JP2017222613A - Method for refining antibody and method for cleaning carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for suppressing reduction of the dynamic connection capacity of carrier for affinity chromatography.SOLUTION: A refining method of antibody includes: a process of equilibrating a column filled with carrier for affinity chromatography; a process of adding liquid containing antibody to the equilibrated column; a process of cleaning contaminant contained in liquid containing the antibody; a process of eluting the antibody from the column; and a process of cleaning the carrier for affinity chromatography by basic liquid, in which the process of cleaning by basic liquid is performed at 20°C or lower.SELECTED DRAWING: None

Description

  The present invention relates to a method for purifying an antibody and a method for washing a carrier.

In recent years, pharmaceuticals containing antibodies (antibody drugs) have been used for the treatment of cancer and infectious diseases. Antibodies used for antibody pharmaceuticals are obtained by culturing antibody-expressing cells (for example, Chinese hamster ovary cells) obtained by genetic engineering techniques and then purifying them by affinity chromatography or the like.
In the affinity chromatography, a carrier on which a substance (ligand) capable of specifically adsorbing an antibody is immobilized is generally used. The ligand forms ionic interaction, hydrophobic interaction, hydrogen bond, weak covalent bond, chelate bond and the like in a complex and steric manner with the antibody.
By using affinity chromatography, impurities (for example, proteins derived from cultured cells (HCP (Host Cell Protein)), nucleic acids, membrane components, metabolites, and components derived from culture fluids are contained in liquid samples containing antibodies. It is said that the antibody can be purified to a high purity by removing the ligand and the carrier, etc., but the impurities contained in the liquid sample are adsorbed and remain on the carrier with a weak binding force. This has been a factor that decreases the purity of the antibody and makes it difficult to reuse the carrier (Patent Document 1, and Non-Patent Documents 1 and 2).

JP 2011-206046 A

Journal of Chromatogr. A, 1102, 224-231, 2006 Biotechnol. Prog. 24,1115-11121,2008

  Therefore, in order to eliminate carry-over of impurities such as HCP adsorbed on the carrier, the carrier is washed with a basic liquid after elution of the antibody, but the ligand such as protein A is deteriorated by the basic liquid. However, there is a problem that the dynamic binding capacity gradually decreases each time the cleaning is performed.

  The problem to be solved by the present invention is to provide means for suppressing a decrease in the dynamic binding capacity of a carrier for affinity chromatography.

The above problems have been solved by the following means.
<1> a step of equilibrating a column packed with a carrier for affinity chromatography, a step of adding a liquid containing an antibody to the equilibrated column, and a step of washing impurities contained in the liquid containing the antibody And a step of eluting the antibody from the column and a step of washing the affinity chromatography carrier with a basic liquid, the method for purifying an antibody, the step of washing with the basic liquid A purification method (hereinafter, also referred to as the purification method of the present invention), characterized by being carried out at 20 ° C. or lower.
<2> The purification method according to <1>, wherein the carrier for affinity chromatography is a carrier on which an antibody-adsorbing ligand is immobilized.
<3> A method for washing a carrier comprising a step of washing a carrier for affinity chromatography with a basic liquid, wherein the step of washing with the basic liquid is performed at 20 ° C. or lower. Cleaning method (hereinafter also referred to as the cleaning method of the present invention).
<4> A method for washing a carrier including a step of washing a carrier for affinity chromatography on which an antibody-adsorbing ligand is immobilized with a basic liquid, wherein the step of washing with the basic liquid comprises 20 ° C. or less. A cleaning method characterized by being performed in
<5> The method according to <2> or <4>, wherein the antibody-adsorbing ligand is a protein ligand.
<6> The method according to <5>, wherein the protein ligand is protein A or Fc-binding protein.

  The purification method and the washing method of the present invention can suppress a decrease in the dynamic binding capacity of the carrier for affinity chromatography.

<Purification method>
The purification method of the present invention comprises a step of equilibrating a column packed with a carrier for affinity chromatography (hereinafter also referred to as step A), and a step of adding a liquid containing an antibody to the equilibrated column (hereinafter referred to as step). B), a step of washing impurities contained in the antibody-containing liquid (hereinafter also referred to as step C), a step of eluting the antibody from the column (hereinafter also referred to as step D), A method for purifying an antibody, comprising a step of washing a carrier for affinity chromatography with a basic liquid (hereinafter also referred to as step E), wherein step E is performed at 20 ° C. or lower. is there. In addition, the notation such as “a to b” representing a numerical range and the like includes “a” and “b” in the numerical range unless otherwise specified, and has the same meaning as “a or more and b or less”.
Hereinafter, the purification method of the present invention will be described.

(Affinity chromatography carrier)
The affinity chromatography carrier used in the purification method of the present invention is not particularly limited as long as it is used for affinity chromatography, and examples thereof include a carrier on which an antibody-adsorbing ligand is immobilized.
As the antibody-adsorbing ligand, a protein ligand is preferable. Specific examples include protein A, protein G, protein L, Fc-binding protein, (strept) avidin, lectin, and functional variants thereof. The whole antibody-adsorbing ligand may be used, but a fragment obtained by recombinant, enzyme treatment or the like may be used.
Among these, at least one selected from protein A, protein G, protein L, Fc binding protein and functional variants thereof is preferable, and protein A and Fc binding protein are particularly preferable. Although Fc binding protein is not specifically limited, For example, Fc binding protein etc. of Unexamined-Japanese-Patent No. 2011-206046 etc. are mentioned.

Examples of the material of the carrier include natural polymers such as agarose, dextran, and cellulose; synthetic polymers such as polystyrene-based resins and methacrylate-based resins; silica; glass and the like, and one of these is used alone. Two or more kinds may be used.
Examples of the shape of the carrier include particles, hollow fibers, nonwoven fabrics, filters, and monoliths. Among these, the particulate form is preferable.

(Process A)
Step A is a step of equilibrating the column packed with the affinity chromatography support.

Here, in step A and step C, the pH in the system is preferably 5 or more, more preferably in the range of 5 to 12 from the viewpoint of suppressing denaturation of the ligand and suppressing elution of the antibody in step C. Yes, more preferably in the range of 5-10, particularly preferably in the range of 6-8 (near neutral). Moreover, in order to make pH into such a range, it is preferable to perform process A and C using a buffer solution.
The buffer used in the buffer is not particularly limited, and examples thereof include phosphates such as sodium phosphate, Tris (trishydroxyaminomethane), boric acid, sodium carbonate, sodium bicarbonate and the like. Also, a good buffer buffer such as MES (2-morpholinoethanesulfonic acid) or HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) may be used. Further, phosphate buffered saline (PBS) may be used as a buffer solution. Moreover, the density | concentration of a buffering agent is a final density | concentration, Preferably it is about 10-100 mM (10-100 mmol / L).
Moreover, as said buffer solution, what contains salts is preferable. Examples of the salts include alkali metal chlorides such as sodium chloride. The final concentration of the salt is preferably 20 mM or more, more preferably 30 mM or more, and particularly preferably 50 mM or more, and the final concentration is preferably 1200 mM or less, more preferably 1000 mM or less, particularly preferably 500 mM or less. It is.

  The amount of buffer used in step A and step C is usually 1 to 10 times the column volume, and preferably 3 to 5 times the column volume.

(Process B)
Step B is a step of adding a liquid containing an antibody to the equilibrated column. By step B, the antibody in the liquid sample comes into contact with the carrier and is adsorbed.
Examples of the liquid containing the antibody include a mixed solution of the culture solution and the antibody when the hybridoma is cultured. In this case, components other than the antibody contained in the culture solution are listed as contaminants.
Examples of the antibody in the present invention include an antibody having a protein containing a CH2 / CH3 region (CH2 region and CH3 region) of an IgG antibody, an Fc fusion protein, a recombinant protein, and the like.
In step B, the liquid sample may be diluted with a buffer solution. Examples of the buffer include those similar to those used in step A.

The amount of the liquid containing the antibody in Step B is usually an amount such that the amount of the antibody contained in the liquid is in the range of 50 to 100% by mass of the adsorption capacity of the affinity chromatography carrier packed in the column. Yes, preferably in an amount in the range of 60-90% by mass of the adsorption capacity, more preferably 75-85% by mass. The adsorption capacity varies depending on conditions such as the flow rate of the column when the antibody is adsorbed, and can be calculated as, for example, the dynamic binding capacity (antibody amount mg / column particle volume mL) by the method described in the Examples. What is necessary is just to select said usage-amount according to the quantity of column particle to be used, a flow rate, the density | concentration of an antibody, etc.
In Step B, the state in which the affinity chromatography support and the antibody are in contact with each other may be held. In this case, the holding time is not particularly limited, but is usually 1 to 60 minutes, preferably 1 to 20 minutes, more preferably 2 to 8 minutes.

(Process C)
Step C is an intermediate washing step for washing impurities contained in the liquid containing the antibody.
In Step C, a cleaning solution used for intermediate cleaning may be used by containing one or more selected from the above buffer solution, the above salts, a water-soluble organic solvent, a nonionic surfactant and a hydrophobic amino acid. This makes it easier to control the hydrophobic interaction between the antibody and the affinity chromatography support.

Examples of the water-soluble organic solvent include linear, branched alcohols having about 1 to 6 carbon atoms such as methanol, ethanol, 1-propanol, 2-propanol (isopropanol), butanol, acetonitrile, acetone, benzyl alcohol, and the like. Is mentioned. Of these, one kind may be used alone, or two or more kinds may be used in combination.
When a water-soluble organic solvent is used, the amount used is usually in the range of 0.1 to 25% (v / v), preferably in the range of 5 to 20% (v / v).
Examples of nonionic surfactants include polysorbates (Tween 20, Tween 40, Tween 60, Tween 80 (trade names) and the like), Triton X-100 (trade names), and the like. Of these, one kind may be used alone, or two or more kinds may be used in combination.
In the case of using a nonionic surfactant, the amount used may be added so as to be a normal concentration higher than the critical micelle concentration, and is preferably in the range of 0.01 to 1% (v / v).
Examples of hydrophobic amino acids include leucine, isoleucine, and valine.
In the case of using a hydrophobic amino acid, the amount used may be in a concentration range in which it can be dissolved, and is preferably in the range of 100 mM to 1 M, more preferably in the range of 200 mM to 400 mM.

Further, the cleaning liquid used in Step C may contain a substance that inhibits the formation of hydrogen bonds. This makes it easier to control the strength of hydrogen bonding between the antibody and the carrier for affinity chromatography.
Examples of the substance that inhibits the formation of hydrogen bonds include urea, and guanidinium salts such as guanidine hydrochloride may be used.
When using a substance that inhibits the formation of hydrogen bonds, the amount used is usually 0.3 M or more, preferably 0.5 M or more, and more preferably 0.5 to 3 M in the final concentration. is there.
The cleaning liquid used in Step C may contain a combination of the water-soluble organic solvent, nonionic surfactant, hydrophobic amino acid, and substance that inhibits the formation of hydrogen bonds. Thereby, the washing | cleaning effect of the foreign material by the process C can further be improved.

  In addition, as above-mentioned, the washing | cleaning liquid used at the process C contains what contains salts. The salt concentration is as described above.

(Process D)
Step D is a step of eluting the antibody from the column.
Step D is preferably performed using an elution buffer. The elution buffer may be selected according to the type of carrier, but in the case of a protein ligand such as protein A, an acidic buffer is preferable. The pH of the acidic buffer is preferably in the range of 2-6, more preferably in the range of 3-6, and particularly preferably in the range of 3-5. Examples of the acidic buffer include acidic buffers using organic acid salts such as citric acid and acetic acid. The concentration of the salt of the organic acid is a final concentration, preferably about 10 to 100 mM.

  The amount of elution buffer used in Step D is usually 0.2 to 10 times the column volume, and preferably 1 to 5 times the column volume.

Prior to step E, the column from which the antibody was eluted in step D may be equilibrated. The equilibration may be performed in the same manner as in step A.
Further, the antibody eluted in step D can be recovered from the column. Examples of the method for recovering the antibody include a method using a fraction collector of a chromatography system.

  In addition, although the temperature which implements said process AD is not specifically limited, Preferably it is 1-40 degreeC, More preferably, it is 1-30 degreeC.

(Process E)
Step E is a step of washing the affinity chromatography carrier at 20 ° C. or lower with a basic liquid. Cleaning in the step E is alkaline cleaning, and in view of suppressing a decrease in dynamic binding capacity and ease of operation, cleaning in place (Cleaning-in-place) is preferable.

The basic liquid used in step E is preferably an aqueous solution of a basic compound. Basic compounds are broadly classified into basic inorganic compounds and basic organic compounds, but basic inorganic compounds are preferred from the viewpoint of enhancing the desired effects of the present invention.
Examples of the basic inorganic compound include metal hydroxide, ammonia and the like. Of these, metal hydroxides are preferred from the viewpoint of enhancing the desired effect of the present invention, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; hydroxides of alkaline earth metals such as calcium hydroxide. More preferred are alkali metal hydroxides, and sodium hydroxide is particularly preferred.
As the basic liquid, an aqueous sodium hydroxide solution is particularly preferable.

  The concentration of the basic compound is the final concentration, preferably 10 mM or more, more preferably 30 mM or more, still more preferably 50 mM or more, particularly preferably 100 mM or more, from the viewpoint of enhancing the desired effect of the present invention. From the viewpoint of enhancing the desired effect of the invention, the final concentration is preferably 2M or less, more preferably 1.5M or less, still more preferably 1M or less, and particularly preferably 0.5M or less.

The cleaning temperature in step E is 20 ° C. or lower. With this configuration, it is possible to suppress a decrease in dynamic binding capacity of the affinity chromatography carrier.
The washing temperature in step E is preferably 17.5 ° C. or lower, more preferably 15 ° C. or lower, further preferably 12.5 ° C. or lower, further preferably 10 ° C. or lower, from the viewpoint of enhancing the desired effect of the present invention. A temperature of not higher than ° C is particularly preferred. Moreover, although the washing | cleaning temperature should just exceed the freezing point of a basic liquid, Preferably it is 1 degreeC or more.
Further, when the cleaning temperature is set to 20 ° C. or lower, the entire column container may be cooled to 20 ° C. or lower, and the temperature of the cleaning solution may be directly controlled to 20 ° C. or lower. Moreover, even if it implements all the processes of a refinement | purification method at 20 degrees C or less, you may implement only the process E at 20 degrees C or less.

The usage-amount of the basic liquid in the process E is 1-10 times of column capacity normally, Preferably it is 3-5 times of column capacity.
The washing time in Step E is preferably 1 minute to 60 minutes, more preferably 5 minutes to 40 minutes, and particularly preferably 15 minutes to 30 minutes per step. In addition, the frequency | count of performing the process E may be 1 time or multiple times.

<Washing method>
The washing method of the present invention is a method for washing a carrier including a step of washing a carrier for affinity chromatography with a basic liquid, wherein the step of washing with the basic liquid is performed at 20 ° C. or lower. It is a feature.
The washing step in the washing method of the present invention is the same as step E in the purification method of the present invention. As the affinity chromatography carrier, those listed as those used in the purification method of the present invention can be used.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

[Test example (comparison of decrease in dynamic binding capacity by basic liquid)]
After measuring the dynamic binding capacity (DBC (Dynamic Binding Capacity) of the support for affinity chromatography packed in the column, the inside of the column is replaced with a basic liquid (0.5 M aqueous sodium hydroxide solution), and a predetermined temperature ( Example 1: 20 ° C., Example 2: 15 ° C., Example 3: 10 ° C., Examples 4 and 5: 6 ° C., Comparative Example: 25 ° C.) and allowed to stand for 15 hours. By measuring, the decrease in the amount of dynamic binding due to the basic liquid was compared, and the specific procedure is as follows: In addition, assuming the case of using multiple times (multiple cycles), the basic liquid immersion Later, it was allowed to stand for a long time.

(Initial DBC measurement)
A Tricorn 50/200 column manufactured by GE Healthcare was filled with 4 mL of a carrier on which Protein A was immobilized (Amsphere ^™ A3 manufactured by JSR Life Sciences), and this column was connected to AKTA prime plus manufactured by GE Healthcare.
Next, the column was equilibrated by feeding a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) 5 CV (Column Volume: column packing volume) at a flow rate of 1 mL / min in a 23 ° C. environment.
Thereafter, DBC for an antibody (human IgG antibody, HGG-1000 manufactured by Equitech Bio) at a retention time of 4 minutes was measured. The antibody was diluted to 5 mg / mL with 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5), and sent at a flow rate of 1 mL / min in a 23 ° C. environment. The initial DBC was determined from the antibody capture amount and column packing volume.

Example 1
After the initial DBC measurement, unadsorbed antibody was washed by feeding 5 CV of 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) at a flow rate of 2.0 mL / min in an environment of 23 ° C. Antibody elution was carried out by feeding 5 CV of an aqueous sodium acid solution (pH 3.2) at a flow rate of 1.0 mL / min. Next, the column was equilibrated again by feeding 5 CV of 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) at a flow rate of 2.0 mL / min.
Thereafter, 0.5M sodium hydroxide aqueous solution was passed through the column at a flow rate of 1.0 mL / min for 10 minutes to replace the inside of the column, and left at 20 ° C. for 15 hours, and then the initial DBC was measured. In general, the DBC after immersion in the basic liquid was measured, and the maintenance rate from the initial DBC was calculated.

(Examples 2 to 4)
The same operation as in Example 1 was performed except that the standing temperature after immersion in the basic liquid was changed to 15 ° C. (Example 2), 10 ° C. (Example 3), and 6 ° C. (Example 4). Comparison between DBC and DBC after standing was performed.

(Comparative example)
Except for changing the standing temperature after immersion in the basic liquid to 25 ° C., the same operation as in Example 1 was performed to compare the initial DBC and the DBC after standing.

(Measurement result)
Table 1 shows the measurement results of each example and comparative example.

(Example 5)
The same procedure as in Example 4 was performed except that all the procedures corresponding to Step A to Step E (from equilibration before initial DBC measurement to standing after immersion in basic liquid) were carried out in a refrigerator at 6 ° C. The comparison was made between the initial DBC and the DBC after standing. As a result, almost the same result as in Example 4 was obtained.

Claims (6)

Equilibrating a column packed with a carrier for affinity chromatography;
Adding an antibody-containing liquid to the equilibrated column;
Washing impurities contained in the liquid containing the antibody;
Eluting the antibody from the column;
Washing the carrier for affinity chromatography with a basic liquid, comprising:
The step of washing with the basic liquid is performed at 20 ° C. or lower,
Purification method.   The purification method according to claim 1, wherein the affinity chromatography carrier is a carrier on which an antibody-adsorbing ligand is immobilized. A method for washing a carrier comprising a step of washing a carrier for affinity chromatography with a basic liquid,
The step of washing with the basic liquid is performed at 20 ° C. or lower,
Cleaning method. A method for washing a carrier comprising a step of washing a carrier for affinity chromatography on which an antibody-adsorbing ligand is immobilized with a basic liquid,
The step of washing with the basic liquid is performed at 20 ° C. or lower,
Cleaning method.   The method according to claim 2 or 4, wherein the antibody-adsorbing ligand is a protein ligand.   6. The method of claim 5, wherein the protein ligand is protein A or Fc binding protein.