JPH06100598A - Method for washing column - Google Patents

Abstract

PURPOSE:To obtain a method for washing a column in which the amount of a wash liquid can remarkably be reduced from a conventional one. CONSTITUTION:Syringes 5 and 6 are respectively connected to the inlet and outlet sides of an affinity column 1, which is then washed by alternately making a wash liquid flow into the interior thereof by utilizing the syringes 5 and 6. The amount of the wash liquid is preferably 2-10 times based on the capacity of the column 1; however, washing effects similar to those of a conventional method using the wash liquid in an amount of about 90 times based on the capacity of the column can be obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for washing a column used in an antibody fractionation / purification process using an affinity column.

[0002]

2. Description of the Related Art An apparatus as shown in FIG. 2 has been conventionally used for fractionating and purifying antibodies from mouse ascites or cell culture supernatant. The steps of antibody fractionation / purification by this apparatus are as follows. Mouse ascites fluid containing the antibody is dissolved in the adsorbent buffer. A predetermined amount of adsorbing liquid is flown by the pump 12 to adsorb the antibody to the ligand in the column 11. The washing liquid is caused to flow into the column 11 by the pump 12. The amount of washing liquid is about 90 times the volume of the column 11. The eluent is caused to flow into the column 11 by the pump 12, and the antibody adsorbed on the ligand is eluted and collected.

However, since such a conventional method requires a large amount of washing liquid of 90 times the column volume,
Especially on an industrial scale, the increase in the amount of cleaning liquid has been a serious problem.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems and can sufficiently remove impurities such as contaminating proteins which are not adsorbed by a ligand from the column,
Moreover, the present invention has been completed in order to provide a column cleaning method capable of significantly reducing the amount of cleaning liquid as compared with the conventional method.

[0005]

The column washing method of the present invention, which has been made to solve the above-mentioned problems, comprises connecting syringes to the inlet side and the outlet side of an affinity column for fractionating and purifying antibodies. Then, the adsorbent is flown through the column to adsorb the antibody to the ligand of the column, and then the washing liquid is washed while alternately flowing through the column using these syringes.

[0006]

The present invention will be described in more detail below with reference to the embodiment shown in FIG. In FIG. 1, 1 is an affinity column for separating and purifying antibodies from mouse ascites or cell culture supernatant, and 2 is a pump for supplying an adsorbent and an eluent to the column 1. In the embodiment, syringes 5 and 6 are provided on the inlet side and the outlet side of the column 1 via valves 3 and 4, respectively. By moving these syringes 5 and 6 by valves 7 and 8 and a pump 9 for supplying a cleaning liquid, the cleaning liquid can be alternately flowed to the column 1 in the forward and reverse directions.

First, as in the conventional case, the pump 2 to the column 1
An adsorbing liquid containing an antibody is supplied to, and the antibody in the adsorbing liquid is adsorbed by the ligand in the column 1. Next, the column 1 is washed. First, the washing liquid is supplied to the syringes 5 and 6. The amount of the washing liquid used at this time is preferably 2 to 10 times the column volume.

Next, using the valves 7 and 8 and the pump 9, the piston of one syringe 5 is moved forward, and at the same time, the piston of the other syringe 6 is moved backward. As a result, the cleaning liquid flows from the upper side to the lower side of the column 1, and the inside of the column 1 is washed. When the cleaning liquid in one of the syringes 5 has moved to the syringe 6 side in this way, the piston of the syringe 6 is then moved forward and at the same time the piston of the other syringe 5 is moved backward. As a result, the washing liquid flows from the lower side to the upper side of the column 1, and the inside of the column 1 is washed in the opposite direction.

If the above steps are repeated, column 1
The inside will be washed many times alternately, and even if the amount of the washing solution is 2 to 10 times the column volume, it will not be adsorbed to the ligand as in the conventional case where 90 times the column volume of washing solution was used. Impurities such as contaminating proteins can be sufficiently removed from the column. If the amount of the washing liquid is less than twice the column volume, the concentration of contaminant proteins in the washing liquid will be high, and the washing effect will not be improved. .

When the washing of the column 1 is completed as described above, the pump 2 is operated again to send the eluent to the column 1 to elute the backward adsorbed by the ligand and fractionate the eluate.

[0011]

As described above, according to the column washing method of the present invention, the washing liquid is alternately flowed into the column by alternately moving the syringes connected to the inlet side and the outlet side of the affinity column. Since the column is washed, the inside of the column can be sufficiently washed as in the conventional case only by using a washing solution having a column volume of 2 to 10 times, and the usage amount of the washing solution can be greatly reduced.
Therefore, the present invention has great value when the antibody is fractionated and purified on an industrial scale.

[Brief description of drawings]

FIG. 1 is a piping system diagram illustrating a process of the present invention.

FIG. 2 is a piping system diagram illustrating a conventional process.

[Explanation of symbols]

 1 column 5 syringe 6 syringe

Claims (1)

[Claims] 1. A syringe is connected to each of an inlet side and an outlet side of an affinity column for fractionating and purifying an antibody, and an adsorbing liquid is caused to flow through the column to adsorb the antibody to a ligand of the column, and then a washing liquid is added to these. The method for washing a column is characterized in that washing is performed while alternately flowing into the column by using the above syringe.