JPH10139796A - Regulating system for column purification process of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a system for regulating a column purification process of a protein by which a purification condition of an apparatus and a process flow are determined based on a required objective protein, by regulating an operating condition and study on the flow and to perform by using the minimum term and the minimum resource when applying different proteins to a column purification apparatus. SOLUTION: A sequence control and a fixed value control are performed by using a monitored data obtained by monitoring a data (e.g. a flow rate, a pressure, an absorbance and a turbidity) from a sensor inserted into a column process by using an online expert system and a factory automation system by a computer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein separation process using a column.

[0002]

2. Description of the Related Art Column chromatography used for protein separation includes ion exchanger chromatography, hydrophobic chromatography, and hydrogen bond chromatography. Here, an ion exchange chromatography will be described as an example. The ion-exchange column chromatography separates the target protein by utilizing the difference between the ionic strength of the target protein and the ionic strength of the contaminating protein. Conventionally, protein separation using a column has been performed by the following procedure.

(1) A target protein is adsorbed on a resin in a column. (2) Pass a buffer solution with a constant electrical conductivity with a force smaller than the ionic strength acting between the target protein and the resin of the column, and remove the protein and the adsorbing power that did not adsorb to the resin in the column. Wash weak proteins.

(3) The target protein is eluted with a buffer having a higher ionic strength and is separated in a certain amount.

[0005] (4) The eluate collected in a certain amount is analyzed off-line in order to confirm the presence or absence of the target protein, and the eluate containing a large amount of the target protein is taken out to complete the removal of the target protein.

[0006]

However, the ionic strength of a protein is not constant, and the ionic strength itself varies depending on the type of protein. Therefore, it is necessary to set conditions for removing the target protein and determine an operation flow. Here, since the electrical characteristics of proteins differ depending on the type, the operating conditions and processing flow of column purification differ. Therefore, when different proteins are separated, the operating conditions and the operation must be adjusted and changed in the flow, applied to the process control, and operated.

Therefore, an object of the present invention is to determine the conditions and process flow for separating different proteins by column chromatography, adjust the required target proteins and operating conditions, and examine the flow with minimum time and minimum resources. Feasible,
It is an object of the present invention to provide a control system for a protein column purification process.

[0008]

SUMMARY OF THE INVENTION A control system for a protein column purification process according to the present invention, when performing protein separation using a column, determines operating conditions when determining whether a target protein can be separated in the column purification process. It is characterized in that the operation flow can be modified and changed online.

That is, a control system for a protein column purification process according to the present invention monitors data from a sensor inserted into a column process by using an on-line expert system and a factory automation system using a computer, and performs sequence processing based on the monitored data. Control and constant value control are performed. Here, what is sensed are flow rate, pressure, UV (absorbance), and OD (turbidity).

[0010] Regarding sequence control, a process flow to be sequence-controlled is divided into unit functions. The unit functions include the function of selecting the inlet to flow into the column, the function of selecting the flow to the air trap, the function of controlling the pump, the function of selecting the mode of flow through the column, the function of selecting the flow of the UV meter, and the function of selecting the flow of the pH meter. , Outlet tank selection function, etc. Each unit function has the following operation modes. First, the inlet selection function to flow into the column includes a buffer sharing mode, an enzyme solution supply mode, a NaOH supply mode, a pure water supply mode, and a fully closed mode. The pump control function includes a manual output operation mode, a PID control operation mode, and a stop mode. The column flow-through mode selection function includes a bypass mode and an UP-FLO
There are a W mode, a DOWN-FLOW mode, and a fully closed mode. The UV meter liquid passing selection function includes a liquid passing mode to the UV sensor and a bypass mode. Similarly, the pH meter passage selection function includes a pH meter passage mode and a bypass mode. The outlet tank selection function includes a disposal mode, a recovery mode, and a removal mode.

These unit functions are put together into one icon, an operation mode setting property is created for this icon, and the operation mode can be implemented only by setting the operation mode in this property.

Further, the creation of a sequence program for performing the process control can be realized by combining the collected icons and setting the properties of the icons. By using the functions of the expert system that can be developed online, icons corresponding to each unit function are created, so it is possible to change the property settings without paying particular attention to online and offline, Furthermore, it is possible to freely change the combination of icons without particularly being aware of online and offline. In other words, the process control flow can be modified and changed in any state,
Of course, the corrections and changes can be directly reflected in the process flow control.

That is, it is possible to solve a problem or a defect at the time of studying the operating conditions on the spot, and to quickly reflect the knowledge obtained at the time of studying the operating conditions in the process. be able to. Therefore,
It is possible to develop conditions and equipment flows that enable faster and more stable production, and to construct an online protein separation column system.

On the other hand, in the online extraction of the target protein, first, the extraction start point and the end point of the eluting protein are determined based on the data monitored by the computer. By sequencing the process at the determined unloading start and end points,
Carry out the target protein online.

UV, which is a part of the monitoring data,
Due to its characteristics, there is a correlation with the distribution of the target protein, start,
It can be used as an alternative indicator to determine the end point. By setting the extraction start and end points with this substitute index and controlling the process, the target protein can be automatically sorted. It is assumed that the setting of this extraction point is determined from fixed values, reference values from past results, dynamic prediction values from current data, etc., and these three items are all supported by this column control process. can do.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a diagram showing an example of column equipment in which a control system for a column purification process according to the present invention is used.

Referring to FIG. 1, valves and instrumentation for performing column chromatography have a unit structure. This unit has five types of inlet valves 11, 12, 1 for supplying a protein separation treatment solution, two types of buffers, water, and caustic soda (NaOH) to the column, respectively.
3, 14, 15 and flow through the column in upflow,
Valves 41 and 4 around the column to support three types of flow modes, that is, flow through the down flow and flow through the bypass.
2, 43, 44, 45, and 46, and outlet valves 71, 72, and 73 having three types of discharge, pool, and hold, an air trap 31, a pH meter 51, and a UV meter 61. I have.

The column purification process flow includes an inlet function 10 for passing the liquid through the column and a pump function 20.
, An air trap function 30 immediately before the column, a column flow function 40, a pH meter flow function 50, a UV meter flow function 60, and an outlet function 70, and these functions are combined. This makes it possible to create a sequence program for controlling columns.

Each unit function is encapsulated in an icon as shown in FIG. 2, and a process control sequence can be created by combining the icons.

There are two operations to modify and adjust the process control sequence. One is to replace or add the connection relation of the icons, and the other is to change the operation mode of the unit function icon.

The general operation of these two types of correction and adjustment will be described below. First, as for the method of changing and adding the connection relation of the icons, the connection of the target icon is deleted, the icon is dragged, and the target connection is changed. On the other hand, if you want to add a new icon,
Insert the copied icon and change to the desired connection.
This series of operations can be performed regardless of whether the column process is operating or stopped.

Next, in changing the operation mode of the unit function icon, the operation mode is changed by displaying a menu for setting the mode of the unit function icon and changing the setting. This operation can be set and changed regardless of the state of the column process, as in the case of changing and adding the connection relation of icons.

[0023]

【Example】

(Example 1) According to the present invention, a target protein is started in a column processing unit UV (UV for starting protein removal)
Value) and end UV (UV value at which protein removal ends) to determine whether separation is possible.

By combining the unit functions and setting the mode of the unit functions, a sequence for separating the target protein was prepared. When the protein was eluted from the column, the rise in absorbance was confirmed before the flow of the void amount filled in the column with the enzyme solution, so that it was determined that the protein separation treatment by the column was abnormal. As a countermeasure for the abnormality judgment, the sequence processing after the extraction condition is satisfied,
The exit mode of the unit function was changed from "pool" to "pending". As a result, the separation test was completed by operating the sequence program including the countermeasure without causing a problem without discarding the processing liquid likely to contain the target protein.

(Example 2) As a specific example of the creation of a sequence program, a sequence program for manually outputting 50% to a waste line by upflow of a buffer into a column was created as follows.

Referring to FIG. 1, inlet function 10, air trap function 30, column function 40, pH meter flow function 5
0, the UV meter flow function 60, and the exit function 70 were connected in this order. The icon connection is
The procedure was as follows.

The point B displayed in FIG. 2 was clicked with the mouse, the mouse pointer was moved to the point A of the next icon, and clicked again. The connection between the icons is now possible.

Next, regarding the setting of each function, the target item was changed from "0" to "1" in the setting menu of each icon as shown in FIG. In the default state, all items were "0". That is, in the above description, the item of the enzyme solution in the entrance function icon menu is changed from “0” to “1”, the item of the upflow in the column function icon menu is changed from “0” to “1”, and the exit function is changed. The item of waste liquid was changed from "0" to "1" in the menu, and "50" was described in the item of manual output by the pump function. As a result, a target sequence program could be created.

(Example 3) In Example 2, the liquid was allowed to flow in the upflow. However, since foreign matter was clogged in the lower part of the column, it was necessary to flow in the downflow.

Therefore, the downflow item is changed from “0” to “1” from the column function icon menu, and the upflow item is changed from “1” to “0” from the column function icon menu. The flow through the column could be changed from upflow to downflow without any problems.

(Example 4) In Example 2, a sequence program for adding a function of changing the enzyme solution to water supply after passing the enzyme solution for 10 minutes was prepared.

First, a 10-minute timer was connected at the end of the sequence created in Example 2, and thereafter, an entrance function icon was connected. The water item in the entrance function icon menu was changed from “0” to “1”. With the addition of this function, it was possible to change from the enzyme solution to a water line after 10 minutes. The additional operation of this function could be performed irrespective of whether the sequence created in the second embodiment is operating or stopped.

This additional function was operated without any problem, and the supply of the enzyme solution could be changed to the supply of water.

The above countermeasure method is changed depending on a specific problem or trouble that has occurred, and the present invention is not limited to the above-described specific processing change of the unit function. Further, the specific mode of the unit function depends on the operation method of the unit function, and the present invention is not limited to the above embodiment.

[0035]

As described above, according to the present invention, the process flow and operating conditions for column chromatography of a target protein can be set in a short period of time, and the target protein required for that purpose can be set in a short period of time. It became possible to minimize the sample. Table 1 shows an example of the effect in comparison with the conventional method.

[0036]

[Table 1]

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of column equipment in which a control system for a column purification process according to the present invention is used.

FIG. 2 is a diagram showing an example of an icon for encapsulating a unit function according to the present invention.

FIG. 3 is a diagram showing an example of a menu screen for setting a mode of a unit function according to the present invention.

[Description of Signs] 10 Inlet function 20 Pump function 30 Air trap function 40 Column flow function 50 pH meter flow function 60 UV meter flow function 70 Outlet function

Claims (1)

[Claims] 1. Using an online expert system and a computerized factory automation system, monitor data from a sensor inserted into a column process, and perform sequence control and constant value control from the monitored data. A control system for the protein column purification process.