JPS6396552A - Industrial separation for vital high polymer and apparatus tobe used for said method - Google Patents

Abstract

PURPOSE:To improve the accuracy of gradient and the sepn. performance of a column by mixing precursor-liquids continuously while controlling the flow rates thereof to prepare a moving phase liquid the concn. and/or pH of which changes continuously with time. CONSTITUTION:A sample mixture contg. a vital high polymer is loaded into a sample vessel 5. Buffer solns. having different concns. and/or compsns. are respectively packed into moving phase precursor-liquid vessels 3a, 3b. The sample in the sample vessel 5 is first transferred into the column 1a by operating valves 4, 11, then the liquids in the vessels 3a, 3b are transferred into a mixer 14 while the flow rates thereof are controlled by pump 13a, 13b. The moving phase liquid obtd. in the mixer 14 is fed as the moving phase liquid the concn. and/or pH of which changes continuously with time to the column 1a. The sample component and moving phase liquid eluted from the column 1a are fed to an electrical conductivity meter 6, a pH meter 9 and a UV meter 10 by which the concn., pH and sample components of the moving phase liquid are measured. The gradient of the concn./pH and the presence or absence of the desired component are judged from the results thereof.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to industrial separation using ion-exchange column chromatography, and more specifically, to industrial separation of a large amount of biopolymer-containing mixture samples using column chromatography. The present invention relates to a method for carrying out the method and an apparatus used in the method.

[Conventional technology]

In recent years, advanced separation and purification of biopolymers by ion-exchange column chromatography has been frequently used in many fields such as chemistry, biology, agricultural chemistry, and medicine.

Ion-exchange column chromatography is a type of chromatography that uses a packing material with ion-exchange ability as a stationary phase.
When a sample mixture is applied to the upper end of the lumen and developed with an appropriate electrolyte solution, the adsorption bands of each component separate and fall due to the difference in adsorption of each component ion to the packing material.

The mobile phase, which is a developing agent, is an eluent, and when the developing operation, that is, elution, is continued, components with weak adsorption properties are eluted, and the components can be separated into predetermined fractions.

When separating and purifying biopolymers such as proteins using ion-exchange column chromatography, the difference in isoelectric point of proteins is used to adjust the salt concentration and/or pH of the eluent over time into the column into which the sample is injected. is supplied while changing the pH continuously to form a gradient of salt concentration and/or pH in the length direction of the column to elute each protein (gradient, concentration gradient elution). Therefore, when separating and purifying biopolymers such as proteins using ion-exchange column chromatography, it is necessary to perform a gradient.

In addition, conventional equipment for separating biopolymer samples on an industrial scale by column chromatography consists of a column filled with a packing material having ion-exchange ability and at least one column provided upstream of the column via a flow path. Two types of mobile phase liquid tanks, a sample injection valve provided in the flow path between the column and the mobile phase liquid tank, a sample tank that communicates with the sample injection valve during sample injection, and a sample injection valve provided in the flow path between the column and the mobile phase liquid tank, A fraction collector provided on the downstream side, and a concentration and/or P
This is a separation device consisting of a first detector for H and a second detector for sample components, and as used in bioprocesses, the concentration and pH are
Most of the methods are of the stepwise type, in which three to five adjusted mobile phase liquid tanks are prepared, and when a predetermined time has elapsed, the valves of these tanks are sequentially switched to perform a gradient.

[Problem that the invention seeks to solve]

A method for separating and purifying biopolymers such as proteins using ion-exchange column chromatography with high accuracy (1%
There were no products on an industrial scale that had linearity in concentration/PH gradients (within 10%). In addition, equipment for separating biopolymer samples using high-precision gradient column chromatography is
Until now, all of these were for analytical purposes, and there were no large, large-capacity devices that could be used on an industrial scale. However, with the recent remarkable progress in biotechnology, there is a desire for the development of high performance and reliable biopolymer separation and purification methods and devices.

This invention was made based on the above background,
The purpose is to provide a method and apparatus for separating biopolymers such as proteins on an industrial scale using a high-precision gradient.

[Means for solving problems]

The present inventors have conducted various prototypes and research on methods and devices for separating biopolymer samples using high-precision gradient column chromatography. As a result, the present inventors have developed various methods and devices for separating biopolymer samples using high-precision gradient column chromatography. It has been found that continuous mixing while controlling the flow rate, preferably automatically controlled by a microcomputer, is effective in achieving this objective, and has completed the present invention.

That is, in the industrial separation method of biopolymers using column chromatography of the present invention, a large amount of a biopolymer-containing mixture sample is charged to the upstream side of a column packed with a packing material having ion exchange ability, and at least two kinds of biopolymers are separated. A mobile phase liquid whose concentration and/or pH continuously changes over time is prepared by continuously mixing the mobile phase precursor liquid while controlling each flow rate in a variable flow rate range of 50:1 to 1ooo:1. The components of the mixture are developed in the column by feeding the mobile phase liquid, and the fractions flowing out from the downstream side of the column are separated to separate each component of the mixture. .

Further, according to the device used in the separation method of the present invention, it is an industrial separation device for separating a large amount of biopolymer-containing mixture samples by column chromatography. , at least two kinds of mobile phase precursor liquid tanks provided upstream of the column via a flow path; a sample injection valve provided in the flow path between the column and the precursor liquid tank; and a sample injection valve provided in the flow path between the column and the precursor liquid tank. A sample tank that communicates with the sample injection valve during injection, a fraction collector tank provided on the downstream side of the column, and a concentration and/or a first detector for pH and a second detector for sample components;
A column exchange valve provided in a flow path between the column and the precursor liquid tank, a column regeneration liquid tank that communicates with the column exchange valve during column regeneration, and a column replacement valve provided on the downstream side of each of the precursor liquid tank. A liquid feeding device capable of controlling the flow rate in a flow rate range of 50:1 to 1000; It is characterized by consisting of a mixer for feeding liquid.

Hereinafter, the separation method and apparatus of the present invention will be explained in more detail with reference to a flow sheet showing one embodiment of the separation apparatus according to the present invention in FIG.

The device of this embodiment is an industrial separation device that separates a large amount of biopolymer-containing mixture samples by column chromatography, and includes columns 1a and 1b filled with a packing material having ion exchange ability, and a flow path 2. Two types of mobile phase precursor liquid tanks 3a are provided upstream of the columns 1a and 1b.
and 3b, the columns 1a and 1b, and the precursor tank 3.
a sample injection valve 4 provided in the flow path 2 between columns 1a and 3b, a sample tank 5 communicating with the sample injection valve 4 during sample injection, and a fraction provided on the downstream side of the columns 1a and 1b. a collector 6, first concentration and pH detectors 8 and 9 and a second sample component detector 10 provided in the flow path 7 between the columns 1a and 1b and the fraction collector 6; and the column 1a. and 1b
and a column exchange valve 11 provided in the flow path 2 between the precursor liquid tanks 3a and 3b, and a column regeneration liquid tank 12a that communicates with the column to be regenerated via the column exchange valve 11 during column regeneration. -, 12bll 2 c and 1
2d, and liquid feeding devices 13a and 13b that are provided downstream of each of the precursor liquid tanks 3a and 3b and that can control the flow rate of each precursor liquid in a variable flow rate range of 50:1 to 1000:1.
and a mixer 1 that continuously mixes each precursor liquid sent from each of the liquid sending devices 13a and 13b and sends the liquid to the column.
4 is the main one. Furthermore, in this embodiment, a sample liquid feeding device 16 is provided in the flow path 15 between the sample injection valve 4 and the sample tank 5, and a sample liquid feeding device 16 is provided in the flow path 15 between the sample injection valve 4 and the sample tank 5. A regenerating liquid feeding device is provided in the flow path 17, and a switching pallet 20 is provided on the downstream side of the columns 1a and 1b to communicate the column in the gradient with the fraction collector 6 and communicate the column in the process of column regeneration with the drain 19. The concentration and pH of the first
Detectors 21 and 22 and a second detector 23 of sample components
is provided.

The targets of separation in this invention are biopolymers that can be separated by a gradient of ion exchange chromatography, such as interferon, interleukin, tissue plasminogen, activator (TPA), and coronin stabbing factor. (CSE)
, proteins such as immune human globulin, and various other enzymes.

The filler used in this method and apparatus consists of a filler with ion exchange ability, such as strongly acidic cation exchange type (SP), weakly acidic cation exchange type (CM), strongly acidic cation exchange type (CM), etc. Basic anion exchange type (QAE)
, weakly basic anion exchange type (DEAE). The particle size of the filler is, for example, more than 5 microns, preferably several tens to hundreds of microns, considering that it is carried out on an industrial scale.

The column used in this invention is preferably a large-capacity medium-low pressure chromatography column that can process a large amount of samples, such as a
It is a column of 17χ to 1002χ. This column is the first
As shown in the figure, there may be a plurality of columns, in which case they can be used in series and in parallel to perform sample gradient on one side and column regeneration on the other.

In the separation method of the present invention, the mobile phase liquid is obtained by mixing at least two types of mobile phase precursor liquids, and is usually an aqueous solution, but a residual solvent may be added.

The type of mobile phase liquid is selected appropriately depending on the type of packing material with ion exchange ability, the properties of the sample, etc. For example, an anionic buffer is used for a cation exchange type, and a cationic buffer is used for an anion exchange type. A neutral buffer is used. Examples of buffers that can be used in ion exchange chromatography of biopolymers include Tris-HCl, Tris-phosphate, and phosphate buffers. In ion exchange chromatography, there are subtle changes in ionic strength (concentration), pH, retention value, elution time, etc., and in this invention, at least two types of mobile phase precursor solutions are mixed at a variable flow rate range of 50:1 to 1000:1. A mobile phase liquid whose concentration and/or pH continuously changes over time is prepared by continuously mixing the liquids while controlling the flow rates of each phase.

This adjustment is performed in the embodiment shown in FIG.
b and each precursor solution is 50:1 to 50:1.
Liquid feeding devices 13a and 13b that can control the flow rate in a variable flow rate range of 1000:1, and a mixer 14 that continuously mixes each precursor liquid fed from each of the liquid feeding devices 13a and 13b and sends the liquid to the column. It is carried out with. A preferred embodiment of this liquid feeding device is a diaphragm type pump having a wide range of flow rate (c1iv'lx/min to 117.7 min), non-pulsating flow, and high-pressure quantitative performance.

More preferably, the liquid contact part is made of non-metal and has sanitary properties. Each liquid feeding device has a ratio of 50:1 to 100
Since it has a wide flow rate variable range of 0:1, the linearity of the concentration gradient, that is, the precision of the gradient, is improved to within ±1%, and it is extremely effective for separating proteins with close isoelectric points. Next, the mixer used in the present invention continuously mixes the respective precursor liquids sent from the liquid sending device (pump) and sends the mixture to the column. An example of such a mixer for continuous mixing is a static mixer. Normal mixers have a fixed capacity, so mixers with different capacities must be prepared according to the discharge flow rate.If the internal volume of the mixer is too large for the flow rate, there will be a delay, and vice versa, there will be stagnation inside the mixer. Although the time is short and the mixing is insufficient, a continuous mixer such as a static mixer makes the mixing smooth and there is no delay due to stagnation.

The first detector used in the present invention detects the salt and/or PH level of the mobile phase liquid and sends out the degree as a signal. Such as electrical conductivity meter,
There is also a PFi meter. On the other hand, the second detector detects the degree of presence of sample components, particularly biopolymers, and sends out the degree as a signal.

Such devices include an ultraviolet absorption photometer (UV), a differential refractometer (R1), and a fluorometer (FP).

When separating biopolymers by ion-exchange chromatography repeatedly, operations for reusing the column are performed after gradient execution. This operation includes column regeneration,
There are steps for buffering, washing, and sterilization. Such reagents include organic solvents such as alcohol, sodium hydroxide,
Examples include salts such as hydrochloric acid, Tris-hydrochloric acid, and sodium chloride.

Next, the operation and operation of the apparatus shown in FIG. 1 will be explained.

In preparation for the separation operation, the column 1a is washed, regenerated and buffered, a biopolymer-containing mixture sample is loaded into the sample tank 5, and the mobile phase precursor tanks 3a and 3b are loaded with a biopolymer-containing mixture sample.
are each filled with buffer solutions of different concentrations and/or compositions. First, the valves 4 and 11 are set so that the sample tank 5 and the column 1a are in communication with each other, and the pump 16 is operated to charge the sample from the sample tank 5 to the upper end of the column 1a via the channels 2 and 15. do. Next, the valve 4 is changed so that the mobile phase precursor liquid tanks 3a and 3b communicate with the column 1a. The pumps 13a and 13b are operated and the respective moving precursor liquids are sufficiently mixed in the mixer without stagnation while controlling the flow rate. The mobile phase liquid obtained from the mixer is sent to column 1a via channel 2 and valves 4 and 11.

Since the flow rate is controlled by the pumps 13a and 13b, the concentration and/or pH of the mobile phase liquid changes continuously over time. The sample will therefore be subjected to a gradient in the column. The sample components and mobile phase liquid eluted from the column 1a are passed through the valve 20 and the flow path 7 to a conductivity meter (CD) 8, a PH meter 9, and a UV meter 10, where the concentration of the mobile phase liquid and the PH1 sample component are measured. be measured. From this measurement result, the gradient of concentration/PH and the presence or absence of the target component can be determined. When the target component is eluted, a fraction containing the target component is collected into a fraction collector 6. Unnecessary fractions are discharged to drain 19.

In this device, a column 1b is provided in parallel with column 1a. Therefore, the column 1b can be subjected to a process such as column regeneration for reuse. For example, methanol for sterilization is placed in the column regeneration liquid tank 12a, purified water is placed in the column regeneration liquid tank 12b, sodium hydroxide solution for removing impurities is placed in the column regeneration liquid tank 12c, and sodium chloride liquid for buffering is placed in the column regeneration liquid tank 12d. The liquid is stored and sent to the column 1b by the pump 18 according to each process of column regeneration, buffering, washing, and sterilization. Each step of column regeneration, buffering, and washing is monitored using an electrical conductivity meter (CD) 21, a PH meter 22, and a UV meter 23 upstream of the drain 19. If the gradient in column 1a needs to be terminated and regenerated, and at the same time a subsequent gradient is to be carried out in column 1b, this can be done by changing the state of valves 11 and 20.

Next, an embodiment of a separation device using a microcomputer will be shown.

A flow sheet for this embodiment is shown in FIG. In this embodiment, a microcomputer control system is further added to the separation apparatus shown in FIG. That is, the flow rate control means 2 controls the output of the pumps 13a and 13b by the flow rate control signal.
00, the gradient control means 210 sends the flow rate control signal to the flow rate control means 200 according to the detection signal from the first detector and/or the set value, and the gradient control means 210 sends the flow rate control signal to the flow rate control means 200 according to the detection signal from the first detector and/or the set value, A peak determination means 230 that evaluates component peaks and sends out a fractionation signal, and a fractionation valve 2 that switches the flow to the fraction collector 6 according to the fractionation signal.
4, and valve 2 of the column regeneration liquid tank according to the regeneration control signal.
Column reuse control means 2 for controlling 5 and pump 18
40, a column reuse control means 250 that evaluates the detection signals from the detectors 21 to 23, determines the end of each column reuse process, and sends out the regeneration control signal; process switching determination means 270 that determines whether to switch to the sample injection process, gradient process, and column reuse process in response to the regeneration control signal and sends a switching signal; and valves 4.11 and 20 in response to the switching signal. and switching control means 260 for controlling the output of the switching pump 16.

An example of a flowchart for controlling the gradient of the separation apparatus of this embodiment is shown in FIG.

Following this control procedure ensures that the separation device is ready for gradient start-up. Next, the gradient control settings are manually set. These set values include the flow rates of the pumps 13a and 13b, their operating times, and the like. The device starts after entering the setpoint. The switching signal activates the pump 16 to load the column 1a. Furthermore, the valve 4 is switched by the switching signal, and the pump 13 is switched by the flow rate control means 200.
a and 13b are started and the mobile phase liquid is loaded onto the column. Eluate exits the column and is detected by a second detector. The component peaks in the eluate are evaluated from this detection signal,
A preparative signal is sent from the peak determination means to the preparative valve,
A desired fraction is collected into the fraction collector 19 by switching the separation valve. The peak determination can also be performed, for example, in level mode, peak mode, or a combination thereof. As shown in the flow sheet, the gradient can be changed midway through the gradient, or the operating conditions can be changed so that the concentration is held once midway.

Incidentally, processes such as column regeneration, buffering, washing, etc. are controlled in the same way, and the process switching to these processes is also controlled in the same way.

〔Effect of the invention〕

The following effects can be obtained by this invention.

Since the flow rate variable range is wide, the precision of the gradient increases accordingly, and the separation performance of the column also improves.

As an aspect of this invention, a salt/PH1 sample component detector is also provided in the flow path of the eluate during column regeneration, and is monitored and controlled by a microcomputer, so that it is possible to monitor not only the separation state during gradient execution but also the regeneration of the column. etc. can be performed automatically, and chromatographic separation and purification can be performed automatically and repeatedly over a long period of time.

Biopolymers are separated in a sterile cold room (approximately 4
℃), but since it is monitored and controlled by a microcomputer, it can be operated remotely, reducing the number of trips into and out of the sterile cold room, and increasing work efficiency.

As another aspect of the present invention, by further providing sensors for abnormal pressure, liquid shortage, etc., safe separation can be performed.

In a preferred embodiment of the present invention, a plurality of columns can be used to perform operations such as gradient operation on one side and column regeneration on the other side, and furthermore, automatic control can be performed, so that the time for separation and purification can be significantly shortened.

Further, as a preferred embodiment of the present invention, the initial separation conditions can be controlled by computer so that they can be changed during the process, so failures in separation and purification can be reduced and loss of valuable samples can be prevented as much as possible.

[Brief explanation of the drawing]

FIG. 1 shows a flow sheet of an example of the separation device of the present invention, and
The figure shows a flow sheet of another example of a separation device according to the present invention.
The figure shows a flowchart. DESCRIPTION OF SYMBOLS 1... Column, 2... Channel, 3... Mobile phase precursor liquid tank, 4... Sample injection valve, 5... Sample tank, 6
... Fraction collector, 7... Channel, 8...
・CD meter, 9...PH meter, 10...UV meter, 11.
...Column replacement valve, 12...Column regeneration liquid tank,
13... Liquid feeding device, 14... Mixer, 15... Channel, 16... Pump, 17... Channel, 18... Pump, 19... Drain, 20...・Switching valve,
21...CD meter, 22...PH meter, 23...UV
Total.

Claims (1)

[Claims] 1. A large amount of biopolymer-containing mixture sample is loaded into the upstream side of a column packed with a packing material having ion exchange ability, and at least two types of mobile phase precursor solutions are mixed at a ratio of 50:1. ~1000:1
A mobile phase liquid whose concentration and/or pH continuously changes over time is prepared by continuous mixing while controlling each flow rate in a variable flow rate range, and then the obtained mobile phase liquid is sent to form a mixture. 1. An industrial method for separating biopolymers by column chromatography, which comprises developing the components in the column and separating each component of the mixture by fractionating a fraction eluted from the downstream side of the column. 2. An industrial separation device that separates a large amount of biopolymer-containing mixture samples by column chromatography. at least two types of mobile phase precursor liquid tanks; a sample injection valve provided in a flow path between the column and the precursor liquid tank; and a sample tank that communicates with the sample injection valve during sample injection. , a fraction collector provided on the downstream side of the column, a first concentration and/or pH detector and a second sample component detector provided in a flow path between the column and the fraction collector; A column exchange valve provided in a flow path between the column and the precursor liquid tank, a column regeneration liquid tank that communicates with the column exchange valve during column regeneration, and a downstream side of each of the precursor liquid tank. and each precursor solution at a ratio of 50:1 to 1000
An industrial separation device comprising: a liquid feeding device that can control the flow rate in a flow rate variable range of 1; and a mixer that continuously mixes each precursor liquid fed from each of the liquid feeding devices and sends the liquid to the column. 3. Flow rate control means for controlling the output of the liquid feeding device using a flow rate control signal, and gradient control for sending the flow rate control signal to the flow rate control means according to the detection signal from the first detector and/or the set value. means, a peak determination means for evaluating the component peak in the eluate from the detection signal of the second detector and sending out a fractionation signal, and a fractionation valve for switching the flow to the fraction collector in accordance with the fractionation signal. , a column reuse control means that controls the valve and pump of the column regeneration liquid tank in accordance with the regeneration control signal; and a column reuse control means that evaluates the detection signal from the detector to determine the end of each column reuse process and outputs the regeneration control signal. Column reuse control means for sending out, and process switching that determines whether to switch to the sample injection step, gradient step, and column regeneration step, respectively, according to the flow rate control signal, the fractionation signal, and the regeneration control signal, and sends a switching signal. 3. The industrial separation apparatus according to claim 2, further comprising a determining means and a switching control means for switching the valve and controlling the output of the pump in accordance with the switching signal.