A method for filling a gas-blown mixed filler in a chromatography column and its application

Abstract

The present application provides a method for filling a chromatography column filler with improved column efficiency, characterized in that the method includes an air blowing step, in which air is blown to the bottom of the chromatography column through one or more air pipes to fully mix the filler in the chromatography column. The filling method of the present application can improve the success rate of column filling, enhance column efficiency, and the filled chromatography column is suitable for antibody separation.

Description

Technical Field

[0001] The invention belongs to the field of liquid chromatography separation, and in particular relates to a method for filling a filler into a chromatography column and an application of the method in antibody purification. Background Art

[0002] Liquid chromatography (LC), also known as liquid chromatography, is a commonly used analysis, separation and purification method in laboratories and industrial production. In liquid chromatography technology, the substances to be separated in the liquid mobile phase have different structural components, sizes, hydrophobicity, and charge distribution. When flowing through the stationary phase (i.e., the filler in the chromatography column), there are differences in the interaction between the filler and the substance to be separated. Therefore, the substances to be separated flow out of the chromatography column in different retention time sequences, thereby achieving the purpose of analytical separation. Liquid chromatography can be divided into adsorption chromatography, distribution chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. according to different separation principles.

[0003] There are many kinds of fillers (also called resins, matrices or media) suitable for various types of chromatography. In terms of their chemical structure skeleton, they include inorganic materials represented by silica gel, alumina, porous glass matrix, etc.; natural polysaccharide modified polymers represented by cellulose, dextran, agarose, cross-linked polysaccharides, etc.; and synthetic polymers represented by polyacrylates, polystyrene, polyacrylamide, etc.

[0004] The uniform and compact state of the packing in the chromatography column is crucial to the separation performance. If the packing is not compressed evenly, bubbles and / or faults are present, which is manifested as the packing height far exceeding the expected value and the column efficiency (i.e., the number of theoretical plates) is low, the separation process of the substance to be separated in the chromatography column will be inconsistent, and the retention time will be inconsistent with the requirements in the process specifications, resulting in band broadening, affecting the separation efficiency of the chromatography column and even the product quality. In this case, the technicians will have to reload the column, which is time-consuming and labor-intensive.

[0005] Currently, the more commonly used column packing methods can be divided into dry packing and wet packing.

[0006] Dry packing is to add the filler directly into the chromatography column in the form of solid powder or granules, and use its own gravity or external pressure to ensure the tightness between the fillers. Wet packing is to first mix the filler with liquid to form a slurry, then add it to the chromatography column, and use the flushing effect of the column liquid under normal pressure or with the help of external pressure to make the filler tighter than dry packing.

[0007] Patent application CN102989198A discloses a dry column packing method in which solid fillers are poured into a preparation column in batches, each batch is stirred with a stick and compacted with external pressure. This method requires that the pressure in the column of the previous pressurized column be completely released before the next batch of fillers can be added, and then pressurization, release pressure, and refilling are repeated, which increases the work intensity and is therefore time-consuming, labor-intensive, and costly to operate.

[0008] Invention patent CN103487533B uses alkaline salt buffer to replace purified water and gel to form a suspension, and performs wet column packing with the help of alkaline salt buffer under external pressure. This method uses alkaline salt buffer to punch the column, and requires external pressure, which has high column packing cost.

[0009] Utility model patent CN205109614U discloses a tower packing gas-blowing distributor and its application, which performs air-blowing on the dry-loaded packing to replace manual leveling to achieve automatic feeding and uniform distribution of packing. This method requires the air-blowing distributor to be removed from the tower after packing before the tower can be started, which is complicated to operate. Moreover, air blowing makes the inherent disadvantages of dry loading more prominent, that is, there is friction between the packing particles. The use of an air source pressure of 0.1-0.5MPa (equivalent to 1-5bar, because 1MPa=10bar) will increase friction and cause a large number of bubbles, which will inevitably cause bubbles and / or faults in the packing, and the actual loading effect is questionable. In addition, the utility model patent document does not indicate the role of air blowing in improving column efficiency, nor does it disclose air blowing conditions such as air pipe specifications and air blowing time.

[0010] Invention patent CN102077087B discloses a separation medium slurry tank, which performs air blowing on separation media of different types and particle sizes that are transferred from multiple storage tanks to an intermediate tank, so that the medium slurry becomes a homogeneous mixture. The slurry is then degassed as needed before being transferred to a separation column. This method first ventilates and degases the separation medium in the intermediate tank before transferring it to the separation column. The steps are complicated, and bubbles are inevitably introduced during the slurry transfer process, so the actual filling effect is questionable. In addition, the patent document does not indicate the role of air blowing in the chromatography column in improving the column efficiency, nor does it disclose air blowing conditions such as air pipe specifications, air blowing pressure, and air blowing time.

[0011] Therefore, how to improve the success rate of column packing, load the filler into the chromatography column evenly and tightly, make the filler height reach the expected value, and improve the column efficiency has always been a technical problem to be solved urgently in this field. SUMMARY OF THE INVENTION

[0012] On the one hand, the present invention provides a method for filling a chromatography column with filler, characterized in that the method includes an air blowing step, in which air is blown to the bottom of the chromatography column through one or more air pipes to fully mix the filler in the chromatography column.

[0013] In some embodiments, the one or more gas tubes are connected to the bottom of the chromatography column.

[0014] In some embodiments, the gas tubes are multiple gas tubes connected to the bottom of the chromatography column at equal distances relative to the central point of the bottom of the chromatography column.

[0015] In some embodiments, in the gas blowing step, one or more gas tubes extend through the top of the chromatography column into a position near the bottom of the chromatography column.

[0016] In some embodiments, the air is compressed air, such as compressed air of 0.3-0.5 bar, preferably 0.5 bar.

[0017] In some embodiments, the one or more tubes are connected to the bottom of the chromatography column, the air is compressed air, and the filler is a filler slurry.

[0018] In some embodiments, the filler slurry concentration is 50%-60%, preferably 50%.

[0019] In some embodiments, the trachea is a hard trachea with specifications of 6#, 8# or 10#, preferably 10# trachea.

[0020] In some embodiments, the air blowing time is 10-30 minutes, preferably 30 minutes.

[0021] In some embodiments, the gas blowing step further includes a step of stirring to break up the settled fillers in the chromatography column.

[0022] In some embodiments, the filler is an inorganic material or a synthetic high molecular polymer material, for example, an inorganic filler selected from silica gel, alumina, and a porous glass matrix.

[0023] In some embodiments, the porous glass matrix is Series of matrices, preferably Ultra Plus or vA series matrix.

[0024] In some embodiments, the filler is Ultra Plus, homogenate concentration is 50%, air blowing pressure is 0.5 bar, air tube specification is 10#, and air blowing time is 30 minutes.

[0025] On the other hand, the present invention relates to the use of the chromatography column filled with the aforementioned method in antibody separation. Exemplary antibodies include adalimumab, alirocumab, benmelstobart monoclonal antibody, bevacizumab, bimekizumab, daratumumab, denosumab, evolocumab, guselkumab, ipilimumab, ixekizumab, metoprolol, selenosumab ... Mepolizumab, nivolumab, obinutuzumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, romosozumab, secukinumab, trastuzumab, or ustekinumab. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides a method for filling a chromatography column filler, characterized in that the method comprises an air blowing step, in which air is blown to the bottom of the chromatography column through one or more air pipes to fully mix the filler in the chromatography column.

[0028] In some embodiments, the one or more gas tubes are connected to the bottom of the chromatography column.

[0029] In some embodiments, the gas tubes are multiple gas tubes connected to the bottom of the chromatography column at equal distances relative to the central point of the bottom of the chromatography column.

[0030] In some embodiments, in the gas blowing step, one or more gas tubes extend through the top of the chromatography column into a position near the bottom of the chromatography column.

[0031] In some embodiments, the air is compressed air, such as compressed air of 0.3-0.5 bar, preferably 0.5 bar.

[0032] In some embodiments, the one or more air tubes are connected to the bottom of the chromatography column, the air is compressed air, and the filler is a filler homogenate.

[0033] In some embodiments, the air pipe is preferably a hard air pipe, and the air pipe specification is 6#, 8# or 10# (corresponding to an outer diameter of 6, 8 or 10 mm), preferably 10#. These are industry standard air pipes, and common suppliers include, for example, Festo.

[0034] In some embodiments, the air blowing time is 10-30 minutes, preferably 30 minutes.

[0035] The type of filler used in the method of the present invention can be selected according to the properties of the substance to be separated and the type of chromatography, preferably an inorganic material or a synthetic high molecular polymer material. The inorganic filler can be silica gel, alumina, a porous glass matrix, such as A series of protein A affinity chromatography porous glass matrices, preferably Ultra Plus or vA protein A affinity chromatography porous glass matrix. The synthetic high molecular polymer material can be polyacrylate, polystyrene, polyacrylamide, etc.

[0036] The filler supplied in solid form can be directly prepared with the column packing liquid into a slurry with a concentration of 50%-60%, preferably 50%, and then transferred into the chromatography column manually or by a diaphragm pump or a peristaltic pump.

[0037] For fillers supplied in liquid form, if the storage liquid and the column packing liquid are inconsistent, it is necessary to replace the storage liquid with the column packing liquid (usually purified water, but it can also be other buffers compatible with the filler). Buffer exchange can be performed in a liquid storage tank, by directly pouring or otherwise removing the supernatant, and then adding the column packing liquid and stirring to make a 50%-60% homogenate. Let it stand for about half an hour to allow the filler to settle, remove the supernatant, and add fresh column packing liquid again, and repeat this 2-3 times to ensure sufficient replacement. Buffer exchange can also be performed using a chromatography column. The filler stored in the storage liquid can be evenly poured or pumped into the chromatography column in advance, and then rinsed with about 3 times the column volume of the column packing liquid for replacement, as long as the filler concentration after replacement is guaranteed to account for 50%-60%, preferably 50%.

[0038] Regardless of the form in which the filler is added to the chromatography column, if sedimentation occurs, it can be stirred manually or automatically with a stirring rod to break up the settled filler to avoid agglomeration, and then air blowing can be performed to achieve the best mixing effect. Those skilled in the art are familiar with the stirring operation. For example, a slow and uniform circle can be drawn in one direction around the inner wall of the chromatography column close to the bottom. The force should be sufficient to break up the settled filler. Stirring can be stopped after 1-5 minutes.

[0039] After completing the air blowing and mixing step described in the present invention, the conventional wet filling method can be followed, and the operation can be performed according to the column packing guide or filling instructions recommended by the filler supplier. Generally, after the filler is naturally settled, it is repeatedly rinsed with the column liquid at the recommended speed and duration under normal pressure or pressurized conditions until the column bed height reaches the expected value or no longer decreases. Optionally, in order to further compact the filler and eliminate the bubbles or holes that may remain in the filler, the column can be knocked with a rubber hose, a wooden stick or a stirring rod during the interval of the column liquid flushing according to the recommendation of the supplier (for laboratory use or small columns with a column diameter of less than 100 mm), or the column (pilot or production column, such as a column with a column diameter greater than or equal to 100 mm) is pressurized and shaken by an external oscillator. Optionally, ultrasonic vibration can also be used to assist in compacting the filler or removing bubbles.

[0040] The packing method of the present invention is particularly suitable for a chromatography column for pilot and / or production, such as a chromatography column with a column diameter of 100-600 mm, preferably a chromatography column with a column diameter of 100, 140, 150, 300, 450 or 600 mm. Based on the disclosure and method of the present invention, those skilled in the art can adjust parameters such as filler slurry concentration, air pipe specifications, air blowing pressure and air blowing time according to actual needs in pilot and production to obtain the most ideal column packing effect.

[0041] In a preferred embodiment, the method of the present invention comprises the following steps:

[0042] (1) an air blowing step, wherein compressed air is blown upward through an air pipe connected to the bottom of the chromatography column to fully mix the filler slurry in the chromatography column; and

[0043] (2) Column packing step: pack the column by repeatedly flushing with the packing solution.

[0044] Optionally, in the column packing step, flushing is performed using a column packing liquid under external pressure.

[0045] Optionally, in the column packing step, an external oscillator is used to pressurize and oscillate the column to assist in column packing.

[0046] Optionally, in the column packing step, ultrasonic vibration is used to assist the column packing.

[0047] Optionally, the method of the present invention further comprises: (3) a column efficiency detection step, using 1-4M sodium chloride (NaCl) solution to load and test the column efficiency.

[0048] The beneficial technical effects of the present invention are: (1) The column packing method of the present invention has high repeatability, with a column packing success rate of up to 100%, achieving a one-time column packing success rate, and overcoming the defect that the average column packing success rate of the traditional method is only 20% to 50% and requires repeated column packing. (2) The column packing method of the present invention enables the filler height to reach the expected value, overcoming the problem of excessive filler volume caused by the traditional method. (3) The column efficiency obtained by the column packing method of the present invention is high. (4) The air blowing step adopted by the method of the present invention realizes standardized and automated operation for the first time, fully ensuring the consistency of chromatographic separation and purification results of different production batches. (5) The column packing method of the present invention has a small column packing volume, thereby avoiding the occurrence of insufficient flushing volume, excessive elution volume, and incompatibility with existing equipment in the production process of the traditional column packing method, thereby reducing the risks brought about in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 . Schematic diagram of the five sampling locations, namely the center point of the column on the packing surface, two locations on both sides of the column symmetrically close to the column wall, and two locations below the middle of the column close to the column wall.

[0050] Figure 2 .Comparison of standard deviations of different homogenate concentrations.

[0051] Figure 3 .Comparison of standard deviations of different air blowing mixing times.

[0052] Figure 4 .Comparison of standard deviations of different trachea specifications.

[0053] Figure 5 .Comparison chart of standard deviation of different air blowing pressures. DETAILED DESCRIPTION

[0054] For the sake of clarity, the present invention is further illustrated by examples, but the examples are not intended to limit the scope of the present invention. The reagents used in the present invention are generally commercially available and can be used without further purification.

[0055] Example 1 Comparative Example - Column Packing According to the Conventional Column Packing Method Recommended by the Manufacturer

[0056] This example adopts Merck Millipore's Ultra Plus Protein A affinity chromatography medium (PUP medium), refer to the manufacturer's instructions ( Ultra Plus Affinity Chromatography Media OPERATING INSTRUCTIONS, 2012) was loaded. The chromatography platform system used was from Cytiva Process TM Protein chromatography system: The chromatography column is a SAC-Bio-450-550-10 chromatography column from Lisui Technology Co., Ltd., with an inner diameter of 450 mm, a column height of 550 mm, and a target column volume of 21.5 L.

[0057] 1.1 Preparation before column loading - PUP filler buffer replacement / transfer to chromatography column

[0058] According to the guideline, since PUP medium is stored in a 0.1 M sodium acetate storage solution containing 1% benzyl alcohol, pH 5.2 ± 0.5, buffer exchange should be performed first using a reservoir or chromatography column.

[0059] If the liquid storage tank is used for replacement, the volume of liquid required for 21.5L of pure PUP filler is calculated based on the concentration of PUP filler in the storage liquid, and the supernatant is removed by direct pouring or other means, and then the column packing liquid (for PUP filler, purified water can be used) is added and stirred to form a 50%-70% homogenate. Let it stand for about half an hour to allow the filler to settle, remove the supernatant, and add fresh column packing liquid again. Repeat the steps of settling, removing the supernatant, and adding column packing liquid at least twice to ensure sufficient replacement. After the PUP filler is replaced with the buffer solution, the filler is mixed and poured or pumped into the chromatography column.

[0060] If the replacement is performed using a chromatography column, based on the concentration of the PUP filler in the stock solution, take a liquid equivalent to 21.5 L of pure PUP filler, mix it and pour or pump it into the chromatography column, use about 65 L of purified water at a linear speed of 100 cm / h to replace the buffer solution, and make the filler slurry concentration reach 50%-70%.

[0061] 1.2 Manual stirring and mixing of PUP filler

[0062] PUP filler is a porous glass matrix. Compared with other chromatography fillers, its sedimentation rate is relatively faster. Therefore, according to the manufacturer's operating instructions, the filler in the chromatography column that has been replaced with buffer needs to be stirred with a manual stirring rod for 30 minutes to mix it thoroughly, and then allowed to settle naturally. When the supernatant level reaches 2-3 cm, the following formal column loading operation can be started.

[0063] 1.3 Column packing operation

[0064] Adjust the sealing ring to a semi-tight state, press down the column head evenly, make sure there are no bubbles on the upper sieve plate, and then completely release the air pressure of the sealing ring until the sealing ring is fully tight. Press down the column head to 2-3cm above the rubber surface, and keep the rubber surface stable during the pressing process.

[0065] Use purified water to press forward at a linear speed of 300 cm / h for 5-10 minutes until the column bed height stops decreasing.

[0066] The column direction was set to Bypass (a bypass mode that does not pass through the chromatography column position), and 30 psi of compressed air was applied to the column for 1 min through two oscillators symmetrically distributed on the steel plate at the chromatography column base.

[0067] Use purified water to press forward at a linear speed of 300 cm / h for 1 min, then turn off the pump and set the column direction to Bypass.

[0068] Apply 30 psi of compressed air to the shaker to shake the column for 1 min, then stop shaking and cancel the Bypass setting.

[0069] Repeat the above steps of pressing with purified water for 1 min and then shaking on an oscillator for 1 min 22 times.

[0070] Press the column head down to 2-3cm above the rubber surface.

[0071] Use purified water to press forward at a linear speed of 300 cm / h for 1 min, then turn off the pump and set the column direction to Bypass.

[0072] Apply 30 psi of compressed air to the shaker to shake the column for 1 min, then stop shaking and cancel the Bypass setting.

[0073] Repeat the above steps of pressing with purified water for 1 min and then shaking on an oscillator for 1 min 8 times.

[0074] Use purified water to press at a linear speed of 300 cm / h for 3-5 minutes until the column bed height stops decreasing.

[0075] Press down on the column head at a constant speed until it can no longer be pressed down.

[0076] The column bed was positively flushed with purified water at a linear velocity of 300 cm / h for 10 min.

[0077] The column bed was reversely flushed with purified water at a linear velocity of 300 cm / h for 10 min.

[0078] Press the column head down to the target height and record the actual column volume. For PUP packing, the acceptable actual column volume should not be higher than the theoretical column volume by 10%. The closer the actual value is to the theoretical value, the better.

[0079] 1.4 Balancing Operations

[0080] The packing was equilibrated in the forward direction with 1-3 column volumes of 0.1 M sodium chloride solution at a linear velocity of 100 cm / h.

[0081] 1.5 Column efficiency detection and parameter calculation

[0082] The column efficiency was tested by loading 1-2% column volume of 1 M sodium chloride solution.

[0083] The two parameters for evaluating column efficiency, namely the number of theoretical plates N (number of theoretical plates) and the asymmetry factor AS (Asymmetry Factor), are calculated based on the obtained chromatogram.

[0084] Where N = 5.54 × t 2 / d 2 , where t is the retention time of the test sample (here sodium chloride) and d is the half peak width.

[0085] AS = b / a, where b is the right peak width at 10% peak height, and a is the left peak width at 10% peak height.

[0086] For PUP fillers, the acceptable column efficiency parameter range is N ≥ 800 and AS = 0.8-1.8. The larger the N value, the better, and the closer the AS is to 1, the better.

[0087] PUP was loaded in three batches according to the above steps. The volume and column efficiency results of the three loadings are shown in Table 1 below:

[0088] Table 1: Column volume and column efficiency results of traditional methods

[0089]

[0090] It can be seen that, using the traditional packing method, although the asymmetry coefficient (1.37-1.49) and the theoretical plate number (1505.1-2319.3) are within the acceptable parameter range (AS = 0.8-1.8, N ≥ 800), the actual column volume is high, far higher than 10% of the theoretical column volume, and does not reach the expected value. Therefore, the three column packings all ended in failure.

[0091] Example 2 Optimization of air blowing conditions

[0092] The column packing method for improving column efficiency of the present invention is improved in that an air blowing step is added during the wet packing process, and compressed air is blown upward through an air pipe connected to the bottom of the chromatography column to fully mix the filler slurry in the chromatography column.

[0093] This embodiment explores and optimizes the air blowing mixing conditions, and the method is as follows:

[0094] 2.1 Combination optimization of air pipe specifications (6#, 8#, 10#), homogenization ratio (50%, 60%, 70%), compressed air (0.3bar, 0.4bar, 0.5bar), and mixing time (10min, 20min, 30min)

[0095] The 50% homogenate ratio of 21.5L PUP filler was subjected to buffer exchange / transfer to the chromatography column as described in Example 1. If the PUP filler settles during the transfer process, a stirring rod can be used to stir clockwise along the bottom of the chromatography column wall to break up the settled filler to avoid filler agglomeration, and the stirring rod can be quickly removed.

[0096] Immediately use the 6# gas pipe to connect the bottom of the chromatography column and introduce 0.3 bar compressed air into the column. Stir for 10 min, 20 min, and 30 min respectively. Figure 1 Take 12 ml of sample from the five locations shown in the figure and put them into 15 ml centrifuge tubes respectively. Let them settle naturally for 12 hours. Observe the conditions in the centrifuge tubes and record the height of the homogenate (i.e. the total height of the column liquid + the sedimentation filler) and the height of the sedimentation filler (cm). Then replace the 8# and 10# air tubes and repeat the above steps. The results are summarized in Table 2 below.

[0097] Table 2: Sampling results of trachea of ​​different specifications after homogenization with 50% and 0.3 bar compressed air

[0098]

[0099] The compressed air pressure was changed to 0.4 bar and 0.5 bar respectively, and the aforementioned sampling and sedimentation steps were repeated. The homogenate and filler height (cm) data were recorded, as described in Tables 3 and 4 below.

[0100] Table 3: Sampling results of trachea of ​​different specifications after homogenization with 50% and 0.4 bar compressed air

[0101]

[0102] Table 4: Sampling results of trachea of ​​different specifications after homogenization with 50% and 0.5 bar compressed air

[0103]

[0104] The homogenization ratio of the filler was changed to 60% and 70%, respectively, and the aforementioned sampling and sedimentation steps were repeated. The homogenization and filler height (cm) data were recorded, as described in the following Tables 5-10.

[0105] Table 5: Sampling results of trachea of ​​different specifications after homogenization with 60% and 0.3 bar compressed air

[0106]

[0107] Table 6: Sampling results of different specifications of trachea homogenate after mixing with 60% and 0.4 bar compressed air

[0108]

[0109] Table 7: Sampling results of trachea of ​​different specifications after homogenization with 60% and 0.5 bar compressed air

[0110]

[0111] Table 8: Sampling results of different specifications of trachea homogenate after mixing with 70% and 0.3 bar compressed air

[0112]

[0113] Table 9: Sampling results of different specifications of trachea homogenate after mixing with 70% and 0.4 bar compressed air

[0114]

[0115] Table 10: Sampling results of different specifications of trachea homogenate after mixing with 70% and 0.5 bar compressed air

[0116]

[0117] 2.2 Statistical analysis of standard deviation

[0118] For the experiment in 2.1, the ratio between the height of the filler after sedimentation and the height of the sampled homogenate was calculated, the average value of the ratio was obtained, and the standard deviation of each group of five ratio data was calculated accordingly, and the results shown in the following Table 11 were obtained.

[0119] Because the filler homogenization ratio is 70%, under the condition of 0.3 bar air blowing pressure, no matter what kind of air tube model is used and how long the blowing time is, the filler visible to the naked eye cannot be well dispersed, so the experimental data statistical analysis under this condition is not carried out.

[0120] Similarly, when the homogenization ratio is 70% and the air blowing pressure is 0.4 bar, the air blowing is performed using a 6# air pipe. It can be judged from the appearance conditions that the mixing effect is not obvious under this condition, so this condition is also abandoned.

[0121] Table 11: Standard deviation statistics under different homogenization ratios, air blowing pressures, air tube specifications, and mixing times

[0122]

[0123]

[0124] It can be seen from the standard deviation statistics in Table 11 that the smaller the standard deviation, the smaller the discreteness between each group of 5 data, which means the smaller the difference between the five points, and also that the filler mixing concentration between the five points is relatively close.

[0125] 2.3 Scatter plot of standard deviation

[0126] The standard deviations of Table 11 were further plotted against homogenate concentration, air blowing time, trachea specifications and air blowing pressure. Figure 2-5 .

[0127] like Figure 2 As shown, under certain air blowing pressure, air tube specifications and air blowing stirring time, the higher the homogenate concentration, the greater the standard deviation, indicating that under the conditions of the three selected initial homogenate concentrations, a homogenate concentration of 50% is most conducive to mixing.

[0128] like Figure 3 As shown in the figure, under the same homogenate concentration conditions, comparing the same air blowing pressure and air tube specifications, as the air blowing stirring time increases, the standard deviation between each group of data becomes smaller and smaller, indicating that the longer the time, the more uniform the filler is stirred. Therefore, in terms of stirring time, the best air blowing time is 30 minutes.

[0129] like Figure 4 As shown in the figure, by comparing the same homogenate concentration, the same air blowing stirring time and a certain air blowing pressure, it is found that the larger the air tube specification, the smaller its standard deviation. Therefore, the best air tube specification is 10#.

[0130] like Figure 5 As shown in the figure, under the same homogenization ratio, air blowing stirring time and air pipe specifications, by comparing the data, it can be found that the greater the air blowing pressure, the smaller the standard deviation. Therefore, the best selection condition for air blowing stirring pressure is 0.5 bar.

[0131] In summary, the best air blowing conditions are: air pipe specification 10#, air blowing pressure 0.5 bar, homogenization ratio 50%, and air blowing time 30 min. Based on the results in Table 11, it is also found that under this condition, the smaller the standard deviation, it can be judged that this condition is the best mixing effect condition under this test condition.

[0132] Example 3 Air Blowing Wet Filling Test

[0133] According to the optimized parameters determined in Example 2 (10# air pipe, air blowing pressure of 0.5 bar, homogenization ratio of 50%, air blowing time of 30 min), air blowing wet filling was carried out to replace the manual stirring means in the traditional filling method shown in Example 1.

[0134] In brief, 50% homogenate ratio 21.5L PUP filler was buffer exchanged / transferred into the chromatography column as described in Example 1. If the PUP filler settles during the transfer process, a stirring rod can be used to stir at a low speed and uniform speed in one direction around the inner wall of the chromatography column and close to the bottom to break up the settled filler and avoid filler agglomeration, and the stirring rod can be quickly removed.

[0135] Immediately use a 10# air pipe to connect the bottom of the chromatography column, and introduce 0.5 bar compressed air into the column, stirring for 30 minutes to fully mix the filler slurry. If foam appears, use a pipette to suck out the foam in time. Then allow natural sedimentation. When the supernatant level is 2-3 cm, according to the operating instructions provided by the manufacturer, perform the column loading, equilibration and column efficiency testing steps according to the methods described in steps 1.3 to 1.5 of Example 1.

[0136] According to the above steps, the PUP was filled by air blowing method in two batches. The volume and column efficiency results of the two column fillings are shown in Table 12 below:

[0137] Table 12: Gas blowing and mixing column packing volume and column efficiency results

[0138]

[0139] It can be seen that compared with the conventional method of Example 1, the actual column volume (22.4 or 22.5) can reach the expected value, that is, the theoretical column volume (21.5) is within the acceptable range of about 4% or 5%. In addition, the asymmetry coefficient (0.99 or 1.12) is within the range of 0.8-1.8 and is closer to 1; and the theoretical plate number (6537.3 or 7476.0) is much higher than the acceptable standard (N≥800) and is significantly improved. It can be seen that the two column loadings were 100% successful.

[0140] Example 4 Isolation of Antibodies

[0141] The PUP filler was loaded as described in Example 3, and then the antibody sample was processed and chromatographed according to the following procedures, referring to the operating instructions provided by the manufacturer.

[0142] 4.1 Sample filtration processing

[0143] The antibody sample was dissolved in a loading buffer with a pH of 7.0–8.0 and a salt concentration of 0.15 M (PBS buffer at pH 7.4; or 25 mM Tris, 150 mM NaCl, pH 7.5 buffer), and passed through a 0.22 μm or 0.45 μm filter to remove cell debris or other impurities before loading.

[0144] 4.2 Column equilibration

[0145] Before loading the sample, equilibrate the column with 5 column volumes of equilibration buffer (PBS buffer or Tris·NaCl buffer in step 4.1).

[0146] 4.3 Sample loading

[0147] The sample was loaded by a peristaltic pump at an optimal flow rate for the separated antibody.

[0148] 4.4 Washing

[0149] Wash the column with 5-10 column volumes of loading buffer to remove unbound or weakly bound substances in the column.

[0150] 4.5 Elution and collection of antibodies

[0151] The bound antibody was eluted with up to 3 column volumes of elution buffer (0.1 M glycine / HCl, pH 3.0) and the target antibody was collected.

[0152] 4.6 Column Regeneration

[0153] After the chromatography, first pass 3-5 column volumes of regeneration buffer (150 mM H3PO4, pH 1.5) through the column, then pass 5 column volumes of equilibration buffer through the column to regenerate the column for later use.

[0154] This method has been used to successfully separate monoclonal antibodies for use in scientific research or production, including Adalimumab, Alirocumab, Benmelstobart, Bevacizumab, Bimekizumab, Daratumumab, Denosumab, Evolocumab, Guselkumab, Ipilimumab, Ixekizumab, and umab, mepolizumab, nivolumab, obinutuzumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, romosozumab, secukinumab, trastuzumab, or ustekinumab.

[0155] According to the contents disclosed in the present invention, although the method of the present invention is described according to a preferred embodiment, it is apparent to those skilled in the art that the method described herein and the steps or the order of steps of the method may be changed without departing from the concept, spirit and scope of the present invention.

[0156] The disclosures of all documents cited herein are incorporated herein by reference, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Claims

1. A method for filling a chromatography column filler with air blowing to mix and improve column efficiency, characterized in that The method comprises firstly mixing the filler liquid and pouring or pumping it into the chromatography column, and then performing an air blowing step, wherein compressed air is blown upward toward the bottom of the chromatography column through an air pipe to fully mix the filler in the chromatography column; the air pipe is connected to the bottom of the chromatography column; wherein the filler is a ProSep Ultra Plus protein A affinity chromatography porous glass matrix, the filler slurry concentration is 50%, the air blowing pressure is 0.5 bar, the air pipe outer diameter is 10 mm, and the air blowing time is 30 minutes; the chromatography column is a SAC-Bio-450-550-10 chromatography column, with an inner diameter of 450 mm and a column height of 550 mm; the target column volume of the chromatography column is 21.5 L; wherein before the air blowing step, a step of stirring and breaking up the settled filler in the chromatography column is also included; and wherein after the air blowing step, an external oscillator is used to pressurize and oscillate the column to assist in column loading or ultrasonic oscillation is used to assist in column loading.

2. A method for separating antibodies using a chromatography column loaded by the method of claim 1, wherein the antibody is selected from the group consisting of adalimumab, alirocumab, bemosubishizumab, bevacizumab, bimegizumab, daratumumab, denosumab, eloumab, guselkumab, ipilimumab, ixekizumab, mepolizumab, nivolumab, obinutuzumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, lomosozumab, secukinumab, trastuzumab, or ustekinumab.

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