JP2014002008A - Method for purifying antibody and antibody purification column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody purification column with high adsorption efficiency, and to sharply shorten time required for antibody purification.SOLUTION: An antibody purification column comprising an adsorbent formed by fixing protein A on the surface of a porous carrier 5 having a skeleton body 7 consisting of silica gel or silica glass with a three-dimensional net structure and pores 9 dispersedly formed on the surface of the skeleton body 7 and penetrated from the surface up to the inside of the skeleton body, in which a center diameter of each pore 9 measured by a nitrogen adsorption method is 40 to 70 nm, is used. Under a condition that contact time between an unpurified solution containing an antibody to be purified and the adsorbent is 40 sec or less, the unpurified solution is passed into the antibody purification column and the antibody in the unpurified solution is adsorbed to the adsorbent to purify the antibody.

Description

  The present invention relates to an antibody purification column provided with an adsorbent formed by immobilizing protein A, and an antibody purification method performed using the antibody purification column.

  In addition to being widely used as an antibody drug, antibodies are also widely used in biochemical research, and it is desired that antibody purification be performed with high efficiency. An immunoglobulin antibody (Ig) has a polypeptide chain of two identical heavy chains and two identical light chains, and a heavy chain and a light chain arranged in a Y-shape. It has a structure. Since immunoglobulin antibodies share the same basic structure, it is common practice to perform a crude purification process followed by a final purification process using affinity chromatography utilizing biological affinity for antibody purification. Yes.

  For purification of an immunoglobulin antibody, an affinity column equipped with an adsorbent formed by liganding protein A having affinity for a constant site of the antibody as a carrier can be used. Conventionally, porous carriers having a large number of pores such as bead-like silica gel, agarose gel, and cellulose gel have been used as carriers used in affinity columns.

  Further, recently, in Patent Document 1 below, it has been proposed to use an inorganic monolith as a porous carrier used for an affinity column. In Patent Document 1, an inorganic monolith has a macropore (through hole) having a diameter of 0.1 μm to 10 μm and a skeleton body forming a co-continuous structure, and mesopores (pores) having a diameter of 2 nm to 200 nm exist in the skeleton body. And is defined as an inorganic porous continuous body mainly composed of silica.

JP 2012-1462 A

  Affinity columns for antibody purification using bead-shaped porous cellulose gel, etc. that are currently in practical use as a carrier, have the disadvantage that the adsorption capacity decreases at high linear velocity, or consolidation occurs at high linear velocity. It has been pointed out that use of an inorganic monolith as a carrier is considered to compensate for this drawback (see Patent Document 1). The defects of these bead-like porous carriers can be analyzed as follows.

  First, the pre-purification solution containing the antibody to be purified that has been passed through the antibody purification column flows through the gaps between the beads, but the movement of the antibody to the pore surface within the beads is diffused. Furthermore, since the carrier is bead-shaped and its diameter is larger than the pore diameter, the antibody is adsorbed only on the pore surface near the bead surface, and the entire pore surface area extending from the vicinity of the bead surface to the deep part thereof. Cannot be used effectively.

  Second, if the bead diameter is reduced in order to improve the first problem, the gap between each bead-like carrier is narrowed to increase the channel resistance of the pre-purification solution and increase the pressure loss. The flow rate of the previous solution cannot be increased. Assuming that the bead-shaped carrier is packed tightly in the column container, the diameter of the flow path passing through the narrowed portion of the gap between each bead-shaped carrier is narrowed to about 15% of the bead diameter, and the narrowed portion is It is considered that the pressure loss increases because the channel surrounded by the bead-shaped carrier from four directions and the flow path entering the constricted portion from one direction is largely bent by the facing bead-shaped carrier.

  Furthermore, in soft gels such as porous cellulose gel, the pressure loss is further high, and when the pre-purification solution is passed at a high linear velocity, consolidation occurs and the pre-purification solution does not flow above a certain flow rate.

  Therefore, by using an inorganic monolith as a carrier, it is expected that the reduction in adsorption capacity and consolidation at high linear speeds will be improved to some extent.

  However, it has been clarified by the inventor's earnest research that the decrease in the adsorption capacity is not simply caused by the high linear velocity. Even in inorganic monoliths, the adsorption capacity decreases when the linear velocity is increased, to some extent, is the same as the conventional bead-shaped carrier, and is designed taking into consideration other factors other than the high linear velocity. Without it, it was found that a high adsorption capacity could not be obtained at a high linear velocity.

  The pore diameter of the inorganic monolith disclosed in Patent Document 1 is distributed over a wide range of 2 nm to 200 nm, and the degree of freedom in setting the pore diameter is extremely large. On the other hand, the immunoglobulin antibody molecule to be adsorbed has a flat Y-shape, and the distance between the vertices of the Y-shape structure (hereinafter referred to as “antibody size”) is about 10 to 12 nm. is there. That is, the pore diameter of the inorganic monolith is distributed over a wide range from 1/6 to 20 times the size of the antibody.

  As the pore diameter increases, the surface area per pore volume decreases and the adsorption efficiency decreases. Conversely, when the pore diameter is reduced, the diffusion rate in the pores is slowed down, so that the antibody is adsorbed only on the surface of the pores in the vicinity of the surface, and the adsorption efficiency is lowered. Therefore, it is considered that the adsorption efficiency is lowered by using a pore diameter that is too large or too small with respect to the size of the antibody. If the pore diameter of the inorganic monolith is distributed over a wide range of 2 nm to 200 nm, the adsorption efficiency is reduced. A range of pore diameters that decrease is included, and there is room for improvement.

  Through the diligent research of the inventor of the present application, it was found that there is a significant correlation between the adsorption efficiency and the pore diameter, and by controlling the pore size distribution tighter than the conventional inorganic monolith, It was confirmed that it can be greatly improved. In fact, Patent Document 1 does not consider any relationship between the adsorption efficiency and the pore diameter, and does not provide any examination or suggestion for improving the adsorption efficiency by controlling the pore diameter distribution. .

  The present invention has been made in view of the above-described problems of affinity columns for antibody purification, and the object thereof is to provide a column for antibody purification with high adsorption efficiency and to greatly reduce the time required for antibody purification. There is.

  In order to achieve the above object, the present invention provides a skeleton made of silica gel or silica glass having a three-dimensional network structure, and pores penetrating from the surface to the inside of the skeleton formed by being dispersed on the surface of the skeleton. And a column for purifying an antibody comprising an adsorbent formed by immobilizing protein A on the surface of a porous carrier having a pore diameter measured by nitrogen adsorption method of 40 nm to 70 nm. The pre-purification solution is allowed to flow through the antibody purification column under a condition that the contact time between the pre-purification solution containing the antibody to be purified and the adsorbent is 40 seconds or less, and the antibody in the pre-purification solution is removed. Provided is a method for purifying an antibody, wherein the antibody is purified by being adsorbed on the adsorbent.

  More preferably, in the antibody purification method having the above characteristics, the contact time is 2 seconds or longer and 40 seconds or shorter.

  More preferably, in the antibody purification method having the above characteristics, the time required for one purification step comprising solvent replacement, adsorption, washing, elution, and post-washing steps is 120 seconds or less.

  More preferably, in the antibody purification method of the above feature, the antibody purification column is washed without using a strong alkali solution having a pH of more than 13 in the post-washing step, and the purification step is performed 100 times or more within 2 hours. repeat.

  In order to achieve the above object, the present invention provides a skeleton made of silica gel or silica glass having a three-dimensional network structure, and pores penetrating from the surface to the inside of the skeleton formed by being dispersed on the surface of the skeleton. Purification of an antibody comprising an adsorbent formed by immobilizing protein A on the surface of a porous carrier having a pore diameter of 40 nm to 70 nm measured by a nitrogen adsorption method. Provide a column for use.

  More preferably, in the antibody purification column having the above characteristics, the pre-purification solution is passed through the adsorbent under a condition where the contact time between the pre-purification solution containing the antibody to be purified and the adsorbent is 2 seconds to 40 seconds. When the antibody in the pre-purification solution is adsorbed to the adsorbent, the adsorption per unit volume of the adsorbent exceeds 10% breakthrough in the adsorption breakthrough curve obtained using ultraviolet light having a wavelength of 280 nm. The dynamic adsorption capacity defined by the added amount of the antibody has an adsorption performance exceeding 10 mg / mLgel.

  In the antibody purification method and antibody purification column having the above characteristics, the surface of the porous carrier includes the inner wall surface of the pore in addition to the surface of the skeleton.

  According to the antibody purification method or antibody purification column having the above characteristics, by using silica monolith as a carrier and using a central pore diameter in the optimum range for antibody adsorption, as described later, at a high linear velocity, A high dynamic adsorption capacity (dBC, also called dynamic binding capacity) can be realized, and the time required for the adsorption step can be 40 seconds or less, and antibody purification can be carried out with high efficiency and in a short time.

The block diagram which shows typically the schematic structure in 1st Embodiment of the column for antibody purification which concerns on this invention. Cross-sectional view of relevant parts schematically showing the structure of an adsorbent of an antibody purification column according to the present invention Cross-sectional view of relevant parts schematically showing the structure of a porous carrier constituting an adsorbent of an antibody purification column according to the present invention SEM photograph of porous carrier constituting adsorbent of antibody purification column according to the present invention Adsorption breakthrough curve diagram showing the results of measuring the relationship between the linear velocity and the dynamic adsorption capacity of the evaluation column packed with the adsorbent of the antibody purification column according to the present invention The figure which shows the relationship between the linear velocity obtained from the adsorption breakthrough curve of FIG. 5, dynamic adsorption capacity, and contact time The figure which shows the result of having comparatively evaluated the adsorption | suction performance of four types of evaluation columns from which the center pore diameter of the porous support | carrier of the column for antibody refinement | purification which concerns on this invention differs. The block diagram which shows typically the schematic structure in 2nd Embodiment of the column for antibody purification which concerns on this invention.

  Embodiments of an antibody purification column according to the present invention (hereinafter, appropriately referred to as “this column”) and an antibody purification method using this column will be described with reference to the drawings.

<First Embodiment>
As an example, the main column 10 of the first embodiment is configured such that a disk-like or columnar adsorbent 1 is accommodated in a cylindrical container 2 as shown in FIG. Openings 3 and 4 are formed on the respective end surfaces of the cylindrical container 2. One of the openings 3 and 4 serves as a solution inlet through which each of the purification steps to be described later passes, and the other serves as a solution outlet after the passage.

  As schematically shown in FIG. 2, the adsorbent 1 which is a main component of the column 10 has an immune target substance adsorbed on the surface of an inorganic porous carrier 5 formed into a disk shape or a column shape. Protein 6 that specifically binds to the constant region of a globulin antibody is surface-modified and immobilized. In this embodiment, protein A is used as the protein 6.

  As shown schematically in FIG. 3, the inorganic porous carrier 5 constituting the adsorbent 1 has a three-dimensional network-like monolithic skeleton 7 and an average pore diameter formed in the gap between the skeleton 7. It has three-dimensional network-like through-holes 8 in the range of 4 μm or less, and furthermore, pores 9 having a central pore diameter in the range of 40 nm to 70 nm are dispersed and formed on the surface of the skeleton 7. Has been. In addition, the hole diameter of the through-hole 8 is larger than the hole diameter of the pore 9, for example, 0.5 micrometer or more. Therefore, the porous carrier 5 used in this column 10 has a double pore structure composed of two kinds of pores (through holes 8 and pores 9) having different pore diameters. FIG. 4 shows an example of an SEM (scanning electron microscope) photograph of the porous carrier 5 used in the column 10. The through holes are also called macropores, and the pores are also called mesopores.

  Next, regarding the method for producing this column provided with the adsorbent 1 in which protein A is immobilized as the protein 6 having antibody-binding ability on the surface of the porous carrier 5, the production of an evaluation column used for the adsorption performance evaluation described later. The process will be described as an example.

  First, 1.2 g of polyethylene glycol (molecular weight 100000) and 0.9 g of urea are dissolved in 10 ml of 10 mM (volume molar concentration) nitric acid aqueous solution, 5 ml of tetraethoxysilane is added under ice cooling, and the mixture is stirred for 30 minutes to obtain a uniform solution. Get. The obtained uniform solution is filled into a polypropylene tube having a diameter of 8 mm and gelled at 30 ° C. Thereafter, the obtained gel is put into a sealed container, heat-treated at 120 ° C. for 20 hours, rinsed and dried with distilled water, and fired at 650 ° C. for 5 hours to form a silica monolith porous carrier 5 (made of silica gel or silica glass). Corresponding to a porous carrier). The method for synthesizing the porous carrier 5 used in this embodiment uses a spinodal decomposition sol-gel method.

  Under the above synthesis conditions, a silica monolith having an average pore diameter (measured by mercury porosimetry) of about 1.7 μm and a pore center diameter (center pore diameter, measured by nitrogen adsorption method) of about 50 nm is obtained. By adjusting the addition amount of the above-mentioned polyethylene glycol, the pore diameter of the through hole can be controlled, and by adjusting the heat treatment temperature after gelation, the central pore diameter can be controlled. In this embodiment, the central pore diameter is a value obtained by differentiating the pore volume (V) in the pore distribution measured by the nitrogen adsorption method with the common logarithm (logD) of the pore diameter (D) (dV / d (logD)) is defined as the pore diameter showing the maximum peak of the curve plotted against the pore diameter (D).

  Subsequently, the obtained silica monolith porous carrier is cut into a diameter of 8 mm, loaded into a hard glass tube, and heated from the outer periphery with a gas burner to closely adhere the silica monolith porous carrier and the glass tube to obtain a glass clad column. The obtained glass-clad column is cut into a predetermined length (10 mm in the present embodiment) with a diamond cutter to obtain an evaluation column before protein A is immobilized.

  Subsequently, the obtained evaluation column was filled with a toluene solution of 5% γ aminopropyltriethoxysilane, and heated at 100 ° C. for 24 hours. After the reaction, 2-propanol was passed through the evaluation column. And dried at 80 ° C. Then, after passing through an acetonitrile solution of 1% succinimidyl carbonate, washing with acetonitrile, drying under reduced pressure, passing through a 5 mg / mL protein A HEPES buffer solution, an evaluation column having protein A immobilized thereon was prepared. obtain.

  In addition, the dimension, the molding method, etc. described in the above-described production method are values according to the shape and size of the evaluation column, and depending on the shape and size of the porous carrier 5 of the column 1 to be put to practical use, It is changed appropriately.

  Next, an evaluation procedure and evaluation results for evaluating the adsorption performance of the immunoglobulin antibody (IgG) of this column using the evaluation column prepared as described above will be described.

  In the evaluation of the adsorption performance of this column, the dependence of the dynamic adsorption capacity (dBC) on the central pore diameter of the porous carrier and the dependence on the linear velocity were examined. In the evaluation of the dependence on the central pore diameter, four types of samples having a central pore diameter of 30 nm, 50 nm, 70 nm, and 100 nm of a porous carrier were prepared, and the dynamic adsorption capacity at a linear velocity of 90 cm / h was measured. In the evaluation of the linear velocity dependence, a sample with a central pore diameter of 50 nm of a porous support was prepared, and the motion at four linear velocities of linear velocity 90 cm / h, 360 cm / h, 720 cm / h, and 1800 cm / h was prepared. The adsorption capacity was measured.

  In any evaluation, the dynamic adsorption capacity was measured as follows. The evaluation column was connected to a high performance liquid chromatography apparatus. After the evaluation column was equilibrated with 10 mM phosphate buffered saline (pH 7.4), 1 mg / mL IgG phosphate buffered saline solution (phosphate concentration 10 mM, pH 7.4) was adjusted to a predetermined linear velocity. Then, the solution was passed through the evaluation column, and the solution at the outlet of the evaluation column was measured online using an ultraviolet-visible absorptiometer at an ultraviolet absorption wavelength of 280 nm.

  In the measurement of the dynamic adsorption capacity, the amount of added IgG per unit volume of the gel (the amount of IgG solution passed through × the IgG concentration / the support volume of the column for evaluation, unit: mg / mLgel) is plotted on the horizontal axis. The detection intensity ratio of IgG phosphate buffered saline at the ultraviolet absorption wavelength of 280 nm of the outlet solution (the intensity at which phosphate buffered saline solution is detected at 1 mg / mL is defined as 100%) is plotted on the vertical axis. An adsorption breakthrough curve was drawn, and the point at which IgG added per gel volume exceeded 10% (10% breakthrough) was defined as the dynamic adsorption capacity.

  FIG. 5 shows an adsorption breakthrough curve of an evaluation column in which the central pore diameter of the porous carrier is 50 nm at linear velocities of 90 cm / h, 360 cm / h, 720 cm / h, and 1800 cm / h. The dynamic adsorption capacity for each linear velocity is derived from the 10% breakthrough of the adsorption breakthrough curve shown in FIG. 5, and the contact from the inlet to the outlet of the IgG solution evaluation column is determined from each linear velocity and the length of the evaluation column. Time is calculated. FIG. 6 summarizes the contact time and dynamic adsorption capacity for each linear velocity.

  As shown in FIG. 6, when the linear velocity is increased to 90 cm / h, 360 cm / h, 720 cm / h, and 1800 cm / h, the contact time is shortened to 40 seconds, 10 seconds, 5 seconds, and 2 seconds. The volume drops to 38 mg / mLgel, 33 mg / mLgel, 27 mg / mLgel, 20 mg / mLgel. Linear velocity and contact time are inversely related. Here, it should be noted that even if the linear velocity is increased and the contact time is reduced from 40 seconds to 2 seconds to 1/20, the dynamic adsorption capacity is reduced to about half from 38 mg / mLgel to 20 mg / mLgel. It is only a drop. That is, it can be seen that the basic adsorption performance of this column is high, so that the contact time can be further shortened from 40 seconds by increasing the linear velocity without causing an extreme decrease in adsorption capacity.

  FIG. 7 shows the dynamic adsorption capacities of four types of evaluation columns having a central support diameter of 30 nm, 50 nm, 70 nm, and 100 nm at a linear velocity of 90 cm / h. From FIG. 7, when the central pore diameter is less than 40 nm and 30 nm, the dynamic adsorption capacity decreases to about 13% when the central pore diameter is 50 nm, and conversely, the central pore diameter exceeds 70 nm and reaches 100 nm. As a result, the dynamic adsorption capacity decreases to about 26% when the central pore diameter is 50 nm, and there is an optimum central pore diameter range in the vicinity of the central pore diameter of 50 nm, generally in the range of 40 nm to 70 nm. I understand. The widths indicated by the horizontal arrows in FIG. 7 indicate the distribution ranges of the pore diameters of the four types of evaluation columns, and each is distributed in a range of approximately ± 10 nm.

  Since the size of the immunoglobulin antibody is about 10 to 12 nm, when the central pore diameter is about 30 nm, the antibody can bind to protein A immobilized near the surface of the pore skeleton, It is presumed that the protein A immobilized on the inner surface is not sufficiently bound. From FIG. 7, it is presumed that the center pore diameter is preferably 40 nm or more. Further, it is considered that as the central pore diameter increases from 70 nm to 100 nm, the surface area per pore volume decreases, and the dynamic adsorption capacity decreases. From the above, the center pore diameter of the porous carrier is preferably set in the range of 40 nm to 70 nm, more preferably in the range of 45 nm to 60 nm.

  Next, the processing procedure of the antibody purification method using this column will be described. In the present embodiment, the antibody purification method is composed of five steps of solvent replacement, adsorption, washing, elution, and post-washing.

  First, in the solvent replacement step, 10 mM phosphate buffered saline (pH 7.4) is passed through the column for equilibration.

  Next, in the adsorption step, IgG phosphate buffered saline solution (pH 7.4) or a solution containing immunoglobulin antibodies such as serum, ascites, cell supernatant, etc. is passed through this column to adsorb the antibodies. Adsorb to the body.

  Next, in the washing step, 10 mM phosphate buffered saline (pH 7.4) is passed through the column to wash away the solution containing the immunoglobulin antibody in the adsorbent.

  Next, in the elution step, 100 mM sodium citrate buffer (pH 3.0) or 100 mM glycine + hydrochloric acid buffer (pH 3.0) is passed through the column to make the adsorbent acidic and adsorbed on the adsorbent. Elute immunoglobulin antibodies. Thereby, the immunoglobulin antibody adsorbed on the adsorbent can be recovered.

  Next, in the post-cleaning step, 10 mM phosphate buffered saline (pH 7.4) is passed through the column to wash away the acidic solution in the adsorbent used in the elution step, and the adsorbent is neutralized. Let

  In the present embodiment, a single purification step comprising the above five steps of solvent replacement, adsorption, washing, elution, and post-washing can be performed within 120 seconds in the following manner. In this embodiment, based on the volume of the adsorbent, the volume of the solution to be passed through in each step is doubled in the solvent replacement step, tripled in the adsorption step, doubled in the washing step, and quadrupled in the elution step. It assumes 1 time in the cleaning process. In addition, the volume ratio of the solution which passes at each process is not limited to the said ratio.

  Assuming the use of an adsorbent in which protein A is immobilized on a cylindrical silica monolith porous carrier having a diameter of 8 mm and a length of 10 mm, which is the same as the column for evaluation described above, the flow rate of each solution through a single purification step is 0. When liquid was passed at 648 mL / min (corresponding to a linear velocity of 360 cm / h), 200 μL was eluted in the solvent replacement step for 17.5 seconds, 300 μL in the adsorption step was 26.3 seconds, and 200 μL was eluted in the washing step for 17.5 seconds. 400 μL is passed through for 35.1 seconds in the process, and 100 μL is passed through for 8.8 seconds in the post-cleaning step, and one purification step is completed in about 105 seconds.

  In addition, when the antibody concentration in the solution containing the immunoglobulin antibody passed through in the adsorption step is 10 mg / mL, an adsorption amount of 30 mg / mL gel per unit volume of the adsorbent can be obtained.

  In the above assumption, a linear velocity of 360 cm / h was assumed. However, from the evaluation results shown in FIGS. 5 and 6, the linear velocity can be made faster than 360 cm / h, so that the time required for one purification step is further longer than 105 seconds. Can be shortened. For example, if the time required for one purification step is shortened to 72 seconds, the purification step can be repeated 100 times in 2 hours. Even if the time required for one purification step is 120 seconds, the above purification step can be repeated 60 times in 2 hours.

  It should be noted here that the time required for one purification process is as short as 1 to 2 minutes, so that a strong alkaline solution (pH 13 or more) is used in the post-washing process for each purification process. There is no need to clean and sterilize the substance. In the conventional affinity column, it is known that the adsorption performance is reduced by the strong alkali treatment, and if the sterilization by the strong alkali treatment is not necessary, the above purification process is continuously repeated without reducing the adsorption performance. be able to.

  By repeating the purification step continuously, antibody purification can be carried out with high efficiency, and protein A used in this column is expensive. Therefore, by repeating the purification step 100 times or more, cost performance can be improved.

  On the other hand, with an affinity column using a conventional bead-shaped agarose gel currently in practical use, the total processing time required for antibody purification often exceeds 2 hours. In the meantime, of course, sterilization is not performed. In other words, even if the purification process is continuously repeated using this column, if the total treatment time is within 2 hours, there is no need for sterilization treatment as in the prior art, and a strong alkaline solution (pH 13 or higher) is used in the post-washing process. It may not be used.

Second Embodiment
A second embodiment of this column will be described. As shown in FIG. 8, the main column 20 of the second embodiment is configured by accommodating a cylindrical adsorbent 11 in a cylindrical container 12. Openings 13 and 14 are formed in the respective end faces of the cylindrical container 12, and one of the openings 13 and 14 serves as a solution inlet through which the liquid is passed in each step of the above-described purification process, and the other after the liquid is passed through. It becomes the outlet of the solution.

  As shown in FIG. 8, the cylindrical outer surface 11 a of the adsorbent 11 is separated from the inner wall surface of the cylindrical container 12, and a solution flow path 15 is formed therebetween. The left and right passages 15 in FIG. 8 are annular and communicate with each other. Further, the space inside the cylindrical shape of the adsorbent 11 also serves as a solution flow path 16. The opening 13 communicates with the flow path 16, and the opening 14 communicates with the flow path 15. When the opening 13 serves as the inlet and the opening 14 serves as the outlet, the solution flows in the order of the opening 13, the passage 16, the adsorbent 11, the passage 15, and the opening 14.

  The adsorbent 11 of this column 20 is processed into a cylindrical shape, but has an integral structure having a double pore structure composed of two types of pores (through-holes and pores) having different average pore diameters. Protein A is immobilized on the surface of the carrier and is the same as the adsorbent 1 of the first embodiment. Further, the average pore diameter of the through holes and the center diameter of the pores of the porous carrier are the same as in the case of the porous carrier 5 of the first embodiment. Therefore, since the production method of the adsorbent and the porous carrier and the adsorption performance thereof are the same as those in the first embodiment, a duplicate description is omitted.

  In the second embodiment, since each solution is passed from one of the cylindrical outer surface 11a and the cylindrical inner surface 11b of the cylindrical adsorbent 11 to the other, the cylindrical outer surface 11a and the cylindrical inner surface 11b. The distance between them is the thickness of the adsorbent 11. In the first embodiment, since each solution is passed from one of the upper surface and the lower surface of the disk-shaped or columnar adsorbent 1 to the other, the distance between the upper surface and the lower surface of the adsorbent 1 is the thickness of the adsorbent 1. It becomes.

  Next, another embodiment of this column will be described.

  <1> In each of the above embodiments, no specific numerical value is specified for the thickness of the adsorbents 1 and 11, but the thickness of the adsorbents 1 and 11 is the same as that of the evaluation column described in the first embodiment. Similarly, by setting it to 10 mm, in the adsorption step, liquid can be passed at a linear velocity of 90 cm / h or more, so that processing in a short time within 40 seconds can be performed. From the evaluation results shown in FIG. 5 and FIG. 6, the linear velocity can be made faster than 90 cm / h, so even if the thickness of the adsorbents 1 and 11 is increased in proportion to the linear velocity, the contact time is less than 40 seconds. Processing in time becomes possible.

  <2> In the first embodiment, a circular shape and a circular shape are assumed as the cross-sectional shapes of the adsorbent 1 and the cylindrical container 2, but the cross-sectional shape is not limited to a circular shape and a circular shape, and may be a polygonal shape. Absent. In the second embodiment, an annular shape is assumed as the sectional shape of the adsorbing body 11 and the cylindrical container 12, but the sectional shape is not limited to the annular shape, and may be a polygonal shape.

  <3> In each of the above embodiments, the average pore diameter (measured by the mercury intrusion method) of the through holes of the porous carrier is assumed to be 4 μm or less, and the evaluation column described in the first embodiment is 1.7 μm. However, it has been confirmed that the adsorption performance of this column does not greatly depend on the average pore diameter in the range where the average pore diameter of the through holes is 4 μm or less. Note that when the average pore diameter of the through-holes exceeds 4 μm, the pore surface area also decreases, and the adsorption performance also decreases.

  The antibody purification method and the antibody purification column according to the present invention can be used for the purification of immunoglobulin antibodies.

1: Adsorbent 2: Cylindrical container 3, 4: Opening (inlet, outlet)
5: Porous carrier 6: Protein A
7: Skeletal body 8: Through hole 9: Pore 10: Column for antibody purification according to the present invention 11: Adsorbent body 11a: Cylindrical outer surface of the adsorbent body 11b: Cylindrical inner surface of the adsorbent body 12: Cylindrical container 13, 14: Opening (inlet, outlet)
15: Flow path (outside of adsorbent)
16: Flow path (inside the adsorbent)
20: Antibody purification column according to the present invention

Claims (6)

It has a skeleton made of silica gel or silica glass having a three-dimensional network structure, and pores penetrating from the surface of the skeleton to the inside formed by being dispersed on the surface of the skeleton, and measured by a nitrogen adsorption method Using an antibody purification column comprising an adsorbent formed by immobilizing protein A on the surface of a porous carrier having a pore central diameter of 40 nm to 70 nm,
The pre-purification solution is allowed to flow through the antibody purification column under a condition where the contact time between the pre-purification solution containing the antibody to be purified and the adsorbent is 40 seconds or less, and the antibody in the pre-purification solution An antibody purification method characterized by adsorbing to an adsorbent and purifying the antibody.   2. The antibody purification method according to claim 1, wherein the contact time is 2 seconds or more and 40 seconds or less.   The method for purifying an antibody according to claim 1 or 2, wherein the time required for one purification step comprising the steps of solvent substitution, adsorption, washing, elution and post-washing is 120 seconds or less.   The said purification | cleaning column is wash | cleaned without using the strong alkali solution exceeding pH13 at the said post-washing process, and the said refinement | purification process is repeated 100 times or more within 2 hours. Antibody purification method. Proteins are formed on the surface of a porous carrier having a skeleton made of silica gel or silica glass having a three-dimensional network structure, and pores penetrating from the surface to the inside of the skeleton formed by being dispersed on the surface of the skeleton. An adsorbent formed by fixing A,
A column for antibody purification, wherein the pore has a center diameter of 40 nm or more and 70 nm or less as measured by a nitrogen adsorption method.   The pre-purification solution is passed through the adsorbent under conditions where the contact time between the pre-purification solution containing the antibody to be purified and the adsorbent is not shorter than 2 seconds and not longer than 40 seconds. Dynamic adsorption defined by the amount of the antibody added per unit volume of the adsorbent exceeding 10% in the adsorption breakthrough curve obtained using ultraviolet light having a wavelength of 280 nm when adsorbed on the adsorbent. 6. The antibody purification column according to claim 5, wherein the column has an adsorption capacity exceeding 10 mg / mLgel.