A method for purifying polypeptides

Abstract

The present invention proposes a method for purifying polypeptides, which uses a high-precision high-performance liquid chromatography system and is also equipped with a control cabinet, an axially pressurized liquid chromatography column (DAC), an eluent delivery system, a sample loading device, an online detection device, and an automatic fraction collection system; the flow error of the pump of the eluent delivery system is within 1.5%; the chromatographic column filler is a spherical mesoporous silica matrix reversed-phase chromatography filler, which is a type of specially constructed silica gel, and has undergone special bonding modification, which effectively improves the effect and yield of polypeptide separation and purification, increases the sample loading amount of the filler, and solves the technical problems existing in the polypeptide purification process such as the large number of micropores, low lifespan, and alkali resistance of traditional silica gel. At the same time, through a closed-loop fully automatic chromatographic purification method that integrates online solvent recovery, online liquid preparation, and UHPLC online detection / automatic fraction collection, the problems of purification, fraction collection, and solvent recovery of large molecular weight polypeptides are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of a method for purifying polypeptides, and in particular to a high performance liquid chromatography method for purifying polypeptides. Background Art

[0002] Peptide molecules, especially those with larger molecular weight (above 2000D), are difficult to separate and purify due to the high similarity between the structure and properties of impurity molecules and target molecules; and peptide drugs are mainly injections, and single impurities usually need to be controlled below 0.1%. Based on the above characteristics of peptides, peptide chromatographic purification usually has a low sample load, resulting in high costs; high requirements for the stability and precision of purification conditions are required to ensure accurate separation. Higher automation and more precise control are required on the equipment to improve efficiency and the yield of high-purity qualified products. On the other hand, especially for peptides with larger molecular weight (above 2000D), not only is it difficult to separate the target from impurities with similar structure and composition, but such peptides are more likely to form severe tailing on the chromatographic column. In addition, if the peptide is prepared by fermentation, the crude product for chromatographic separation often contains a small amount of protein, and these proteins will be adsorbed and denatured on the chromatographic filler. In the end, the chromatographic filler in the column can only be cleaned with an alkaline aqueous solution to restore the separation effect. To sum up, for large molecular weight peptides, the filler needs to provide materials with higher selectivity, higher sample loading, reduced tailing (adsorption loss), pressure resistance and corrosion resistance to improve efficiency, increase yield and reduce overall cost; therefore, in the development and production of peptide drugs, new chromatographic purification solutions are urgently needed to achieve innovation in equipment, fillers and methods to improve more efficient and stable peptide purification methods, especially chromatographic purification of peptides with a molecular weight of more than 2000D.

[0003] It is generally known to those skilled in the art that for relatively high molecular weight polypeptides (above 2000D), such as insulin, semaglutide, etc., the crude product contains a large amount of impurities with very similar molecular weight and structure, which are difficult to separate; and these polypeptide molecules will form different three-dimensional forms (such as ion pair polymers, nanospheres) under different conditions. The irregular micropores in silica gel not only have no contribution to the separation of such larger molecules, but also cause negative effects such as tailing or even dead adsorption; therefore, the selection of a chromatographic filler with a suitable silica gel structure has a crucial influence on the separation and purification of relatively large molecular weight polypeptides.

[0004] The molecular weight of peptides such as insulin and liraglutide is about 5000D. The length of a compound with a molecular weight of 5000D is close to 500Å. In the process of chromatographic separation, 10% is the limiting pore, that is, 50Å. A pore size below 50Å cannot achieve chromatographic separation and will form a dead pore. Considering the influence of the C8 / C18 bonded phase, the effective pore volume and diameter are reduced, and the inner diameter is reduced by about 15-20Å. Therefore, it is defined as 7nm, that is, the micropore content below 70Å affects the purification and separation recovery rate of peptide compounds. Furthermore, smaller pores (<40 Å) not only have no separation effect on peptides, but also cause more serious tailing and reduce the ability of the filler to withstand pressure and alkaline water corrosion.

[0005] On the other hand, many large molecular weight polypeptides are produced by biological methods, so protein fiber deposition will form on the surface of the chromatographic packing during chromatographic purification, so the chromatographic packing needs to be cleaned frequently with sodium hydroxide solution, so it is necessary to improve the mechanical strength and alkali resistance of the packing.

[0006] It is generally known to those skilled in the art that small particles can increase the number of plates and resolution, but at the same time make column packing difficult and require higher operating pressure; at the same time, a high column pressure drop will be formed. The increased column pressure drop will cause more friction heat to be generated when the mobile phase passes through the chromatographic column, and friction heat is an important factor causing the instability of the chromatographic column. In addition, friction heat is difficult to diffuse in a large-diameter chromatographic column, so reducing the friction heat effect is very important for large-diameter chromatographic columns.

[0007] By optimizing separation materials and developing an intelligent, precise and efficient liquid chromatography purification system and the corresponding purification method, the separation effect can be improved, the efficiency can be increased, the purification cost can be reduced, and the production quality risks and losses can be reduced. This has very important practical significance for the production of high-purity and high-value products such as polypeptide raw materials. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for purifying polypeptides, especially polypeptides with a molecular weight between 2000-6000D. To achieve the above objectives, the present invention adopts the following technical means:

[0009] A method for purifying a polypeptide, using a high-precision high-efficiency preparative liquid chromatography system, the system is equipped with a control device, an axially pressurized liquid chromatography column, an eluent delivery system, a sample loading device, an online detection device, and an automatic fraction collection system;

[0010] The control device consists of a remote control center and a control cabinet to realize data transmission and electrical control; the remote control center realizes communication connection with the control cabinet, and realizes data transmission and command transmission with the control cabinet; the control cabinet adopts positive pressure explosion-proof to ensure the explosion-proof of electrical components for operation of each device of the control system and data transmission.

[0011] The eluent delivery system consists of a solvent storage tank, an infusion pump, a mixer, an electrically programmable valve, a flow meter, etc.; one or two precision constant flow pumps can be used, and the flow error of the pump must be within 1.5%; the flow meter data can be sent to the control system in real time to adjust the flow in real time to further ensure the accuracy of the flow; the control system can also control the flow of the two pumps according to the pre-set gradient program to achieve various gradient elution modes.

[0012] The loading device can be composed of a sample solution storage tank and a loading pump, equipped with corresponding control valves, and can also be equipped with a flow meter for accurate quantitative injection. The loading device can also be equipped with a manual injection valve so that samples or reagents can be added manually when necessary. The loading can also be achieved by the control system to implement linkage control of the injection pump, the infusion pump and the switching valve in front of the column to achieve rapid alternating entry of the sample and the eluent. This method can be used for: 1-reducing the diffusion of the sample solution in the chromatographic column; 2-realizing multi-stage separation of the sample in the same column to greatly improve efficiency. The loading device further includes a short column filled with large-particle chromatographic filler (20um-100um), which is between the axial pressure column and the injection pump. The front and rear ends of the short column have multi-channel switching valves, and the switching valve is connected to the control device by data transmission and electrical control lines, and is controlled by the control software through the control device.

[0013] The chromatographic column filler is a spherical mesoporous silica matrix reverse phase chromatographic filler, the column filling height is 20-100 cm, preferably 20-80 cm; the total content of other metals in the filler except silicon does not exceed 100ug / g, the average pore size range is 90Å-150Å, and the porosity is 0.8-1.0cm 3 / g, specific surface area is 280-380m 2 / g, the average particle size ranges from 5 microns to 30 microns; the proportion of micropores with a pore size range below 70Å is less than 15%, and the proportion of micropores below 40Å is less than 5%; the eluent used for separation is an aqueous solution, which contains water-soluble acids, bases or salts, or organic solvents that can be dissolved in water.

[0014] Furthermore, polar groups are embedded on the surface of the chromatographic filler to achieve hybrid modification.

[0015] Furthermore, the polar group is selected from polar nitrogen-containing groups and / or carboxyl groups; the polar nitrogen-containing groups include amino groups, amine groups, amide groups, aminocarbonates, urea groups; more specifically, such as: aminopropyldimethylsiloxy, aminoethyldimethylsiloxy, dodecylamidoethyldimethylsiloxy, etc.

[0016] The surface of the silica-based ball of the chromatographic filler (including the surface in the pores) is modified by hybridization to improve the filler's alkali resistance and salt resistance. The hybridization can be achieved by coating a mixed hydrolyzate containing ethylene hexamethoxysilane and tetraethoxy or tetramethoxysilane.

[0017] Furthermore, the chromatographic filler can improve the separation effect of compounds with similar structures by bonding with two or more silane groups. The two or more silane groups bonded here do not include tail capping, and can be bonded simultaneously or in steps; the silane group without polar groups contains no less than 5 carbon atoms, and the proportion of the silane group with the lowest content is not less than 10%.

[0018] Furthermore, the silyl group refers to an alkylsilyl group containing a polar group or not containing a polar group, and the silyl group includes a silyl group modified with a polar group;

[0019] Also included are: octadecyldimethylsilyloxy, octadecyldiisopropylsilyloxy, octadecyldiisobutylsilyloxy, hexadecyldimethylsilyloxy, dodecyldimethylsilyloxy, decyldimethylsilyloxy, octyldimethylsilyloxy, hexyldimethylsilyloxy, butyldimethylsilyloxy, propyldimethylsilyloxy, isopropyldimethylsilyloxy, isobutyldimethylsilyloxy, aminopropyldimethylsilyloxy, and aminoethyldimethylsilyloxy.

[0020] Further, the pH of the aqueous solution is between 1 and 12, and the added acid, base or salt includes trifluoroacetic acid, acetic acid, phosphoric acid, citric acid, sodium acetate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate, sodium carbonate, sodium bicarbonate, ammonium bicarbonate, ammonium acetate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, triethylamine, triethylammonium carbonate, and triethylammonium acetate.

[0021] Furthermore, a high-precision mass flow meter monitors the flow rate accurately in real time and feeds back to the control cabinet; the control cabinet instantly adjusts the flow rate to the set value to ensure the flow rate accuracy; and a high-precision mass flow meter is used to monitor the density of the recovered solvent in real time and feed back to the control cabinet. The control cabinet controls the solvent tank to add the required volume of the corresponding solvent according to the density of the recovered solvent, so that the density of the mixed solvent reaches the set value of the eluent density.

[0022] Furthermore, the temperature of the chromatographic column and the eluent is controlled between 10°C and 30°C, and the chromatographic column and the eluent are kept constant and the temperature is consistent, and the temperature difference between the eluent and the chromatographic column is within ±3°C.

[0023] Furthermore, the axially pressurized chromatographic column adopts an oil-free cylinder type chromatographic column.

[0024] Further, the fractions flowing out of the chromatographic column are tested, the fractions without the target polypeptide are subjected to solvent recovery, and the fractions with the target polypeptide are subjected to target collection;

[0025] The fractions containing the target polypeptide are tested for target purity, and the fractions whose target purity reaches the preset standard are subjected to vacuum freeze drying to remove the solvent to obtain the target solid; the fractions whose target purity does not reach the preset standard but exceeds the preset purity of the recoverable fraction are subjected to solvent recovery.

[0026] Furthermore, the fractions without the target peptide are proportion-confirmed by a high-precision mass flow meter, and the fractions that meet the set ratio of the eluent are directly recovered; the fractions that do not meet the set ratio of the eluent are adjusted to the set ratio of the eluent and then recovered;

[0027] The fraction whose purity reaches the preset standard is evaporated by vacuum in a closed system, and the solvent in the fraction is separated by vacuum evaporation, and then condensed into a liquid solvent mixture after cooling; the proportion is confirmed by a high-precision mass flow meter, and the mixture is recovered after adjusting to the set proportion of the eluent.

[0028] Furthermore, the eluent is a homogeneous solution containing a volatile organic solvent and water, and the organic solvent is methanol, ethanol, acetonitrile, acetone or tetrahydrofuran.

[0029] Furthermore, the eluent is pure water or an aqueous solution, to which an acid, a base or a salt is added, and the acid, a base or a salt is formic acid, acetic acid, trifluoroacetic acid, ammonia water, triethylamine, ammonium acetate, ammonium carbonate or triethylammonium acetate.

[0030] Furthermore, the proportion of micropores with a pore size range below 40Å is less than 5%.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] For polypeptides with a molecular weight of more than 2000D, the target and impurities have high similarity, are difficult to separate, and are prone to tailing and self-aggregation. In order to achieve accurate separation of large molecular weight polypeptides (2000D-6000D) and improve sample loading, separation and yield, the present invention has made significant improvements in separation and purification efficiency, continuous working life, separation and yield through systematic integrated innovation in the overall purification method, purification related devices and chromatographic fillers. Its main innovation is the integration of the following key points, focusing on improving the separation effect, separation accuracy and intelligence of the entire process:

[0033] (1) In the process of the present invention, the inventors have found out the influence of micropores on large molecular polypeptides (greater than 2000D) through a large number of experimental designs and analysis and comparison of a large number of experimental data generated, and discovered the serious harm of micropores (less than 70Å) and ultramicropores (less than 40Å) to the chromatographic separation of such large molecular polypeptides; in particular, ultramicropores (less than 40Å) not only cannot provide separation for large molecular polypeptides, but also cause tailing, and even reduce the pressure resistance and alkali resistance of the filler. By reducing micropores below 70Å, especially ultramicropores below 40Å, and improving the uniformity of pore size, not only the tailing of polypeptides on the filler is reduced, the separation is improved, and the yield is increased, but also the alkali resistance, pollution resistance (strong adsorption of small molecular impurities caused by micropores) and mechanical strength of the filler are improved, solving the technical problems existing in the polypeptide purification process such as the large number of micropores, low lifespan, and alkali resistance of traditional silica gel. Based on a large number of experimental results and data analysis, the present invention creatively provides the characteristics of the preferred filler for the purification of large molecular polypeptides, develops and prepares the corresponding filler, and successfully applies the filler with this characteristic, thereby inventing a complete new method for the purification of large molecular polypeptides (2000D-6000D) with multiple characteristics.

[0034] The present invention further uses two or more silanes for mixed bonding, and utilizes the difference in the different effects of different silanes on the target and impurities, that is, the effects of polar silanes on the target and impurities are different from those of non-polar silanes on the target and impurities. The sum of the effects will improve the separation effect and selectivity, and further enhance the separation of chromatographic filler fixed relative to large molecular polypeptides and other target and impurity mixtures with very similar composition and structure. The silanes described here do not include tailing agents with no more than 4 carbons used for silica gel tailing (although some tailing agent molecules themselves contain more than 5 carbons, the carbon number of the siloxy group after bonding with silica gel does not exceed 4, and they are still tailing agents, not the bonding silanes described in the present invention).

[0035] (2) The present invention also effectively reduces tailing, improves peak shape and separation effect by ensuring consistency and accuracy of chromatographic column temperature and eluent temperature (the temperature can be selected between 10°C and 30°C, and the temperature difference between the eluent and the chromatographic column is within ±3°C).

[0036] (3) The intelligent fraction collection and online solvent recovery based on online ultra-high performance liquid chromatography (column efficiency exceeding 150,000 plates / m) detection of the present invention forms the first closed-loop fully automatic system in this field, which not only improves efficiency but also reduces the loss of target products during collection, inspection and post-processing, and also reduces solvent loss and environmental pollution during the entire purification and solvent recovery process.

[0037] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0039] Figure 1 This is a diagram for preparing semaglutide by chromatography using a silica gel packing material embedded with amino polar groups in Example 9;

[0040] Figure 2 This is the spectrum of Example 6;

[0041] Figure 3 It is the spectrum of comparative example 6;

[0042] Figure 4 This is the chromatogram of Example 7;

[0043] Figure 5 It is the spectrum of comparative example 7;

[0044] Figure 6 This is the fitting curve for the conventional C18 separation of salidroside in Example 10;

[0045] Figure 7 Fitting curve for separating salidroside by embedding amide group C11 in Example 10;

[0046] Figure 8 It is a schematic diagram of oil-free cylinder type chromatography column;

[0047] Fig. 9 This is the AA cross-section of the oil-free cylinder type chromatographic column;

[0048] Fig.10 The diagram of Example 2;

[0049] Fig.11 It is the spectrum of comparative example 1;

[0050] Fig.12 It is the spectrum of comparative example 2;

[0051] Fig.13 It is the spectrum of comparative example 11;

[0052] Fig.14 It is the spectrum of comparative example 9;

[0053] Fig.15 It is the spectrum of comparative example 10;

[0054] Fig.16 This is the spectrum of Example 12;

[0055] Fig.17 This is the insulin separation spectrum 1 of Example 4;

[0056] Fig.18 This is the second insulin separation spectrum of comparative example 3;

[0057] Fig.19 This is the third insulin separation spectrum of comparative example 4;

[0058] Fig. 20 This is the spectrum of Example 8;

[0059] Fig.21 It is the spectrum of comparative example 8;

[0060] Fig. 22 The chromatogram of Example 5;

[0061] Fig.23 It is the spectrum of comparative example 5;

[0062] Fig.24 The chromatogram of Example 5;

[0063] Fig.25 This is the spectrum of Comparative Example 5. DETAILED DESCRIPTION

[0064] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0065] As a basic embodiment of the present invention, the present invention provides a method for purifying a polypeptide, wherein the molecular weight of the polypeptide is between 2000-6000D;

[0066] It uses a high-precision high-performance liquid chromatography system, and is also equipped with a control cabinet, an axially pressurized liquid chromatography column (DAC), an eluent delivery system, a sample loading device, an online detection device, and an automatic fraction collection system;

[0067] Among them, the eluent delivery system consists of one or two pumps, and the flow error of the pump is within 1.5%;

[0068] The chromatographic column filler is a spherical mesoporous silica matrix reverse phase chromatographic filler, the column filling height is 20-100 cm, preferably 20-40 cm; the total content of other metals in the filler except silicon does not exceed 50 μg / g, the average pore size ranges from 90Å to 150Å, and the porosity is 0.8-1.0cm 3 / g, specific surface area is 280-380m 2 / g, with an average particle size ranging from 5 microns to 30 microns;

[0069] The proportion of micropores with a pore size below 70Å is less than 15%, and the proportion of micropores below 40Å is less than 5%;

[0070] The eluent used for separation is an aqueous solution containing a water-soluble acid, base or salt, or an organic solvent soluble in water.

[0071] Semaglutide was separated and purified by Examples 1-3 and Comparative Examples 1-2, and the effects of different micropore contents on sample purity, yield and filler life were compared.

[0072] Silica gel preparation with less than 15% of pores smaller than 70Å and less than 5% of pores smaller than 40Å;

[0073] Heat 10kg of silica sol to 50℃, add 15ml of concentrated nitric acid and 800g of urea, and stir to dissolve. Add 1000ml of formaldehyde under rapid stirring and continue stirring for 60s. Let it stand and a white precipitate will precipitate. After filtration, monodisperse silica microspheres are obtained. Tests are carried out by calcining at 650℃ for 5h and 800℃ for 10h. The particle size of the silica microspheres is 10μm, the pore size is 10nm, and the specific surface area is 382m 2 / g.

[0074] Take 100 g of conventionally calcined silica microspheres, add 1000 ml of 3% ammonia solution, stir at room temperature for 72 hours, cool down and wash with pure water until neutral, and bake at 100°C for 16 hours.

[0075] The baked silica gel was added with 1000 ml of 10% phosphoric acid aqueous solution, treated at 95°C for 24 h, washed with pure water to neutralize, and baked at 100°C for 16 h.

[0076] The desired silica microspheres are obtained after treatment with ammonia and acid.

[0077]

[0078] Semaglutide was separated and purified using the silica gel filler prepared by the above preparation method. The separation methods and conditions of Examples 1-3 and Comparative Examples 1-2 are shown in Table 2:

[0079]

[0080] The results are shown in Table 3:

[0081]

[0082] It can be seen from Examples 1, 2 and 3 that within the set main parameter ranges of column filling height, average particle size, average pore size, specific surface area and porosity, a relatively satisfactory separation effect can be obtained, which is reflected in purity and yield; while the lifespan (anti-pollution) is greatly affected by the particle size and pore size. The larger the particle size and pore size, the longer the lifespan.

[0083] Example 2 ( Fig.10 ) and Comparative Example 1 ( Fig.11 )、Comparative Example 2( Fig.12 ) comparison shows that: with the increase of the proportion of micropores, the separation effect becomes worse, which is ultimately reflected in the significant reduction in yield under the same purity requirements; on the other hand, due to the increase in micropores, the residual contamination of the filler increases, the number of times the filler is used decreases, that is, the life of the filler is shortened.

[0084] The mechanical strength (pressure resistance and shear resistance) of the filler in the preparative HPLC column for large molecular weight polypeptides in this embodiment is usually higher than that of the filler for analysis, so as to facilitate repeated use. Therefore, it is necessary to select a filler with high mechanical strength, long life and alkaline resistance.

[0085] The silica gel-based balls used in the present invention have been specifically improved in structure and manufacturing process, reducing the presence of micropores in the silica gel, which not only improves the separation and tailing of polypeptides, but also increases the skeleton density of the silica gel, ensuring the mechanical strength, life and alkali resistance of the silica gel.

[0086] Example 4, the effect of different micropore ratios on insulin separation (resolution and tailing factor).

[0087] The silica gel filler prepared by the above preparation method was used to separate and purify insulin. The results are shown in Figure 17-19 :

[0088]

[0089] According to Example 4 ( Fig.17 )、Comparative Example 3( Fig.18 ) and Comparative Example 4 ( Fig.19 ) It can be seen that the higher the proportion of micropores, the larger the tailing factor and the worse the separation effect.

[0090] The silica gel matrix chromatographic filler prepared by the above-mentioned embodiment method is a spherical material with an average pore size range of 90Å-150Å and a porosity of 0.8-1.0cm 3 / g, specific surface area is 280-380m 2 / g, and the average particle size range is 5 microns-30 microns; among them, the proportion of micropores with a pore size range of less than 70Å is less than 15%, and the proportion of micropores with a pore size range of less than 40Å is less than 5%. Based on the analysis of a large amount of experimental data, the present invention creatively provides a method and comprehensive parameter characteristics for simultaneously improving the separation effect, mechanical strength and alkali resistance of silica gel chromatographic fillers. Examples 1-4 and Comparative Examples 1-4 clearly show that the parameter optimization combination of silica gel fillers provided by the present invention provides excellent comprehensive results for the purification of large molecular polypeptides in terms of purification results, tailing improvement, strength and alkali resistance.

[0091] The reduction of micropore content in the silica gel skeleton will also reduce the surface area of ​​micropores that cannot be used for separation and purification but will be contaminated and corroded by alkali during use, thereby enhancing the service life and anti-pollution ability, and reducing tailing and even dead adsorption; the increase in the number of effective pores and specific surface area in the pore size range of 90Å-150Å in the filler also improves the purification and separation recovery rate of polypeptide compounds.

[0092] As for the particle size range, the particle size range of the silica gel-based chromatographic filler of the present invention is 5 microns to 30 microns. The use of spherical materials has both higher column efficiency and lower column pressure drop in a large-diameter chromatographic column. The use of a narrower range of particle sizes as fillers is not only easy to pack firmly, but also reduces the column pressure, so it is easier to obtain an efficient chromatographic column.

[0093] Example 5 Comparison of the separation purity, yield and lifespan of polypeptide G (molecular weight 4228 D) using micropores.

[0094] The separation method and conditions are as follows:

[0095]

[0096] The results are shown in Table 6:

[0097]

[0098] The silica gel filler C8-H (Example 5, Fig. 22 , Fig.24 ) and silica gel filler C8-B (Comparative Example 5, Fig.23 , Fig.25 ) on the separation purity, yield and lifespan of polypeptide G (molecular weight 4228 D).

[0099] According to the results in the above table, the silica gel filler of the present invention has a lower micropore ratio and a higher yield of the purified polypeptide G (molecular weight 4228 D), which is increased from 72.2% to 93.3%.

[0100] As another possible implementation, polar groups are embedded in the surface of the chromatographic filler to perform hybrid modification.

[0101] The polar group is selected from polar nitrogen-containing groups and / or carboxyl groups. Polar nitrogen-containing groups include amino groups, amine groups, amide groups, amino carbonate groups, and urea groups.

[0102] Furthermore, the chromatographic filler is bonded with two or more silane groups, wherein the silane groups that do not contain the above-mentioned nitrogen groups and / or polar groups such as carboxyl groups contain not less than 5 carbon atoms, and the proportion of the silane group with the lowest content is not less than 10%.

[0103] Furthermore, the silyl group refers to an alkylsiloxy group containing a polar group or not, including a siloxy group modified with a polar group; it also includes: octadecyldimethylsiloxy, octadecyldiisopropylsiloxy, octadecyldiisobutylsiloxy, hexadecyldimethylsiloxy, dodecyldimethylsiloxy, decyldimethylsiloxy, octyldimethylsiloxy, hexyldimethylsiloxy, butyldimethylsiloxy, propyldimethylsiloxy, isopropyldimethylsiloxy, isobutyldimethylsiloxy, aminopropyldimethylsiloxy, and aminoethyldimethylsiloxy.

[0104] Example 6 C8 isolated polypeptide GL, see Figure 2 .

[0105] Comparative Example 6, C8 / C20 isolated polypeptide GL, see Figure 3 .

[0106] Preparation of C8 / C20 silica gel chromatographic packing:

[0107] 1) Take 100 g of silica gel prepared in Example 1, 20 g of octyldimethylsilyl chloride, 30 g of eicosyldimethylsilane, and 500 ml of toluene, and reflux at 110°C for 16 hours. Cool to room temperature, filter, wash with toluene 3 times, 50 ml each time; wash with methanol 6 times, 50 ml each time; vacuum dry for 100 o C, 8 hours. C%=18.2%.

[0108] 2) Stir dried silica gel, 500 ml toluene for 10 minutes, heat up to dehydrate, cool to room temperature, add 50 g trimethylsilyl chloride, keep reflux at 110°C, reflux time 16 hours. Cool to room temperature, filter, wash with toluene 3 times, 50 ml each time; wash with methanol 6 times, 50 ml each time; vacuum dry at 100°C, 8 hours.

[0109] The separation conditions are shown in Table 7:

[0110]

[0111] The separation effect and yield of polypeptide GL under different bonding fillers are shown in Table 8:

[0112]

[0113] According to the above table and Figure 2 and Figure 3 It can be seen that the selectivity and separation of C8 / C20 mixed bonds and fillers for front and back impurities are significantly better than those of C8 single bond fillers.

[0114] Example 7, C8 10μm 120Å separation of crude hydrophobic decapeptide, see Figure 4 .

[0115] Comparative Example 7, AQ C8 / C20 (C20 12.7%) 10μm 120Å Separation of crude hydrophobic decapeptide, see Figure 5 .

[0116] The bonding method of silica gel is shown in Example 6

[0117] The hydrophobic decapeptide separation adjustment is shown in Table 9:

[0118]

[0119] The selectivity and yield of hydrophobic decapeptides under the action of fillers with different bonding modes are shown in Table 10:

[0120]

[0121] According to the above table and Figure 4 and Figure 5 Yes, mixed bonds and packing materials have significantly better selectivity and resolution for front and back impurities than single bonded packing materials.

[0122] Example 8 (C18-NH2 (NH2 accounts for 20.4%) separation of crude liraglutide peptide, the silica gel treatment and bonding operation refer to Example 6, the separation spectrum is shown in Fig. 20 .

[0123] Comparative Example 8, C18 (C18 bonding) separation of crude liraglutide peptide, see Fig.21 .

[0124] The chromatographic parameters and results of liraglutide separation are shown in Table 11:

[0125]

[0126] According to the above table and Fig. 20 and Fig.21 It can be seen that the mixed C18 bonded stationary phase containing 20.4% amino groups changes the separation of peptides and their isomers, homologues and other impurities, and can obtain better yields.

[0127] Further, the pH of the aqueous solution is between 1 and 12, and the added acid, base or salt includes trifluoroacetic acid, acetic acid, phosphoric acid, citric acid, sodium acetate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate, sodium carbonate, sodium bicarbonate, ammonium bicarbonate, ammonium acetate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, triethylamine, triethylammonium carbonate, and triethylammonium acetate.

[0128] Silica gel with low micropore content uses a new surface modification technology to embed polar groups into the surface of silica gel particles of the filler. The purification filler obtained by the above surface modification technology has shown advantages in separation in the purification of some peptides and can significantly improve the separation of target peptides and impurities. The reasons are:

[0129] 1. According to the principle of like dissolves like, the polar groups modified on the surface of silica gel have better similarity with polypeptides in composition and structure, which will be conducive to increasing the sample loading amount.

[0130] 2. There are unbonded silanol groups and impurity metals such as Al, Fe, Mn, Cr, etc. on the surface of silica gel. These unbonded silanol groups and impurity metals will have an adverse effect on separation and purification. They are generally called exposed sites. Capping these exposed sites with the above-mentioned polar groups can reduce excessive adsorption of peptides by the filler and improve the recovery rate.

[0131] 3. Improve selectivity through multiple selective superposition of different silanes.

[0132] The polar groups of this embodiment are polar nitrogen-containing groups and / or carboxyl groups, and the polar groups are embedded in the surface of the silica gel particles of the filler. The polar nitrogen-containing groups are preferably amino groups, and the amino groups include amino, amine, amide, amino carbonate, etc.

[0133] That is, the filler used in this embodiment can be prepared by embedding nitrogen-containing groups into C4-C21 hydrocarbon chains, or by using the above nitrogen-containing groups to cap the reverse phase filler. The nitrogen-containing groups include -NH-, -NR-, -NR2+, amide groups, urea groups or nitrogen-containing heterocyclic molecules.

[0134] It is understandable that the organic compounds targeted above are not limited to polypeptides, but may also be other organic compounds with a molecular weight of more than 2000D. In other words, the use of the chromatographic fillers of the above embodiments to purify organic compounds with a molecular weight of more than 2000D is within the protection scope of the present invention.

[0135] This embodiment provides a silica-based chromatographic filler with the above-mentioned characteristics, the particle size, pore size range, porosity, and specific surface area are improved compared with conventional silica-based chromatographic fillers, and polar or non-polar groups are embedded on the surface of the chromatographic filler, which can significantly increase the loading amount and purification and separation recovery rate of polypeptide compounds, as shown in the following embodiments and comparative examples:

[0136] Example 9: Chromatographic separation of semaglutide using silica gel packing embedded with amino polar groups.

[0137] In the synthesis of semaglutide, chemical synthesis is a relatively common synthesis method. Its main impurities are solvent residues, process impurities, degradation impurities, metal impurities, etc., which have high purification requirements. Compared with the conventional two-step purification method, the chromatographic filler of the silica matrix of this embodiment can be purified by a one-step purification method. While meeting the purity and yield requirements, it has a very high sample loading capacity, greatly reducing customer costs. The experimental plan is as follows:

[0138] S1. Preparation of C18 silica gel chromatographic packing embedded with amino polar groups

[0139] 1) 100 g of basic silica gel A and B in Example S1 above, 30 g of octadecyltrichlorosilane, 500 ml of toluene, reflux at 110°C for 16 hours. Cool to room temperature, filter, wash with toluene 3 times, 50 ml each time; wash with methanol 6 times, 50 ml each time; vacuum dry for 100 o C, 8 hours. C%=13.2%.

[0140] 2) Stir dried silica gel, 500 ml toluene for 10 minutes, heat up to dehydrate, cool to room temperature, add 50 g aminotrimethoxysilane, keep reflux at 110°C, reflux time 16 hours. Cool to room temperature, filter, wash with toluene 3 times, 50 ml each time; wash with methanol 6 times, 50 ml each time; vacuum dry at 100°C, 8 hours. C% = 15.3%.

[0141] S2. Sample purification

[0142] Dissolution of semaglutide sample: weigh 2 mg sample, add 1 mL pure water to dissolve, add 10 μL ammonia water to promote dissolution, filter and set aside for use.

[0143] Filling: Take the silica gel chromatographic filler 10μm 100Å prepared in S2 and fill it into a 10*250mm chromatographic column, test its column efficiency, and use it after it passes the test.

[0144] Mobile phase preparation: Mobile phase A: 20mM NH4HCO3 aqueous solution

[0145] Mobile phase B: ACN

[0146] Column equilibration: Use mobile phase A to equilibrate the column until the baseline is balanced.

[0147] Sample loading: Load 1% of the column filler volume at 3 mL / min.

[0148] Elution: Elution was performed according to the gradient shown in Table 12.

[0149]

[0150] Main peak collection and peak cutting: Peak cutting and main peak collection were performed according to the 220nm detection signal.

[0151] Fraction analysis: The collected fractions can be analyzed and the qualified fractions can be combined for subsequent operations.

[0152] Experimental results: The preparation of isolated semaglutide is shown in the figure Figure 1 shown.

[0153]

[0154] In this embodiment, the chromatographic filler of the silica gel matrix embedded with polar groups prepared by the basic silica gel B with low micropore content can be used for the purification and separation of semaglutide by a one-step purification method, with a purity of 99.87% and a yield of 76.22%. Compared with the modified silica gel prepared by the basic silica gel A with high micropore content, the purity and yield are greatly improved, which can meet the purity and yield requirements of semaglutide one-step purification, and greatly reduce customer costs.

[0155] Example 10, Separation of Salidroside Using C18 Silica Gel Chromatographic Filler Embedded with Amide Groups:

[0156] S1. Preparation of amide-embedded silica gel chromatographic filler.

[0157] 1) 100 g of the basic silica gel B in Example S1, 60 g of aminopropyltriethoxysilane, and 500 ml of toluene were refluxed at 110°C for 16 hours. The mixture was cooled to room temperature, filtered, and washed with toluene 3 times, 50 ml each time; washed with methanol 6 times, 50 ml each time; and vacuum dried for 100 o C, 8 hours. C%=1.5%.

[0158] 2) Stir dried silica gel, 500 ml toluene for 10 minutes, heat up to dehydrate, cool to room temperature, add 50 g octyl chloride, keep reflux at 110°C, reflux for 16 hours. Cool to room temperature, filter, wash with toluene 3 times, 50 ml each time; wash with methanol 6 times, 50 ml each time; vacuum dry for 100 o C, 8 hours. C%=7.8%.

[0159] S2. Isolation of Salidroside

[0160] like Figure 6 , conventional C18 separation salidroside fitting curve, Figure 7The fitting curve of separation of salidroside by C11 embedded with amide group shows the comparison of separation of salidroside by using conventional C18 silica gel chromatographic filler and C11 silica gel chromatographic filler embedded with amide group provided in this example.

[0161] 1. Comparison of packing parameters, column specifications and purification results

[0162]

[0163] 2. Separation of salidroside using conventional C18 silica gel chromatography filler

[0164]

[0165] 3. Separation of Salidroside Using the Amide-Embedded C11 Silica Gel Chromatographic Filler Provided in This Example

[0166]

[0167] By comparison, it can be seen that when the particle size, pore size, specific surface area, chromatographic column specifications and preparation conditions are the same, the loading amount, recovery rate and purity of the C11 silica gel chromatographic packing embedded with amide groups in the purification and separation of salidroside are 7.0%, 93.8%, and 4.38% respectively, compared with the conventional C18, which are significantly higher than the conventional C18 silica gel chromatographic packing by 3.5%, 81.9% and 90.45%.

[0168] That is to say, by embedding amide groups into the surface of C11 silica gel chromatographic filler particles, the selectivity of the silica gel chromatographic filler is significantly improved due to spatial isomerism.

[0169] Furthermore, a high-precision mass flow meter monitors the flow rate accurately in real time and feeds back to the control cabinet; the control cabinet instantly adjusts the flow rate to the set value to ensure the flow rate accuracy; and a high-precision mass flow meter is used to monitor the density of the recovered solvent in real time and feed back to the control cabinet. The control cabinet controls the solvent tank to add the required volume of the corresponding solvent according to the density of the recovered solvent, so that the density of the mixed solvent reaches the set value of the eluent density.

[0170] It can be understood that after the density of the recovered solvent is measured by a high-precision mass flow meter, it is compared with the density of the eluent and fed back to the control cabinet. The control cabinet calculates the required volume of the solvent based on the comparison result, and the control cabinet controls the valve of the corresponding solvent tank of the solvent tank group to open, and injects the required volume of the corresponding solvent into the solvent mixing tank, adjusts the proportion of the recovered solvent to the set proportion of the eluent, and then transfers it to the eluent tank to achieve solvent recovery and reuse.

[0171] Example 11, comparison between high-precision flow meter and ordinary flow meter in insulin purification and separation.

[0172]

[0173] The introduction of high-precision flow meters improves the stability of the preparation and purification process, resulting in higher purity and yield of purification, and reduces variables introduced into the process.

[0174] Furthermore, by precisely controlling the temperature of the chromatographic column and the eluent and making them consistent, the peak shape and separation effect can be effectively improved, as shown in Example 12.

[0175] Furthermore, the temperature of the chromatographic column and the eluent is controlled between 10°C and 30°C, and the chromatographic column and the eluent are kept constant and the temperature is consistent, and the temperature difference between the eluent and the chromatographic column is within ±3°C.

[0176]

[0177] From Example 12 ( Fig.16 ), Comparative Example 9 ( Fig.14 )、Comparative Example 10( Fig.15 ) and Comparative Example 11 ( Fig.13 ) It can be seen that changes in column temperature will lead to peak broadening. Mobile phase and column temperature control need to be precisely controlled within a certain range to obtain the best separation; increasing the temperature of the column or eluent will weaken the retention of the main peak, and the impurities before the peak cannot be effectively separated; decreasing the temperature of the column or eluent will increase the retention time, prolong the separation time, and deteriorate the separation from the subsequent impurities; if there is no proper column thermostat, a radial temperature gradient will form in the column, affecting the separation efficiency (theoretical plate number, N) and analyte retention. When there is a large temperature difference between the eluent and the chromatographic column itself, the peak will be deformed and the separation will decrease.

[0178] In Example 12, when the chromatographic column and the eluent were both maintained at 20° C., the main peak was separated from both the front impurities and the back impurities.

[0179] In Comparative Example 9, when the temperature of the chromatographic column and the eluent were both lowered to 10° C., the sample retention time increased, and the front impurities were separated and the rear impurities were encapsulated.

[0180] In Comparative Example 10, when the temperature of the chromatographic column and the eluent were both increased to 40° C., the sample retention time decreased, and the main peak and the front and rear impurities were not separated.

[0181] In Comparative Example 11, the eluent temperature was lowered to 5°C, the chromatographic column was maintained at 20°C, the sample retention time increased, but the separation of the impurities before and after became worse.

[0182] Further, such as Figure 8-9 As shown, the axially pressurized chromatographic column adopts an oil-free cylinder type chromatographic column.

[0183] It is understandable that the purpose of separation and purification is to obtain a target polypeptide with high purity, and the oil-free cylinder chromatographic column can effectively avoid contamination of the separated sample caused by oil leakage.

[0184] Further, the fractions flowing out of the chromatographic column are tested, the fractions without the target polypeptide are subjected to solvent recovery, and the fractions with the target polypeptide are subjected to target collection;

[0185] The fractions containing the target polypeptide are tested for target purity, and the fractions whose target purity reaches the preset standard are subjected to vacuum freeze drying to remove the solvent to obtain the target solid; the fractions whose target purity does not reach the preset standard but exceeds the preset purity of the recoverable fraction are subjected to solvent recovery.

[0186] Furthermore, the fractions without the target peptide are proportion-confirmed by a high-precision mass flow meter, and the fractions that meet the set ratio of the eluent are directly recovered; the fractions that do not meet the set ratio of the eluent are adjusted to the set ratio of the eluent and then recovered;

[0187] The fraction whose purity reaches the preset standard is evaporated by vacuum in a closed system, and the solvent in the fraction is separated by vacuum evaporation, and then condensed into a liquid solvent mixture after cooling; the proportion is confirmed by a high-precision mass flow meter, and the mixture is recovered after adjusting to the set proportion of the eluent.

[0188] Furthermore, the eluent is a homogeneous solution containing a volatile organic solvent and water, and the organic solvent is methanol, ethanol, acetonitrile, acetone or tetrahydrofuran.

[0189] Furthermore, the eluent is pure water or an aqueous solution, to which an acid, a base or a salt is added, and the acid, a base or a salt is formic acid, acetic acid, trifluoroacetic acid, ammonia water, triethylamine, ammonium acetate, ammonium carbonate or triethylammonium acetate.

[0190] Furthermore, the proportion of micropores in the chromatographic column packing with a pore size range below 40Å is less than 5%.

[0191] In order to achieve accurate separation of large molecular weight polypeptides (2000D-6000D), the present invention adopts the following key innovations in the purification method:

[0192] 1. By using a precision-controlled eluent infusion pump, the eluent flow rate during purification is precisely regulated to ensure that the flow error is within 1.5%; and the flow rate is accurately measured online through a high-precision mass flow meter, and when the flow rate fluctuates, the flow rate can be accurately adjusted instantly through the control cabinet;

[0193] 3. The present invention also effectively reduces tailing, improves peak shape and separation effect by regulating the consistency and accuracy of the chromatographic column temperature and the eluent temperature (the temperature can be selected between 10°C and 30°C, and the temperature difference between the eluent and the chromatographic column is within ±3°C);

[0194] 4. Through online detection, automatic control of fraction collection and solvent recovery control are realized; online liquid preparation is realized based on precise density monitoring, which improves the accuracy, efficiency and stability of peptide purification.

[0195] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0196] It is not difficult for those skilled in the art to understand that the present invention includes any combination of the invention content and specific implementation methods of the above specification and the various parts shown in the drawings. Due to the limited space and to make the specification concise, the various schemes composed of these combinations are not described one by one. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0197] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for purifying a polypeptide, characterized in that: The molecular weight of polypeptides ranges from 2000-6000D; It adopts a high-precision high-performance liquid chromatography system, and is also equipped with a control cabinet, an axially pressurized liquid chromatography column, an eluent delivery system, a sample loading device, an online detection device, and an automatic fraction collection system; Among them, the eluent delivery system consists of one or two pumps, and the flow error of the pump is within 1.5%; The chromatographic column filler is a spherical mesoporous silica matrix reverse phase chromatographic filler, and the column filling height is 20-100 cm; the total content of other metals in the filler except silicon does not exceed 100ug / g, the average pore size ranges from 90Å to 150Å, and the porosity is 0.8-1.0cm 3 / g, specific surface area is 280-380m 2 / g, with an average particle size ranging from 5 microns to 30 microns; The proportion of micropores with a pore size range below 70Å is less than 15%; the proportion of micropores with a pore size range below 40Å is less than 5%; The temperature of the chromatographic column and the eluent is controlled between 10℃-30℃, and the chromatographic column and the eluent are kept constant and the temperature is consistent. The temperature difference between the eluent and the chromatographic column is within ±3℃; The eluent used for separation is an aqueous solution containing a water-soluble acid, base or salt, or an organic solvent soluble in water.

2. The method for purifying a polypeptide according to claim 1, characterized in that: Polar groups are embedded in the surface of the chromatographic filler and hybridized and modified, and polar nitrogen-containing groups are selected as polar groups; the chromatographic filler is bonded with two or more silane groups, among which the silane groups that do not contain the above polar groups contain not less than 5 carbon atoms, and the proportion of the silane group with the lowest content is not less than 10%.

3. The method for purifying a polypeptide according to claim 2, characterized in that The polar nitrogen-containing group is selected from an amino group and an amide group.

4. The method for purifying a polypeptide according to claim 2, characterized in that: The silyl group is selected from octadecyldimethylsiloxy, octadecyldiisopropylsiloxy, octadecyldiisobutylsiloxy, hexadecyldimethylsiloxy, dodecyldimethylsiloxy, decyldimethylsiloxy, octyldimethylsiloxy, hexyldimethylsiloxy, butyldimethylsiloxy, propyldimethylsiloxy, isopropyldimethylsiloxy, isobutyldimethylsiloxy, aminopropyldimethylsiloxy, and aminoethyldimethylsiloxy.

5. The method for purifying a polypeptide according to claim 1, characterized in that: The pH of the aqueous solution is between 1 and 12, and the added acid, base or salt includes trifluoroacetic acid, acetic acid, phosphoric acid, citric acid, sodium acetate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate, sodium carbonate, sodium bicarbonate, ammonium bicarbonate, ammonium acetate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, triethylamine, triethylammonium carbonate, and triethylammonium acetate.

6. The method for purifying a polypeptide according to claim 1, characterized in that: The high-precision mass flow meter monitors the flow rate accurately in real time and feeds back to the control cabinet; the control cabinet immediately adjusts the flow rate to the set value to ensure the flow rate accuracy; and the high-precision mass flow meter monitors the density of the recovered solvent in real time and feeds back to the control cabinet. The control cabinet controls the solvent tank to add the required volume of the corresponding solvent according to the density of the recovered solvent, so that the density of the mixed solvent reaches the set value of the eluent density.

7. The method for purifying a polypeptide according to claim 1, characterized in that: The axially pressurized chromatographic column adopts an oil-free cylinder type chromatographic column.

8. The method for purifying a polypeptide according to claim 1, characterized in that: The fractions flowing out of the chromatographic column are tested, and the fractions without the target polypeptide are used for solvent recovery, and the fractions with the target polypeptide are used for target collection; The fractions containing the target polypeptide are tested for target purity, and the fractions whose target purity reaches the preset standard are subjected to vacuum freeze drying to remove the solvent to obtain the target solid; the fractions whose target purity does not reach the preset standard but exceeds the preset purity of the recoverable fraction are subjected to solvent recovery.

9. The method for purifying a polypeptide according to claim 8, characterized in that: The fractions without the target peptide are confirmed by a high-precision mass flow meter. The fractions that meet the set ratio of the eluent are directly recovered; the fractions that do not meet the set ratio of the eluent are adjusted to the set ratio of the eluent and then recovered; The fraction whose purity reaches the preset standard is evaporated by vacuum in a closed system, and the solvent in the fraction is separated by vacuum evaporation, and then condensed into a liquid solvent mixture after cooling; the proportion is confirmed by a high-precision mass flow meter, and the mixture is recovered after adjusting to the set proportion of the eluent.

10. The method for purifying a polypeptide according to claim 1, characterized in that: The eluent is a homogeneous solution containing a volatile organic solvent and water, and the organic solvent is methanol, ethanol, acetonitrile, acetone or tetrahydrofuran.

11. The method for purifying a polypeptide according to claim 1, characterized in that: The eluent is pure water or an aqueous solution, to which an acid, a base or a salt is added, and the acid, a base or a salt is formic acid, acetic acid, trifluoroacetic acid, ammonia water, triethylamine, ammonium acetate, ammonium carbonate or triethylammonium acetate.

Images