6～32 integer;
And wherein parent Regular Insulin 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
B29The insulin human.
3. the chromatogram purification method of lipid acid list acylated insulin according to claim 2, it is characterized in that: described lipid acid list acylated insulin is (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.
4. the chromatogram purification method of lipid acid list acylated insulin according to claim 1, it is characterized in that: described mesolow chromatographic system is that operating pressure is the chromatographic system of 2～25 bar.
5. the chromatogram purification method of lipid acid list acylated insulin according to claim 1, it is characterized in that: described organic polymer reverse-phase chromatography material is polymethacrylate or polystyrene-divinylbenzene.
6. the chromatogram purification method of lipid acid list acylated insulin according to claim 5, it is characterized in that: the mean particle size of described organic polymer reverse-phase chromatography material is 5～300 μ m.
7. the chromatogram purification method of lipid acid list acylated insulin according to claim 6 is characterized in that: described organic polymer reverse-phase chromatography material is a kind of among MCI GEL CHP, Source15, Source30, CG161, CG300, CG1000 and the Uni PS or at least two kinds.
8. the chromatogram purification method of lipid acid list acylated insulin according to claim 1 is characterized in that: described can be that acetonitrile, ketone or carbonatoms are the alcohol of C1～C4 with the miscible organic solvent of water.
9. the chromatogram purification method of lipid acid list acylated insulin according to claim 1, it is characterized in that: described buffer material is phosphoric acid salt, acetate or Citrate trianion.
10. the chromatogram purification method of lipid acid list acylated insulin according to claim 1, it is characterized in that: described pH value is 3.0～3.5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN105658325A (en) * | 2013-10-10 | 2016-06-08 | 通用电气医疗集团生物工艺研发股份公司 | Method for production of a chromatography material |
| CN109748948A (en) * | 2018-11-27 | 2019-05-14 | 珠海冀百康生物科技有限公司 | A kind of purification process of palmityl tetrapeptide -7 |
| CN111518193A (en) * | 2020-06-11 | 2020-08-11 | 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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| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467593A (en) * | 2013-09-05 | 2013-12-25 | 杭州诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN103467593B (en) * | 2013-09-05 | 2015-05-13 | 杭州阿德莱诺泰制药技术有限公司 | Purification method of thymalfasin |
| CN105658325A (en) * | 2013-10-10 | 2016-06-08 | 通用电气医疗集团生物工艺研发股份公司 | Method for production of a chromatography material |
| CN109748948A (en) * | 2018-11-27 | 2019-05-14 | 珠海冀百康生物科技有限公司 | A kind of purification process of palmityl tetrapeptide -7 |
| CN111518193A (en) * | 2020-06-11 | 2020-08-11 | 南京赛诺生物制药有限公司 | Purification method of fatty acid acylated insulin and GLP-1 polypeptide |
| 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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