JP5392782B2 - Liquid chromatography equipment - Google Patents

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, as shown in the flow path system shown in FIG. 1, a separation / purification apparatus in which two 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 by liquid chromatography. This apparatus is suitable for concentration recovery and separation / purification of proteins consisting of a large amount solution at a very low concentration.

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.
Target proteins such as recombinant proteins can be obtained by removing other contaminating proteins and other contaminants from a mixed solution of proteins such as cell-free extract obtained by disrupting host cells such as E. coli. Purification is performed, but the method used at that time is to adsorb the target protein together with the contaminating protein or only the target protein to the chromatographic support, and then distinguish it from the contaminating protein or contaminants by an appropriate elution method. It is very effective to perform elution and recovery, i.e., separation by adsorption chromatography. In liquid chromatography used for protein separation and purification, in addition to adsorption chromatography, methods such as molecular sieving (or gel filtration) such as differences in protein molecular size are also used. Adsorption chromatography is considered to be optimal as a method that can be recovered in a more concentrated form than before processing.
Separation by adsorption-type liquid chromatography, which is mainly used in the separation and purification of proteins, is widely applied by so-called batch-type separation. Here, the batch type is completed by a series of operations that use a single packed column and equilibrate the column, adsorption by injecting the sample into the column, elution of the target substance from the column, and regeneration and washing of the column. Indicates the operation to be performed.
When batch type adsorption chromatography is applied, the size (capacity) of the separation column is determined according to the sample to be separated. In other words, a column that can guarantee a capacity (binding capacity) that allows the target protein to be completely adsorbed onto the column is required.
Therefore, when a large amount of sample is separated and purified by liquid chromatography, it is required to use a huge separation column according to the sample. If a huge column cannot be prepared, it is necessary to divide the sample into several parts and repeat the liquid chromatography operation. In the former case, the cost of the column due to the use of a huge column and the equipment cost such as the need to prepare equipment suitable for the size are major obstacles. On the other hand, in the latter case, by dividing into an appropriate size and performing batch operation, the above cost problem can be solved, but the operation time by repeating the batch operation is required in proportion to the number of divisions, It takes a long time to process a large amount of samples.
Already, the present inventors have devised adsorption chromatography by continuously injecting a sample in order to eliminate the above-mentioned obstacles, and have developed a liquid chromatography apparatus in which three separation columns are arranged in parallel. Then, a patent application was filed that made it possible to improve the efficiency of protein purification in large quantities (see Patent Document 1).

The liquid chromatography apparatus in which three separation columns developed in parallel by the present inventors are arranged in a separation carrier having a very high adsorption property to a target protein such as an affinity column, and in a mixed solution of proteins. Developed with the goal that the content of the target protein can be broadly applied from very high to low, eliminating waste and simplifying operations in terms of cost reduction of purification, simplicity and reliability of equipment, etc. In addition, it was considered that the element of the further idea was included in the reliability improvement. In particular, it was considered necessary to optimize the response to the actual state of protein separation and purification.

The present invention relates to a continuous technique for adsorption chromatography used for protein separation, but several attempts have already been made in connection with the continuous technique for chromatography. For example, 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. A system (simulated moving bed (SMB) method) in which a target component is continuously separated by moving in the opposite direction with respect to the flow of the mobile phase has been developed and used (see Patent Documents 2 and 3). ). 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. A feature of this method is that it is used to separate a solution containing two or more components into two fractions according to the difference in the moving speed of each component, and consists of 8 to 16 separation columns. Although high separation accuracy and efficiency can be obtained based on the principle, the equipment configuration and control become complicated and the construction cost becomes expensive.
In recent years, for purification and separation of proteins, affinity chromatography using an affinity column on which a protein ligand is immobilized and various columns for adsorption chromatography are widely used. For example, by using affinity chromatography, only a specific protein can be selectively purified. Therefore, a complicated separation system is constructed by applying the SMB method developed for the purpose of separating two components having poor separability. There is no need. That is, the SMB method has not been applied so much due to the complexity of the apparatus and the development of a separation carrier having good separation characteristics.
On the other hand, in the analysis using liquid chromatography, a device for improving the efficiency of repeated measurement has been developed, and multiple columns are parallelized for the purpose of reducing the complexity of repeated measurement and reducing the total analysis time. By doing so, development of an apparatus that realizes continuous analysis has been performed (see Patent Documents 4 and 5). 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. In liquid chromatography using parallel columns for the purpose of continuous analysis or efficiency, it is not necessary to inject samples continuously because the amount of sample used for analysis is small. Although an apparatus has been developed to efficiently perform analysis using parallel columns while introducing samples intermittently, the purpose of continuous sample introduction and purification separation, that is, a large amount of protein It is a device that cannot be diverted for refining. In general, when introducing samples continuously, it is considered that it is necessary to use a lot of columns, and the time (waiting time) between sample introduction and introduction should be virtually zero. Is not a device that can be used for such purposes. Therefore, the method invented by the present inventors was the only one of the adsorption-type liquid chromatography devices devised that the time required for purification can be obtained by continuously injecting the sample into the column (patents). Reference 1).

Japanese Patent Application No. 2009-104625 Special table 2008-539395 JP-T-2004-533919 JP-T 2002-40008 JP 2003-329660 A

When considering the protein production process, we often encounter situations where the protein is diluted or the amount of the target protein is extremely small in a large amount of sample as a raw material. In addition, in order to obtain a highly pure protein, several separation operations must be performed. When adsorption chromatography is applied in the process, the target protein must be in a solvent condition that can be adsorbed on the carrier to be used. Need to adjust. This is a so-called buffer exchange operation. When handling a small volume of protein solution, it is easy to change the solvent by dialysis or the like, but in the case of a large volume of sample solution, its execution becomes difficult and the time required for dialysis is long. The problem of being. The simplest method for solving the solvent exchange problem is a dilution method. By diluting the original sample solution with the target solvent, for example, 100 times, the target solvent exchange of the adsorption chromatography is substantially achieved. Further, the time required for dilution is much less than the time required for other solvent exchanges such as dialysis, and only the time required for mixing, so that the operation can be completed substantially instantaneously.
However, for example, if dilution is performed 100 times, the volume of the target sample solution becomes 100 times, and when purifying this by ordinary liquid column chromatography, a large volume column is required. A new problem arises. As a method of processing such a large volume sample with as small a column as possible, it is effective to use the apparatus already invented by the present inventors, and a large volume sample solution is as small as possible. This is in accordance with the development purpose of being developed for the purpose of purification using a column (see Patent Document 1).
The purpose of the present invention is to develop an apparatus for purifying a sample with a very low target protein concentration in a large amount of highly diluted protein sample or a large amount of mixed solution while efficiently concentrating it by adsorption-type liquid chromatography. This is a result of intensive studies for the development of a method and apparatus more suitable for purifying and concentrating a large volume of low-concentration protein solution quickly and at low cost.

Already, the present inventors paralleled an adsorption chromatography column represented by an affinity carrier, and washed and separated in the second column within the time required to saturate the adsorption of the protein sample in the first column. In addition, the column, regeneration and initial equilibration can be completed in the third column. By continuously changing the role of the three columns, the protein sample can be continuously injected. We have developed a liquid chromatography device that allows separation and purification while continuously injecting a sample (Patent Document 1).
When performing such continuous injection, when using a diluted protein solution, the time required for the adsorption of the protein sample in the first column is the washing and separation in the second column, and the third column. Compared with the time required for the regeneration and initialization in the above, it took an extremely long time, so it was found that there was no need to separate the second column step and the third column step. That is, in the parallelization of the column, there is a case where there is no necessity to use three, but separation and purification of proteins using a liquid chromatography apparatus that performs separation and purification while continuously injecting according to the inventors' invention. It became clear by doing. This also led to the need to clarify in terms of applying continuous injection chromatography whether the protein solution is dilute or not.

In the present invention, whether or not a target protein sample solution is dilute will be organized as follows.
In order to discuss whether or not the protein is diluted when attempting purification / concentration using adsorption chromatography, (1) the maximum binding capacity of the protein indicated by the applied column carrier per unit volume for the separation column. (This is expressed as Q unit: mg / mL-column), (2) The total amount of various solutions required for operations other than protein adsorption (this is expressed as W (unit: mL)) (3) Total amount of protein in the target protein solution (this will be expressed as P (unit: mg)), (4) Total volume of the target protein solution (this will be expressed as V ( If the value of Q × W> P × V is satisfied, the target protein solution is simply applied to the separation column per volume. It is possible that it is sufficiently dilute. Alternatively, if the protein concentration in the protein solution (which is expressed as C (mg / mL) (= P / V)) is used, the value Z = Q / C (mL / mL-column) is calculated. However, if operations other than protein adsorption in adsorption chromatography can be completed within the Z column volume, it can be said that the protein solution of interest is dilute.
This means that whether or not a protein solution is diluted greatly varies depending on characteristics of a separation column used, various solvents used, chromatography equipment used, and other factors.
Therefore, the present inventors aimed to develop an apparatus that can be widely applied under the condition of being as lean as possible.

  For this purpose, various studies have been conducted on a device in which two identical separation columns are arranged in parallel, which is the simplest parallel column device, and those having the flow path configuration shown in FIG. A conclusion was reached that the device was a device, a device having this flow path configuration was manufactured, and by confirming its operation confirmation, it was shown that a continuous process was achieved, and the present invention was completed. The outline of the study for that is shown below.

In the adsorption column chromatography, the column chromatography process can be divided into a plurality of stages according to the characteristics of the solution to be passed. 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).
If the protein solution satisfies the condition that it is diluted, the amount of the solution used for equilibration of the column, washing, elution of the target component, and regeneration is sufficiently smaller than the sample volume to be injected. As already noted by the present inventor, even when the liquid flow is performed at the same flow rate, the initialization (equilibration), washing, elution, and regeneration operations can be performed in combination within the time required for sample injection. This is possible (see Patent Document 1). Thus, by using two columns in parallel, the objective was achieved by remodeling the sample continuous injection chromatography device using the parallel column already developed by the present inventors into a more simplified device. it can.
FIG. 2 shows a flow chart of a device composed of three columns already produced by the present inventors. By removing the third column portion from this flow chart, the flow chart shown in FIG. 3 according to the present invention can be devised.
Here, the major difference between using two columns and three columns is that, in the case of two columns, the operations of initialization (equilibration), washing, elution, and regeneration are performed within the time required for sample application. However, in the case of three columns, all operations can be completed in half the maximum time. That is, in the case of two columns, only half the concentration of the sample in the three columns can be applied, and the condition of dilution becomes severe. Therefore, by devising the device, we studied to shorten the time required for the operations of initialization (equilibrium), washing, elution and regeneration.

  The time required for the operations of initialization (equilibration), washing, elution and regeneration has characteristics for each separation column used, and cannot be changed by a chromatography apparatus using such characteristics itself.

  On the other hand, in actual chromatography, in order to continuously pass various solutions, it takes a certain amount of time for the solutions to be completely switched in the flow path after the solutions are switched. This is a particular problem in that the solution before the switching and the solution after the switching are mixed in the piping forming the flow path, resulting in a solution gradient. This not only affects the separation characteristics, but also greatly affects the time required for the operations of initialization (equilibration), washing, elution and regeneration. For example, even when the solution in the column is completely switched by passing a solution that is 1 to 2 times the column volume, the mixing of the solution before and after in the pipe is actually more effective in completely switching the solution. In the case where the operation is continuously repeated as in the present invention, it is necessary to secure a sufficient operation time in order to ensure the reproducibility. This indicates that the actual time required for initialization, washing, elution, and regeneration operations can be shortened by improving the flow path system.

  As a method for minimizing the influence on the separation column, which is caused by the mixing of the solution before and after in the pipe, it is conceivable to make the pipe from RT1 to the column shown in FIG. 3 as short as possible. It is not possible to do it. Therefore, in order to eliminate the influence more reliably, the present inventor can install a switching valve immediately upstream of the column and perform an operation of preventing the switching valve from flowing into the column as waste liquid when switching the solution. As a device, a method (so-called channel purge) was adopted in which the solution in the pipe was completely switched through the solution after switching for a certain period of time, and then the valve was switched again to pass the solution to the column. Since the switching valve can be directly connected to the column, the effect of switching the solution before and after the switching of the solution can be minimized. The flow path system realizing this effect is the present invention shown in FIG. If the purging is not performed in the apparatus shown in FIG. 1, the configuration shown in FIG. 1 naturally includes the configuration shown in FIG.

  A feature of the present invention is that by using only two identical adsorption-type separation columns, purification and concentration treatments consisting of continuous adsorption and elution can be performed as long as the durability of the columns allows. The processing time is determined by the volume of the sample to be purified and the speed at which the sample is sent to the apparatus per unit time, that is, the flow rate, regardless of the column size. That is, approximately the same time as the time when all the samples have been injected into the apparatus, more precisely, the time for initialization at the start of operation and the time required for cleaning, elution, regeneration, and initialization after the final injection is completed. It is time to complete all operations. It is clear that the processing time can be shortened when using one column compared to the time required for initialization at the start of operation, the time for sample solution injection, and the time required for washing, elution, regeneration, and initialization. It is. When using a single column, unlike the case of the present invention, it is necessary to use a large column because the required column volume is sufficient to adsorb all the target protein in the sample. There must be. Compared to this, for example, when the repetition is performed 100 times, it is only necessary to use a column having a capacity of 1/100 that is performed in one operation with one column. This means that the use of the device of the present invention can greatly reduce the effort of scaling down the column to be used or repetitive batch purification, contributing to the achievement of cost reduction and labor saving without changing the purification accuracy. can do. 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.

The present invention employs a flow channel system as shown in FIG. 1 in order to parallelize two columns. On the other hand, it is considered that the same effect can be expected by controlling the inflow of the sample by parallelizing two chromatography apparatuses. When considering using two chromatographic apparatuses as they are, paralleling the apparatuses, and achieving the continuous injection of the sample which is the object of the present invention, the flow chart shown in FIG. 4 is considered. In FIG. 4, for example, the configuration downstream of the pump P1 is merely a connection of the purge valve of the present invention and the recovery valve to a universal column chromatography apparatus. On the other hand, upstream of the pump P1, the S liquid, the R1 liquid, the R2 liquid, the R3 liquid, and the R4 liquid are allowed to flow using a flow path switching valve. The major feature of the flow path system shown in FIG. 4 in which the upstream side of the pump is modified in order to connect the liquid flow chart shown in FIG. It is used only for liquid transfer. In protein purification, many samples for separation are crude. Therefore, cleaning of the flow path and the pump becomes a problem, but in the case of the flow path system of the present invention, since only the pump 1 is in contact with the sample, the pump 2 does not need to worry about contamination of the protein sample. Is raised. Further, in the flow path system shown in FIG. 1 of the present invention, the pump 1 and the pump 2 have no problem even if they have completely different specifications. However, in the flow path shown in FIG. 4, synchronization (synchronization) by a parallel column is achieved. Therefore, it is necessary that the pumps have exactly the same specifications.
In the present invention, as a requirement that enables continuous operation with two parallel columns, it has been described that whether or not a protein solution for separation is dilute is an important factor. By taking the above, it is possible to achieve that all protein solutions are dilute by appropriately changing the flow rate of the pump P1. That is, the upper limit of the volume of the sample solution that can be passed is determined by the binding capacity of the separation column. For example, the optimal time (T (optimum or set)) required for the initialization (equilibration), washing, elution, and regeneration operations via the pump P2 is the characteristics of the protein to be purified, the characteristics of the separation column used, and each operation. Determined by the solution used. On the other hand, the flow rate of the sample can be arbitrarily selected as long as the performance of the pump allows. Therefore, the optimal time required for the initialization (equilibration), washing, elution, and regeneration operations via the pump P2 × the flow rate of the pump P1 × the protein concentration of the sample solution is the characteristics of the pump P1 that is the characteristic of the separation columns C1 and C2. If the flow rate of the pump P1 is set to be equal to or less than the value of the dynamic binding capacity at the flow rate, the protein solution for separation can be handled as being dilute. Moreover, efficient separation and purification can be achieved by appropriately setting the flow rate of the pump P1 in consideration of the optimal time required for the operations of initialization (equilibrium), washing, elution, and regeneration via the pump P2.

FIG. 1 shows a flow chart of the liquid chromatography apparatus of the present invention. In the figure, S, R1, R2, R3, and R4 indicate solution reservoirs, SV1 and SV2 indicate selection valves, and numbers therein indicate flow paths that can be selected by the valves, SW1, WS3, and SW3. , SW4 indicate switch valves, numerals in them indicate the flow paths to be switched, P1 and P2 indicate liquid feed pumps, and C1 and C2 indicate separation columns. Also shown in the figure is a reservoir and UV monitor for collection of the purified sample. In the figure, black circles indicate that the flow paths are connected. FIG. 2 shows a flow chart of a liquid chromatography apparatus that the inventors have already invented (see Patent Document 1), in which three columns are arranged in parallel to enable continuous sample injection. In the figure, S, R1, R2, R3, and R4 indicate solution reservoirs, SV1, SV2, SV3, SV4, and SV5 indicate selection valves, and the numbers in them indicate flow paths that can be selected by the valves. , SW1 and WS3 indicate switch valves, numbers in them indicate flow paths to be switched, P1, P2 and P3 indicate liquid feed pumps, and C1, C2 and C3 indicate separation columns. Also shown in the figure is a reservoir and UV monitor for collection of the purified sample. In the figure, black circles indicate that the flow paths are connected. FIG. 3 is a flow chart having no purge function when switching solutions. In the figure, S, R1, R2, R3, and R4 indicate solution reservoirs, SV1 and SV2 indicate selection valves, numbers therein indicate flow paths that can be selected by the valves, and SW3 and SW4 are A switch valve is shown, the number in it shows the flow path to switch, P1 and P2 show the liquid feeding pump, and C1 and C2 show the separation column. Also shown in the figure is a reservoir and UV monitor for collection of the purified sample. In the figure, black circles indicate that the flow paths are connected. FIG. 4 shows a flow chart in the case where the achievement of continuation is considered simply using two liquid chromatography apparatuses. In the figure, S, R1, R2, R3, and R4 indicate solution reservoirs, SV1 and SV2 indicate selection valves, and numbers therein indicate flow paths that can be selected by the valves, SW1, WS3, and SW3. , SW4 indicate switch valves, numerals in them indicate the flow paths to be switched, P1 and P2 indicate liquid feed pumps, and C1 and C2 indicate separation columns. Also shown in the figure is a reservoir and UV monitor for collection of the purified sample. In the figure, black circles indicate that the flow paths are connected. FIG. 5 shows the change over time in the conductivity of the eluate coming out of the column when the solution is switched from R1 which is a high salt concentration solution to R2 which is a low salt concentration solution. Plotted as time passing through the solution column switched to R2 at time zero. In the figure, the horizontal axis represents the elapsed time (unit: minutes), and the vertical axis represents the conductivity (arbitrary unit) of the solution coming out of the column. The curve indicated by the dotted line represents the change in conductivity when the purge time is 0, and the curve indicated by the solid line is the change in conductivity when the purge time is 30 seconds. FIG. 6 shows the recovery of the protein obtained in the above experiment monitored by absorption at 280 nm. In the figure, the horizontal axis represents elapsed time in minutes, and the vertical axis represents absorbance at 280 nm (arbitrary unit). The data of the absorbance sampling interval is plotted every 5 seconds.

The apparatus constituting the present invention can be shown by the flow chart shown in FIG.
That is, the liquid chromatography apparatus of the present invention comprises two separation columns (referred to as column C1 and column C2, respectively), two liquid feed pumps (referred to as pump P1 and pump P2, respectively), Minimum 5 types of solution reservoirs (referred to as Reservoir S, Reservoir R1, Reservoir R2, Reservoir R4, Reservoir R4, respectively), 6 flow path switching site devices (this are switch valve SW1, switch valve, respectively) SW2, switch valve SW3, switch valve SW4, selection valve SV1, and selection valve SV2), and piping components connecting them, and the configuration of the piping is represented by the flow chart shown in FIG. Thus, the solution in the reservoir S is switched by the selection valve SV2 via the pump P1. It is selected whether it is sent to the piping on the switch valve SW1 side or the piping on the switch valve SW2 side, and the solution in the reservoir R1, reservoir R2, reservoir R4, and reservoir R4 is connected to the pump P2 by the selection valve SV1. In the case where it is selected whether or not to be connected, it is sent via the pump P2 to the pipe on the switch valve SW1 side or to the pipe on the switch valve SW2 side via the pump P2. When the selection valve SV2 is selected and the piping from the pump P1 side is selected to the switch valve SW1, the piping on the pump P2 side is simultaneously selected to the switch valve SW2 side, or the pump P1 When piping from the side is selected on the switch valve SW2 side The pipe on the pump P2 side is simultaneously selected on the switch valve SW1 side, and either the pipe on the column C1 side or the pipe on the waste liquid side is selected in the switch valve SW1, and the switch valve SW2 , Either the pipe on the column C2 side or the pipe on the waste liquid side is selected, the pipe on the opposite side of the pipe connecting the switch valve SW1 side and the column C1 is connected to the switch valve SW3, and the switch valve SW2 side and the column C2 are connected. Is connected to the switch valve SW4. In the switch valve SW3, either the recovery container side pipe or the waste liquid side pipe is selected. In the switch valve SW4, the recovery container side pipe is selected. Alternatively, the flow path is piped so that either the waste liquid side pipe is selected. All the pumps and flow path switching devices are controlled so that they are operated according to a pre-programmed order so that liquid chromatography can be performed automatically and continuously. Further, at least four types of reservoirs connected to the pump P2 by the selection valve SV1 are required, but it is possible to increase the number of reservoirs. When six types of reservoirs (which are referred to as R1, R2, R, R4, R5, and R6, respectively) are installed in advance, a maximum of six types of solutions can be selected. When 12 types of reservoirs are installed, a maximum of 12 types of solutions can be selected.
In FIG. 1, C1 and C2 are separation columns, respectively. Immediately upstream of the separation columns C1 and C2, switch valves SW1 and SW2 are provided, respectively. The switch valves SW1 and SW2 flow the solution that passes through the separation column to the waste liquid side in advance, and switch the solution that passes through the column. In this case, the mixing of the solution before and after is minimized. Immediately downstream of the separation columns C1 and C2, switch valves SW3 and SW4 are provided, respectively. The switches SW3 and SW4 allow the solution coming out of each separation column to pass through the waste reservoir container or the recovery container. Change whether to pass liquid. S, R1, R2, R3, and R4 are various solution reservoirs. S contains a sample solution to be separated first, and is supplied to the selection valve SV2 via the liquid feed pump P1. R1 contains a column initialization solution, R2 contains a washing solution, R3 contains an elution solution, R4 contains a regeneration solution, and R1, R2 are selected by a selection valve SV1. , R3, R4 are switched and supplied to the selection valve SV2 via the liquid feed pump P2.
The selection valve SV2 is a valve for switching the solution flowing from the P1 side and the solution flowing from the P2 side while selecting the flow path while switching the C1 side or the C2 side so as not to select the same column. For example, when the flow from the P1 side flows to the C1 column side, the flow from the P2 side flows to the C2 side, and the flow from the P1 side flows to the C2 column side. In this case, the selection valve has a function of simultaneously switching the flow from the P2 side to the C1 side. Each switch valve SW1, SW2, SW3, SW4 and each selection valve SV1, SV2 are automatically controlled by a control device (not shown). The purified protein separated by the column C1 or C2 is intermittently recovered by sending it to the recovery container only when it is desired to recover it with the switch valves of SW3 and SW4. 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.
The case where only four kinds of R1, R2, R3, and R4 are used as the solution of the reservoir is illustrated, but by changing the valve shown in SV1 and changing to the desired number, using four or more kinds of solutions, Chromatography can be performed. In the present invention, as a solution required in the adsorption chromatography, an initialization solution, a washing solution, an elution solution, and a regeneration solution are necessary. It is described. Therefore, it is obvious that the present invention also includes a case where four or more types of solutions are connected and controlled upstream of SV1.
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. 1, the minimum configuration of the chromatography apparatus of the present invention includes five solution reservoirs (indicated by S, R1, R2, R3, and R4), and two liquid pumps (P1, P2 having the same performance). 2) same separation column (indicated by C1, C2), 4 switch valves (indicated by SW1, SW2, SW3, SW4), 2 selection valves (indicated by SV1, SV2), waste reservoir, for recovery Consists of containers and piping connecting them. A UV monitor for monitoring the protein recovery status should be equipped as much as possible, but is not essential. In the figure, the intersection of the lines indicated by black circles indicates that the pipes are connected there, but those having a function of preventing backflow are desirable. The operation of the present apparatus is automatically performed by switching a valve programmed in advance (switched at regular intervals). The parameters necessary for the program are very simple, and the switch valves SW1, SW2, SW3 and SW4 are programmed to issue a control signal at regular intervals and switch the flow path to 1 or 2 in the figure. The selection valves SV1 and SV2 are programmed to issue a control signal at regular time intervals and selectively select one of the flow paths indicated by numerals.

In order to perform purification by adsorption chromatography using the C1 and C2 columns by simply passing the sample S continuously through the pump P1 using the apparatus shown in FIG. A time program for switching SW1, SW2, SW3, SW4, SV1, and SV2 may be created and operated according to the procedure described.
The parameters for creating the time program include the time required for purging the flow path at the time of solution switching, and the time for passing the solutions R1, R2, R3, R4, which is T (P), respectively. , T (R1), T (R2), T (R3), and T (R4), the time from the passage of the elution solution R2 to the start of elution of the purified protein from the column is expressed as T ( S), the time from passing the elution solution R2 until the purified protein is completely eluted from the column, and T (E). By using it, a time program can be created.
Since the present apparatus repeats a normal purification process performed by one, it is only necessary to determine a time program for the nth cycle and to repeat this.
In normal chromatography using a single column, the sample solution in the reservoir S is passed through the initialized column for a time T (S), and then the R2 solution (washing solution). Is passed through T (R2) to wash out non-specifically adsorbed substances, and then R3 solution (solution for elution) is passed through T (R3) to elute the adsorbed protein to be purified. At this time, since the target protein is eluted at time T (S), the recovery operation is started. Thereafter, since the elution of the target protein is completed at time T (E), the recovery operation is terminated. After passing the R3 liquid, the R4 liquid (regeneration solution) is passed through T (R4) to regenerate the column, and then the R1 liquid (initialization solution) is replaced with T (R1). The sample solution is passed through and the column is initialized, so that the operation of passing the sample solution for the next purification cycle can be started.
Therefore, of the two separation columns (C1, C2), as the nth purification cycle, the sample solution is passed through the C1 column and at the same time T (S) passing through the C1 column. In the same time, in the C2 column, R2 liquid flow, purge, R3 liquid flow, sample recovery start, sample recovery end, R4 liquid flow, R1 solution flow are all performed, and then SV2 is switched. At the same time as passing the sample solution through the C2 column, at the same time as the time T (S) during which the sample is passed through the C2 column, the R2 liquid flow, purge, and R3 liquid flow through the C1 column. , Sample collection start, sample collection end, R4 liquid flow and R1 solution flow are all performed, so that one purification operation in the C1 column is completed and It is, so that the liquid passage of the sample can be finished. If a valve operation that keeps repeating this operation is programmed, it can be operated automatically.
Here, since the relationship T (S) = T (R2) + T (P) + T (R3) + T (P) + T (R4) + T (P) + T (R1) holds, it is necessary in the time program to be created. As parameters, the number of repeated operations N and T (P), T (R1), T (R2), T (R3), T (R4), T (S), and T (E) are sufficient. By setting, it is possible to create a program that automatically operates the various valves shown in FIG. 1, and by executing the created program, chromatography using this apparatus characterized by continuous injection is performed. Can be implemented.

  The use of a liquid chromatography apparatus that enables protein concentration and purification while continuously injecting a sample having the flow path system shown in FIG. 1 of the present invention is widely used in protein concentration and purification. It can be used. A typical application example will be described below, but it can be widely applied to chromatography using an adsorption type column.

[Typical use example 1]: Concentration / recovery of protein using diluted protein solution When a protein to be concentrated in a diluted protein solution is used, the target protein is adsorbed / By using a column that can bind, adsorption is performed in one column of adsorption-elution-column regeneration-initialization, and elution-column regeneration-initialization is performed in the other column. By performing the replacement operation automatically with this apparatus, it is possible to perform concentration that maximizes the binding capacity of the column used. The column used depends on the characteristics of the target protein, but various affinity groups with low molecular functional groups such as anion exchange carrier, cation exchange carrier, hydrophobic carrier, hydroxyapatite carrier, nickel chelate carrier, etc. Various affinity carriers bound with protein ligands such as carriers and protein A columns are suitable.

[Typical application example 2]: Affinity purification from cell-free extract Affinity purification can be performed by combining various tagged proteins such as histag expressed in E. coli and an affinity carrier that binds the tag portion. In the Examples, purification examples of histag proteins are shown. Since the expression level of many tag proteins in E. coli is about 100 mg / L, and the binding capacity of the affinity carrier for purification for tags is about 1000 mg / mL, which is a thousand times, expression in E. coli The chromatographic apparatus of the present invention, which can treat proteins as sufficiently dilute in tag purification, can be easily applied.

[Typical use example 3]: Affinity purification of monoclonal antibodies expressed in CHO cells Protein A and protein G, which are proteins that specifically recognize and bind to the Fc region of antibodies in purification of monoclonal antibodies expressed in CHO cells Alternatively, it is effective for purification using an affinity carrier on which a protein ligand such as a derivative thereof is immobilized. In Examples, purification examples of human monoclonal antibodies in which protein ligands of protein A derivatives developed by the present inventors are immobilized are shown. Although the expression of monoclonal antibodies using CHO cells is 1 g / L in many cases, high expression of 10 g / L or more has been achieved by improving the CHO cell expression culture method. Even in this case, the chromatography apparatus of the present invention can be applied by reducing the flow rate during sample application to about 1/10.

[Typical use example 4]: Purification of subsequent chromatography in multi-step purification In order to obtain a highly pure protein, several separation operations must be performed, that is, a plurality of chromatographic techniques are used. Multi-stage purification is performed. At that time, when the adsorption chromatography is continuously performed, it is necessary to adjust the sample solution purified and recovered in the former stage chromatography to a condition that can be adsorbed to the carrier used in the latter stage. This is a so-called buffer exchange operation. When handling a small volume of protein solution, it is easy to change the solvent by dialysis or the like, but in the case of a large volume of sample solution, its execution becomes difficult and the time required for dialysis is long. The problem of being. The simplest method for solving the solvent exchange problem is a dilution method. By diluting the original sample solution with the target solvent, for example, 100 times, the target solvent exchange of the adsorption chromatography is substantially achieved. Further, the time required for dilution is much less than the time required for other solvent exchanges such as dialysis, and only the time required for mixing, so that the operation can be completed substantially instantaneously. However, since the amount of solution to be handled greatly increases due to dilution, it is necessary to process a large amount of sample. For that purpose, such as whether to use a large column or repeatedly perform subsequent purification using a small column, etc. The procedure of sample dilution has not been used in practice. The apparatus shown in FIG. 1 of the present invention is the most suitable method for processing a large amount of diluted sample, and it is obvious that the apparatus can be used for subsequent purification and concentration in multi-stage purification.

[Typical use example 5]: Application to a removal type chromatography in which extra components are adsorbed without adsorbing the target protein on the adsorption type column, the apparatus of the present invention continuously injects a sample. In the flow chart shown in FIG. 1, for example, when the sample is injected from the P1 side into the column C1, the switch valve SW3 is used as the recovery side, and excess components are adsorbed and removed from the column C1. The sample was recovered as a purified sample, and at the same time, on the column C2 side, various solutions were passed from the pump P2 side, so that the adsorbed excess components were eluted and the column was regenerated and initialized, and the initialization was completed. At this point, the selection valve SV2 is used to change the flow path upstream of the column C1 and the column C2, thereby continuously injecting the sample and purifying and collecting Can. In this case, unlike the case where the target protein is adsorbed on the column, the protein can be continuously purified and recovered.

[Typical use example 6]: Performance evaluation related to repeated use of the column (measurement of lifetime, etc.)
The apparatus of the present invention makes it possible to repeatedly perform liquid chromatography operations while continuously injecting samples. Therefore, this apparatus can be utilized for an apparatus for performing a durability test such as deterioration in repeated measurement of a column. In such a case, there is also an advantage that two durability tests such as deterioration under the same chromatographic separation conditions can be simultaneously performed by using different carriers for the columns C1 and C2.

  As described above, by using the apparatus of the present invention, it can be used in a wide range in liquid chromatography.

[Example 1]
As an example of this apparatus satisfying the configuration of the continuous injection chromatography apparatus represented by the flow chart of FIG. 1, necessary parts were produced using commercially available parts. As parts constituting this apparatus, the liquid feed pumps P1 and P2 use SP-12-13 manufactured by Flour, and the selection valve SV1 is an automatic rotary valve 401-74 (1-6 direction manufactured by Flour). Rotary, the selection valve SV1 is an automatic four-way switching valve 401-140 manufactured by FROM, and the switch valves SW1, SW2, SW3, SW4, and SW5 are TAKASAGO ELECTRIC, Inc. The separation column C1 and C2 are the same for the two separation columns C1 and C2, but selected for each target to be separated (depending on the protein to be purified). , UV-MII manufactured by GE Healthcare, using normal glass beakers as solution reservoirs R1, R2, R3, R4, and R5, and piping connecting each device using normal pressure tubes, On the other hand, as a control device for transmitting and operating a selection or switching signal, a signal is sent to the pump, selection valve, and switch valve according to a time program, pump on / off, selection valve position, switch valve flow path selection I made a device for PC control.
As described below, the operation of the manufactured device was confirmed to operate normally in each purification example.
Since the automatic rotary valve 401-74 manufactured by FROM, which is used as the selection valve SV1, is a 1-6-way rotary, six types of solution reservoirs can be used. Furthermore, it was also confirmed that by using a 1-12-way rotary valve and an automatic rotary valve 401-117 manufactured by Flour, the solution reservoir can be appropriately increased to 12 kinds, and fine separation / purification conditions can be set.

[Example 2]
(Effect of flow path purge)
In the present invention, as shown in FIG. 1, the switch valves SW1 and SW2 are arranged immediately upstream of the column, and the purpose is to prevent the mixing of the preceding and following solutions as much as possible when switching the solution to be passed. The effect was measured by changing the salt concentration in the solution and measuring the conductivity. A conductivity meter for measuring the conductivity is installed at the UV monitor of FIG. 1, 10 mM sodium phosphate, pH 7.4, 1M NaCl as R1, and 10 mM sodium phosphate, pH 7.4 as R2. Conductivity change when connecting anion exchange column HiTrap QXL (1 mL) (GE Healthcare Prepack column) as a separation column using 5 mM NaCl and switching between R1 and R2 at a flow rate of 1 mL / mL Was measured. As a result, as shown in FIG. 5, when the purge time was set to 0, it was confirmed that there was a delay in the complete exchange of the solution in the column compared to the case where the purge time was 30 seconds. That is, it was shown that the solution exchange can be performed more quickly by performing the purge, and the effect of the purge was confirmed.

  When using the apparatus of the present invention, as the items to be set in advance, the type and size of the separation column to be used, the type of sample and the concentration of the target protein therein, the initialization solution (R1), the washing solution (R2), and the elution solution (R3), regeneration solution (R4), each type and composition, purge time (X), flow time for each of R1, R2, R3, R4, T (R1), T (R2), T (R3), It is necessary to determine T (R4), the recovery start time (Y) after passing the elution solution, the recovery time (Z), the number of repetitions (n), and the liquid feeding speed of each of the pumps P1 and P2. In the following examples, this parameter and its results are described in various enrichment experiments.

[Example 3]
(Concentration and recovery of protein using diluted protein solution)
In this example, a highly purified protein preparation is used as a low-concentration protein solution diluted with an initialization solution, and the apparatus of the present invention is used for continuous injection experiments using the diluted protein solution. went.

[Concentration of histag protein using nickel chelate column as affinity column]
Protein preparation: Purified protein PAA3T (a recombinant protein encoded by the plasmid pAA3T described in Japanese Patent Application Laid-Open No. 2008-115151, which has been developed by the present inventors and has an antibody binding ability (hereinafter referred to as “PAA3T”) ) Highly refined preparation)
Protein concentration: 0.1 mg / mL (measured by BCA method)
Total amount of protein solution injected: 588 mL (59 mg)
Separation column: Nickel chelate column HisTrap HP (1 mL) (GE Healthcare pre-packed column)
R1: 10 mM sodium phosphate, pH 7.4, 50 mM NaCl, 5 mM imidazole R2: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 75 mM imidazole R3: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 250 mM Imidazole R4: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 1M imidazole T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 3 min
Number of repetitions (n) = number of elution sample collection: 7 times Flow rate of pump P1 (sample injection rate): 2 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, a chromatogram as shown in FIG. 5 was obtained.
The total volume of the collected sample was 21 mL, and the protein concentration was 2.5 mg / mL. As a result, 53 mg of protein PAA3T could be recovered in a 25-fold concentrated form at a recovery rate of 90%. When the area of each elution peak was determined, the average value was 360 (arbitrary unit), and the standard deviation was 12 (arbitrary unit). It was shown that the recovery was very reproducible. In addition, the total time concerning the chromatography of this experiment was less than 5 hours including the initialization (automatic operation) time of the apparatus.

[Concentration of histag protein using ion exchange column]
Protein preparation: Purified protein PAA3T (a recombinant protein encoded by the plasmid pAA3T described in Japanese Patent Application Laid-Open No. 2008-115151, which has been developed by the present inventors and has an antibody binding ability (hereinafter referred to as “PAA3T”) ) Highly refined preparation)
Protein concentration: 0.05 mg / mL (measured by BCA method)
Total amount of protein solution injected: 966 mL (48 mg)
Separation Column: Anion Exchange Column HiTrap QXL (1 mL) (GE Healthcare Prepack Column)
R1: 10 mM sodium phosphate, pH 7.4, 5 mM NaCl
R2: 10 mM sodium phosphate, pH 7.4, 250 mM NaCl
R3: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl
R4: 10 mM sodium phosphate, pH 7.4, 1M NaCl
T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 2 min
Number of repetitions (n) = number of elution sample collection: 23 times Flow rate of pump P1 (sample injection speed): 1 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, the total volume of the collected sample was 46 mL and the protein concentration was 1.0 mg / mL. As a result, 46 mg of protein PAA3T was recovered in a 20-fold concentrated form with a recovery rate of 96%. . The total time for chromatography in this experiment was less than 18 hours including the initialization (automatic operation) time of the apparatus.

[Concentration of polyclonal antibody with protein A carrier as affinity column]
Protein preparation: Human polyclonal antibody (purchased from Sigma, IgG from Human Serum, Technical Grade)
Protein concentration: 0.2 mg / mL (measurement by BCA method)
Total amount of protein solution injected: 966 mL (193 mg)
Separation column: Affinity carrier HiTrap Protein A HP (1 mL) (a prepacked column manufactured by GE Healthcare)
R1: 100 mM sodium phosphate, pH 7.2
R2: 5 mM sodium phosphate, pH 7.4, 1M NaCl
R3: 100 mM sodium citrate, pH 3.5
R4: 100 mM glycine-HCl, pH 2.5
T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 3 min
Number of repetitions (n) = number of elution sample collection: 23 times Flow rate of pump P1 (sample injection speed): 1 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, the total volume of the collected sample was 69 mL and the protein concentration was 2.6 mg / mL. As a result, 178 mg of human polyclonal antibody could be recovered in a 13-fold concentrated form with a recovery rate of 92%. It was. The total time for chromatography in this experiment was less than 18 hours including the initialization (automatic operation) time of the apparatus.

[Example 4]
(Affinity purification from cell-free extract)
Affinity purification can be performed by combining various tagged proteins such as histag expressed in E. coli and an affinity carrier that binds the tag portion. In this example, purification examples of histag proteins expressed in E. coli are shown. In addition, since the expression level of many tag proteins in E. coli is about 100 mg / L or less, and the binding capacity of the affinity carrier for purification for tags is about 1000 mg / mL, which is 1000 times, the expression level in E. coli The expressed protein could be treated as sufficiently dilute in tag purification.

[Purification of Histag protein, RRRRG]
As a histag protein, a protein already invented by the present inventors and having an antibody binding ability (hereinafter referred to as “RRRRRG”, a recombinant protein encoded by the plasmid pAA-RRRG described in JP-A-2008-115151). 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 disrupted with a French press apparatus, and then centrifuged at high speed for 20 minutes (20,000 rotations per minute) to separate the supernatant. did. 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, 1008 mL was subjected to a continuous injection purification experiment under the following setting conditions. On the other hand, 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 3 times separately, and about 1.7 mg of purified protein was obtained each time.
Separation column: Nickel chelate column HisTrap HP (1 mL) (GE Healthcare pre-packed column)
R1: 10 mM sodium phosphate, pH 7.4, 50 mM NaCl, 5 mM imidazole R2: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 75 mM imidazole R3: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 250 mM Imidazole R4: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 1M imidazole T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 3 min
Number of repetitions (n) = Number of elution sample collection: 12 times Flow rate of pump P1 (sample injection speed): 2 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, the total volume of the collected sample was 36 mL, and the protein concentration was 2.4 mg / mL. As a result, 86.4 mg of protein RRRRG could be recovered. The total time for chromatography in this experiment was less than 10 hours including the initialization (automatic operation) time of the apparatus.
In this continuous experiment, 86.4 mg of purified protein (ie, 0.086 mg / mL sample) was recovered using 1008 mL of sample solution, and in a single column experiment, from 5 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 was equal to or better than that of a single column.

[Example 4]
(Affinity purification of monoclonal antibodies expressed in CHO cells)
Protein preparation: Anti-IL8 human monoclonal antibody (culture supernatant obtained by culturing ATCC CRL-12445 CHO strain)
Protein concentration: Estimated to contain about 0.096 mg / mL IgG by titration Total amount of protein solution injected: 1008 mL (about 97 mg)
Separation column: Affinity carrier (1 mL) in which the protein PAA3T, which is the protein A variant described above, is immobilized on a silica monolith surface-modified with an amino group
R1: 100 mM sodium phosphate, pH 7.2
R2: 5 mM sodium phosphate, pH 7.4, 1M NaCl
R3: 100 mM sodium citrate, pH 3.5
R4: 100 mM glycine-HCl, pH 2.5
T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 2 min
Number of repetitions (n) = elution sample collection number: 12 times Flow rate of pump P1 (sample injection speed): 2 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, the total volume of the collected sample was 24 mL and the protein concentration was 3.7 mg / mL. As a result, 88 mg of anti-IL8 monoclonal antibody could be recovered at a recovery rate of 91%.
As a control experiment, 20 mL (presumed to contain about 1.92 mg of IgG) of the same culture supernatant was used, and an affinity carrier HiTrap Protein A HP (1 mL) (a prepack column manufactured by GE Healthcare) was used as a single column. When purification was performed under the same separation solution conditions, 1.76 mg of anti-IL8 monoclonal antibody was recovered (recovery rate 92%).

[Example 5]
(Purification of subsequent chromatography in multistage purification)
About RRRRG as a histag protein used in Example 2, in the first-stage purification using a cell-free extract of Escherichia coli, as shown in Example 2, purification using a nickel chelate column was performed and recovered. The mixture was diluted 20 times with water and subjected to continuous injection chromatography using an anion exchange column HiTrap QXL (1 mL) (a prepacked column manufactured by GE Healthcare). The eluate from the nickel chelate column is 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 250 mM imidazole. By diluting this 20 times with water, 0.5 mM sodium phosphate, pH 7.4, The solution conditions can be such as 25 mM NaCl and 12.5 mM imidazole that can be adsorbed on the used anion exchange carrier. Using this diluted solution, a continuous injection chromatography experiment was performed under the following conditions.
Protein concentration: 0.12 mg / mL (measured by BCA method)
Total amount of protein solution injected: 714 mL (86 mg)
Separation Column: Anion Exchange Column HiTrap QXL (1 mL) (GE Healthcare Prepack Column)
R1: 10 mM sodium phosphate, pH 7.4, 5 mM NaCl
R2: 10 mM sodium phosphate, pH 7.4, 250 mM NaCl
R3: 10 mM sodium phosphate, pH 7.4, 500 mM NaCl
R4: 10 mM sodium phosphate, pH 7.4, 1M NaCl
T (R1): 10 min
T (R2): 10 min
T (R3): 10 min
T (R4): 10 min
X: 0.5 min
Y: 1 min
Z: 2 min
Number of repetitions (n) = Number of elution sample collection: 17 times Flow rate of pump P1 (sample injection speed): 1 mL / min
Flow rate of pump P2 (purification processing speed): 1 mL / min
Continuous chromatography was performed under the above conditions.
As a result, the total volume of the collected sample was 34 mL, the protein concentration was 2.3 mg / mL, and as a result, 75 mg of protein RRRRG could be recovered with a recovery rate of 87%. The total time for chromatography in this experiment was a little less than 14 hours including the initialization (automatic operation) time of the apparatus.

  Various verification experiments were conducted using an affinity carrier and an ion exchange carrier as the adsorption separation carrier. The apparatus of the present invention is widely applicable to various separation columns, and the purification process can be continued. In this way, a batch process using chromatography can be converted into a continuous process, and a technology that leads to innovation in the manufacturing process is provided.

Claims (2)

Two separation columns (hereinafter referred to as “column C1” and “column C2”, respectively), two liquid feed pumps (hereinafter referred to as “pump P1” and “pump P2”, respectively) ), At least 5 types of solution reservoirs (hereinafter referred to as “reservoir S”, “reservoir R1”, “reservoir R2”, “reservoir R4”, and “reservoir R4”), 6 or 6 respectively. (Referred to as “switch valve SW1”, “switch valve SW2”, “switch valve SW3”, “switch valve SW4”, “selection valve SV1”, and “selection valve SV2”, respectively) ), And a liquid chromatography apparatus composed of piping parts connecting them,
As for the configuration of the pipe, it is selected whether the solution in the reservoir S is sent to the switch valve SW1 side pipe or the switch valve SW2 side pipe via the pump P1 by the selection valve SV2.
Whether the solution of the reservoir R1, the reservoir R2, the reservoir R4, or the reservoir R4 is connected to the pipe connected to the pump P2 is selected by the selection valve SV1, and when it is selected to be connected, the pump P2 The selection valve SV2 selects whether the pipe is sent to the switch valve SW1 side pipe or the switch valve SW2 side pipe.
In the selection of the selection valve SV2, if the piping from the pump P1 side is selected to the switch valve SW1 side, the piping on the pump P2 side is simultaneously selected to the switch valve SW2 side, or the piping from the pump P1 side is When the switch valve SW2 side is selected, either the pump P2 side pipe is selected at the same time on the switch valve SW1 side,
In the switch valve SW1, either the column C1 side pipe or the waste liquid side pipe is selected,
In the switch valve SW2, either the pipe on the column C2 side or the pipe on the waste liquid side is selected, and the pipe on the opposite side of the pipe connecting the switch valve SW1 side and the column C1 is connected to the switch valve SW3, and the switch valve SW2 side And the pipe on the opposite side of the pipe connecting column C2 is connected to switch valve SW4,
In the switch valve SW3, either the recovery container side pipe or the waste liquid side pipe is selected,
In the switch valve SW4, either the recovery container side pipe or the waste liquid side pipe is selected.
The flow path is piped as
Liquid chromatography apparatus characterized in that liquid chromatography can be performed automatically and continuously by being controlled so that all pumps and flow path switching devices are operated according to a pre-programmed order. .   2. The liquid chromatography apparatus according to claim 1, wherein the type of the solution reservoir selected to be sent to the pump P2 by the selection valve SV1 is any one of 5 to 12 types. .