CN110540587B - Chromatographic method for effectively improving purification yield of synthetic peptide - Google Patents
Chromatographic method for effectively improving purification yield of synthetic peptide Download PDFInfo
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Abstract
The invention belongs to the field of medicine preparation, relates to a chromatographic method for improving the purification yield of synthetic polypeptide, and particularly relates to a method for improving the purification yield of liraglutide, which can effectively remove impurities with small difference between the physicochemical properties and target objects, such as achiral enantiomer impurities and the like, and is particularly suitable for purifying samples with complex impurity spectrums; meanwhile, the technical problems of small sample loading amount, large single impurity, low purity, low efficiency and the like of a polypeptide product can be solved.
Description
Technical Field
The invention belongs to the field of medicine preparation, and particularly relates to a preparation and purification method of liraglutide.
Background
Liraglutide (Liraglutide) is the first long-acting human glucagon-like peptide-1 (GLP-1) analogue developed by danish norand norder, having 97% homology to GLP-1 and having the peptide sequence: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (γ -Glu-Palmitoyl) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; the liraglutide has various effects of reducing blood sugar, promoting islet cell regeneration, slightly prolonging gastric emptying and the like, and has a wide application prospect. The liraglutide serving as a new generation of hypoglycemic drugs based on incretins has long action time, fully retains multiple physiological activities of natural GLP-1, can safely and effectively reduce blood sugar and can protect multiple cardiovascular hazard factors. The liraglutide injection is administered by subcutaneous injection, the peak time of blood concentration is 20-14 hours, the half-life period is 11-13 hours, the injection is performed once a day, 24-hour blood sugar control is provided, and the pharmacokinetic characteristic of the liraglutide injection is not influenced by gender or age. Liraglutide is absolutely a revolutionary drug in the field of the treatment of type 2 diabetes.
In the application field of reverse phase preparative chromatography, a high-pressure preparative liquid phase system and a dynamic axial compression column, the maximum height of a column barrel is usually 650mm, the normal use pressure of a common commercialized reverse phase chromatographic packing is not more than 100bar, and the packing height of a standard packing is 250 mm; under the condition of a certain height of a column bed, the work of separating complex samples by a mobile phase and an elution gradient in the development of a preparative chromatography method is limited, and if the work is carried out on the aspects of the length of a chromatographic column and packing, a better effect can be obtained, for example, 2 chromatographic columns are commonly used in series in analysis to obtain a higher column length so as to realize the separation of impurities difficult to separate. Therefore, a tandem chromatography application concept is applied to the development of preparative chromatography, a single or multiple gradient elution method is adopted to improve the separation and removal of impurities so as to improve the purification yield, and the technical problems of small sample loading amount, low purity and low yield of the liraglutide in the purification process are solved.
Disclosure of Invention
In view of the above, the invention discloses a chromatographic method for effectively improving the purification yield of synthetic peptide, and particularly relates to a method for improving the purification yield of liraglutide reversed phase chromatography, which relates to the application concept of separating complex samples by using a series chromatographic column, is applied to a single chromatogram, and is combined with single or multiple gradient elution to achieve the purpose of improving the impurity removal capacity under the same chromatographic condition. The specific implementation principle is that the filling height of a single chromatographic column filler is increased, the separation degree of the single chromatographic column on impurities in the synthesized polypeptide is improved, the purification yield of a sample is improved, and the technical problems of small sample loading amount, large single impurity, low purity, low efficiency and the like of a polypeptide product can be solved; the above strategy is suitable for dynamic axial compression columns.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a purification method of liraglutide, which comprises the following steps:
(1) taking a target polypeptide crude product, and pretreating a sample;
(2) a first step of HPLC purification;
(3) second step HPLC purification;
the pretreatment method of the crude target polypeptide product comprises the following steps: and dissolving the liraglutide sample in acetonitrile water solution, filtering the solution by using a 0.22 mu m filter membrane after the liraglutide sample is completely dissolved, and collecting the filtered crude liraglutide peptide water solution for later use.
The first step HPLC purification method is reversed phase high performance liquid chromatography, wherein octadecyl or octyl bonded silica gel filler is used as a stationary phase.
Preferably, the packed bed height of the chromatography column is greater than 250 mm.
Preferably, the height of the packed column bed of the chromatographic column is 260-400 mm.
Preferably, the height of the packed column bed of the chromatographic column is 280-400 mm.
Preferably, the height of the packed column bed of the chromatographic column is 300-400 mm.
Preferably, the size of the chromatographic column filled with stationary phase octadecyl or octyl bonded silica gel is 8-20 μm.
Preferably, the mobile phase A is selected from one or more of phosphate, sulfate, perchlorate, acetate, trifluoroacetate and Tris.
Preferably, the mobile phase B is selected from one or more of acetonitrile, methanol, isopropanol and ethanol.
Preferably, the mobile phase A is 0.1% ammonium sulfate (pH3.3) and the mobile phase B is acetonitrile.
Preferably, the cation of the buffer salt used is selected from the group consisting of hydrogen ion, sodium ion, potassium ion, ammonium ion, triethylamine ion.
Preferably, the elution step is a single gradient elution or a multiple gradient elution.
Fractions containing the liraglutide sample collected after elution were partially acetonitrile removed using rotary evaporation.
Preferably, the water bath temperature of the rotary evaporator is 30-35 ℃, and the vacuum degree is-0.09 MPa.
The second step HPLC purification method is reversed phase high performance liquid chromatography, wherein octadecyl or octyl bonded silica gel filler is used as a stationary phase.
Preferably, the height of the packed column bed of the chromatographic column is 260-400 mm.
Preferably, the size of the chromatographic column filled with stationary phase octadecyl or octyl bonded silica gel is 8-20 μm. Preferably, the mobile phase A is selected from one or more of phosphate, sulfate, perchlorate, acetate, trifluoroacetate and Tris.
Preferably, the mobile phase B is selected from one or more of acetonitrile, methanol, isopropanol and ethanol.
Preferably, the mobile phase A is 0.1% ammonium sulfate (pH6.5), the mobile phase B is acetonitrile, and multiple gradient elution is performed.
Preferably, the cation of the buffer salt used is selected from the group consisting of hydrogen ion, sodium ion, potassium ion, ammonium ion, triethylamine ion.
Fractions containing the liraglutide sample collected after elution were partially acetonitrile removed using rotary evaporation.
Preferably, the water bath temperature of the rotary evaporator is 30-35 ℃, and the vacuum degree is-0.09 MPa.
The liraglutide is
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(γ-Glu-Palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH；
Preferably, the above purification step may be performed by, but not limited to, one purification, or two purifications, or three purifications.
The invention discloses a purification method of liraglutide and/or somaglutide, which specifically comprises the following steps:
1. liraglutide purification method
And carrying out liraglutide purification by using octadecyl or octyl bonded silica gel filler as a fixed phase and a chromatographic column packed column bed with the height of 280-400 mm.
(1) And dissolving the target polypeptide crude product in acetonitrile water solution to obtain the liraglutide crude peptide water solution.
(2) The liraglutide aqueous solution was filtered through a 0.22 μm filter to remove insoluble particles. Collecting the filtrate for later use.
(3) Octadecyl or octyl bonded silica gel filler is used as a fixed phase (8-20 mu m), and the height of a packed column bed of a chromatographic column is as follows: 280-400 mm; taking 0.1% ammonium sulfate (1000 ml water, adding 1ml ammonium sulfate, mixing well, adjusting pH to 3.3 with ammonia water) as mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; fractions containing the liraglutide sample were collected.
(4) And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a first-step sample solution of liraglutide, wherein the sample solution is acidic.
(5) Taking the sample in the step (4), taking octadecyl or octyl bonded silica gel filler as a stationary phase (8-20 mu m), wherein the height of a packed column bed of the chromatographic column is as follows: 280-400 mm; taking 10mmol/L ammonium acetate solution (ammonia water for adjusting pH to 6.5) as a mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; fractions containing the liraglutide sample were collected.
(6) And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a liraglutide sample solution which is nearly neutral.
2. Purification method of Somalutide
And (3) taking octadecyl or octyl bonded silica gel filler as a fixed phase, and purifying the Somaloude by using a chromatographic column packed column bed with the height of 280-400 mm.
(1) And dissolving the target polypeptide crude product in acetonitrile water solution to obtain a crude peptide water solution of the somaglutide.
(2) The aqueous solution of somaglutide was filtered through a 0.22 μm filter to remove insoluble particles. Collecting the filtrate for later use.
(3) Octadecyl or octyl bonded silica gel filler is used as a fixed phase (8-20 mu m), and the height of a packed column bed of a chromatographic column is as follows: 280-400 mm; using 0.1% TFA (1000 ml water, using ammonia water to adjust pH value to 2.3) as mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; recovering the fractions containing the sample of somaglutide.
(4) And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. Obtaining a first-step sample solution of the Somaltulipide, wherein the sample solution is acidic.
(5) Taking the sample in the step (4), taking octadecyl or octyl bonded silica gel filler as a stationary phase (8-20 mu m), wherein the height of a packed column bed of the chromatographic column is as follows: 280-400 mm; 0.1% phosphoric acid (1000 ml of water is taken, and the pH value is adjusted to 7.5 by ammonia water); acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; recovering the fractions containing the sample of somaglutide.
And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. Obtaining the somaglutide sample solution which is near neutral.
The invention has the beneficial effects that the octadecyl or octyl bonded silica gel filler is used as the stationary phase, and the height of the chromatographic column packed column bed is as follows: 260-400 mm, taking phosphate, or sulfate, or perchlorate, or chloride, or acetate, or trifluoroacetate, or Tris as a mobile phase A, taking acetonitrile, methanol, isopropanol, or ethanol as a mobile phase B, eluting by single gradient or multiple gradients, collecting fractions containing target polypeptide samples, and purifying once, twice or three times to achieve the target of producing high-purity target polypeptide products, wherein the purity is more than 99.0%, and the maximum single impurity is less than 0.10%. With the increase of the height of the packed bed of the packing material and the same mobile phase and gradient, the total purification yield is greatly improved. In addition, under the condition of the same yield, the purification steps can be reduced, and the maximum purification yield is up to more than 95.0 percent, so that the problem of low yield when the crude synthetic polypeptide product is purified to reach high purity (more than 99.0 percent) and low single impurity (less than 0.10 percent) is solved.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
fig. 1 is a chromatogram of crude liraglutide.
FIG. 2 is a chromatographic chart of the primary purification of liraglutide at a bed height of 300 mm.
FIG. 3 is a chromatogram of a secondary purification of liraglutide at a bed height of 300 mm.
FIG. 4 is a chromatographic chart of the primary purification of liraglutide at a column bed height of 350 mm.
FIG. 5 is a chromatogram of a secondary purification of liraglutide at a bed height of 350 mm.
FIG. 6 is a chromatographic chart of the primary purification of liraglutide at a bed height of 400 mm.
FIG. 7 is a chromatogram of a secondary purification of liraglutide at a bed height of 400 mm.
Fig. 8 is a somaluo peptide crude peptide chromatogram.
FIG. 9 is a single purification chromatogram of somaglutide at a bed height of 300 mm.
FIG. 10 is a second purification chromatogram of Somalulptin at a bed height of 300 mm.
FIG. 11 is a column height 350mm Somalglutide primary purification chromatogram.
FIG. 12 is a somaltulin secondary purification chromatogram with a bed height of 350 mm.
FIG. 13 is a column height 400mm Somalglutide primary purification chromatogram.
FIG. 14 is a column height 400mm Somalglutide secondary purification chromatogram.
FIG. 15 is a line graph showing the correspondence between the yield and the stationary phase height.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, which are not intended to limit the present invention.
Example 1 purification of liraglutide
1.1 bed height: 300mm
Taking a target liraglutide crude product
Sample treatment: a150.0 g sample of liraglutide was dissolved in aqueous acetonitrile and filtered through a 0.22 μm filter after complete dissolution. Collecting the filtered crude liraglutide peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (100mm multiplied by 250mm, 8-20 μm) is used as a chromatographic column; taking 0.1% ammonium sulfate (1000 ml water, adding 1ml ammonium sulfate, mixing well, adjusting pH to 3.3 with ammonia water) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 15.0 g.
Elution is performed with the following table elution gradient.
Fractions of the liraglutide sample with a purity greater than 95.0% were recovered. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a first-step sample solution of liraglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler is used as a fixed phase (8-20 mu m), and a chromatographic column comprises: 100mm × 250 mm; taking 10mmol/L ammonium acetate solution (ammonia water for adjusting pH to 6.5) as a mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; the amount of the sample was 15.0 g.
Elution is performed with the following table elution gradient.
Collecting the fraction of the liraglutide sample with the purity of more than 99.5%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining the solution to contain 120.9g of liraglutide by using a reference substance, wherein the yield reaches 80.6%.
1.2 bed height: 350mm
Taking a crude liraglutide product
Sample treatment: a sample containing 150.0g of liraglutide was dissolved in an aqueous acetonitrile solution, and after complete dissolution, the sample was filtered through a 0.22 μm filter. Collecting the filtered crude liraglutide peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (100mm multiplied by 350mm, 10 mu m) is taken as a chromatographic column; taking 0.1% ammonium sulfate (1000 ml water, adding 1ml ammonium sulfate, mixing well, adjusting pH to 3.3 with ammonia water) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 200mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 15.0 g.
Elution is performed with the following table elution gradient.
Fractions of the liraglutide sample with a purity greater than 95.0% were recovered. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a first-step sample solution of liraglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler is used as a fixed phase (10 mu m), and a chromatographic column comprises: 100mm × 350 mm; taking 10mmol/L ammonium acetate solution (ammonia water for adjusting pH to 6.5) as a mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; the amount of the sample was 15.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the liraglutide sample having a purity greater than 99.0%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining the solution to contain 137.7g of liraglutide by using a reference substance, wherein the yield reaches 91.8%.
1.3 bed height: 400mm
Taking a crude liraglutide product
Sample treatment: a sample containing 150.0g of liraglutide was dissolved in an aqueous acetonitrile solution, and after complete dissolution, the sample was filtered through a 0.22 μm filter. Collecting the filtered crude liraglutide peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (100mm multiplied by 400mm, 10 mu m) is taken as a chromatographic column; taking 0.1% ammonium sulfate (1000 ml water, adding 1ml ammonium sulfate, mixing well, adjusting pH to 3.3 with ammonia water) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 200mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 15.0 g.
Elution is performed with the following table elution gradient.
Fractions of the liraglutide sample with a purity greater than 95.0% were recovered. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a first-step sample solution of liraglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler is used as a fixed phase (10 mu m), and a chromatographic column comprises: 100mm × 400 mm; taking 10mmol/L ammonium acetate solution (ammonia water for adjusting pH to 6.5) as a mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; the amount of the sample was 15.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the liraglutide sample having a purity greater than 99.0%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining 143.55g of liraglutide in the solution through a reference substance, wherein the yield reaches 95.7%.
Example 2 purification of Somalutide
2.1 bed height: 300mm
Taking crude soxhlet peptide
Sample treatment: dissolving a sample containing 12.0g of the somaltulin in acetonitrile water solution, and filtering the solution by using a 0.22 mu m filter membrane after the sample is completely dissolved. Collecting the filtered crude soxhlet peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 300mm, 10 mu m) is taken as a chromatographic column; using 0.1% TFA (1000 ml water, using ammonia water to adjust pH value to 2.3) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of the sample loaded on a single needle was 3.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 90.0%. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. Obtaining a first-step sample solution of the somaglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 300mm, 10 mu m) is taken as a chromatographic column; 0.1% phosphoric acid (1000 ml water, ammonia water to adjust pH value to 7.5) is used as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 2.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 99.0%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining the solution to contain 9.7g of the somaglutide through a reference substance, wherein the yield reaches 81.2%.
2.2 bed height: 350mm
Taking crude soxhlet peptide
Sample treatment: dissolving a sample containing 12.0g of the somaltulin in acetonitrile water solution, and filtering the solution by using a 0.22 mu m filter membrane after the sample is completely dissolved. Collecting the filtered crude soxhlet peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 350mm, 10 mu m) is taken as a chromatographic column; using 0.1% TFA (1000 ml water, using ammonia water to adjust pH value to 2.3) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of the sample loaded on a single needle was 3.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 90.0%. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. Obtaining a first-step sample solution of the somaglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 350mm, 10 mu m) is taken as a chromatographic column; 0.1% phosphoric acid (1000 ml water, ammonia water to adjust pH value to 7.5) is used as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 2.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 99.0%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining the content of the solution containing the somaglutide by 10.3g through a reference substance, wherein the yield reaches 86.3%.
2.3 bed height: 400mm
Taking crude soxhlet peptide
Sample treatment: dissolving a sample containing 12.0g of the somaltulin in acetonitrile water solution, and filtering the solution by using a 0.22 mu m filter membrane after the sample is completely dissolved. Collecting the filtered crude soxhlet peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 400mm, 10 mu m) is taken as a chromatographic column; using 0.1% TFA (1000 ml water, using ammonia water to adjust pH value to 2.3) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of the sample loaded on a single needle was 3.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 90.0%. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. Obtaining a first-step sample solution of the somaglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (50mm multiplied by 400mm, 10 mu m) is taken as a chromatographic column; 0.1% phosphoric acid (1000 ml water, ammonia water to adjust pH value to 7.5) is used as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 2.0 g.
Elution is performed with the following table elution gradient.
Recovering a fraction of the sample of somaglutide having a purity greater than 99.0%. Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa, and quantitatively determining the solution to contain 11.1g of the somaglutide through a reference substance, wherein the yield reaches 92.8%.
From the experimental results of example 1 and example 2, it can be seen that as the height of the bed increases, the yield of liraglutide and somagluteptide increases under the same chromatographic conditions. During the experiment, which was accompanied by an increase in column pressure, the following experiment was further conducted to obtain more detailed data in order to further explore the effect of bed height on yield, as detailed in example 3.
Example 3
Taking a crude liraglutide product
Sample treatment: a150.0 g sample of liraglutide was dissolved in aqueous acetonitrile and filtered through a 0.22 μm filter after complete dissolution. Collecting the filtered crude liraglutide peptide aqueous solution for later use.
First step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler stationary phase (100mm multiplied by 300mm, 8-20 μm) is used as a chromatographic column; taking 0.1% ammonium sulfate (1000 ml water, adding 1ml ammonium sulfate, mixing well, adjusting pH to 3.3 with ammonia water) as mobile phase A; acetonitrile is used as a mobile phase B; the flow rate is 20mL per minute, multiple gradient elution is carried out, and the detection wavelength is 230 nm; the amount of sample loaded on a single needle was 15.0 g.
Elution is performed with the following table elution gradient.
Fractions of the liraglutide sample with a purity greater than 95.0% were recovered. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa. And obtaining a first-step sample solution of liraglutide.
Second step HPLC purification
Chromatographic conditions are as follows: octadecyl or octyl bonded silica gel filler is used as a fixed phase (8-20 mu m), and a chromatographic column comprises: 100mm × 300 mm; taking 10mmol/L ammonium acetate solution (ammonia water for adjusting pH to 6.5) as a mobile phase A; acetonitrile is used as a mobile phase B; the detection wavelength is 230 nm; performing multiple gradient elution; the amount of the sample was 15.0 g.
Elution is performed with the following table elution gradient.
Collecting the fraction of the liraglutide sample with the purity of more than 99.5%. And removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 30-35 ℃ and the vacuum degree of below-0.09 MPa.
Height of stationary phase: 260-270 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 2.1g of liraglutide quantitatively by a reference substance, and the yield reaches 14.00 percent.
Height of stationary phase: 270-280 mm, collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 3.2g of liraglutide quantitatively by a reference substance, and the yield reaches 21.33 percent.
Height of stationary phase: 280-290 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 4.5g of liraglutide quantitatively by a reference substance, and the yield reaches 30.00 percent.
Height of stationary phase: 290-300 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 6.1g of liraglutide quantitatively by a reference substance, and the yield reaches 40.67%.
Height of stationary phase: 300-310 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 13.1g of liraglutide quantitatively by a reference substance, and the yield reaches 87.33%.
Height of stationary phase: and (3) collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent at 310-320 mm. The solution contains 13.2g of liraglutide quantitatively by a reference substance, and the yield reaches 88.00%.
Height of stationary phase: 320-330 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 13.3g of liraglutide quantitatively by a reference substance, and the yield reaches 88.67 percent.
Height of stationary phase: 330-340 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 13.4g of liraglutide quantitatively by a reference substance, and the yield reaches 89.33%.
Height of stationary phase: 340-350 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 13.3g of liraglutide quantitatively by a reference substance, and the yield reaches 88.67 percent.
Height of stationary phase: 350-360 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 13.6g of liraglutide quantitatively by a reference substance, and the yield reaches 90.67%.
Height of stationary phase: and (3) collecting the fraction of the liraglutide sample with the purity of more than 99.0% and the single impurity of less than 0.1% at the length of 360-370 mm. The solution contains 13.8g of liraglutide quantitatively by a reference substance, and the yield reaches 92.00 percent.
Height of stationary phase: 370-380 mm, collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 14.1g of liraglutide quantitatively by a reference substance, and the yield reaches 94.00 percent.
Height of stationary phase: 380-390 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 14.2g of liraglutide quantitatively by a reference substance, and the yield reaches 94.67%.
Height of stationary phase: 390-400 mm, and collecting the fraction of the liraglutide sample with the purity of more than 99.0 percent and the single impurity of less than 0.1 percent. The solution contains 14.3g of liraglutide quantitatively by a reference substance, and the yield reaches 95.33 percent.
The specific data collated are as follows:
| height of stationary phase | Sample loading amount | Standard of final qualified product | Amount of withdrawal | Yield of |
| 260～270mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 2.1g | 14.00％ |
| 270～280mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 3.2g | 21.33％ |
| 280～290mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 4.5g | 30.00％ |
| 290～300mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 6.1g | 40.67％ |
| 300～310mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.1g | 87.33％ |
| 310～320mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.2g | 88.00％ |
| 320～330mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.3g | 88.67％ |
| 330～340mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.4g | 89.33％ |
| 340～350mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.3g | 88.67％ |
| 350～360mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.6g | 90.67％ |
| 360～370mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 13.8g | 92.00％ |
| 370～380mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 14.1g | 94.00％ |
| 380～390mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 14.2g | 94.67％ |
| 390～400mm | 15g | The purity is more than 99.0 percent, and the single impurity is less than 0.1 percent | 14.3g | 95.33％ |
The yield and stationary phase height are plotted in FIG. 15.
The experimental data show that when the length of the chromatographic stationary phase is increased from 260-270 mm to 300-310 mm, the yield is increased from 14.00% to 87.33%, the yield is obviously increased between 290mm and 300mm of the column length, and the yield is slowly increased after 300 mm.
Claims (6)
1. A method of purifying a synthetic peptide, comprising: the method comprises the following steps:
(1) taking a target polypeptide crude product, and carrying out sample pretreatment;
(2) a first step of HPLC purification, wherein the first step of HPLC purification is reverse phase high performance liquid chromatography, octadecyl or octyl bonded silica gel filler is used as a stationary phase, a single chromatographic column is adopted, the height of a packed column bed is 350-400 mm, and the size of the octadecyl or octyl bonded silica gel filler of the stationary phase packed by the chromatographic column is 8-20 mu m;
(3) performing HPLC purification, wherein the HPLC purification method is reversed-phase high performance liquid chromatography, octadecyl or octyl bonded silica gel filler is used as a stationary phase, the height of a column bed filled with a chromatographic column is 350-400 mm, and the size of the octadecyl or octyl bonded silica gel filled with the stationary phase is 8-20 mu m;
the polypeptide is liraglutide or somagluteptide;
in the first and second HPLC purification steps:
the mobile phase B is selected from one or more of acetonitrile, methanol, isopropanol and ethanol;
when the liraglutide is purified, the mobile phase A in the HPLC purification method in the first step is ammonium sulfate, and the pH value is 3.3; in the second step of HPLC purification method, the mobile phase A is ammonium acetate, and the pH value is 6.5;
when the Somalutide is purified, the mobile phase A in the first step of HPLC purification method is trifluoroacetic acid, and the pH value is 2.3; the mobile phase A in the second HPLC purification process is phosphoric acid, pH 7.5.
2. The method of claim 1, wherein: the pretreatment method of the crude target polypeptide product comprises the following steps: and (3) dissolving the polypeptide sample in acetonitrile water solution, filtering the solution by using a 0.22 mu m filter membrane after the polypeptide sample is completely dissolved, and collecting the filtered crude liraglutide peptide water solution for later use.
3. The method of claim 1, wherein: the mobile phase a in the first HPLC purification process was 0.1% ammonium sulfate, ph 3.3.
4. The method of claim 1, wherein: in the first step HPLC purification and the second step HPLC purification, the elution step is single gradient elution or multiple gradient elution.
5. The method of claim 1, wherein: in the first-step HPLC purification and the second-step HPLC purification, the fraction containing the liraglutide sample collected after elution is subjected to rotary evaporation to remove part of acetonitrile, the water bath temperature of the rotary evaporator is 30-35 ℃, and the vacuum degree is-0.09 MPa.
6. The method of claim 1, wherein: the mobile phase A in the second HPLC purification method is 10mmol/L ammonium acetate, pH 6.5.
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| CN114605523A (en) * | 2020-12-03 | 2022-06-10 | 中国科学院大连化学物理研究所 | Method for preparing liraglutide through reversed phase chromatography purification |
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