Method for filling thin and high chromatographic column

Abstract

The invention relates to the technical field of separation, purification and analysis, in particular to a method for filling a thin and high type chromatographic column, the method can improve the column filling success rate of the thin and high type chromatographic column, the filler reaches the ideal height, meanwhile, the good column efficiency is achieved, and the method is simple, easy to implement, efficient, stable and low in cost.

Description

Technical Field

[0001] This application relates to the technical field of separation, purification and analysis, and relates to a method for packing a chromatography column. More specifically, this application relates to a method for packing a slender and tall chromatography column. Background Art

[0002] Liquid chromatography (also known as liquid chromatography) is a commonly used means of analysis, separation and purification in the early-stage R & D of products and industrial production. In liquid chromatography technology, due to the different physicochemical properties of the substances to be separated dissolved in the mobile phase, such as structure, size, charge, hydrophobicity, etc., the interactions with the packing material when flowing through the stationary phase (i.e., the packing in the chromatography column) are also different in terms of size and strength, that is, the retention time of the substances to be separated in the packing is different. Therefore, the substances to be separated flow out of the packing successively, so as to achieve the purpose of analysis and separation. According to the different forms of the stationary phase, liquid chromatography is divided into column chromatography, paper chromatography, thin-layer chromatography, etc. According to the different adsorption forces, liquid chromatography can be divided into adsorption chromatography, partition chromatography, gel permeation chromatography, ion exchange chromatography, affinity chromatography, etc.

[0003] Column chromatography is widely used in the separation and purification of biological products such as monoclonal antibodies, recombinant proteins and polypeptides. The stationary phase of column chromatography plays an indispensable role in separation and purification. There is a rich variety of packing materials for column chromatography on the market, and commonly used ones include silica gel packing, chiral packing, ion exchange chromatography packing, polymer packing and other inorganic packing materials, etc. According to the physicochemical properties of the substances to be separated, selecting a suitable chromatography packing will make the sample separation and purification get twice the result with half the effort. In addition, the packing effect of the chromatography column is the key factor determining the separation efficiency of the chromatography column. Poor packing effect will lead to low resolution of the chromatography column, poor yield and quality of the separated substances, etc.

[0004] In the early development stage of biopharmaceutical R & D, in order to reduce the material cost, a chromatography column with a smaller inner diameter (for example, a slender and tall chromatography column) can be selected for analysis, separation and purification. However, there are many challenges in the use of slender and tall chromatography columns. For example, during the packing process of the packing material in the slender and tall chromatography column, conventional packing liquid is prone to the phenomenon of packing sticking to the wall. At the same time, the wall effect formed by the conventional packing method is also extremely serious, and it is difficult for the packing material to achieve a uniform and compact effect in the chromatography column, resulting in low column efficiency; in addition, the pressure in the slender and tall chromatography column is prone to be too high, affecting the separation effect.

[0005] To solve the deficiencies of the prior art, this application provides a method for packing a slender and tall chromatography column, which can improve the packing success rate of the slender and tall chromatography column, and make the packing material reach the ideal height while having good column efficiency. Summary of the Invention

[0006] The technical problem to be solved by this application is to provide a method for loading a tall and slender chromatography column in view of the deficiencies in the prior art. The method can make the packing in the chromatography column evenly distributed, significantly improve the column efficiency and separation efficiency, is simple and easy to implement, and has low cost. In some embodiments, the method includes a gas blowing and / or suction step, for example, gas blowing and / or suction is carried out into the chromatography column through a blowing and suction device.

[0007] In some specific embodiments, the method for loading a tall and slender chromatography column includes but is not limited to the following steps:

[0008] (1) Prepare a packing slurry with a packing preservation liquid and the packing.

[0009] (2) Add the packing slurry from the upper end of the chromatography column, and use a blowing and suction device to blow air into the packing in the chromatography column.

[0010] (3) Add the packing slurry from the upper end of the chromatography column, and use a blowing and suction device to suck the solution in the chromatography column.

[0011] Among them, the steps (1)-(3) are carried out in sequence.

[0012] In some specific embodiments, the method for loading a tall and slender chromatography column includes but is not limited to the following steps:

[0013] (1) Prepare a packing slurry with a packing preservation liquid and the packing, and the homogenization ratio of the packing slurry is 30%-70%.

[0014] (2) Add the packing slurry from the upper end of the chromatography column, and use a blowing and suction device to blow air into the packing in the chromatography column.

[0015] (3) Add the packing slurry from the upper end of the chromatography column, and use a blowing and suction device to suck the solution in the chromatography column, and repeat the steps until all the packing is added to the chromatography column.

[0016] (4) Add the equilibration liquid from the upper end of the chromatography column, and use a blowing and suction device to suck the solution in the chromatography column until there is no obvious dispersion on the upper gel surface of the packing.

[0017] Among them, the steps (1)-(4) are carried out in sequence.

[0018] In some specific embodiments, the method for loading a tall and slender chromatography column includes but is not limited to the following steps:

[0019] (1) Use the equilibration liquid to remove the air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom, and fill the lower column head of the chromatography column with the equilibration liquid.

[0020] (2) Prepare a packing homogenate with a ratio of packing preservation liquid to packing of 30%-70%, and connect the blowing and suction device to the lower end of the pipeline at the bottom of the lower column head;

[0021] (3) Add the packing homogenate from the upper end of the chromatography column, and use the blowing and suction device to blow air into the packing in the chromatography column;

[0022] (4) Add the packing homogenate from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column. Repeat the above steps until all the packing is added to the chromatography column;

[0023] (5) Add the equilibration liquid from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column until there is no obvious dispersion on the upper gel surface of the packing;

[0024] (6) Use the equilibration liquid to remove the air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with the equilibration liquid;

[0025] (7) Press down the upper column head to a position 1-2 cm away from the upper gel surface, and use the equilibration liquid to press the upper gel surface of the packing forward until the upper gel surface of the packing no longer drops and a clear solution layer appears between the upper gel surface of the packing and the upper column head, then stop;

[0026] (8) Use the equilibration liquid to press the lower gel surface of the packing backward until the upper gel surface of the packing moves up and a clear solution layer appears between the lower column head and the lower gel surface of the packing, then stop;

[0027] (9) Press up the lower column head;

[0028] (10) Use the equilibration liquid to press the upper gel surface of the packing forward, and press down the upper column head to make the height of the packing reach the target column packing height;

[0029] (11) Use the equilibration liquid to press the upper gel surface of the packing forward;

[0030] Among them, the steps (1)-(11) are carried out in sequence. Among them, the steps (1) and (2) can be interchanged.

[0031] In some specific embodiments, the packing material may be selected from affinity chromatography packing materials, hydrophobic chromatography packing materials, ion exchange chromatography packing materials, composite mode packing materials, and gel filtration packing materials. Ion exchange chromatography packing materials and affinity chromatography packing materials are preferred, and MabSelect SuRe packing material, MabSelect SuRe LX packing material, SP Sepharose FF packing material, Novo-A Diamond packing material, AT Protein A Diamond Plus packing material, MabPurix packing material, ProSep Ultra Plus packing material, VIIISelect packing material, Capto Adhere packing material, POROS 50HQ packing material, POROS XS packing material, Fractogel EMD TMAE packing material, Q-Sepharose High Performance packing material, Capto Butyl ImpRes packing material, Capto adhere packing material, NM90 Agarose HAM packing material, UniHR Phenyl-60s packing material, Diamond Q packing material, NanoGel-50SP HP packing material, NanoGel-50SP packing material, NanoGel-50Q packing material, MegaPoly 50XS packing material, NanoGel-50Q HC packing material, or MaXtar MMC packing material are further preferred.

[0032] In some specific embodiments, the equilibration solution may be selected from sodium chloride solutions, acetate solutions, phosphate solutions, Tris buffers, water for injection, sodium chloride solutions containing ethanol solutions, acetate solutions containing sodium chloride, phosphate solutions containing sodium chloride, or Tris buffers containing sodium chloride. In some specific embodiments, the equilibration solution may be selected from sodium chloride solutions with a concentration less than or equal to 1 M (e.g., 0.05 M or 0.1 M sodium chloride solution), acetate solutions with a concentration less than or equal to 1 M (e.g., 0.2 M sodium acetate solution, 0.1 M sodium acetate solution), phosphate solutions with a concentration less than or equal to 1 M (e.g., 0.05 M phosphate solution), Tris buffers, water for injection, acetate solutions containing sodium chloride, phosphate solutions containing sodium chloride, sodium chloride solutions containing ethanol solutions (e.g., 0.2 M - 0.5 M sodium chloride solution containing 20% ethanol), or Tris buffers containing sodium chloride. In some specific embodiments, the equilibration solution is water for injection. In some specific embodiments, the equilibration solution is a sodium chloride solution with a concentration less than or equal to 1 M. In some specific embodiments, the equilibration solution is 0.1 M sodium chloride solution.

[0033] In some specific embodiments, the homogenization ratio is 30%-70%, such as 30%, 32%, 34%, 35%, 36%, 38%, 40%, 42%, 44%, 45%, 46%, 48%, 50%, 52%, 54%, 55%, 56%, 58%, 60%, 62%, 64%, 65%, 66%, 68%, 70% or the range formed by any of these values. In some specific embodiments, the homogenization ratio is 40%-60%. In some specific embodiments, the homogenization ratio is 40-50%.

[0034] In some specific embodiments, the rate of air blowing is 1-10 mL / min, such as 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min or the range formed by any of these values. In some specific embodiments, the rate of air blowing is 1-5 mL / min. In some specific embodiments, the rate of air blowing is 3-5 mL / min.

[0035] In some specific embodiments, the number of times of air blowing is 1-10 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or the range formed by any of these values. In some specific embodiments, the number of times of air blowing is 1-5 times. In some specific embodiments, the number of times of air blowing is 1-3 times.

[0036] In some specific embodiments, the gas blown out in the air blowing step is air or oxygen.

[0037] In some specific embodiments, the rate of suction is 1-10 mL / min, such as 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min or the range formed by any of these values. In some specific embodiments, the rate of suction is 1-5 mL / min. In some specific embodiments, the rate of suction is 3-5 mL / min.

[0038] In some specific embodiments, the rate of adding the balance liquid is 1 - 10 mL / min, such as 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min or the range formed by any of these values. In some specific embodiments, the rate of adding the balance liquid is 1 - 5 mL / min. In some specific embodiments, the rate of adding the balance liquid is 3 - 5 mL / min.

[0039] In some specific embodiments, the rate of the "forward stamping" is a linear velocity of 10 - 500 cm / h, such as 10 cm / h, 20 cm / h, 30 cm / h, 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 220 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 280 cm / h, 300 cm / h, 350 cm / h, 400 cm / h, 450 cm / h, 500 cm / h or the range formed by any of these values. In some specific embodiments, the rate of the "forward stamping" is a linear velocity of 50 - 150 cm / h. In some specific embodiments, the rate of the "forward stamping" is a linear velocity of 100 cm / h.

[0040] In some specific embodiments, the rate of the "reverse stamping" is a linear velocity of 10 - 500 cm / h, such as 10 cm / h, 20 cm / h, 30 cm / h, 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 220 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 280 cm / h, 300 cm / h, 350 cm / h, 400 cm / h, 450 cm / h, 500 cm / h or the range formed by any of these values. In some specific embodiments, the rate of the "reverse stamping" is a linear velocity of 50 - 150 cm / h. In some specific embodiments, the rate of the "reverse stamping" is a linear velocity of 100 cm / h.

[0041] In some specific embodiments, the "upper pressing on the column head" is to press the column head down by 1 - 10 cm or until slight damping appears and then stop. For example, press the column head down by 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4.5 cm, 5 cm, 5.5 cm, 6 cm, 6.5 cm, 7 cm, 7.5 cm, 8 cm, 8.5 cm, 9 cm, 9.5 cm, 10 cm or stop after the range formed by any of the above values. In some specific embodiments, the "upper pressing on the column head" is to press the column head down by 1 - 5 cm, 1 - 4 cm, 1 - 3 cm, 1 - 2 cm or until slight damping appears and then stop. In some specific embodiments, the "upper pressing on the column head" is to press the column head down by 1 - 5 cm or until slight damping appears and then stop.

[0042] In some specific embodiments, the blowing and suction device is separable from the chromatography column.

[0043] In some specific embodiments, the blowing and suction device is a syringe. In some specific embodiments, the blowing and suction device is a 10 mL disposable sterile syringe, and the syringe does not include a needle when used in this application.

[0044] In some specific embodiments, the blowing and suction device is connected to the lower end of the pipeline at the bottom of the lower column head.

[0045] In some specific embodiments, the target column filling height is 5 - 100 cm, such as 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 31 cm, 32 cm, 33 cm, 34 cm, 35 cm, 36 cm, 37 cm, 38 cm, 39 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 95 cm or 100 cm or the range formed by any of the above values. In some specific embodiments, the target column filling height is 5 - 80 cm. In some specific embodiments, the target column filling height is 5 - 60 cm. In some specific embodiments, the target column filling height is 5 - 40 cm. In some specific embodiments, the target column filling height is 5 - 35 cm. In some specific embodiments, the target column filling height is 5 - 25 cm.

[0046] In some specific embodiments, after the above steps, column efficiency testing is performed.

[0047] In some specific embodiments, the method for packing a tall and slender chromatography column includes, but is not limited to, the following steps:

[0048] (1) Use the equilibration solution to remove the air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom, and fill the lower column head of the chromatography column with the equilibration solution;

[0049] (2) Prepare a slurry of packing material with a homogenization ratio of 30%-70% using the packing preservation solution and the packing material, and connect the blowing and suction device to the lower end of the pipeline at the bottom of the lower column head;

[0050] (3) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to blow air into the packing in the chromatography column at a blowing rate of 1-10 mL / min;

[0051] (4) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column at a suction rate of 1-10 mL / min. Repeat the above steps until all the packing material is added to the chromatography column;

[0052] (5) Add the equilibration solution from the upper end of the chromatography column at a rate of 1-10 mL / min, and use the blowing and suction device to suck the solution in the chromatography column at a suction rate of 1-10 mL / min until there is no obvious dispersion on the upper gel surface of the packing;

[0053] (6) Use the equilibration solution to remove the air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with the equilibration solution;

[0054] (7) Press down the upper column head to a position 1-2 cm away from the upper gel surface, and use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 10-500 cm / h until the upper gel surface of the packing no longer descends and there is an obvious clear solution layer between the upper gel surface and the upper column head, then stop;

[0055] (8) Use the equilibration solution to press the lower gel surface of the packing backward at a linear velocity of 10-500 cm / h until the upper gel surface of the packing moves upward and there is an obvious clear solution layer between the lower column head and the lower gel surface of the packing, then stop;

[0056] (9) Press the lower column head upward by 1-10 cm or until a slight damping appears, then stop;

[0057] (10) Use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 10-500 cm / h, and press down the upper column head to make the height of the packing reach the target column packing height;

[0058] (11) Use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 10-500 cm / h;

[0059] Among them, the steps (1)-(11) are carried out in sequence. Among them, the steps (1) and (2) can be reversed.

[0060] In some specific embodiments, the method for packing a slender chromatography column includes, but is not limited to, the following steps:

[0061] (1) Use the equilibration solution to remove the air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom, and fill the lower column head of the chromatography column with the equilibration solution;

[0062] (2) Prepare a packing slurry with a homogenization ratio of 40%-60% using the packing preservation solution and the packing, and connect the blowing and suction device to the lower end of the pipeline at the bottom of the lower column head;

[0063] (3) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to blow the packing in the chromatography column, with a blowing rate of 3-5 mL / min;

[0064] (4) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column, with a suction rate of 3-5 mL / min. Repeat the above steps until all the packing is added to the chromatography column;

[0065] (5) Add the equilibration solution from the upper end of the chromatography column at a rate of 3-5 mL / min, and use the blowing and suction device to suck the solution in the chromatography column, with a suction rate of 3-5 mL / min, until there is no obvious dispersion on the upper gel surface of the packing;

[0066] (6) Use the equilibration solution to remove the air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with the equilibration solution;

[0067] (7) Press down the upper column head to a position 1-2 cm away from the upper gel surface, and use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 100 cm / h until the upper gel surface of the packing no longer descends and a clear solution layer appears between the upper column head and the packing, and then stop;

[0068] (8) Use the equilibration solution to press the lower gel surface of the packing backward at a linear velocity of 100 cm / h until the upper gel surface of the packing moves upward and a clear solution layer appears between the lower column head and the lower gel surface of the packing, and then stop;

[0069] (9) Press up the lower column head by 1-5 cm or until a slight damping appears and then stop;

[0070] (10) Use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 100 cm / h, and press down the upper column head to make the height of the packing reach the target column packing height;

[0071] (11) Use the equilibration solution to press the upper gel surface of the packing forward at a linear velocity of 100 cm / h;

[0072] Among them, the steps (1)-(11) are carried out in sequence. Among them, the steps (1) and (2) can be reversed.

[0073] In some specific embodiments, the method for loading a slender chromatography column includes but is not limited to the following steps:

[0074] (1) Use water for injection to remove air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom, and fill the lower column head of the chromatography column with water for injection;

[0075] (2) Prepare a packing slurry with a packing preservation liquid and packing at a slurry ratio of 40%-60%, and connect the blowing and suction device to the lower end of the pipeline at the bottom of the lower column head;

[0076] (3) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to blow air into the packing in the chromatography column at a blowing rate of 3-5 mL / min;

[0077] (4) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column at a suction rate of 3-5 mL / min. Repeat the above steps until all the packing is added to the chromatography column;

[0078] (5) Add water for injection from the upper end of the chromatography column at a rate of 3-5 mL / min, and use the blowing and suction device to suck the solution in the chromatography column at a suction rate of 3-5 mL / min until there is no obvious dispersion on the upper side of the packing;

[0079] (6) Use water for injection to remove air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with water for injection;

[0080] (7) Press down the upper column head to a position 1-2 cm away from the upper packing surface, and use water for injection to punch the upper side of the packing surface in the positive direction at a linear velocity of 100 cm / h until the upper side of the packing surface no longer drops and a clear solution layer appears between the upper side of the packing surface and the upper column head, then stop;

[0081] (8) Use water for injection to punch the lower side of the packing surface in the reverse direction at a linear velocity of 100 cm / h until the upper side of the packing surface moves up and a clear solution layer appears between the lower column head and the lower side of the packing surface, then stop;

[0082] (9) Press up the lower column head by 1-5 cm or until a slight damping appears, then stop;

[0083] (10) Use water for injection to punch the upper side of the packing surface in the positive direction at a linear velocity of 100 cm / h, and press down the upper column head to make the height of the packing reach the target column loading height;

[0084] (11) Press the upper rubber surface of the packing forward at a linear velocity of 100 cm / h with 0.1 M sodium chloride solution;

[0085] Among them, the steps (1)-(11) are carried out in sequence. Among them, the steps (1) and (2) can be interchanged.

[0086] In some specific embodiments, the blowing and suction device is separable from the chromatography column.

[0087] In some specific embodiments, the blowing and suction device is a syringe. In some specific embodiments, the blowing and suction device is a 10 mL disposable sterile syringe, and the syringe does not include a needle when used in this application.

[0088] In some specific embodiments, the number of times of air blowing is 1-10 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or the range formed by any of these values. In some specific embodiments, the number of times of air blowing is 1-5 times. In some specific embodiments, the number of times of air blowing is 1-3 times.

[0089] In some specific embodiments, the target column packing height is 5-100 cm, such as 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 31 cm, 32 cm, 33 cm, 34 cm, 35 cm, 36 cm, 37 cm, 38 cm, 39 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 95 cm or 100 cm or the range formed by any of these values. In some specific embodiments, the target column packing height is 5-80 cm. In some specific embodiments, the target column packing height is 5-60 cm. In some specific embodiments, the target column packing height is 5-40 cm. In some specific embodiments, the target column packing height is 5-35 cm. In some specific embodiments, the target column packing height is 5-25 cm.

[0090] In some specific embodiments, after the above steps, column efficiency testing is carried out.

[0091] The beneficial effects of this application are as follows:

[0092] (1) The method for packing a slender and tall chromatography column in this application can make the packing distribution uniform, with a high success rate of column packing. It enables the packing to reach the ideal height while having good column efficiency, solving the problem of low success rate caused by wall effects and excessive column pressure in the traditional chromatography column packing method, and overcoming the defects of low success rate and poor column efficiency of slender and tall chromatography columns.

[0093] (2) The method for packing a slender and tall chromatography column in this application provides a simple, easy-to-implement, efficient, stable and low-cost means for the early research stage.

[0094] Glossary of Terms

[0095] Unless otherwise defined, the meanings of the technical and scientific terms described in this application are the same as those commonly understood by those of ordinary skill in the art. In the case of multiple definitions for a term in the present invention, the definitions described in this section shall be adopted unless otherwise defined.

[0096] "Packing" is sometimes also referred to as "gel" in this application. The "packing" or "gel" in this application includes, but is not limited to, materials for realizing ion exchange chromatography separation, affinity chromatography separation and reverse phase chromatography separation.

[0097] "Chromatography column" in this application refers to a structure for separating the components of a mixture by bringing the mixture into contact with a solid or liquid exchange medium. The "tall and thin chromatography column" in this application is known or can be determined by those skilled in the art, that is, a chromatography column with an aspect ratio greater than or equal to 5:1, and the inner diameter of the column body of the chromatography column is less than or equal to 50 mm, for example, less than or equal to 40 mm, less than or equal to 35 mm, less than or equal to 30 mm, less than or equal to 25 mm, less than or equal to 20 mm. Also, for example, the inner diameter is 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, 50 mm or a range formed by any of the above values; also, for example, the inner diameter is 2.1 mm, 4.6 mm, 5.7 mm, 6.6 mm, 7.8 mm, 8.9 mm, 12.9 mm, 15.6 mm, 19.4 mm, 23.5 mm, 26.4 mm, 49.4 mm or a range formed by any of the above values.Common suppliers and applicable specifications include, but are not limited to, Lisui Technology Co., Ltd. (specifications of chromatography columns such as: length 200mm, inner diameter 15mm; length 400mm, inner diameter 15mm; length 600mm, inner diameter 15mm; length 800mm, inner diameter 15mm; length 1000mm, inner diameter 15mm; length 200mm, inner diameter 25mm; length 400mm, inner diameter 25mm; length 600mm, inner diameter 25mm; length 800mm, inner diameter 25mm; length 1000mm, inner diameter 25mm; length 200mm, inner diameter 40mm; length 400mm, inner diameter 40mm; length 600mm, inner diameter 40mm; length 800mm, inner diameter 40mm; length 1000mm, inner diameter 40mm; length 400mm, inner diameter 50mm; length 600mm, inner diameter 50mm; length 800mm, inner diameter 50mm; length 1000mm, inner diameter 50mm, etc.), Hanbang Technology Co., Ltd. (specifications of chromatography columns such as: length 400mm, inner diameter 16mm; length 200mm, inner diameter 16mm; length 200mm, diameter 26mm, etc.), Shanghai Chenqiao Biotechnology Co., Ltd. (specifications of chromatography columns such as: length 200mm, inner diameter 10mm; length 330mm, inner diameter 10mm), Suzhou Sepax Technologies Co., Ltd. (specifications of chromatography columns such as: length 250mm, inner diameter 10mm; length 400mm, inner diameter 6.6mm; length 250mm, inner diameter 6.6mm, etc.), Bailinke Pharmaceutical Equipment Technology Co., Ltd. (specifications of chromatography columns such as: length 400mm, inner diameter 10mm; length 400mm, inner diameter 16mm; length 400mm, inner diameter 26mm; length 400mm, inner diameter 50mm, etc.), Omnifit (specifications of chromatography columns such as: length 250mm, inner diameter 10mm; length 250mm, inner diameter 6.6mm, etc.), etc.

[0098] In this application, the "rubber surface" refers to the uppermost or lowermost layer of the packing material in the chromatography column during the packing process of the chromatography column.

[0099] "About" In this application, "about" means fluctuating within the range of ±0.05 of the given specific numerical range, preferably within the range of ±0.02, and more preferably within the range of ±0.01. For example, a distance of about 0.5 cm means a distance of 0.5 ± 0.05 cm, preferably a distance of 0.5 ± 0.02 cm, and more preferably a distance of 0.5 ± 0.01 cm.

[0100] "Column efficiency" refers to the packing effect of the chromatography column, which is mainly measured by two indicators: "the number of theoretical plates per meter N / m" (number of theoretical plates) and "asymmetry factor AS" (Asymmetry Factor). The "number of theoretical plates" mainly reflects the separation effect of the chromatography column. N depends on the type and properties of the stationary phase (particle size, particle size distribution, etc.), packing condition, column length, type and flow rate of the mobile phase, and the properties of the substance used to measure the column efficiency. The "asymmetry factor" refers to the ratio of the front and rear half-peak widths at 10% of the peak height, which mainly reflects the symmetry of the peak.

[0101] Wall Effect refers to the phenomenon that the separation zone diffuses and bends at the edge due to the uneven flow of the solution near the peripheral wall of the chromatography column, which is caused by the flow difference between the contact part of the inner wall of the chromatography column and the packing and the contact part between the packings. The wall effect of a chromatography column with a smaller inner diameter will lead to problems such as high column pressure and low column efficiency. Example

[0102] To better prove the applicability of this method, tall and thin chromatography columns and packings with different separation principles are selected below, and the present application is further described in combination with specific examples. However, the examples do not limit the scope of the present invention. Where specific conditions are not indicated in the examples, they are carried out according to the conventional conditions in the art or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments used are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0103] Example 1. Packing method of affinity chromatography packing

[0104] In this example, the packing used is MabSelect SuRe affinity chromatography packing from Cytiva Sweden AB; the chromatography system used is the SCG chromatography workstation from Suzhou Sepax Technologies Co., Ltd.; the chromatography column used is the 202000-0640-AA model chromatography column from Suzhou Sepax Technologies Co., Ltd., with an inner diameter of 6.6 mm and a column height of 400 mm.

[0105] The target packing height of this example is 31 cm, and the target packing volume is 10.6 mL. The packing method includes the following steps:

[0106] 1. Drain the air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom with injection water, and fill the lower column head of the chromatography column with injection water;

[0107] 2. Prepare a packing slurry with a slurry ratio of 40%-60% by mixing the MabSelect SuRe affinity chromatography packing preservation solution and the packing, and stir until completely mixed;

[0108] 3. Connect a 10 mL needleless sterile syringe to the lower end of the pipeline at the bottom of the lower column head. Slowly add the packing slurry from the upper end of the chromatography column, and use the syringe to blow air into the packing in the chromatography column through the pipeline at a blowing rate of 3 - 5 mL / min;

[0109] 4. Slowly add the packing slurry from the upper end of the chromatography column, and use the syringe to aspirate the solution in the chromatography column at an aspiration rate of 3 - 5 mL / min. Repeat the above steps until all the packing slurry is added to the chromatography column;

[0110] 5. Add water for injection from the upper end of the chromatography column at a flow rate of 3 - 5 mL / min, and use the syringe to aspirate the solution in the chromatography column at an aspiration rate of 3 - 5 mL / min until there is no obvious dispersion on the upper rubber surface of the packing;

[0111] 6. Use water for injection to exhaust the air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with water for injection;

[0112] 7. Press down the upper column head to a position 1 - 2 cm away from the upper rubber surface, and use at least 1 column volume of water for injection to punch the upper rubber surface of the packing at a linear velocity of 100 cm / h in the forward direction until the upper rubber surface of the packing no longer descends and there is an obvious clear solution layer between the packing and the upper column head, then stop;

[0113] 8. Use at least 1 column volume of water for injection to punch the lower rubber surface of the packing at a linear velocity of 100 cm / h in the reverse direction until the upper rubber surface of the packing moves upward and there is an obvious clear solution layer between the lower column head and the lower rubber surface of the packing, then stop;

[0114] 9. Press up the lower column head by 1 - 5 cm or until a slight damping appears, then stop;

[0115] 10. Use at least 1 column volume of water for injection to punch the upper rubber surface of the packing at a linear velocity of 100 cm / h in the forward direction, and press down the upper column head to make the height of the packing reach the target column filling height of 31 cm;

[0116] 11. Use 2 column volumes of 0.1 M sodium chloride solution to punch the packing in the forward direction at a linear velocity of 100 cm / h for equilibration;

[0117] 12. Load 0.16 mL of 1 M sodium chloride solution, and use 3 column volumes of 0.1 M sodium chloride solution to punch in the forward direction at a linear velocity of 100 cm / h to measure the column efficiency. The results are shown in Table 1.

[0118] Table 1 Parameters and column efficiency of the slender chromatography column filled with affinity packing

[0119] Packing Name Column Height (cm) Column Volume (mL) Column Inner Diameter (cm) Number of Theoretical Plates per Meter (N / m) Asymmetry Factor MabSelect SuRe 31 10.6 0.66 2443.20 0.991

[0120] Example 2. Method for packing cation exchange chromatography packing

[0121] In this embodiment, the packing material used is the SP Sepharose FF affinity chromatography packing material of Cytiva Sweden AB; the chromatography system used is the SCG chromatography workstation of Suzhou Sepure Instruments Co., Ltd.; the chromatography column used is the chromatography column of model 202000-0640-AA of Suzhou Sepax Technologies Co., Ltd., the inner diameter of the chromatography column is 6.6 mm, and the column height is 400 mm.

[0122] The target packing height of this embodiment is 24 cm, and the target packing volume is 8.21 mL. The packing method includes the following steps:

[0123] 1. Drain the air bubbles in the lower end head of the chromatography column and the pipeline connected to its bottom with injection water, and fill the lower end head of the chromatography column with injection water;

[0124] 2. Prepare a packing slurry with a uniform slurry ratio of 40%-60% by mixing the preservation solution of the SP Sepharose FF cation exchange chromatography packing material and the packing material, and stir until completely mixed;

[0125] 3. Connect a 10 mL sterile syringe without a needle to the lower end of the pipeline at the bottom of the lower end head, slowly add the packing slurry from the upper end of the chromatography column, and blow air into the packing in the chromatography column with the syringe through the pipeline, and the air blowing rate is 3-5 mL / min;

[0126] 4. Slowly add the packing slurry from the upper end of the chromatography column, and aspirate the solution in the chromatography column with the syringe, and the aspiration rate is 3-5 mL / min. Repeat the above steps until all the packing slurry is added to the chromatography column;

[0127] 5. Add injection water from the upper end of the chromatography column at a flow rate of 3-5 mL / min, and aspirate the solution in the chromatography column with the syringe, and the aspiration rate is 3-5 mL / min until there is no obvious dispersion on the upper side gel surface of the packing;

[0128] 6. Drain the air bubbles in the upper end head of the chromatography column and the pipeline connected to its top with injection water, and fill the upper end head of the chromatography column with injection water;

[0129] 7. Press down the upper end head to a position 1-2 cm away from the upper gel surface, and use at least 1 times the column volume of injection water to punch the upper side gel surface of the packing at a linear velocity of 100 cm / h until the upper side gel surface of the packing no longer drops and a clear solution layer appears between the upper side gel surface and the upper end head, and then stop;

[0130] 8. Use at least 1 times the column volume of injection water to punch the lower side gel surface of the packing at a linear velocity of 100 cm / h until the upper side gel surface of the packing moves up and a clear solution layer appears between the lower end head and the lower side gel surface of the packing, and then stop;

[0131] 9. Press the upper pressing head down by 1 - 5 cm or until slight damping appears and then stop;

[0132] 10. Use injection water with a linear velocity of 100 cm / h to press the upper rubber surface of the packing in the positive direction with at least 1 times the column volume, and press the upper pressing head down to make the packing height reach the target column packing height of 24 cm;

[0133] 11. Use 0.1 M sodium chloride solution with 2 times the column volume to press the packing in the positive direction at a linear velocity of 100 cm / h for equilibration;

[0134] 12. Load 0.13 mL of 1 M sodium chloride solution, and use 0.1 M sodium chloride solution with 3 times the column volume to press in the positive direction at a linear velocity of 100 cm / h to measure the column efficiency. The results are shown in Table 2.

[0135] Table 2 Parameters and column efficiency of the slender chromatography column packed with cation exchange packing

[0136] Packing Name Column Height (cm) Column Volume (mL) Column Inner Diameter (cm) Number of Theoretical Plates per Meter (N / m) Asymmetry Factor SP Sepharose FF 24 8.21 0.66 2338.63 1.000

[0137] As described above, it is only the preferred embodiment of the present application. The above embodiments are only illustrative of the principles and effects of the present application, rather than any formal and substantial limitations on the present application. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the method of the present application, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present application. For those skilled in the art, without departing from the spirit and scope of the present application, any equivalent changes such as slight modifications, refinements, and evolutions made by using the technical content disclosed above are all equivalent embodiments of the present application; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the essential technology of the present application still fall within the scope of the technical solution of the present application.

Claims

1. A method for loading a tall and slender chromatography column, wherein, The method includes using a blowing and suction device to blow air and / or suck in the chromatography column.

2. The method according to claim 1, wherein The method includes but is not limited to the following steps: (1) Use the equilibration solution to remove air bubbles in the lower column head of the chromatography column and the pipeline connected to its bottom, and fill the lower column head of the chromatography column with the equilibration solution; (2) Prepare a packing slurry with a homogenization ratio of 30%-70% using the packing preservation solution and the packing, and connect the blowing and suction device to the lower end of the pipeline at the bottom of the lower column head; (3) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to blow air into the packing in the chromatography column; (4) Add the packing slurry from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column. Repeat the above steps until all the packing is added to the chromatography column; (5) Add the equilibration solution from the upper end of the chromatography column, and use the blowing and suction device to suck the solution in the chromatography column until there is no obvious dispersion on the upper side of the packing; (6) Use the equilibration solution to remove air bubbles in the upper column head of the chromatography column and the pipeline connected to its top, and fill the upper column head of the chromatography column with the equilibration solution; (7) Press down the upper column head to a position 1-2 cm away from the upper side of the packing, and use the equilibration solution to press the upper side of the packing forward until the upper side of the packing no longer descends and a clear solution layer appears between the upper side of the packing and the upper column head, then stop; (8) Use the equilibration solution to press the lower side of the packing backward until the upper side of the packing moves upward and a clear solution layer appears between the lower column head and the lower side of the packing, then stop; (9) Press up the lower column head; (10) Use the equilibration solution to press the upper side of the packing forward, and press down the upper column head to make the height of the packing reach the target column packing height; (11) Use the equilibration solution to press the upper side of the packing forward.

3. The method according to any one of claims 1-2, wherein, The steps (1)-(11) are carried out in sequence, and the steps (1) and (2) can be interchanged.

4. The method according to any one of claims 1 to 3, wherein, The equilibration solution can be selected from sodium chloride solution, acetate solution, phosphate solution, Tris buffer solution, water for injection, acetate solution containing sodium chloride, phosphate solution containing sodium chloride, Tris buffer solution containing sodium chloride or sodium chloride solution containing ethanol solution; optionally, the equilibration solution is sodium chloride solution with a concentration less than or equal to 1 M, preferably 0.1 M.

5. The method according to any one of claims 1 to 4, wherein The packing can be selected from affinity chromatography packing, hydrophobic chromatography packing, ion exchange chromatography packing, composite mode packing and gel filtration packing, preferably ion exchange chromatography packing and affinity chromatography packing; optionally, the homogenization ratio is 40%-60% or 40%-50%.

6. The method according to any one of claims 1-5, wherein, The rate of air blowing is 1-10 mL / min, 1-5 mL / min or 3-5 mL / min; optionally, the rate of suction is 1-10 mL / min, 1-5 mL / min or 3-5 mL / min; optionally, the equilibration solution in step (5) is added at a rate of 1-10 mL / min, 1-5 mL / min or 3-5 mL / min.

7. The method according to any one of claims 1-6, wherein, In step (9), the lower column head is pressed up by 1-10 cm, 1-5 cm, 1-4 cm, 1-3 cm, 1-2 cm or until slight damping appears and then stop.

8. The method according to any one of claims 1-7, wherein, The rate of the "forward stamping" is a linear velocity of 10 - 500 cm / h or 50 - 150 cm / h; optionally, the rate of the "reverse stamping" is a linear velocity of 10 - 500 cm / h or 50 - 150 cm / h.

9. The method according to any one of claims 1-8, wherein, The blowing and sucking device is a syringe.

10. The method according to any one of claims 1-9, wherein, The target column filling height is 5 - 100 cm, 5 - 80 cm, 5 - 60 cm, 5 - 40 cm, 5 - 35 cm or 5 - 25 cm; optionally, the length - to - diameter ratio of the chromatography column is greater than or equal to 5:1, and the inner diameter of the chromatography column is less than or equal to 50 mm.