JP2010271300A - Protein separating and refining device using liquid chromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable continuous infusion by a device for controlling a refining step repeatedly that a protein separating and refining step is divided into three steps by shifting each three parallel columns. <P>SOLUTION: The liquid chromatography for separating and refining protein includes a parallel process using three columns in order to continuously conduct a protein refining process including five steps of absorbing objective protein to a column, a cleaning non-specific adsorbate, eluting an objective component, regenerating a column (including the elution of an adsorbate except for the objective component), and balancing (initializing), in which one of the three columns is assigned for the adsorption process of the objective protein to the column, one of the remaining two columns is assigned for the processes of cleaning the non-specific adsorbate and eluting the objective component following the cleaning, and the third column is assigned for the processes of regenerating a column and balancing process following the regeneration, which are time-programmed to repeat the five steps sequentially. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a field related to separation and purification of substances in a solution, and relates to an apparatus for separating and purifying substances in a solution using liquid chromatography. In particular, a separation / purification apparatus in which three columns of the same liquid chromatography are connected in parallel, and the operation of injecting the sample into the liquid chromatography is continuously performed. The present invention relates to an apparatus for separating and purifying proteins.

Since liquid chromatography has excellent performance such as ease of handling, a wide range of analysis objects, and high separation efficiency, it has become an effective means for separation and purification of substances in a solution. In particular, with the recent expansion of the protein pharmaceutical market, it has been widely used for purification of recombinant proteins expressed in host cells such as E. coli.
The separation by liquid chromatography mainly used in the separation and purification of proteins is a so-called batch separation. Here, the batch type uses a single packed column, and the operation of the chromatography is performed by the operation of equilibration of the column, injection of the sample into the column, elution of the target substance from the column, and regeneration and washing of the column. Indicates that the next separation (batch) step is performed after the process is completed. Therefore, when separating and purifying a large amount of sample by liquid chromatography, use a huge separation column according to the sample, or divide the sample into several parts and repeat the operation of liquid chromatography. Or, a combination thereof is performed. However, it is difficult to perform separation and purification by continuously injecting a sample, that is, to perform a continuous operation. On the other hand, as represented by antibody drugs and the like, the use of proteins as drugs has become widespread, and in the production of large quantities of proteins, the efficiency of separating and purifying proteins expressed and produced in cultured cells has been increased. It is desired. Proteins used in pharmaceuticals and the like require a high-purity sample, and therefore require separation and purification by a plurality of liquid chromatography. As described above, batch separation is used in protein separation by liquid chromatography, so it is difficult to operate the entire production process continuously. This is one of the reasons why efficiency in pharmaceutical protein production does not advance. This is one factor.

In the field of liquid chromatography, in order to increase the resolution, a plurality of columns are connected in an endless circle, the supply port and the extraction port are switched in the flow direction of the mobile phase, and the stationary phase is simulated as the mobile phase. A system (simulated moving bed (SMB) method) that moves in the opposite direction to the flow of the gas and continuously separates the target component has been developed and used (see Patent Documents 1 and 2). This method is a system in which a large number of columns are connected in series, and a rotary valve or the like is arranged in the middle of a connection pipe between each liquid tank and the column. The rotary valve is switched one after the other at regular intervals (period time), and the liquid inlet and outlet are sequentially moved to the left one by one. The feature of this method is that it is used for separation to separate into two fractions from a solution containing two or more components according to the difference in the moving speed of each component, and is composed of 8 to 16 separation beds. High separation accuracy and efficiency can be obtained based on the process principle, but the construction and control of the equipment are complicated and the construction cost is high.
In recent years, affinity chromatography using an affinity column on which a protein ligand is immobilized has been widely used for purification and separation of pharmaceutical proteins. Since it is possible to selectively purify only specific proteins by using affinity chromatography, it is necessary to construct a complicated separation system by applying the SMB method developed for the purpose of separating two components with poor separation. The SMB method has not been applied much.
On the other hand, in the analysis using liquid chromatography, there are cases where measurement is repeated, and for the purpose of reducing the complexity and total analysis time, the analysis can be continued by paralleling multiple columns. Devices have been developed (Japanese translations of PCT publication Nos. 2008-539395 and 2004-533919). In measurement using a parallel column, separation analysis is performed on one column, and operations such as column washing and equilibration are performed on other columns that are parallelized within the time. However, in a liquid chromatography apparatus for such an analysis, the introduction of the sample is intermittent and cannot be diverted for the purpose of introducing the sample continuously and performing purification separation, or introducing the sample. In the case of continuous operation, it is necessary to use a large number of columns, and it is necessary to reduce the time (latency) between sample introductions to zero, which causes a problem that the apparatus must be very complicated.

Special table 2008-539395 JP-T-2004-533919

As the use of protein drugs including antibody drugs expands, the inefficiency of the batch process can be considered, and it is thought that the process efficiency can be improved by switching to a continuous process. In particular, when a large amount of sample is processed, it is desirable to process the sample continuously in a large amount using as little separation carrier as possible. On the other hand, the operability, control, etc. are complicated in the chromatography which enables the continuous process so far represented by SMB.
Under such circumstances, the present inventors have come to consider that it is possible to develop an apparatus that can easily achieve continuation by using affinity chromatography with good selectivity. In other words, when affinity chromatography with good selectivity is used, the time required for washing and elution after protein sample adsorption, that is, the separation time, can be significantly shortened compared to separation columns with poor selectivity. Thus, within the time required for saturation of protein sample adsorption in the first column, washing and separation can be completed in the second column, and column, regeneration and initial equilibration can be completed in the third column. Thus, by cyclically changing the role of these three columns, the protein sample can be continuously injected.
Based on the above idea, as a result of diligent study, a continuous injection separation system was proposed that can use three affinity columns in parallel to continuously process a solution containing protein components to be separated. It came to do. Such parallelization is considered to be achieved by simply using three liquid chromatography apparatuses and switching protein injection with a selection valve or the like as shown in the configuration diagram 1 (configuration diagram 1). However, in such a flow channel system, the protein sample is passed through all the liquid pumps, and when handling different types of proteins, dirt from the protein sample may be removed from all the flow channels. (Operation called CIP) is required. On the other hand, the present inventors have devised a flow path system as shown in the configuration diagram 2 and devised to specify a solution to be fed by each of the three pumps (configuration diagram 2). As a result, the flow path derived from the protein sample can be specified, and countermeasures against contamination can be reduced. Based on this idea, a separation / purification device was prepared and its operation was confirmed to show that a continuous process was achieved, thereby completing the present invention.

By limiting to affinity column chromatography or chromatography that performs adsorption / elution stepwise, the column chromatography process can be divided into a plurality of stages. When a single column is used, the chromatography steps include (i) sample application (adsorption of the target protein to the column), (ii) washing of non-specific adsorbate, etc. (iii) elution of the target component, ( It is divided into five steps: iv) column regeneration (including elution of adsorbate other than the target component) and (v) equilibration (initialization).
Considering the case where the adsorption capacity of the column is large, the amount of the solution used for equilibration of the column, washing, elution of the target component, and regeneration can be reduced compared to the sample volume to be injected. Paying attention to this, even if liquids are passed at the same flow rate, it is possible to perform a combination of initialization (equilibration), washing, elution, and regeneration operations within the time required for sample injection. I came up with an idea.
The inventors pay attention to this, and divide the chromatography step (time domain) into three. As the first domain, (i) sample application (adsorption of the target protein to the column) step, The domain is separated by (ii) washing non-specifically adsorbed substances and the like, (iii) elution step of target components, and the third domain is divided into (iv) column regeneration and (v) equilibration steps. It was invented that the sample solution to be purified can be continuously injected into the apparatus and the purification operation by chromatography can be performed automatically and simply. By using three affinity columns of the same size, the first column operates the first domain, the second column operates the second domain, and the third column uses the third domain. When the time domain is completed, it has been devised that the domain of each column can be sequentially operated in order of 1 → 2 → 3 →. According to this device, the sample can be continuously injected, and the protein purified intermittently at the elution step of the second domain can be recovered, so that a continuous injection purification separation system is constructed. In the present invention, the simplified operation does not mean that purified protein can be obtained continuously, but it is easy to construct a continuous process as long as sample injection can be performed continuously in the purification process. Therefore, it is considered that there is no practical disadvantage in intermittently recovering purified protein.

In the liquid chromatography apparatus for protein separation and purification, the present invention provides various solution reservoirs, the same three separation columns connected in parallel to the various solution reservoirs via valves, and the three separation columns. In a liquid chromatography apparatus for protein separation and purification comprising a waste liquid reservoir and a collection container connected in parallel via each of the above and a control device for controlling the valve, a protein separation and purification process by the liquid chromatography apparatus, The target protein is divided into five steps: adsorption to the column, washing non-specific adsorbate, elution of the target component, column regeneration, and equilibration. Adsorption onto the surface, followed by washing of non-specific adsorbate, etc. as the second domain Elution of the target component is assigned to each domain as column regeneration and subsequent equilibration as the third domain, and the time required for each domain is set to be the same. One has the first domain, one of the remaining two separation columns has the second domain, the remaining third separation column has the third domain, and each separation column has Controls the valve so that the first domain, the second domain, and the third domain are sequentially repeated, and injection of the sample solution for adsorption of the target protein to the column is performed in any of the three separation columns. It is characterized in that it is carried out sequentially one by one.
The present invention is also directed to the above-described liquid chromatography apparatus for protein separation and purification, wherein the various solution reservoirs include five solution reservoirs R1, R2, R3, R4, and R5 used for the five steps, respectively. Consists of switching valves SW1, SW2, SW3, SW4, SW5 and selection valves SV1, SV2, SV3. R1 is connected to selection valves SV1, SV2, SV3 via a liquid feed pump P1, and is connected to switch valve SW1. Is switched between R2 and R3 and supplied to and connected to the selection valves SV1, SV2 and SV3 via the liquid feed pump P2, and is switched between R4 and R5 via the switch valve SW2 and selected via the liquid feed pump P3. , SV2 and SV3 and connected to the selection valves SV1, SV2 and SV3. The three separation columns C1, C2, and C3 are connected to the flow side, respectively, and the waste liquid is connected to the downstream side of the separation columns C1, C2, and C3 via switch valves SW3, SW4, and SW5, respectively. The reservoir and the collection container are switchably connected.

  Since the affinity column is expensive, when the purification scale becomes large, it is necessary to increase the column size or to repeat small batch purification many times, but by using the apparatus of the present invention, The labor in scale-down or repeated batch purification can be greatly reduced, and cost reduction and labor saving can be achieved without changing the precision of purification. Furthermore, in protein purification separation, the only solution that cannot be sterilized in advance is a protein sample. Therefore, in protein purification of protein pharmaceuticals and the like, it is always a heavy burden to wash the apparatus after passing through the protein solution. In the case of this device, the flow path for handling the protein sample is clear, and in particular, only one specific pump out of the three liquid feed pumps feeds the protein solution. As a result, the effect of reducing the burden of CIP can be obtained as compared with the case where a simple parallel operation or a plurality of liquid chromatographies are simultaneously operated.

FIG. 1 shows a flow path diagram in the case of considering the achievement of continuity by simply using three liquid chromatography apparatuses. FIG. 2 shows a flow chart of the present invention. FIG. 3 is a time chart showing a valve switching time program in the apparatus shown in FIG. FIG. 4 is a diagram showing various conditions in Experimental Examples 1 to 5.

The apparatus constituting the present invention can be shown by the flow chart shown in FIG.
In the figure, C1, C2, and C3 are separation columns, respectively. On the downstream side of the separation columns C1, C2, and C3, switch valves SW3, SW4, and SW5 are provided, respectively. The switch valves SW3, SW4, and SW5 pass the solution coming out of each separation column to the waste liquid reservoir container. Or whether to pass through the collection container. R1, R2, R3, R4, and R5 are various solution reservoirs, and R1 contains a sample solution used in the first domain (domain 1), and is supplied to the selection valves SV1, SV2, and SV3 via the liquid feed pump P1. Supplied. R2 contains the washing solution used in the second domain (domain 2), and R3 contains the elution solution used in domain 2. The switch valve SW1 switches between R2 and R3 and sends them. It is supplied to the selection valves SV1, SV2 and SV3 via the liquid pump P2. R4 contains the regenerating solution used in the third domain (domain 3), R5 contains the equilibrating solution used in domain 3, and R4 and R5 are switched by the switch valve SW2, It is supplied to the selection valves SV1, SV2 and SV3 via the liquid feed pump P3. Separation columns C1, C2, and C3 are connected to the downstream sides of the selection valves SV1, SV2, and SV3. The selection valves SV1, SV2, and SV3 are connected to each domain according to which domain the separation column is in. Select a solution for use and pass through each separation column. Each switch valve SW1, SW2, SW3, SW4, SW5 and each selection valve SV1, SV2, SV3 are controlled by a control device (not shown) according to a time program shown in FIG.
In the interlocking of the devices, liquids sent by the pumps (P1, P2, P3) from the reservoirs of various solutions illustrated by R1, R2, R3, R4, and R5 are used using selection valves indicated by SV1, SV2, and SV3. , C1, C2, and C3, and select and pass through the separation columns as programmed in advance, and the separated purified protein is collected by the SW1, SW2, SW3, SW4, and SW5 switch valves. It is intermittently collected by sending it to the collection container only occasionally. In domain 2 and domain 3, since a plurality of solutions are switched and sent, the switch valves indicated by SW1 and SW2 are used for the switching, so that they can be selected and communicated by programming in advance. Can be liquid. In addition, in order to monitor whether collection | recovery is performed normally, chromatography monitoring can be performed by attaching an ultraviolet monitor (UV monitor) in the middle of a collection line. Pump on / off, flow rate and valve switching can be controlled by programming in advance with a computer or the like. Therefore, by using this apparatus, after starting the liquid feeding of the sample, it is continuously operated until the sample runs out, and the protein can be purified by full automation.

  As shown in FIG. 2, the minimum configuration of the chromatography apparatus of the present invention includes five solution reservoirs (indicated by R1, R2, R3, R4, and R5) and three liquid pumps (P1, P2 having the same performance). , P3), three identical separation columns (denoted by C1, C2, C3), five switch valves (denoted by SW1, SW2, SW3, SW4, SW5), three select valves (denoted by SV1, SV2, SV3). As shown), a waste liquid reservoir, a collection container, and a pipe connecting them. A UV monitor for monitoring the protein recovery status should be equipped as much as possible, but is not essential. The connector indicated by the cross in the figure indicates that the pipe is connected there, but it is desirable that the connector has a function of preventing backflow. The operation of the present apparatus is automatically performed by switching a valve programmed in advance (switched at regular intervals). The parameters required for the program are very simple. In the switch valves SW1, SW2, SW3, SW4, SW5, the program outputs a signal at regular intervals and switches the flow path to the 1 side or 2 side in the figure. The selection valves SV1, SV2 and SV3 may be programmed to select one side, two sides or three sides of the three flow paths at regular time intervals. good. Any type of valve can be used as long as it can control and switch the flow path, such as a rotary valve or a solenoid valve.

  FIG. 3 shows a time program for switching between SW1, SW2, SW3, SW4, SW5, SV1, SV2 and SV3. Considering one of the three separation columns (C1, C2, C3), for example, the C1 column, the domain 1 operation is performed at time tn0 as the nth purification cycle, and the SV1 valve is 1 side is selected, SW3 is selected as 1 side, at time tn1, the SV1 valve is selected as 1 side, the SW1 valve is selected as 1 side, and the SW3 valve is selected as 1 side. Is selected and the operation of domain 2 is started. In this domain 2, at time tn11, the SW1 valve is switched to the 2 side, and the solvent for elution flows. Thereafter, at time tn12, the SW3 valve is switched to the 1 side, and the protein eluted from the column is recovered. At the time when protein elution / recovery is completed, that is, at time tn13, the SW3 valve is switched to the 1 side, and the recovery ends. Thereafter, at time tn2, the respective valve positions are selected such that the SV1 valve is selected on the 3 side, the SW2 valve is selected on the 1 side, and the SW3 valve is selected on the 1 side, and the operation of the domain 3 is started. . In domain 3, at time tn21, the SW2 valve is switched to the 2 side, and this state continues until time tn3. All operations from domain 1 are completed, and the next purification cycle (n + 1 cycle) is started. For the columns C2 and C3, the purification cycle by the equivalent valve selection operation is performed only by shifting the domain phases.

It is necessary to set several parameters as necessary parameters in this time program. When the operations from domain 1 to domain 3 are defined as one cycle, the necessary number of cycles, N, is set. Since the operation times of the domain 1, domain 2, and domain 3 need to be the same, the operation unit time of each domain, Δt (= tn1-tn0, = tn2-tn1, = tn3-tn2) is set equal. . In domain 2 and domain 3, a time from switching the solution to be fed on the way to switching is set by using SW 1 or SW 2, Δta (= tn01−tn0 = tn11−tn1 = tn21−tn2) is set. . In domain 2, it is necessary to use SW3, SW4 or SW5 to switch between recovering the solution coming out of the column or turning it into waste liquid, but in order to start liquid feeding for protein elution Since there is a difference in the time from when the position of the SW1 valve is switched to the right line in FIG. 1 until the target protein is eluted from the column, Δtb (= tn02− tn01 = tn12−tn11 = tn22−tn21), and after this time, SW3, SW4 or SW5 is switched to the right side of FIG. Next, SW3, SW4, or SW5 is switched to the left side of FIG. 1 in order to end the recovery when the target protein begins to elute from the column and the elution is completed. A time required for recovery, Δtc (= tn03−tn02 = tn13−tn12 = tn23−tn22), is set, and after this time, SW3, SW4 or SW5 is switched to the left side of FIG.
In this way, by setting N, Δt, Δta, Δtb and Δtc as parameters, it is possible to create a program for automatically operating the various valves shown in FIG. Can be used to carry out chromatography using the apparatus characterized by continuous injection.

A feature of the present invention is that by using only three identical separation columns, any volume of sample can be continuously processed as long as the durability of the column allows. The processing time is determined by the speed at which the liquid is fed to the apparatus per unit time, that is, the flow rate, regardless of the column size. For example, even with a short contact time, by using a separation carrier having high binding characteristics, the time required for one cycle, 3 × Δt, can be set short, and a large amount of solution can be processed with a very small amount of separation carrier. It becomes possible to do. In particular, protein-ligand affinity columns are expensive, so if the purification scale increases, the column size must be increased or small batch purification must be repeated many times. By doing so, it is possible to greatly reduce the labor in scale down or repeated batch purification of the column to be used, and it is possible to contribute to the achievement of cost reduction and labor saving without changing the purification accuracy. In addition, it is possible to design the number of cycles by considering the life of the carrier, and it is possible to use a small-capacity column in a disposable manner, which occurs when using a protein sample in a different lot or a different protein. It is also possible to avoid operations such as cross-contamination and column cleaning for each purification batch (CIP operation) to prevent it. In particular, in recent antibody drug production, culturing on the scale of tens of thousands of liters is performed, and a huge separation column is used. By using the apparatus of the present invention, purification can be performed. Not only can it be scaled down at least 100 times without changing the accuracy, but also the automation and labor saving of the refining operation becomes possible.
In the description of the present invention, the operation of affinity chromatography was mainly described. However, the use of this apparatus is not limited to affinity chromatography based on the principle, and the protein in the first column is not limited. In the time required for the sample adsorption to saturate, it is possible to set the conditions for the completion of washing and separation in the second column and the completion of column regeneration and initial equilibration in the third column. It is clear that the present invention can also be applied to chromatography utilizing a carrier capable of elution, for example, ion exchange chromatography, hydrophobic chromatography, and reverse phase chromatography.

As an example of this apparatus satisfying the configuration of the continuous injection chromatography apparatus represented by the flow chart of FIG. 2, necessary parts were produced using commercially available parts. As parts constituting this device, the liquid feed pumps P1, P2 and P3 use SP-12-13 manufactured by From Corporation, and the selection valves SV1, SV2 and SV3 and the switch valves SW1 and SW2 serve as From Corporation. A rotary valve 401-249 type is used, and switch valves SW3, SW4, SW5 are manufactured by TAKASAGO ELECTRIC, Inc. The separation column C1, C2, C3 is the same for all three separation columns C1, C2, and C3, but is selected according to the target to be separated (depending on the protein to be purified), and the UV monitor. As the solution reservoirs R1, R2, R3, R4, and R5, a normal glass beaker is used as the solution reservoirs R1, R2, R3, R4, and R5. As a control device for transmitting a selection or switching signal in response to the operation, an FP-X C60 manufactured by Panasonic was used.
As described below, the operation check of the manufactured device actually works by purifying the purified protein PAA3T, the purified protein RRRRG, and the purified protein human polyclonal antibody, and comparing them with the purified data by the batch process. Judgment was made and the result of normal operation was obtained.

(Continuous injection purification experiment using nickel chelate column of purified protein PAA3T)
A nickel chelate column (Histrap HP manufactured by GE Healthcare, column size 5 mL) widely used for purification of histag protein was used as an affinity carrier. As the histag protein, a protein having an antibody binding ability (hereinafter referred to as “PAA3T”, a recombinant protein encoded by the plasmid pAA3T described in JP-A-2008-115151), which has been already developed by the present inventors, is used. A highly purified sample was used. The protein used was dissolved in 10 mM phosphate buffer (pH 7.4) containing 0.05 M NaCl and 5 mM imidazole. As the protein sample to be injected, the protein concentration, the total injected protein amount, and the total injected protein solution amount were set as shown in FIG. 4, and the amount of recovered protein, that is, the recovery rate was measured under various conditions.
In the present invention, three parallel columns are used. When each of the columns is an A, B, C column, when the A column is in the first domain, the B column is the second domain, and the C column is It is in the third domain. The time (Δt) of each domain is 10 minutes, and when that time is exceeded, the next domain is transferred by a solvent switch or the like. The protein sample injection in the first domain in this purification experiment was 20 mL each, and the injection was performed at a flow rate of 2 mL / min. That is, the protein sample was fed for 10 minutes, and the operation of the second domain was moved by the solvent switch.
The second domain is composed of two steps, washing and elution. The washing uses 10 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and 20 mM imidazole at a flow rate of 2 mL / min and 10 mL. The liquid was fed. Next, by switching the solvent, 10 mL of solution (5 minutes) was sent at a flow rate of 2 mL / min using a 10 mM phosphate buffer solution (pH 7.4) containing 0.5 M NaCl and 250 mM imidazole and eluted. The protein solution eluted for 90 seconds from the time when the absorbance reached 0.01 was recovered (3 mL was recovered). The absorption at 280 nm of the solution coming out from the column in the second domain is constantly monitored. Thereafter, the operation of the third domain was started by a solvent switch.
The third domain consists of regeneration and rebalancing. For regeneration, a 10 mM phosphate buffer solution (pH 7.4) containing 0.5 M NaCl and 500 mM imidazole was used, and 10 mL of liquid was fed (5 minutes) at a flow rate of 2 mL / min. Next, 10 mM phosphate buffer (pH 7.4) containing 0.05 M NaCl and 5 mM imidazole was used, and re-equilibration was performed by feeding 10 mL (5 minutes) at a flow rate of 2 mL / min. .
With this, the cycle was completed once. Next, the solvent switch was used to move to the first domain and to the next step. This process was repeated until the sample solution to be injected was exhausted, and the recovered protein was measured. The results are summarized in FIG. As shown in FIG. 4, it was confirmed that the affinity purification process can be operated with a recovery rate of 90% or more even under various conditions.

(Continuous injection purification experiment using nickel chelate column of histag protein RRRRG expressed in E. coli)
The same operation as “(Continuous injection purification experiment using nickel chelate column of purified protein PAA3T)” was performed using a histag protein expressed in E. coli. As the histag protein, a protein having an antibody binding ability already invented by the present inventors (hereinafter referred to as “RRRRRG” and encoded by the plasmid pAA-RRRG described in JP-A-2008-115151) is used. Using.
E. coli strain JM109 transformed with recombinant plasmid pAA-RRRG was grown overnight at 35 ° C. in 2 liters of medium (containing 20 g sodium chloride, 20 g yeast extract, 32 g tryptone, 100 mg ampicillin sodium). Cultured. Thereafter, the culture broth was centrifuged at a low speed for 20 minutes (5,000 revolutions per minute) to obtain bacterial cells having a wet weight of about 5 g. This was suspended in 20 mL of 10 mM phosphate buffer (pH 7.0), the cells were crushed with a French press apparatus, and then centrifuged at high speed (20,000 rotations per minute) for 20 minutes. separated. Streptomycin sulfate was added to the resulting supernatant to a final concentration of 2%, stirred for 20 minutes, and then centrifuged at high speed for 20 minutes (20,000 rotations per minute) to separate the supernatant. Thereafter, ammonium sulfate treatment was performed, and a 10 mM phosphate buffer solution (pH 7.4) containing 0.05 M NaCl and 5 mM imidazole was added to the resulting supernatant to make the total volume 1100 mL. Of these, 960 mL was subjected to a continuous injection purification experiment in exactly the same manner as in the above experimental example "(Continuous injection purification experiment using a nickel chelate column of purified protein PAA3T)". Obtained in the state of.
As a control experiment, purification using a single column was performed using 20 mL of the total 1100 mL sample solution. Purification on a single column was performed using the conditions for the first domain, the second domain, and the third domain in the above experimental example ((Continuous injection purification experiment using a nickel chelate column of purified protein PAA3T)). Purification on a single column was performed 3 times separately, and about 1.7 mg of purified protein was obtained each time.
In continuous injection purification experiments, 86 mg of purified protein (ie, 0.089 mg / mL sample solution) is recovered using 960 mL of sample solution, and in a single column experiment, from 60 mL of sample solution to 5 .1 mg of purified protein (ie, 0.085 mg / mL sample solution) was recovered, and even with continuous injection purification, the recovery amount was equal to or better than that of a single column.

(Continuous injection purification experiment using protein A affinity column of commercially available human polyclonal antibody purified protein)
As an affinity carrier, a protein A affinity carrier (MabSelect Sure carrier manufactured by GE Healthcare, filled with 1 mL of Triron 10/20 column (id = 1 cm)) widely used for purification of human antibodies was used. A commercially available human polyclonal antibody (purchased from Sigma, IgG from Human Serum, Technical Grade) was used as the antibody protein. The antibody protein was dissolved in 20 mM phosphate buffer (pH 7.0) and the concentration was adjusted to 0.2 mg / mL.
In this purification experiment, the time (Δt) of each of the three domains is 37.5 minutes, and when that time is exceeded, the next domain is transferred by a solvent switch or the like. The protein sample injection in the first domain in this purification experiment was 15 mL each, and the injection was performed at a flow rate of 0.4 mL / min. That is, the protein sample was fed for 37.5 minutes, and the operation was shifted to the second domain by a solvent switch.
The second domain consists of two steps, wash and elution. The wash uses 20 mM phosphate buffer (pH 7.0) containing 0.25 M arginine hydrochloride, 0.75 M NaCl, and a flow rate. 10 mL (25 minutes) of liquid feeding was performed at 0.4 mL / min. Next, by switching the solvent, using 1.0 M arginine acetate buffer (pH 3.5), feeding 5 mL (12.5 minutes) at a flow rate of 0.4 mL / min, the eluted protein By monitoring the absorbance at 280 nm, the protein solution eluted for 7 minutes was recovered from the time when the absorbance reached 0.01 (2.1 mL was recovered). The absorption at 280 nm of the solution coming out from the column in the second domain is constantly monitored. Thereafter, the operation of the third domain was started by a solvent switch.
The third domain consists of regeneration and rebalancing. For regeneration, 0.1 M glycine buffer (pH 2.5) was used, and 5 mL of liquid was fed (12.5 minutes) at a flow rate of 0.4 mL / min. Next, using a 10 mM phosphate buffer (pH 7.0), re-equilibration was performed by feeding 10 mL (25 minutes) at a flow rate of 0.4 mL / min.
With this, the cycle was completed once. Next, the solvent switch was used to move to the first domain and to the next step. This process was repeated until the sample solution to be injected was exhausted, and the recovered protein was measured. 79 mg of human polyclonal antibody was recovered in the elution fraction at pH 3.5. Since the total amount of the input protein was 90 mg, the recovery rate was determined to be about 88%. Even in a purification experiment using a single column, a recovery rate of 85 to 90% was obtained, and no decrease was observed in the recovery rate by continuous injection.
Thus, it has been found that continuous injection chromatography functions effectively even when the combination of protein and carrier is changed.

In the above purification experiment, a purification experiment was performed on a combination of a nickel chelate column and a histag protein, and a combination of a protein A column and a human antibody, but it can also be applied to combinations of other purification columns and various proteins. .
In particular, the present invention provides a technology that can convert a batch process using chromatography into a continuous process, leading to innovation in the manufacturing process.

C1-C3 separation column P1-P3 Liquid feed pump R1-R5 Solution reservoir SV1-SV3 Selection valve SW1-SW5 Switch valve

Claims (2)

Various solution reservoirs, the same three separation columns connected in parallel to the various solution reservoirs via valves, and a waste liquid reservoir and a collection container connected in parallel to the three separation columns via valves, respectively. And a liquid chromatography device for protein separation and purification comprising a control device for controlling the valve,
The protein separation and purification process using a liquid chromatography apparatus is divided into five steps: adsorption of the target protein to the column, washing of nonspecific adsorbate, elution of the target component, column regeneration, and equilibration. The first domain is the adsorption of the target protein to the column, the second domain is the nonspecific adsorbate washing and the subsequent elution of the target component, the third domain is the column regeneration and the subsequent equilibration Are assigned to each domain, and the time required for each domain is set to be the same, and the control device assigns the first domain to one of the three separation columns and the other two separation columns. One has a second domain, the remaining third separation column has a third domain, and each separation column has a first domain, a second domain. The valve is controlled so that the third and third domains are sequentially repeated, and the sample solution for the adsorption of the target protein to the column is sequentially and sequentially injected into one of the three separation columns. A liquid chromatographic apparatus for protein separation and purification, which is characterized by the above.   The various solution reservoirs include five solution reservoirs R1, R2, R3, R4, and R5 used for the five steps, respectively. The valves are switching valves SW1, SW2, SW3, SW4, and SW5, and selection valves. SV1, SV2, and SV3. R1 is connected to the selection valves SV1, SV2, and SV3 via the liquid feed pump P1, and is selected via the liquid feed pump P2 by switching between R2 and R3 by the switch valve SW1. Supplyed and connected to the valves SV1, SV2 and SV3, and switched between R4 and R5 by the switch valve SW2, and supplied and connected to the selection valves SV1, SV2 and SV3 via the liquid feed pump P3. The three separation columns C1, C2, and C3 are connected to the downstream side, 2. The protein according to claim 1, wherein downstream of the separation columns C1, C2, and C3 are switchably connected to a waste liquid reservoir and a collection container through switch valves SW3, SW4, and SW5, respectively. Liquid chromatography device for separation and purification.