JP2021148455A - Continuous column chromatography unit - Google Patents

Abstract

To provide a continuous column chromatography unit capable of swiftly finding and replacing abnormal columns by monitoring the condition of the columns used for purification to obtain a highly pure and stable purified protein in the continuous protein purification process.SOLUTION: There is provided a continuous purification method in which, when one column performs the biopolymer purification process in a column switching method using two independent columns, pattern analysis of the concentration of the evaluation sample is performed after passing through the column following the washing and regeneration process on the other column.SELECTED DRAWING: Figure 1

Description

The present invention relates to a continuous protein purification apparatus by chromatography using a plurality of columns.

In the purification of biopharmaceuticals, generally, a host such as an animal cell, yeast, or bacterial cell capable of expressing a target substance by gene recombination is cultured, and cells or cells or cells or cells or cells or cells or cells or cells or cells or cells are used from a raw material secreted into the host cell or culture medium. After removing solids such as precipitates, it is often purified through a purification step by chromatography in 2 to 3 steps. Further, in these chromatographic purifications, since the amount of the target product to be purified by one operation is limited depending on the size of the column packed with the separating agent, it is often purified by a plurality of batch treatments. Therefore, a more efficient method is required because it is necessary to use a large column in order to obtain a large amount of the target product, low efficiency due to batch processing, and economic problems of the separating agent to be used.

In addition, conventional biopharmaceuticals have been produced by a discontinuous method such as a batch method or a fed batch culture method in the protein production process by culturing the production cells, which is the upstream of the production, but the perfusion culture method is used. There is a demand to switch to the continuous production method due to the progress of protein production technology. For this reason, even in the downstream purification method, continuous processing that can cope with it is required.

However, it is difficult to guarantee the reproducibility of the process and the quality of the purified object by the conventional separation and purification method by simply repeating column chromatography. Under such circumstances, the following methods are attracting attention as new purification methods.

A so-called pseudo-moving bed (SMB) type separation / purification method is known in which separation / purification, washing / regeneration, and the like are continuously performed while continuously flowing a process liquid through a plurality of columns (Non-Patent Document 1). Since the separation method by SMB can continuously process a large amount of process solution by increasing the number of columns, it is used for the purpose of capturing the so-called downstream first half to recover the target substance from the culture solution. However, questions remain about the reproducibility of the process and the safety of the column.

In the latter half of the downstream, so-called polishing, a high-performance separation ability capable of separating a small amount of impurities is required, so a purification method of purifying using a column finely packed with a high-performance separating agent is adopted. As a continuous precision purification method of this purification method, a column switching method has been developed in which a plurality of high-performance separation columns are used to sequentially process the process liquid, and separation / purification and washing are alternately repeated to continuously produce the target product. An apparatus for purifying has already been developed (Non-Patent Document 2 and Patent Document 1).

Further, in liquid chromatography in which a sample separated by one separation column is recirculated to the same separation column again, two flow paths each having a separation column and two flow paths for connecting and disconnecting the flow paths. Liquid chromatography is known, which is provided with a switching valve and is characterized in that the introduction direction of sample reintroduction between two separation columns is switched by switching the flow path switching valve at the time of recycling separation (patented). Document 2).

However, the column generally used in such a column switching method is used after inactivating microorganisms and endotoxins and confirming the filling state of the separating agent to confirm its suitability before starting purification. However, once continuous purification is started, the filling state is not confirmed even if purification and washing / regeneration are repeated, and the eligibility is not confirmed in repeated purification.

As a method of confirming the filling state of the separating agent packed in the column and confirming its suitability, a low molecular weight sample (for example, a high-concentration salt solution) is added to the column in a pulsed manner, and the theory of the column is based on the elution position and shape. A method of measuring the number of stages (N / m) and peak symmetry (Af), and a method of linearly differentiating the signal from the monitor for these measurements and continuously measuring the number of theoretical plates from the set of the first derivative curves. Is known (Non-Patent Document 3). Using this technology, in the conventional discontinuous protein production, the production is stopped regularly, the theoretical plate number and peak symmetry are measured, and the column eligibility is checked, but it is continuous. It is not possible to check column eligibility without stopping production in various production methods.

For this reason, in continuous refining, if an abnormality is found by regular inspection after the end of continuous production, it is unknown at what stage the abnormality occurred, and the problem is that a large number of products must be suspended from shipment. be.

In the continuous purification step of protein, in order to always obtain a highly pure and stable purified protein, a method of monitoring the state of the column used for purification, rapidly finding an abnormal column, and using it for continuous purification is desired. ..

JP 2014-001979 JP 2006-201039

Rahul Godawat et al., Journal of Biotechnology, 2015, 213, pp. 13-19 Seiki Sugiyama et al., Journal of Japanese Food Engineering, 2018, Vol. 19, No. 1, pp. 35-41 Yuan Yuan Cui et al., "Using Direct Transition Analysis in Chromatography", Bio Pharma International (Biofarm International, January 31, 2018, Vol. 1, 2018), January 20 Page 40 (searched January 21, 2020), Internet http://www.biopharminternational.com/using-direct-transition-analysis-chromatography

In the precision separation and purification method, a separating agent with a relatively small particle size is required because it requires high-performance separation ability such as separating substances with very similar physical and chemical properties such as trace impurities and impurities derived from the target object. Closely packed columns are often used. Therefore, the separation performance changes when the filling state of the separating agent in the column changes. In the purification method of continuously purifying using a repeating column, the packed state of the column is a very important factor for obtaining a product of a certain standard. Especially in the manufacture of pharmaceuticals, there is a concern that contamination with impurities may cause serious side effects, so reproducibility in the manufacture is a very important factor.

In the continuous separation and purification method by the conventional column switching method, the filling state of the separating agent is not confirmed every time after the washing and regeneration operation of the column, and the first and last purification treatments of the continuous purification, or the continuous purification treatment is performed 10 times. The filling state was confirmed after performing the times at regular intervals such as 20 times. However, the filling state of the separating agent in the column may change, and in the conventional method of confirming the packing material of the separating agent in the column in continuous purification, when an abnormality is found, the abnormality is found from any point in the past. It became difficult to determine whether it had occurred, and a large production loss was caused, such as discarding all the produced lots.

The present invention
[1] A purification system provided with two independent columns A and B in which the introduction port is connected to the purification sample supply pipe or the cleaning liquid supply pipe and the corresponding outlets are connected to separate outlet pipes is used. When one column is subjected to the biopolymer purification step, the other column is provided in the cleaning liquid supply pipe in the cleaning and regeneration step in the continuous purification method of the biopolymer, which comprises performing the washing and regeneration step. A continuous purification method characterized by pattern analysis of the concentration of the evaluation sample supplied by the evaluation sample supply device after passing through the column.
[2] The continuous purification method according to [1], wherein the lead-out pipe is connected to a detection device and branches into a purification sample discharge pipe and a cleaning waste liquid discharge pipe.
[3] The continuous purification method according to [1] or [2], wherein the pattern analysis of the concentration of the evaluation sample after passing through the column is used for process control.
[4] The continuous purification method according to any one of [1] to [3], wherein the replacement time of the column can be grasped by pattern analysis of the concentration of the evaluation sample after passing through the column.
[5] The continuous purification method according to [4], wherein the system is automatically stopped when an abnormality in the column state is detected by pattern analysis of the concentration of the evaluation sample after passing through the column.
[6] The continuous purification method according to any one of [1] to [5], wherein the individual result of column purification and the status of the column are automatically recorded in the continuous production of pharmaceutical products.
[7] Process control by pattern analysis of the concentration of the evaluation sample after passing through the column in the washing and regenerating process is the measurement result of the theoretical plate number of the chromatogram pattern and the symmetry of the separated peaks by the detection device connected to the lead-out tube. The continuous purification method according to [3], which is carried out by evaluating the packed state of the column based on the above.
[8] The continuous purification according to any one of [1] to [7], wherein the purification step is a step of adsorbing the purification sample to the resin in the column after being added to the column. Method,
[9] The purification step comprises one or more purification conditions, a step in which a purification sample is added to the column and adsorbed on the resin in the column, and unadsorbed impurities and an excess purification sample are washed with a buffer solution. The continuous purification method according to any one of [1] to [7], which is a step in which at least one of a step, a purification sample elution step, and a washing elution step is combined.
[10] The continuous purification method according to any one of [1] to [7], wherein the purification step is a step in which impurities are adsorbed on the resin while the purification sample passes through the column.
[11] The washing and regenerating step is a step in which unadsorbed impurities on the column and an excessive purification sample are washed with a buffer solution, a purification sample elution step or a washing elution step, and a washing with injection water or sterile purified water or a buffer solution. Any one of [1] to [10], which is a combination of one or more steps selected from a step, a column regeneration cleaning step, and a resin equilibrium step, and a test step using a test sample. The continuous purification method described in one,
[12] The washing and regenerating step combines at least one of a column washing and elution step, a washing step with water for injection or sterile purified water or a buffer, a column reclaiming and washing step, a resin equilibrium step, or a test step using a test sample. The continuous purification method according to any one of [1] to [10], which is characterized by the above-mentioned step.
[13] The continuous purification method according to any one of [1] to [12], wherein the purification sample is a solution containing a biopolymer.
[14] The continuous purification method according to [13], wherein the biopolymer is an antibody, and [15] the evaluation sample has absorption in a sodium chloride solution, a high-concentration buffer solution for testing, or ultraviolet rays. The method according to any one of [1] to [14], which comprises a solution containing a low molecular weight compound or a high concentration low molecular weight solution capable of measuring a refractive index.

Furthermore, the present invention
[16] In a purification apparatus provided with two independent columns A and B, the introduction port of one column is connected to the purification sample supply pipe via the first four-way switching valve, and the other column is connected. A purification sample discharge pipe and a cleaning waste liquid discharge which are connected to a cleaning liquid supply pipe which can be connected to the evaluation sample supply device via the first four-way switching valve and whose corresponding outlets are respectively via the second four-way switching valve. A purification device, characterized in that it branches into a tube and is separately connected to two lead-out tubes provided with a pattern detection device for evaluation sample concentration.
[17] The sample supply pipe for purification can be connected to any one or more or a combination of a biopolymer-containing solution introduction pipe, an equilibrium buffer introduction pipe, an eluate introduction pipe, and a buffer solution introduction pipe for a washing elution step. The purification apparatus according to [16],
[18] The cleaning liquid supply pipe is an equilibrium buffer introduction pipe, an eluent introduction pipe, a cleaning dissolution buffer introduction pipe, injection water or sterile purified water or a buffer solution introduction pipe, a column regeneration cleaning liquid introduction pipe, and a resin. The purification apparatus according to [16] or [17], which can be connected to any one or more or a combination of equilibrium buffer introduction tubes.
[19] The purification device according to any one of [16] to [18], wherein the evaluation sample introduction device is connected to a cleaning liquid introduction tube by a sample injection valve.
[20] The purification apparatus according to any one of [16] to [19], wherein the four-way switching valve is a four-way rotary valve.
[21] The evaluation sample concentration pattern detecting device is a device including one or more or a combination of an electrical conductivity measuring device, an ultraviolet ray, a visible part absorption measuring device, and a refractive index measuring device. The purification apparatus according to any one of [16] to [20], and [22] a lure-lock type connector or a triclamp, wherein the column, in-line filter and detection apparatus are suitable for single use. The purification apparatus according to any one of [16] to [21], which is coupled to the apparatus by a TC) type connector.

The purification apparatus according to the present invention purifies an object to be purified by a continuous chromatographic purification method by repeating operations in a column switching method by measuring the theoretical plate number and peak symmetry after each cleaning and regeneration treatment. Quality can be guaranteed.

In the purification apparatus according to the present invention, not only the purification conditions developed by using one column can be used as they are, but also the cleaning and regeneration conditions of the column are the same. It is possible to think in the same way as the method of. Furthermore, since the filling status of the columns can be checked each time the two columns are switched and regenerated and washed, the operation can be stopped before the purification is started using the column in which the abnormality has occurred. Therefore, the equivalence can be ensured for the quality of the purified target product.

The following items can be mentioned as the effects of the present invention.
(1) The apparatus of the present invention can connect a plurality of chromatography columns to continuously process the process liquid, and at the same time, the other columns not treated with the process liquid are washed, regenerated and column efficiency (theoretical). It is a device that can measure the number of stages and peak symmetry).
(2) In order to carry out the function of (1) in the present invention, the liquid feeding system including the pump and the monitor for separating and purifying the process liquid and the liquid feeding system for cleaning, regenerating the column and measuring the column efficiency are separately separated. It is equipped and can be switched with a valve.
(3) In the apparatus of the present invention, after the separation and purification operation is completed by adding a predetermined amount of the process liquid to the column that has been washed, regenerated and balanced in advance, the cleaning, regeneration and column are performed by switching the valve. It is possible to switch to a liquid feeding system that measures efficiency (theoretical plate number and peak symmetry). On the other hand, the column which has been washed, regenerated, and equilibrated in advance and newly switched can be switched to a liquid feeding system for separation and purification, and a predetermined amount of process liquid can be added to perform the separation and purification operation.
(4) In the apparatus of the present invention, in order to perform the function of (3), the column efficiency (theoretical plate number and peak symmetry) can be measured in the liquid feeding system for cleaning and regenerating the column. Equipped with a sample injection valve, the column efficiency can be measured after cleaning is completed.
(5) Further, since the apparatus of the present invention performs the function of (3), the column efficiency (theoretical plate number and peak symmetry) is measured via a valve provided with a sample loop for adding a predetermined amount of sample. However, if the amount of sample to be added is large, the sample can be added directly from the pump via the valve.
(6) In the apparatus of the present invention, the column subjected to separation and purification can be automatically measured for column efficiency (theoretical plate number and peak symmetry) after washing, regeneration, and equilibration each time.
(7) The column connected to the apparatus of the present invention differs depending on the separation mode, and is filled with affinity, ion exchange, hydrophobicity, reverse phase, normal phase, hydroxyapatite, multimode, etc., as well as activated carbon or diatomaceous earth adsorbent. And any separation medium such as membrane chromatography separation medium can be used.
(8) The separation / purification mode is an adsorption / separation type (B / E type) in which the target object is once bound to a separation medium such as a filler and then the target substance and impurities are separated by changing various elution conditions. Other than the separation method The target object can be used in any separation method such as a flow-through type (FT type) separation method in which impurities are adsorbed without being adsorbed on a separation medium such as a filler.
(9) In the adsorption / separation type (B / E type) separation method, stepwise elution is performed by gradually changing the elution conditions such as the concentration of the salt in the eluate, the concentration of the adsorption inhibitor, or the pH and temperature. It can be used for basically any separation method such as a method or a gradient type elution method in which the mixture is continuously changed.
(10) The apparatus of the present invention is continuous in any separation and purification step such as a clarification process liquid containing a large amount of impurities from which solids have been removed, a crude purification process liquid in which the target product is partially purified, or a final purification process liquid. It can be used as a purification method.
(11) Since the apparatus using a plurality of columns of the present invention can faithfully reproduce the optimized conditions using one column, the column cleaning conditions, the column regeneration conditions, and the addition of the process liquid It is possible to establish a continuous purification method that makes use of conventional process development methods such as quantity, separation condition setting, and column life.

FIG. 1 shows a purification apparatus by a 2-column switching method using a B / E type cation exchange column. FIG. 2 shows a purification apparatus by a 2-column switching method using an FT type anion exchange column. FIG. 3 shows a purification device by a column switching method using a B / E type protein A affinity column, which is designed for single use. FIG. 4 shows the results regarding the following (a) to (f) of the 6-cycle step by column switching in the continuous 6-cycle purification using the protein A affinity column of the antibody. (A) UV280 absorption measurement in the purification step, (b) theoretical number of steps measurement by UV280 absorption measurement in the cleaning step, (c) pH measurement in the purification step, (d) pH measurement in the cleaning step, (e) purification step The number of theoretical stages is measured by measuring the electrical conductivity in (f) and measuring the electrical conductivity in the cleaning process. FIG. 5 shows the results regarding the following (a) to (f) of the 6-cycle process by column switching in the continuous 6-cycle purification using the cation exchange column of the antibody. (A) UV280 absorption measurement in the purification step, (b) theoretical number of steps measurement by UV280 absorption measurement in the cleaning step, (c) pH measurement in the purification step, (d) pH measurement in the cleaning step, (e) purification step The number of theoretical stages is measured by measuring the electrical conductivity in (f) and measuring the electrical conductivity in the cleaning process. FIG. 6 shows the results regarding the following (a) to (f) of the three-cycle process by column switching in the continuous three-cycle purification using the anion exchange column of the antibody. (A) UV280 absorption measurement in the purification step, (b) theoretical number of steps measurement by UV280 absorption measurement in the cleaning step, (c) pH measurement in the purification step, (d) pH measurement in the cleaning step, (e) purification step The number of theoretical stages is measured by measuring the electrical conductivity in (f) and measuring the electrical conductivity in the cleaning process.

The biopolymer in the method for continuously purifying a biopolymer of the present invention means a sugar, a protein, a nucleic acid, etc., and is particularly preferably used for purification of a protein. The continuous purification method is not a batch-based purification method, but may be any method as long as it continuously purifies a sample for purification. For example, in the production of biopharmaceuticals, a culture step such as perfusion culture may be used. Either the integration method performed in combination with continuous production or the method of processing a large amount of stocked samples for purification by continuous purification may be used.

In the method of the present invention, two independent columns A and B in which the inlet is connected to the purification sample supply pipe or the cleaning fluid supply pipe and the corresponding outlet is connected to the purification sample discharge pipe or the cleaning waste liquid discharge pipe. In the purification system provided with, two independent column A and column B inlets can be fluidly connected to the purification sample supply pipe or cleaning liquid supply pipe via the first four-way switching valve. In addition, the system in which the outlets of the column A and the column B are connected to the purified sample discharge pipe or the cleaning waste liquid discharge pipe via the second four-way switching valve is shown. It is preferable to use a four-way rotary valve as the four-way switching valve.

By using the purification system, when one column performs the biopolymer purification step, the other column can perform the washing and regeneration step, and the continuous production of the biopolymer can be smoothly carried out. It will be possible.

In the present invention, the sample supply pipe for purification means a means for supplying a sample to be purified by the purification means of the present invention, and the culture tank for producing the sample or the purification obtained by performing crude purification of the sample produced in the culture tank. The piping that is fluidly connected to the means is shown. Further, depending on the aspect of the present invention, various cleaning solutions, eluates of the purification sample, etc. may be fluidly connected to the purification sample supply pipe via a valve, and these solutions may be supplied to the column if necessary. Specifically, the purification sample supply pipe is one or more or a combination of a biopolymer-containing solution introduction pipe, an equilibrium buffer introduction pipe, an eluate introduction pipe, and a buffer solution introduction pipe for a washing elution step. It suffices if it can be connected.

The sample for purification in the present invention may be any chemical substance that requires purification using a column as long as it is a biopolymer that requires continuous purification, but a protein is preferable, and for example, angiotensin, etc. Otacoids such as brazikinin and endoserine, interleukins, hematopoietic factors, interferons, tumor necrosis factors, growth factors, cytokines such as chemokine and their receptors and antibodies, other immunoglobulins and their humanized antibodies, humanoid antibodies, FAB and (FAB) ) Examples thereof include fragment antibodies such as _{2 and variants such as bifunctional antibodies.}

Further, in the continuous purification method of the present invention, it is preferable to use a buffer solution, and any buffer solution used for purifying biopolymers may be used, but various concentrations of sodium phosphate buffer solution and phosphoric acid may be used. Potassium acetate buffer, sodium citrate buffer, Tris hydrochloride buffer or Tris acetate buffer, or metal salts such as calcium chloride, calcium carbonate, magnesium chloride, magnesium carbonate, ethylenediamine tetraacetic acid (EDTA), etc. In addition to a buffer solution containing a metal chelating agent such as glycol ether diamine tetraacetic acid (EGTA), a buffer solution containing amino acids such as glycine and arginine, ethylene glycol, and various thioethers is used to ensure the stability of the protein. ..

In the present invention, the cleaning liquid supply pipe is fluidly connected to these solutions via a valve so that various cleaning liquids and eluents of purification samples used in the purification step and the cleaning and regeneration step of the present invention can be supplied. The piping is shown. One of the features of the present invention is that an evaluation sample supply device is connected to the cleaning liquid supply pipe via a sample injection valve, and has a function of supplying an evaluation sample in the regeneration cleaning process and detecting the status of the column. It is one. Specifically, the cleaning solution supply pipe is an equilibrium buffer solution introduction pipe, an eluent introduction pipe, a cleaning dissolution buffer introduction pipe, injection water or sterile purified water, or a buffer solution introduction pipe, a column regeneration cleaning liquid introduction pipe, and a column. Any one or more or a combination of resin equilibrium buffer introduction pipes may be connected.

In the present invention, the outlet pipe refers to two pipes that can be fluidly connected separately from the outlets of the two columns, and these outlet pipes are branched as necessary to the purified sample discharge pipe, the cleaning liquid discharge pipe, and the like. It has a structure that can be fluidly connected. The purified sample discharge pipe has a structure that can be fluidly connected to the following purification means. One of the features of the present invention is that an evaluation sample detection device is connected to at least one of the two lead-out tubes to have a function of detecting the status of the column.

In the method of the present invention, analyzing the concentration pattern of the evaluation sample supplied by the test sample supply device provided in the cleaning liquid supply pipe in the cleaning / regeneration step after passing through the column means that the cleaning liquid is supplied in the cleaning / regeneration step. The evaluation sample supplied by the evaluation sample supply device provided in the tube enters the column through the inlet of the column connected to the cleaning liquid supply tube and is eluted from the outlet of the column. Regarding the concentration, it is shown that the filling state of the column is evaluated from the measured value of the symmetry of the separated peaks and the theoretical plate number of the chromatogram pattern measured by the detection device provided in the elution tube.

Since the evaluation value of the column filling state such as the theoretical plate number of the chromatogram pattern measured by the above method and the measured value of the symmetry of the separated peaks is automatically recorded, and the operator can always check it on the screen. It can be used for purification process control. Furthermore, if the number of continuous purifications in which the evaluation value of the packed state of the column is disturbed, such as the theoretical plate number of the chromatogram pattern measured empirically and the measured value of the symmetry of the separated peaks, the column replacement time can be determined in advance. Can be grasped. Further, when the measured value of the symmetry of the peak separated from the theoretical plate number of the chromatogram pattern deviates from the preset reference value range, it can be detected as an abnormality. When the detection device detects an abnormality, it transmits a signal for stopping the work of the purification process in which the purification system is incorporated. The evaluation sample supply device is a fluid from a container storing a test sample such as a high-concentration NaCl solution of about 0.5 M to 2.0 M or a high-concentration buffer solution, which is an evaluation sample, via a cleaning liquid introduction pipe and a valve. It is connected and can be added via a valve with a sample loop for adding a predetermined amount of sample, but if the amount of sample to be added is large, it can be added directly from the pump via the valve. Samples can be added.

As another aspect of the present invention, as a purification step in one column fluidly connected to the purification sample supply pipe, a step of adding the purification sample to the column and then adsorbing it to the resin in the column is performed, and the other is performed. In the column, as the washing and regenerating step, a step of washing the unadsorbed impurities and excess of the purified sample of the column with a buffer solution, an elution step of the purified sample adsorbed on the resin or a washing and elution step, water for injection or sterile purified water. Alternatively, at least one of a cleaning step using a buffer solution, a column regeneration cleaning step, a resin equilibrium step, or a test step using a test sample may be continuously performed in combination with other steps.

Further, as another aspect of the present invention, as a purification step in one column fluidly connected to the purification sample supply pipe, a step of adding a purification sample to the column and adsorbing it on the resin in the column, unadsorbed impurities, and the like. When a step of washing the excess purified sample with a buffer solution, a step of elution of the purified sample, or a step of combining at least one of the washing and elution steps is continuously performed, the other column is used as a washing and regenerating step of the column. Continuously combine at least one of a washing and elution step, a washing step with water for injection or sterile purified water or a buffer, a column regeneration washing step, a resin equilibrium step or a test step with a test sample, and other steps. Just go.

The purification method in the protein purification step of the present invention differs depending on the separation mode, and is a column packed with affinity, ion exchange, hydrophobicity, reverse phase, normal phase, hydroxyapatite, multimode, etc., as well as activated charcoal or diatomaceous earth adsorbent. And any separation medium such as membrane chromatography separation medium can be used. The separation mode is an adsorption / separation type (B / E type) separation method in which the target object is once bound to a separation medium such as a filler and then the target object and impurities are separated by changing various elution conditions. The target object can be used in any separation method such as a flow-through type (FT type) separation method in which impurities are adsorbed without being adsorbed on a separation medium such as a filler. In the adsorption / separation type (B / E type) separation method, a stepwise elution method or continuous elution method in which elution conditions such as salt concentration in the eluate, concentration of adsorption inhibitor, pH and temperature are changed stepwise to separate and purify. It can be used for basically any separation method such as a gradient type elution method in which the substance is changed.

The cleaning solution used in the present invention is a separating agent based on polysaccharides such as dextran, agarose, and cellulose. In addition to salts such as sodium hydroxide and sodium sulfate, organic solvents such as methyl alcohol, ethyl alcohol, n-propyl alcohol, and iso-propyl alcohol, and solutions mixed with various ionic and nonionic surfactants are pumped through the column. Liquid and washed. On the other hand, in separating agents using inorganic substrates such as silica gel, glass beads or crushed glass and controlled pore glass, in addition to acids such as hydrochloric acid and phosphoric acid, organic acids such as acetic acid and citric acid are used and they are used. A solution or the like in which various organic solvents such as various salts and alcohols or various surfactants are mixed with the acid of the above is used.

In the washing and regenerating step of the present invention, there is a system that automatically measures the column efficiency (theoretical plate number and peak symmetry) as a means for continuously confirming the filling status of the column each time. Specifically, an evaluation sample supply device for testing the filling state of the column is arranged in the cleaning liquid supply piping system into which the cleaning liquid is introduced. The evaluation sample supply device is fluid-connected via a cleaning liquid introduction pipe and a sample injection valve. After the cleaning of the column is completed by installing the evaluation sample supply device, the column efficiency can be measured by injecting the evaluation sample into the column. Further, in order to measure the concentration of the evaluation sample after the column of the injected sample is derived, in the apparatus of the present invention, a sample detection device is provided in the cleaning waste liquid discharge pipe of the branched outlet pipe. The detection device measures the symmetry of the peak separated from the theoretical plate number from the chromatogram pattern of the evaluation sample, and evaluates the packed state of the column. If the detector evaluates that the column is poorly packed, it sends an automatic signal to the device that manages the continuous purification process, interrupting the continuous purification operation of the biopolymer. Columns that are judged to have a bad evaluation during this interruption can be replaced with new columns.

The sample detection device in the present invention may be any device as long as it can measure the symmetry of the peak separated from the theoretical plate number from the chromatogram pattern of the test sample, and the number of theoretical plates repeatedly detected and the number of theoretical plates. When an abnormality is found in the symmetry of the peak, it is sufficient as long as it can transmit a signal in which the abnormality is recognized. By using such an apparatus, the filling state of the column is automatically and continuously evaluated. Specifically, if the detection device evaluates that the column is poorly filled, any device that can send an automatic signal to the device that manages the continuous purification process may be used, but a high-concentration salt solution or high-concentration buffer can be used. When the solution is used as a test sample, an electric conductivity meter manufactured by Toa DDK (product name: MM-43X2-1A00), a conductivity meter manufactured by HORIBA, Ltd. (product name: HE-960HS) and the like are suitable.

In the present invention, two independent columns A and B are provided, and the introduction port of one column is connected to the purification sample supply pipe via a first four-way switching valve, preferably a four-way rotary valve, and the other. Column is connected to a cleaning fluid supply pipe that can be connected to the evaluation sample supply device via a first four-way switching valve, and the corresponding outlets are each via a second four-way switching valve, preferably a four-way rotary valve. The present invention relates to a purification device which is branched into a purification sample discharge pipe and a cleaning waste liquid discharge pipe and is separately connected to two outlet pipes provided with a pattern detection device for evaluation sample concentration.

In the purification apparatus of the present invention, the purification sample supply tube is not only for the purpose of introducing the biopolymer for the purpose of purification into the column, but also for the balance buffer solution, eluate, wash elution step buffer solution and the like column. It has a function of transferring all the solutions necessary for the purification operation of the biopolymer in the column to the column. Therefore, the sample supply pipe for purification is connected to one or more or a combination of a biopolymer-containing solution introduction pipe, an equilibrium buffer introduction pipe, an eluent introduction pipe, and a buffer solution introduction pipe for a washing / elution process. It is connected to a rotary valve connected to these introduction pipes or a branch valve having a plurality of introduction ports and one outlet and allowing the introduction port to be arbitrarily selected.

In the purification apparatus of the present invention, the cleaning solution supply pipe is used for cleaning columns such as equilibrium buffer solution, eluate, cleaning elution buffer solution, injection water, sterile purified water, buffer solution, and regenerated cleaning solution used for column cleaning. It has the function of supplying the solution to the column. Therefore, an equilibrium buffer introduction tube, an eluent introduction tube, a washing elution buffer introduction tube, injection water or sterile purified water or a buffer introduction tube, a column regeneration cleaning solution introduction tube, and a resin balancing buffer introduction. Connected to a rotary valve connected to these inlet pipes to connect any one or more of the pipes or a branch valve of the type that has multiple inlets and one outlet and the inlet can be selected arbitrarily. There is.

The evaluation sample introduction device, which is a feature of the present invention, is characterized in that it branches into a purification sample discharge pipe and a cleaning waste liquid discharge pipe, and is separately connected to two outlet pipes provided with a pattern detection device for evaluation sample concentration. Regarding the purification equipment to be used. It is connected to the sample injection valve that is connected to the evaluation sample storage tank and the cleaning liquid introduction pipe. Therefore, the evaluation sample is introduced into the column through the cleaning liquid introduction tube.

In the purification apparatus of the present invention, of the two outlet tubes connected via the column outlet and the four-way switching valve, preferably the four-way rotary valve, the purified sample outlet tube is the following biopolymer purified by the column. It is connected to the equipment of the refining process, and the cleaning waste liquid discharge pipe is directly connected to the discharge port. The evaluation sample concentration pattern detection device is connected to these outlet tubes. The evaluation sample concentration pattern detecting device is a device provided with a detecting means for any one or more or a combination of an electric conductivity measuring device, an ultraviolet ray, a visible part absorption measuring device, and a refractive index measuring device.

The apparatus of the present invention enables real-time confirmation of the filling state of the column, recording of the filling state along the time axis, and storage thereof in the process of continuous purification of the in vivo polymer.

Further, in the device of the present invention, the components required for safety management single use are joined by a joint suitable for the purpose so that each member is suitable for single use, and can be easily replaced. Is designed for. Specifically, for the joints of equipment that is important for refining operations such as columns, filters or detectors, and their measurements, keep them aseptic or clean with luer lock type fittings or sterile single-use fittings. Use a fitting that can be connected or replaced as a joint. For columns, filters or detectors, luer lock type fittings, sanitary fittings (TC clamps) or sterile single use fittings may be used for the fittings of any one of the devices, all of which are used in the fittings. Is also good. As for the joints of the other devices, any fitting may be used as long as the aseptic condition can be maintained, and a bamboo shoot type cable tie or the like may be used.

[Example 1]
[Purification device by 2-column switching method using B / E type cation exchange column]
A purification apparatus by a 2-column switching method using a B / E type cation exchange column was manufactured (Fig. 1). The apparatus can perform a purification step including adsorption, washing, and elution of proteins on one of the two columns, and a column regeneration washing step on the other. The outline of this apparatus will be described below with reference to the apparatus numbers in the drawings.

In FIG. 1, the antibody used as the purification sample is supplied from the upstream process of the purification system to the purification sample supply pipe (2) via the branch valve (1). The process liquid containing the purification sample is adjusted to be adsorbed on the B / E type chromatography column, and then sucked by the pump (13) via the air trap (12), and is sucked by the first four-way switching valve (3). ) Is connected. The column switching valve consists of two valves, a first four-way switching valve (3) and a second four-way switching valve (4), and two columns, column A (5) and column B (6) between the two valves. ) Is connected.

When the process liquid is connected so as to flow to one column A (5), operations such as adsorption, washing, and elution of the purification sample in the purification step are continuously performed in the column A. At this time, the other column B (6) is connected to the evaluation sample supply tank (15) for measuring column efficiency, which is connected to the cleaning liquid supply pipe (14) via the sample injection valve (7). .. Further, a plurality of different solutions are connected to the pump (13) via a manifold (8) with a valve. When each valve is opened and closed according to the elution conditions, the liquid is sent to the column and used for purification steps such as adsorption, cleaning, and elution. On the other hand, in the sample injection valve (7), an evaluation sample for measuring column efficiency is added from the evaluation sample supply tank (15) to the sample loop (22) by the sample addition pump (9). Further, in the cleaning liquid supply pipe (14), the cleaning liquid, the regenerating liquid, the equilibrium buffer liquid, etc. used in the cleaning and regenerating process are air trapped (20) from the manifold (16) with a branch valve via the sample injection valve (7). The column B (6) is cleaned by being supplied via the pump (21) and the pump (21). After the column is washed, an evaluation sample for measuring column efficiency is added to the column by the above-mentioned flow path and measured. Two lead-out tubes (10) and (11) are connected to the outlet of the second four-way switching valve (4), and detection devices for purification samples and evaluation samples [UV monitor (17) and pH monitor (18), respectively. ), Electrical conductivity meter (19)] is connected, the status of separation and purification and the status of washing and regeneration column efficiency measurement are monitored, and the solution discharged from the flow path (10) connected to the separation and purification step is the next step. However, the solution discharged from the flow path (11) for measuring the efficiency of the washing / regeneration column becomes waste liquid.

[Example 2]
[Purification device by 2-column switching method using FT type anion exchange column]
A purification apparatus by a 2-column switching method using an FT type anion exchange column was prepared (Fig. 2).
The apparatus can perform a purification step of adsorbing proteins on one of the two columns and a protein wash, elution and regeneration wash steps on the other column. The outline of this apparatus will be described below with reference to the apparatus numbers in the drawings.

In FIG. 2, the antibody used as the purification sample is supplied to the purification sample supply tube (2) from the upstream process of the purification system. The process liquid containing the purification sample is adjusted to be adsorbed on the FT type chromatography column, and then sucked by the pump (13) via the air trap (12) to the first four-way switching valve (3). Be connected. The column switching valve consists of two valves, a first four-way switching valve (3) and a second four-way switching valve (4), and two columns, column A (5) and column B (6) between the two valves. ) Is connected.

When the process liquid is connected so as to flow to one column A (5), the adsorption operation of the purification sample in the purification step is continuously performed in the column A. At this time, the other column B (6) is connected to the evaluation sample supply tank (15) for measuring column efficiency, which is connected to the cleaning liquid supply pipe (14) via the sample injection valve (7). ..

On the other hand, an evaluation sample for measuring column efficiency is added to the sample injection valve (7) by the sample addition pump (9) to the sample loop (22). Further, in the cleaning liquid supply pipe (14), a solution used for cleaning and elution of proteins from a manifold (16) with a branch valve via the sample injection valve (7), a cleaning liquid and a regenerating liquid used in the cleaning and regeneration step, and equilibrium. A buffer solution or the like is supplied via the air trap (20) and the pump (21), and the column B (6) is washed.

After the column is washed, an evaluation sample for measuring column efficiency is added to the column by the above-mentioned flow path and measured. Two lead-out tubes (10) and (11) are connected to the outlet of the second four-way switching valve (4), and detection devices for purification samples and evaluation samples [UV monitor (17) and pH monitor (18), respectively. ), Electrical conductivity meter (19)] is connected, the status of separation and purification and the status of washing and regeneration column efficiency measurement are monitored, and the solution discharged from the flow path (10) connected to the separation and purification step is the next step. However, the solution discharged from the flow path (11) for measuring the efficiency of the washing / regeneration column becomes waste liquid.

[Example 3]
[Single-use device]
FIG. 3 shows a drawing of a device in which a connecting device is devised so that the biopharmacy production device can be used for single use even in continuous purification. Specifically, the parts of the connection joint where dirt may accumulate after long-term use, the connection parts such as the two columns, the in-line filter, and the wetted parts of various sensors are aseptic or relatively clean. An HFC39 luer lock type connector (manufactured by Colder Products Company (CPC)) that allows parts to be replaced in the state was adopted. In addition, ID3 / 8 "and TC3 / 4" type sanitary joints (manufactured by Ultrapharma) were used for buffer inlet joints and waste liquid outlet joints, and bamboo shoot type joints were used for other connections and fixed with cable ties.

[Example 4]
Alternately two columns using the 2-column switching purification apparatus equipped with the Protein A affinity column in B / E type separation mode described in Example 1 (please let us know if there are any changes in FIG. 1). The column eligibility was confirmed by 6 cycles of continuous antibody purification (continuous operation for about 37 hours) and column efficiency (N / M, Af) measurement. The capacity of the sample to be used and the time required for various operations are expressed based on the column capacity (CV).

As a sample, a culture supernatant containing a recombinant IgG1 antibody produced by culturing CHO cells (lot number: M-L006, 40 Mg, 33.3 CV / time) was used, and two columns were protein A affinity columns (commodity). Name: HiTrap MabSelect SuRe, 1 mL, manufactured by GE Healthcare Co., Ltd.) was used. As an operation of the antibody sample purification step, the antibody sample was injected into a column, adsorbed, and then the sample adsorbed on the column was equilibrated at 10 CV / time of 0.05 M NaCl-containing 50 mM Na acetate buffer (pH 3.5). Then, it was eluted with 0.1 M glycine HCl buffer (pH 2.5) at 10 CV / time. Further, the column was washed with 0.15 M NaCl 20 mM Na phosphate buffer (pH 7.0) at 10 CV / time.

While the antibody sample was purified in one column, the other column was washed, and the number of theoretical plates and peak symmetry were measured for regeneration and confirmation of column eligibility. Specifically, the column was washed with 0.05 M NaCl-containing 50 mM Na acetate buffer (pH 3.5) 3 CV / time, then washed with 0.1 M NaOH, 1 CV / time, and 0.15 M NaCl-containing 20 mM Na phosphate. The column was equilibrated with buffer (pH 7.0), 10 CV / dose. Column with 20 mM Na phosphate buffer (pH 7.0) for HETP measurement to which 100 μL / dose of 20 mM Na phosphate buffer (pH 7.0) containing 2% acetone + 0.4 M NaCl was added as a theoretical equivalent height (HETP) measurement sample. After washing, the column was rebalanced with 20 mM Na phosphate buffer (pH 7.0) containing 0.15 M NaCl, 17.3 CV / time. The above step was repeated for 6 cycles, that is, 12 purification steps alternately with two columns. This method shows a method in which the batch purification method is continuously repeated although the processing and purification of the sample are intermittent.

FIG. 4 shows the results of the following (a) to (f) in the step of 6 cycles (12 times in total) by column switching in the continuous 6 cycles (12 times in total) purification using the protein A affinity column of the antibody. ..
(A) UV280 absorption measurement in the purification step,
(B) Measurement of the number of theoretical plates by UV280 absorption measurement in the cleaning process,
(C) pH measurement in the purification process,
(D) pH measurement in the washing process,
(E) Measurement of electrical conductivity in the refining process,
(F) Measurement of theoretical plates by measuring electrical conductivity in the cleaning process

In the UV280 absorption measurement in the cleaning step of FIG. 4 (b), the peak of acetone was observed as the theoretical plate number or the theoretical plate equivalent height (HETP) measurement sample, and from the electrical conductivity measurement in the cleaning step of FIG. 4 (f), A peak of NaCl was observed as a theoretical plate number or theoretical plate equivalent height (HETP) measurement sample. Comparing the peak patterns of both, peaks of the same shape were confirmed at almost the same position at the same interval, indicating that there was no change in the filling state of the column during the process. From the UV280 absorption measurement results in the purification step of FIG. 4 (a), sharp peaks of the purified antibody were confirmed at regular intervals. When the inflection point analysis was performed on the UV280 absorption measurement result, the reproducibility of purification in continuous steps was confirmed. If the function of inflection point analysis is also added to the device, accurate continuous purification monitoring becomes possible. The ionic strength of the eluate can be measured from the electrical conductivity measurement in the purification step of FIG. 4 (e), but it was confirmed that the eluate was eluted at regular intervals. Inflection point analysis was also performed on this electrical conductivity measurement result, and it was confirmed that the purification was reproducible in a continuous process. By adding the function of inflection point analysis to the device in this way, accurate continuous purification monitoring becomes possible.

As described above, from the various measurement results shown in FIG. 4, it was confirmed that continuous purification was performed in the apparatus of the present invention with high reproducibility and automatic measurement was possible.

[Example 5]
Using a 2-column switching purification device equipped with a cation exchange column in B / E type separation mode, 6 cycles of continuous antibody purification (continuous operation for about 37 hours) using two columns for purification alternately. ) And column efficiency (N / m, Af) were measured to confirm column eligibility. The capacity of the sample to be used and the time required for various operations are expressed based on the column capacity (CV).

As a sample, the recombinant IgG1 antibody produced by culturing the CHO cells was purified by protein A affinity chromatography, diluted with 0.05 M NaCl-containing 50 mM Na acetate buffer (pH 5.0), and the antibody was about 30 Mg / time, 30 CV. Was used. For the two columns, a cation exchange column (trade name: HiTrap Capto Q, 1 mL, manufactured by GE Healthcare Co., Ltd.) was used.

As an operation of the purification step of the antibody sample, the antibody sample was injected into a column, adsorbed, and then the sample adsorbed on the column was equilibrated with 0.05 M NaCl-containing 50 mM Na acetate buffer (pH 5.0) at 10 CV / time. Then, it was eluted with 0.2 M NaCl-containing 50 mM Na acetate buffer (pH 5.0) at 10 CV / time. Further, washing and elution was performed with 1.0 M NaCl-containing 50 mM Na acetate buffer (pH 5.0) at 10 CV / time, and the column was equilibrated at 20 mM Na phosphate buffer (pH 7.0) at 10 CV / time.

While the antibody sample was purified in one column, the other column was washed, and the number of theoretical plates and peak symmetry were measured for regeneration and confirmation of column eligibility. Specifically, the column was washed with 0.05 M NaCl-containing 50 mM Na acetate buffer (pH 5.0) 3 CV / time, then washed with 0.1 M NaOH, 1 CV / time, and 0.15 M NaCl-containing 20 mM Na phosphate. The column was equilibrated with buffer (pH 7.0), 10 CV / dose. Column with 20 mM Na phosphate buffer (pH 7.0) for HETP measurement to which 100 μL / dose of 20 mM Na phosphate buffer (pH 7.0) containing 2% acetone + 0.4 M NaCl was added as a theoretical stage equivalent height (HETP) measurement sample. Was washed, and the column was rebalanced at 14 CV / dose in 50 mM Na acetate buffer (pH 5.0) containing 0.05 M NaCl. The above step was repeated 12 times alternately with 6 cycles, that is, 2 columns. This method shows a method in which the batch purification method is continuously repeated although the processing and purification of the sample are intermittent.

FIG. 5 shows the results of the following (a) to (f) in the step of 6 cycles (chromatography 12 times in total) by column switching in the continuous 6 cycles (12 times in total) purification using the cation exchange column of the antibody. showed that.
(A) UV280 absorption measurement in the purification step,
(B) Measurement of the number of theoretical plates by UV280 absorption measurement in the cleaning process,
(C) pH measurement in the purification process,
(D) pH measurement in the washing process,
(E) Measurement of electrical conductivity in the refining process,
(F) Measurement of theoretical plates by measuring electrical conductivity in the cleaning process

In the UV280 absorption measurement in the cleaning step of FIG. 5 (B), the peak of acetone as a theoretical stage equivalent height (HETP) measurement sample was observed, and from the electrical conductivity measurement in the cleaning step of FIG. 5 (f), the theoretical stage A peak of NaCl was observed as a considerably high (HETP) measurement sample. Comparing the peak patterns of both, peaks of the same shape were confirmed at almost the same position at the same interval, so that there was no significant change in the column filling state during the chromatographic purification process of 12 times in total for 6 cycles. I understand.

[Example 6]
The following test was performed using the flow-through type 2-column switching purification apparatus of the FT type separation mode described in Example 2.

Using a 2-column switching purification device equipped with an anion exchange column in the FT type separation mode, two columns were alternately used for purification, and continuous antibody purification was performed 6 times in 3 cycles (continuous operation for about 7 hours). ) And column efficiency (N / m, Af) were measured to confirm column eligibility. The capacity of the sample to be used and the time required for various operations are expressed based on the column capacity (CV).

As a sample, the recombinant IgG1 antibody produced by culturing the CHO cells was purified by protein A affinity chromatography, diluted with 1.0 M NaCl-containing 25 mM, Tris HCl buffer (pH 8.5), and the antibody was about 70 mg / time. The one set to 70 CV was used.

As the two columns, an anion exchange column (trade name: HiTrap Capto Q, 1 mL, manufactured by GE Healthcare Co., Ltd.) was used.

As an operation of the antibody sample purification step, the antibody sample was injected into the column at a flow rate of 1.0 mL / min.

While the antibody sample was purified in one column, the other column was washed, and the number of theoretical plates and peak symmetry were measured for regeneration and confirmation of column eligibility. Specifically, the column was washed with 1.0 M NaCl-containing 50 mM Tris HCl buffer (pH 8.5) at 10 CV / time, then washed with 0.1 M NaOH, 1 CV / time, and 0.15 M NaCl-containing 20 mM Na phosphate. The column was washed with buffer (pH 7.0), 10 CV / time. 20 mM Na phosphate buffer (pH 7.0) for HETP measurement at 35 CV + 10 CV to which 100 μL / dose of 20 mM Na phosphate buffer (pH 7.0) containing 2% acetone + 0.4 M NaCl was added as a theoretical stage equivalent height (HETP) measurement sample. After washing the column, the column was rebalanced with 25 mM Tris HCl buffer (pH 8.5), 14 CV / dose. The above step was repeated 6 times alternately with 3 cycles, that is, 2 columns.

FIG. 6 shows the results of the following (a) to (f) in the step of 3 cycles (6 times in total) by column switching in the continuous 3 cycles (6 times in total) purification using the antibody anion exchange column. ..
(A) UV280 absorption measurement in the purification step,
(B) Measurement of the number of theoretical plates by UV280 absorption measurement in the cleaning process,
(C) pH measurement in the purification process,
(D) pH measurement in the washing process,
(E) Measurement of electrical conductivity in the refining process,
(F) Measurement of theoretical plates by measuring electrical conductivity in the cleaning process

In the UV280 absorption measurement in the cleaning step of FIG. 6 (B), the peak of acetone acetone as a theoretical stage equivalent height (HETP) measurement sample was observed, and from the electrical conductivity measurement in the cleaning step of FIG. 6 (f), the theory A peak of NaCl was observed as a step equivalent height (HETP) measurement sample. Comparing the peak patterns of both, peaks of the same shape were confirmed at almost the same position at the same interval, indicating that there was no change in the filling state of the column in the series of purification steps and washing steps. ..

(1) Branch valve, (2) Sample supply pipe for purification, (3) First four-way switching valve, (4) Second four-way switching valve, (5) Column A, (6) Column B, (7) Sample injection valve, (8) Manifold with valve, (9) Sample addition pump, (10) Outlet pipe, (11) Outlet pipe, (12) Air trap, (13) Pump, (14) Cleaning liquid supply pipe, (15) ) Evaluation sample supply tank, (16) Manifold with branch valve, (17) UV monitor, (18) pH monitor, (19) Electrical conductivity meter, (20) Air trap, (21) Pump, (22) Sun loop (50) Luer lock joint

Claims (22)

Using a purification system with two independent columns A and B, where the inlet is connected to the purification sample supply or cleaning fluid supply, and the corresponding outlets are each connected to separate outlets, one. In a method for continuously purifying a biopolymer, the other column is subjected to a washing and regenerating step when the column is subjected to a biopolymer purification step. A continuous purification method characterized by performing pattern analysis of the concentration of an evaluation sample supplied by a sample feeder after passing through a column. The continuous purification method according to claim 1, wherein the outlet pipe is connected to a detection device and branches into a purification sample discharge pipe and a cleaning waste liquid discharge pipe. The continuous purification method according to claim 1 or 2, wherein the pattern analysis of the concentration of the evaluation sample after passing through the column is used for process control. The continuous purification method according to any one of claims 1 to 3, wherein the replacement time of the column can be grasped by pattern analysis of the concentration of the evaluation sample after passing through the column. The continuous purification method according to claim 4, wherein the system is automatically stopped when an abnormality in the column state is detected by pattern analysis of the concentration of the evaluation sample after passing through the column. The continuous purification method according to any one of claims 1 to 5, wherein the individual result of column purification and the status of the column are automatically recorded in the continuous production of pharmaceutical products. Process control by pattern analysis of the concentration of the evaluation sample after passing through the column in the washing and regeneration process is based on the measurement result of the theoretical plate number of the chromatogram pattern and the symmetry of the separated peaks by the detector connected to the lead-out tube. The continuous purification method according to claim 3, wherein the method is carried out by evaluating the filling state of the above. The continuous purification method according to any one of claims 1 to 7, wherein the purification step is a step of adsorbing the purification sample to the resin in the column after being added to the column. The purification step consists of one or more purification conditions, a step in which a purification sample is added to the column and adsorbed on the resin in the column, a step in which unadsorbed impurities and excess purification sample are washed with a buffer solution, and purification. The continuous purification method according to any one of claims 1 to 7, wherein the step is a combination of at least one of a sample elution step and a washing elution step. The continuous purification method according to any one of claims 1 to 7, wherein the purification step is a step in which impurities are adsorbed on the resin while the purification sample passes through the column. The washing and regenerating steps include washing the column with unadsorbed impurities and excess purified sample with a buffer, elution of purified sample or washing and elution, washing with water for injection or sterile purified water or buffer, column. The method according to any one of claims 1 to 10, wherein the process is a combination of one or more steps selected from the regeneration cleaning step and the resin equilibrium step of the above, and a test step using a test sample. The continuous purification method described. The washing and regenerating step is a combination of at least one of a column washing and elution step, a washing step with water for injection or sterile purified water or a buffer, a column reclaiming and washing step, a resin equilibrium step, or a test step with a test sample. The continuous purification method according to any one of claims 1 to 10, characterized in that there is. The continuous purification method according to any one of claims 1 to 12, wherein the purification sample is a solution containing a biopolymer. The continuous purification method according to claim 13, wherein the biopolymer is an antibody. The evaluation sample is a sodium chloride solution, a high-concentration buffer solution for testing, a solution containing a low-molecular-weight compound having absorption in the ultraviolet, or a high-concentration low-molecular-weight solution capable of measuring the refractive index. The method according to any one of claims 1 to 14. In a purification apparatus provided with two independent columns A and B, the inlet of one column is connected to the purification sample supply pipe via the first four-way switching valve, and the other column is the first. It is connected to the cleaning liquid supply pipe that can be connected to the evaluation sample supply device via the four-way switching valve, and the corresponding outlets branch to the purified sample discharge pipe and the cleaning waste liquid discharge pipe via the second four-way switching valve, respectively. A purification device characterized by being separately connected to two outlet tubes provided with a pattern detection device for evaluation sample concentration. Claim that the sample supply pipe for purification can be connected to any one or more or a combination of a biopolymer-containing solution introduction pipe, an equilibrium buffer introduction pipe, an eluate introduction pipe, and a buffer solution introduction pipe for a washing elution step. 16. The purification apparatus according to 16. The cleaning liquid supply pipe is an equilibrium buffer introduction pipe, an eluent introduction pipe, a cleaning elution buffer introduction pipe, injection water or sterile purified water or a buffer solution introduction pipe, a column regeneration cleaning liquid introduction pipe, and a resin balancing buffer. The purification apparatus according to claim 16 or 17, wherein any one or more or a combination of liquid introduction pipes can be connected. The purification apparatus according to any one of claims 16 to 18, wherein the evaluation sample introduction apparatus is connected to a cleaning liquid introduction pipe by a sample injection valve. The purification apparatus according to any one of claims 16 to 19, wherein the four-way switching valve is a four-way rotary valve. The feature is that the evaluation sample concentration pattern detecting device is a device provided with a detecting means for any one or more or a combination of an electric conductivity measuring device, an ultraviolet ray, a visible part absorption measuring device, and a refractive index measuring device. The purification apparatus according to any one of claims 16 to 20. The purification apparatus according to any one of claims 16 to 21, wherein the column, the in-line filter and the detection apparatus are coupled to the apparatus by a luer lock type connector so as to be suitable for single use. ..

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