JPH10221323A - Continuously using method and device of adsorption column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a continuously using method and device of an adsorption column capable of suppressing a rise in pressure loss due to clogging and maintaining high efficiency in treatment even in the case of operating at the high velocity of flow. SOLUTION: After the completion of a series of operation of the balancing of an adsorption column, the loading of a liquid to be treated, washing, and the recovery of elution, the adsorption column is washed and regenerated sequentially with (1) a mixture between acetic acid and ethanol, (2) a mixture between sodium hydroxide and ethanol, and (3) an acid solution such as hydrochloric acid. An adsorbing layer 8 of adsorption supports with particle diameters of less than 100μm and an inert unadsorbed-support layer 9 of gel filtration supports, etc., with particle diameters coarser than those adsorption supports are arranged in the same or different column, and a liquid to be treated is brought to pass from the side of the unadsorbed-support layer 9.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for continuously using an adsorption column of a preparative liquid chromatography apparatus used in the production of biopharmaceuticals and the like.

[0002]

2. Description of the Related Art In the production process of biopharmaceuticals, a purification method by liquid chromatography is frequently used.
The raw material solution of the useful protein to be a biopharmaceutical is a cell culture solution obtained by culturing animal cells and the like in a medium, and the culture supernatant obtained by removing cells from the material solution is filled with an ion exchanger, an affinity carrier, and the like. Concentration and purification of useful proteins are performed by passing the solution through an adsorption column and treating. At that time, since the useful protein concentration in the culture supernatant is low, it is necessary to treat a large amount of the culture supernatant at one time. It was started. The column packed with the adsorption carrier is mounted on a protein fractionation apparatus. From the viewpoint of preventing damage to the protein and operability at scale-up, the operating pressure of the protein fractionation apparatus is 5 kgf / cm ² or less. It is carried out with a low pressure loss, so that it is necessary to carry out the adsorption and recovery operations under the condition that the pressure loss in the system is kept low.

However, in reality, the raw material liquid contains medium components and impurities (lipids, cell-derived components, etc.),
When the adsorption column is used, clogging occurs and a sudden increase in pressure loss occurs, which causes a problem that the pressure exceeds the device pressure. In particular, when operated at high flow rates, clogging tends to occur,
The load of the raw material liquid is reduced, and the processing efficiency is reduced. Eventually, the adsorption column becomes unusable beyond the limit pressure of the protein fractionator.

[0004]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems and continuously uses an adsorption column capable of suppressing an increase in pressure loss and maintaining high processing efficiency even when operated at a high flow rate. It has been made to provide methods and apparatus.

[0005]

Means for Solving the Problems According to the first aspect of the present invention, which has been made to solve the above-mentioned problems, a liquid to be treated such as a cell culture supernatant is passed through an adsorption column, and a purification process of a useful substance is repeated. In the continuous operation of the adsorption column, after a series of operations such as equilibration of the adsorption column, loading of the liquid to be treated, washing, and elution recovery, (1) a mixture of acetic acid and ethanol, (2) sodium hydroxide and ethanol And (3) regenerating and washing the adsorption column in the order of an acidic solution such as hydrochloric acid. In this case, it is preferable to perform the equilibration of the adsorption column, the flow direction to the load of the liquid to be treated, and the subsequent washing, elution recovery, and regeneration washing in the opposite directions.

The invention of claim 3 is directed to a method of continuously using an adsorption column for purifying useful substances by passing a sample such as a cell culture supernatant through the adsorption column.
At least one layer of an adsorption carrier having a particle size of 0 μm or less and an inert non-adsorption carrier layer such as a gel filtration carrier having a particle size coarser than the adsorption carrier is installed. The liquid to be treated is passed in the direction from the carrier layer side to the adsorption carrier layer side. The invention of claim 4 is to pass a sample such as a cell culture supernatant through an adsorption column, In continuous use of an adsorption column for purifying substances, an adsorption column equipped with an adsorption layer of an adsorption carrier having a particle size of 100 μm or less, and an inert filter such as a gel filtration carrier with a particle size coarser than this adsorption carrier And an adsorption column provided with such an unadsorbed carrier layer, and the liquid to be treated is passed from the side of the inert carrier layer to the side of the adsorption carrier layer of the adsorption column. According to the third and fourth aspects of the present invention, the thickness of the unadsorbed carrier layer is 3 cm.
As described above, the value of the thickness of the adsorbed carrier layer / the thickness of the non-adsorbed carrier layer is preferably 3 or less. When the number of the non-adsorbed carrier layers is two or more, the thickness of the non-adsorbed carrier layer refers to a total thickness obtained by adding the thicknesses of the non-adsorbed carrier layers. Further, the invention according to claim 6 is characterized in that a switching valve is provided at the upper and lower portions of the adsorption column, and a liquid sending line for passing the regenerating cleaning solution in a direction opposite to the direction of the liquid to be treated is provided. Characterized in that the adsorption column is continuously used by the method described in (1).

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of each invention will be described below. (Invention of Claim 1) FIG. 1 is a view showing the structure of an apparatus for carrying out the invention of claim 1, wherein 1 is an adsorption column, and 2 is a container containing a sample such as a cell culture supernatant. The inside of the adsorption column 1 is filled with an affinity packing material 3. Reference numeral 4 denotes a pump for passing liquid, which can pass an equilibration liquid, an eluent, a regenerating washing liquid and the like to the adsorption column 1 via a solvent switching valve 5 and a deaerator 6. Reference numeral 7 denotes a UV meter for measuring the concentration of the target substance in the effluent from the adsorption column 1 using ultraviolet rays.

According to the first aspect of the invention, after a series of operations of equilibration of the adsorption column 1, loading of the liquid to be treated (sample), washing, and elution and recovery, which are the same as in the prior art, (1) a mixed solution of acetic acid and ethanol (2) A mixed solution of sodium hydroxide and ethanol and (3) an acidic solution such as hydrochloric acid are passed through the adsorption column 1 in this order, and the adsorption column 1 is regenerated and washed. In the present invention, the reason why these three liquids are passed in this order is as follows.

The inventor of the present invention has firstly obtained a general regenerating solution, 6
An attempt was made to regenerate the adsorption column 1 in which the pressure loss was increased using a protein denaturant such as M guanidine hydrochloride, but the pressure loss of the adsorption column 1 which was once increased was not recovered. However, when the mixture was made acidic with a mixture of acetic acid and ethanol, and then changed to the alkali side with a mixture of NaOH and ethanol, the lipid removal effect was large. It was found to recover. That is, when the pressure loss of the adsorption column 1 is increased from the initial pressure 0.35kgf / cm ² after sample loading up to 0.5 kgf / cm ^2, (1) a mixture of acetic acid and ethanol, (2) a mixture of sodium hydroxide and ethanol When the regeneration washing was performed by the combination of the above, the pressure loss recovered to 0.4 kgf / cm ² . However, it is necessary to restore the initial pressure to use the adsorption column repeatedly. (1) A mixture of acetic acid and ethanol, (2) A mixture of sodium hydroxide and ethanol, and (3) An acid such as hydrochloric acid The solution is passed through the backwash flow in this order to reduce the initial pressure to 0.
It was able to recover to 35 kgf / cm ² .

In this case, the equilibration of the adsorption column 1 and the loading of the liquid to be treated are performed in a downward flow as shown in FIG. 2, and the subsequent washing, elution recovery and regeneration washing are performed in an upward direction as shown in FIG. It is preferred to carry out the flow. The reason is that there is a high possibility that the substance causing the clogging of the adsorption column 1 is accumulated from the upper part of the column. Therefore, by performing the backwashing flow from the step after loading the sample, the effect of suppressing the increase in pressure drop can be obtained. It is.

(Invention of Claim 3) FIG. 4 is a view showing an embodiment of the invention of Claim 3, wherein 100 μm
An adsorbing layer 8 of an adsorbing carrier having the following particle diameter and an inert non-adsorbing carrier layer 9 such as a gel filtration carrier having a coarser particle diameter than the adsorbing carrier are provided. As the adsorption carrier constituting the adsorption layer 8, for example, a hard polymer-based or silica-based adsorption carrier that has been developed in recent years and has a high speed and high adsorption capacity can be used.

Thus, the adsorbing layer 8 of the adsorbing carrier and the inert non-adsorbing carrier layer 9 are placed in the adsorption column 1, and the liquid to be treated is passed from the inert carrier layer side to the adsorbing carrier layer side. If liquefied, the medium components and impurities contained in the sample solution are filtered by the inert non-adsorbed carrier layer 9, thereby preventing the adsorption layer 8 of the adsorbed carrier from being clogged as shown in Examples described later. . Preferably, the thickness of the non-adsorbed carrier layer 9 is 3 cm or more, and the value of (thickness of the adsorbed carrier layer 8 / thickness of the non-adsorbed carrier layer 9) is 3 or less. This is because if the thickness of the non-adsorbed carrier layer 9 is less than 3 cm, or if the ratio exceeds 3, the effect of preventing clogging is small.

(Invention of Claim 4) In addition, as shown in FIG. 4, a single adsorption column 1 is provided with an adsorbing carrier adsorption layer 8 and an inert non-adsorbing carrier layer 9, and the adsorbing carrier is adsorbed. Adsorption column 1 packed only with layer 8 and inert unadsorbed carrier layer 9
The same effect can be obtained by connecting in series an adsorption column filled with, and passing the liquid to be treated in a direction from the inert carrier layer 9 side to the adsorption carrier layer 8 side. Examples of each invention will be described below.

[0014]

[Example] (Example 1) The invention of claim 1 is applied to an animal cell culture supernatant (containing human monoclonal IgG).
Sodium chloride was added and dissolved at a concentration of 0.5 mol / L, and then filtered through a 1-micron filter to obtain a sample that was passed through an adsorption column. Affinity packing (Biosepra, trade name Hyper) using protein A, which adsorbs specifically to human IgG in the sample, as the stationary phase (ligand)
D) was packed into a glass column with an inner diameter of 10 mm to a height of 60 mm. As an adsorption carrier, a chromatographic filler having characteristics of high speed and high adsorption capacity, such as ion exchange, affinity, and hydrophobicity, is suitable in terms of a large throughput. For example,
The recently developed high-speed, high-adsorption capacity adsorption carrier is made of a hard polymer-based or silica-based material and has an average particle diameter of 10
Thicknesses below 0 μm are often used. FIG. 5 shows the adsorption column of Example 1.

In the experiment of Example 1, an adsorbent having an average particle diameter of 50 μm was used. In addition, it is preferable to use a filter having a maximum pore diameter with no leakage of the filler in the upper and lower portions of the gel layer in the column.
A micron mesh (such as a polypropylene mesh) was used. After such an adsorption column was mounted on the fractionation apparatus shown in FIG. 1 and equilibrated in advance, the culture supernatant sample was collected at a high flow rate of 600 cm / hr (= 100 CV / hr, 7.85 ml / min.). The liquid was passed downward. The column was washed upward until the UV absorption value at the outlet of the column became parallel to the baseline by switching the valve from the subsequent column washing. Next, the human IgG
After the recovery, in the regeneration step, a regenerating solution 1: 0.5 M acetic acid / 60
% Ethanol, regenerating solution 2: 0.2 M NaOH / 20% ethanol, and regenerating solution 3: 0.1 N HCl were flowed through the column in the stated order by 17 column volumes, 4 column volumes, and 17 column volumes in a backwash flow. The column pressure loss at each stage was measured with a pressure gauge provided in the system.

FIG. 6 shows the results. In the conventional method, the column pressure loss rises remarkably due to repeated use, whereas in the present invention, it is 300 times the column volume (1413 ml, 3
00C.V.) and repeated purification treatments, the column pressure loss rise was suppressed and stable operation was possible. The experimental conditions and methods of Example 1 are summarized in the following table.

[0017]

[Table 1]

[0018]

[Table 2]

[0019]

[Table 3]

Example 2 Inventive Example 3 In the experiment of Example 2, an adsorbent (HyperD) having an average particle diameter of 35 μm was used.
Protein A gel) was used. Otherwise, the same experimental conditions as in Example 1 were used. The average particle size of the adsorption carrier is 35μm
When, the sample load at a higher flow rate of 600 cm / hr was restricted than in the case of the average particle diameter of 50 μm used in Example 1. That is, as shown in FIG. 7, a sharp increase in pressure loss was observed from the time when the sample was flowed for 70 column volumes. When the pressure drop increased, the inside of the column was once washed with the adsorption equilibrium solution, and the pressure drop could be temporarily reduced. However, when the sample was flown again, the pressure drop increased. Therefore, in this case, although the column adsorption capacity has a margin, the processing amount is reduced and the result is that repeated continuous processing cannot be performed.

Therefore, in order to overcome this problem, the upper part of the adsorption carrier layer in the adsorption column has a coarser particle size than the adsorption carrier.
(100μm) gel filtration carrier (trade name Sephadex
G25, manufactured by Pharmacia) as an unadsorbed carrier layer. The effect is shown in FIG.
No pressure increase was observed even after passing through the column volume, and the treatment performance of the adsorbent carrier was maximized. Also in this method, stable continuous use of the adsorption column was possible by combining with the purification treatment method of Example 1. The same effect was obtained when the unadsorbed carrier layer was provided as a separate column and used in connection. In this case, as a method of filling the gel into the column, the gel slurry is poured into the column and the gel is allowed to settle naturally, so that the lower layer has a large particle size and the upper layer has a small particle size distribution. Is desirably passed in upward flow. The experimental conditions and methods of Example 2 are described in the following table.

[0022]

[Table 4]

[0023]

[Table 5]

[0024]

[Table 6] (Experimental method)

Example 3 In the experiment of Example 3, the thickness of the adsorbed carrier layer and the thickness of the non-adsorbed carrier layer were changed as shown in Tables 7 and 8. Other conditions were the same as those in Example 2. First, an experiment was conducted with the thickness of the adsorbed carrier layer fixed and the thickness of the non-adsorbed carrier layer changed, and as shown in the graph of FIG. 9, the effect of preventing clogging was enhanced when the thickness of the non-adsorbed carrier layer was 3 cm or more. When the thickness of the non-adsorbed carrier layer was fixed and the experiment was performed while changing the thickness of the adsorbed carrier layer, when the value of the thickness of the adsorbed carrier layer / the thickness of the non-adsorbed carrier layer became 4 as shown in the graph of FIG. The pressure increased as the unadsorbed carrier layer began to clog. In other words, when the thickness of the non-adsorbed carrier layer is 3 cm or more and the value of the thickness of the adsorbed carrier layer / thickness of the non-adsorbed carrier layer is 3 or less, it is possible to suppress the pressure increase of the column and to operate stably. .

[0026]

[Table 7]

[0027]

[Table 8]

(Embodiment 4-Invention of Claim 6) Claim 6
FIG. 11 shows a flow chart around the column of the preparative liquid chromatography device. In FIG. 11, switching valves 11a, 11b, 11c and 12a, 12b, 12c are installed at upper and lower portions of three adsorption columns 10a, 10b, 10c in series, respectively. Further, supply lines 13 and 14 for the regeneration cleaning liquid are provided. FIG. 11 shows a state in which the liquid to be treated is passed through the adsorption columns 10a, 10b, and 10c to purify useful substances.

FIG. 12 shows a state in which the adsorption column 10c is regenerated and washed. In this case, the switching valve 15 is switched so that the regenerated cleaning liquid flows into the supply line 13, and the switching valve
The regenerating washing liquid is passed through the adsorption column 10c in the direction opposite to the flowing direction of the liquid to be treated via 12c. The regenerating washing liquid flowing out of the adsorption column 10c is discharged via the switching valve 11c and the discharge line 16.

FIG. 13 shows a state where the regeneration of the adsorption columns 10a and 10b is performed. In this case, the regenerating cleaning liquid is supplied from the pump 17, and the supply line 14, the switching valve 12
b, flows through the adsorption column 10b in the direction opposite to the liquid flow direction of the liquid to be treated, and further flows through the switching columns 11b, 12a through the adsorption column 10a in the direction opposite to the liquid flow direction of the liquid to be treated.
It is discharged via a discharge line 18. Washing of any adsorption column is performed by the method described in any one of claims 1 to 5, and continuous use of the adsorption column becomes possible. In this example, three adsorption columns 10a, 10b, and 10c are shown in series, but a single adsorption column may be used.

[0031]

As described above, according to each invention of the present application, even when the adsorption column is operated at a high flow rate, an increase in pressure loss due to clogging can be suppressed, and high processing efficiency can be maintained. For this reason, each invention of the present application has an excellent effect as a method and an apparatus for continuously using an adsorption column used in the production of biopharmaceuticals or the like that requires a large amount of culture supernatant to be treated at one time.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an apparatus for carrying out the invention of claim 1;

FIG. 2 is a diagram showing a state in which a liquid is passed through an adsorption column in a downward flow.

FIG. 3 is a diagram showing a state in which liquid is passed through an adsorption column in an upward flow.

FIG. 4 is a sectional view of an adsorption column showing an embodiment of the third invention.

FIG. 5 is a sectional view showing the adsorption column of Example 1.

FIG. 6 is a graph showing the results of Example 1.

FIG. 7 is a graph showing a relationship between a sample load and a pressure loss when no unadsorbed carrier layer is provided in Example 2.

FIG. 8 is a graph showing the relationship between sample load and pressure loss when a non-adsorbed carrier layer is provided in Example 2.

FIG. 9 is a graph showing the relationship between sample load and pressure loss when the thickness of an unadsorbed carrier layer is changed in Example 3.

FIG. 10 is a graph showing the relationship between sample load and pressure loss when the thickness of the adsorption carrier layer is changed in Example 3.

FIG. 11 is a piping system diagram showing an embodiment of the invention according to claim 6;

FIG. 12 is a piping system diagram showing an embodiment of the invention of claim 6;

FIG. 13 is a piping diagram showing an embodiment of the invention according to claim 6;

[Explanation of symbols]

1 adsorption column, 2 sample containers, 3 affinity packing material, 4 liquid pump, 5 solvent switching valve, 6 deaerator, 7 UV meter, 8 adsorption carrier adsorption layer, 9 inert non-adsorption carrier layer , 10 adsorption column, 11
Switching valve, 12 Switching valve, 13 Regeneration cleaning liquid supply line, 14 Regeneration cleaning liquid supply line, 15 Switching valve, 16 discharge line, 17 pump, 18 discharge line

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tsuyoshi Majima 2-56 Sudacho, Mizuho-ku, Nagoya, Aichi Prefecture Inside Nihon Insulators Co., Ltd. (72) Inventor Saichi Yamada 2nd Sudacho, Mizuho-ku, Nagoya Aichi, Japan No. 56 Nippon Insulators Co., Ltd.

Claims (6)

[Claims] 1. A method for continuously using an adsorption column, in which a liquid to be treated such as a cell culture supernatant is passed through an adsorption column and a purification process of a useful substance is repeated, the equilibration of the adsorption column, loading of the liquid to be treated, and washing. After the end of each series of elution and recovery operations,
(1) A mixture of acetic acid and ethanol; (2) A mixture of sodium hydroxide and ethanol; and (3) A continuous use of the adsorption column, wherein the adsorption column is regenerated and washed in the order of an acidic solution such as hydrochloric acid. . 2. The continuous use method of the adsorption column according to claim 1, wherein the equilibration of the adsorption column, the flow direction up to the load of the liquid to be treated, and the subsequent washing, elution recovery, and regeneration washing are performed in opposite directions. 3. A continuous use of an adsorption column for purifying a useful substance by passing a sample such as a cell culture supernatant through an adsorption column and adsorbing an adsorption carrier having a particle diameter of 100 μm or less in the adsorption column. A layer and at least one inert non-adsorbed carrier layer such as a gel filtration carrier having a particle size coarser than the adsorbent carrier are provided, and the layer is covered in the direction from the inert carrier layer side to the adsorbent carrier layer side of the adsorption column. A method for continuously using an adsorption column, wherein a treatment liquid is passed. 4. An adsorption layer of an adsorption carrier having a particle diameter of 100 μm or less is provided for continuous use of an adsorption column for purifying a useful substance by passing a sample such as a cell culture supernatant through an adsorption column. The adsorption column is connected to an adsorption column provided with an inert non-adsorbed carrier layer such as a gel filtration carrier having a particle size coarser than the adsorption carrier, and the adsorbed carrier layer is placed from the inert carrier layer side of those adsorption columns. Continuous flow of the liquid to be treated in the direction of the side. 5. The method according to claim 3, wherein the thickness of the non-adsorbed carrier layer is 3 cm or more, and the value of (thickness of the adsorbed carrier layer / thickness of the non-adsorbed carrier layer) is 3 or less. 6. The liquid supply line according to claim 1, wherein a switching valve is provided at the upper and lower portions of the adsorption column, and a liquid feed line is provided for passing the regenerating washing liquid in a direction opposite to a liquid flowing direction of the liquid to be treated. Continuous use of an adsorption column by a continuous method.