JP2014094936A - Purification method of antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for purifying an antibody with high purity by effectively removing an impurity included in a solution containing the antibody when purifying the antibody using affinity chromatography from the solution containing the antibody.SOLUTION: A purification method comprises: a step of adding a solution containing an antibody to an equilibrated column filled with carriers for affinity chromatography; a first washing step of washing with the solution used in equilibration of the column; a second washing step of washing with a neutral or alkali buffer containing sodium chloride having the final concentration of 0.2 M or more; and a step of washing an impurity included in the solution containing the antibody. Alternatively, the purification method comprises the steps of: adding sodium chloride to the solution containing the antibody so that the concentration of sodium chloride is 0.2 M or more; adding the solution containing the antibody to the column; washing the impurity included in the solution containing the antibody using the solution used in equilibration of the column; and eluting the antibody using an elution liquid.

Description

  The present invention relates to a method for purifying the antibody with high purity from a solution containing the antibody using affinity chromatography.

  In recent years, pharmaceuticals containing antibodies (antibody drugs) have been used for the treatment of cancer and infectious diseases. The antibody used for the antibody drug is purified to high purity using column chromatography after culturing cells capable of expressing the antibody (eg, CHO (Chinese hamster ovary) cells) obtained by genetic engineering techniques. And manufacture.

  Among column chromatography, affinity chromatography is an adsorbent obtained by immobilizing a substance (ligand) capable of specifically binding to a substance to be adsorbed on an insoluble carrier (hereinafter also referred to as a carrier for affinity chromatography). ). The specific binding force for the substance to be adsorbed is usually obtained by complex and steric formation of ionic interaction, hydrophobic interaction, hydrogen bond, weak covalent bond, chelate bond with metal, etc. Yes. Affinity chromatography is performed by using impurities contained in the solution to be adsorbed (for example, proteins derived from cultured cells (Host Cell Protein, HCP), nucleic acids, membrane components and metabolites, components derived from culture broth, affinity chromatography). And the adsorbed substance can be adsorbed specifically. Therefore, the adsorption target substance can be purified with high purity. However, while the affinity chromatography carrier is adsorbed to the substance to be adsorbed with a specific binding force, impurities contained in the solution to be purified are also adsorbed to the carrier with a weak binding force, which is the object of adsorption. This was a factor in reducing the purification purity of the substance.

  In particular, affinity chromatography is often employed in the capture process, which is the first stage of the purification process, so the solution added to the column packed with the support for affinity chromatography is the most common in the entire purification process. Contains impurities. Therefore, even if a carrier for affinity chromatography with high specificity is used, there is a problem that the purification purity of the substance to be adsorbed decreases due to non-specific adsorption of impurities other than the substance to be adsorbed on the carrier. was there.

  In general, a carrier in which protein A is immobilized as a ligand is used as a carrier for affinity chromatography for purifying an antibody. When purifying an antibody using a column packed with a carrier on which protein A is immobilized, the antibody is usually added by adding a solution containing the antibody near neutrality (for example, a culture solution of cells capable of expressing the antibody). Is adsorbed on the carrier, and then the carrier is washed with a buffer used for equilibration of the column, and finally the antibody adsorbed on the carrier is eluted with an acidic buffer to purify the antibody with high purity. By performing the chromatography operation described above once, the purity of the antibody can be improved to 90% or more. However, the antibody solution obtained by the above operation contains a protein derived from cultured cells (HCP) at a concentration of several hundred to several thousand ppm (Non-patent Document 1). Ingredients such as nucleic acid components, membrane components, metabolic components, and components derived from the culture solution are problematic. In order to solve the above problem, after washing with a buffer used for equilibration of the column and before eluting the antibody, additional washing with a buffer containing urea or isopropanol is performed, so that impurities such as HCP are contained. A method for enhancing the effect of removing water is disclosed (Non-Patent Document 2). However, even with the method disclosed in Non-Patent Document 2, the removal rate of HCP is about 60% at the maximum, and the remaining HCP remains mixed in the purified antibody solution.

  Fc receptors that are a group of protein molecules that bind to the Fc region of IgG antibodies and Fc receptors that have Fc region binding ability as carriers for affinity chromatography to purify antibodies other than the carrier on which protein A is immobilized A carrier in which a partial region (hereinafter collectively referred to as Fc-binding protein) is immobilized as a ligand has been put into practical use (Patent Document 1). The carrier immobilized with the Fc-binding protein can achieve the same high antibody purification purity as the carrier immobilized with protein A, but thousands to tens of thousands of ppm of HCP are mixed in the purified antibody solution. There was a problem to do.

JP 2011-206046 A

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

  An object of the present invention is to provide a method for purifying the antibody with high purity by efficiently removing impurities contained in the solution when the antibody is purified from the solution containing the antibody using affinity chromatography. There is.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have identified a cleaning component capable of efficiently removing impurities contained in a solution containing an antibody, and have completed the present invention.

That is, the first aspect of the present invention is:
A step of equilibrating a column packed with a carrier for affinity chromatography, a step of adding a solution containing an antibody to the equilibrated column, a step of washing impurities contained in the solution containing the antibody, and an eluent Elution of the antibody using a method for purifying an antibody,
The step of washing the contaminants is a first washing step of washing with the solution used for equilibration of the column, and a neutral or basic buffer containing sodium chloride having a final concentration of 0.2 M or more. It is the said purification method which consists of a 2nd washing | cleaning process.

  The second aspect of the present invention is the first aspect wherein the step of adding the antibody-containing solution to the equilibrated column is performed after adding sodium chloride so as to be 0.2 M or more to the antibody-containing solution. The purification method described.

  In the third aspect of the present invention, the buffer used in the second washing step is any one of a water-soluble organic solvent, a nonionic surfactant, a hydrophobic amino acid, and urea having a final concentration of 0.3 M or more. The purification method according to the first or second aspect, further comprising one or more.

  The fourth aspect of the present invention is the purification method according to the third aspect, wherein the water-soluble organic solvent is any one of isopropanol, acetonitrile, and ethanol.

  The fifth aspect of the present invention is the purification method according to the third aspect, wherein the hydrophobic amino acid is any one of valine, leucine and isoleucine.

Furthermore, the sixth aspect of the present invention provides
Equilibrating a column packed with a carrier for affinity chromatography;
Adding sodium chloride so as to be 0.2 M or more with respect to the solution containing the antibody;
Adding a solution containing the antibody to the equilibrated column;
Washing impurities contained in the solution containing the antibody with the solution used for equilibration of the column;
Elution of the antibody with an eluate.
This is a method for purifying antibodies.

  The seventh aspect of the present invention is the purification method according to any one of the first to sixth aspects, wherein the affinity chromatography carrier is a carrier on which an Fc-binding protein or protein A is immobilized. .

  Hereinafter, the present invention will be described in detail.

  One aspect of the purification method of the present invention is included in the step of equilibrating a column packed with a carrier for affinity chromatography, the step of adding a solution containing an antibody to the equilibrated column, and the solution containing the antibody A first washing step including a step of washing impurities and a step of eluting the antibody using an eluate, wherein the step of washing the impurities is washed with the solution used for equilibration of the column. And a second washing step of washing with a neutral or basic buffer solution containing sodium chloride having a final concentration of 0.2 M or more.

  The solution used in the step of equilibrating the column and the first washing step is generally a buffer solution having a pH near neutral (the concentration of the buffer solution is several tens to 100 mM) or PBS (phosphate buffered saline). Used.

  The buffer solution used in the second washing step is characterized by containing sodium chloride having a final concentration of 0.2 M (0.2 mol / L) or more. Since the sodium chloride concentration contained in PBS is 0.137M, the second washing step involves washing with a buffer solution containing a sodium chloride concentration higher than that of PBS. Although there is no particular upper limit to the sodium chloride concentration contained in the buffer used in the second washing step, it is preferably in the final concentration range of 0.2M to 1.2M, more preferably in the final concentration range of 0.3M to 1.0M. Most preferably, the final concentration ranges from 0.35M to 0.6M. The pH of the buffer used in the second washing step may be 7 or more, but if the pH is extremely high (extremely basic), the ligand constituting the affinity chromatography carrier may be denatured. Therefore, it is preferably in the range of pH 7 to pH 12, more preferably in the range of pH 8 to pH 10. The buffer constituting the buffer may be appropriately selected from those commonly used by those skilled in the art depending on the pH to be used. Phosphate, trishydroxyaminomethane (Tris), boric acid, sodium carbonate, heavy An example is sodium carbonate. Also, a good buffer type buffer such as MES (2-Morpholinoethane acid) or HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] etheric acid) may be used.

  If the buffer used in the second washing step further contains (adds) any one or more of a water-soluble organic solvent that increases hydrophobicity, a nonionic surfactant, and a hydrophobic amino acid, the substance and affinity chromatography This is preferable because the hydrophobic interaction with the photographic support can be controlled. Examples of the water-soluble organic solvent include methanol, ethanol, 1-propanol, 2-propanol (isopropanol), butanol, acetonitrile, acetone, and benzyl alcohol. The addition amount of the water-soluble organic solvent may be added so that the adsorptive power of the antibody and the carrier for affinity chromatography does not become weak, and is preferably in the range of 0.1% (v / v) to 25% (v / v). More preferably, it is in the range of 5% (v / v) to 20% (v / v). Examples of the nonionic surfactant include polysorbate (Tween 20, Tween 40, Tween 60, Tween 80 (trade name) and the like) and Triton X-100 (trade name). The addition amount of the surfactant may be added so as to have a normal concentration higher than the critical micelle concentration, and is preferably in the range of 0.01% (v / v) to 1% (v / v). Examples of hydrophobic amino acids include leucine, isoleucine, and valine. The amount of the hydrophobic amino acid added may be in a concentration range in which it can be dissolved, and is preferably in the range of 100 mM to 1M, more preferably in the range of 200 mM to 400 mM.

  It is preferable that the buffer used in the second washing step further contains (adds) a substance that inhibits the formation of hydrogen bonds because the strength of hydrogen bonds can be controlled. A preferable substance that inhibits the formation of hydrogen bonds is urea, and other guanidinium salts such as guanidine hydrochloride can be used. In the case of urea, it is necessary to add at least 0.3 M (0.3 mol / L) or more at a final concentration, preferably 0.5 M or more, more preferably a final concentration in the range of 0.5 M to 3 M. is there.

  The buffer solution used in the second washing step may contain (add to) the above-described water-soluble organic solvent and the above-described substance that inhibits the formation of hydrogen bonds, whereby the second washing The effect of removing impurities by the process can be further enhanced. What is necessary is just to examine suitably the aspect of a combination in the range by which the binding force of the support | carrier for affinity chromatography and an antibody is fully hold | maintained.

  In the purification method of the present invention, when the step of adding the antibody-containing solution to the equilibrated column packed with the carrier for affinity chromatography is performed, 0.2 M (0.2 mol / 0.2 mol / ml) of the antibody-containing solution is performed in advance. L) The above step may be performed after sodium chloride is added so that the concentration is greater than or equal to that, and impurities contained in the solution containing the antibody can be efficiently removed as in the purification method described so far. In addition, when employ | adopting this method, you may abbreviate | omit the 2nd washing | cleaning process mentioned above.

The carrier for affinity chromatography used in the purification method of the present invention is not particularly limited as long as it is a carrier having a ligand capable of specific adsorption to an antibody. For example, a carrier on which protein A is immobilized or an Fc binding protein Examples of the carrier on which is immobilized. Examples of the Fc-binding protein include an Fc-binding protein disclosed in JP2011-206046, and an Fc-binding protein containing the amino acid sequence from the 34th to the 307th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1. Or an amino acid sequence from 34th to 307th of the amino acid sequence described in SEQ ID NO: 1 and at least one of the following (1) to (34) in the 34th to 307th amino acid sequence: An Fc-binding protein (Japanese Patent Application No. 2012-145850) in which one amino acid substitution has occurred can be mentioned.
(1) 37th threonine of SEQ ID NO: 1 is replaced with isoleucine (2) 38th proline of SEQ ID NO: 1 is replaced with serine (3) 53rd leucine of SEQ ID NO: 1 is replaced with glutamine (4) SEQ ID NO: 1st 62nd glutamic acid is replaced with valine (5) 63rd valine of SEQ ID NO: 1 is replaced with alanine or glutamic acid (6) 66th leucine of SEQ ID NO: 1 is replaced with glutamine or proline (7) SEQ ID NO: 1 67 of serine is replaced with proline (8) 69th alanine of SEQ ID NO: 1 is replaced with valine or threonine (9) 71st serine of SEQ ID NO: 1 is replaced with threonine or leucine (10) 78th aspartic acid is replaced with glutamic acid (11) 81st isoleucine of SEQ ID NO: 1 is replaced with valine (12) 84th serine of No. 1 was replaced with threonine (13) 88th phenylalanine of SEQ ID NO: 1 was replaced with tyrosine (14) 95th glutamic acid of SEQ ID NO: 1 was replaced with aspartic acid (15) 119 of SEQ ID NO: 1 The histidine is replaced with glutamine (16) The 127th valine of SEQ ID NO: 1 is replaced with alanine (17) The 146th arginine of SEQ ID NO: 1 is replaced with lysine (18) The 147th aspartic acid of SEQ ID NO: 1 is Substitute asparagine (19) Replace 151st histidine of SEQ ID NO: 1 with tyrosine (20) Replace 178th threonine of SEQ ID NO: 1 with alanine (21) Replace 191st arginine of SEQ ID NO: 1 with lysine (22 ) The 199th threonine of SEQ ID NO: 1 is substituted with alanine (23) The 200th position of SEQ ID NO: 1 Isine replaced with methionine (24) 213 threnin of SEQ ID NO: 1 replaced with alanine (25) 216th valine of SEQ ID NO: 1 replaced with alanine (26) 221th leucine of SEQ ID NO: 1 replaced with arginine (27) 229th serine of SEQ ID NO: 1 is replaced with asparagine (28) 236th isoleucine of SEQ ID NO: 1 is replaced with lysine (29) 244th tyrosine of SEQ ID NO: 1 is replaced with histidine (30) SEQ ID NO: 1 253rd threonine is replaced with alanine (31) 290th arginine of SEQ ID NO: 1 is replaced with glutamine (32) 293rd lysine of SEQ ID NO: 1 is replaced with asparagine (33) 297th position of SEQ ID NO: 1 Lysine replaced with glutamic acid (34) 306th proline of SEQ ID NO: 1 replaced with threonine

  The purification method of the present invention maintains the binding force between the antibody and the carrier for affinity chromatography by adding 0.2 M (0.2 mol / L) or more sodium chloride to the antibody-containing solution and / or washing solution. However, the binding force between the carrier and the contaminants is weakened, and the contaminants contained in the antibody-containing solution can be effectively washed. Therefore, the purification purity of the antibody can be improved as compared with the conventional purification method. Since the purification process load of the latter stage can be greatly reduced by the purification method of the present invention, high-purity antibodies can be produced at low cost as a result.

It is a protocol which shows an example of the purification method of the antibody of this invention. It is a figure which shows the result of having examined the buffer used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use Fc binding protein fixed gel). It is a figure which shows the result of having examined the sodium chloride density | concentration added to the buffer used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use Fc binding protein fixed gel). It is a figure which shows the result of having examined the water-soluble organic solvent added to the buffer solution used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use Fc binding protein fixed gel). iPrOH refers to isopropanol. It is a figure which shows the result of having examined the urea density | concentration added to the buffer solution used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use Fc binding protein fixed gel). It is a figure which shows the result of having examined the buffer used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use Fc binding protein fixed gel). It is a figure which shows the result of the reference example 1. It is a figure which shows the result of having examined the buffer solution used by the 2nd washing | cleaning in the purification method of the antibody of this invention (use protein A fixed vinyl polymer gel). It is a protocol which shows another example of the purification method of the antibody of this invention. It is a figure which shows the result of having examined the component added to the to-be-purified solution in the purification method of the antibody of this invention (use Fc binding protein fixed gel). It is a figure which shows the result of having examined the component added to the to-be-purified solution in the purification method of the antibody of this invention (use protein A fixed vinyl polymer gel). It is a figure which shows a result when the purification method of the antibody of this invention is applied with respect to the solution containing a high concentration of antibody (use Fc binding protein fixed gel). It is a figure which shows a result when the purification method of the antibody of this invention is applied with respect to the solution containing an antibody high concentration (use of protein A fixed agarose gel). It is a figure which shows a result when the purification method of the antibody of this invention is applied with respect to the solution containing a high concentration of antibody (use Fc binding protein fixed gel). It is a figure which shows a result when the purification method of the antibody of this invention is applied with respect to the solution containing a high concentration of antibody (use Fc binding protein fixed gel).

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example and a reference example, this invention is not limited to the said example.

Example 1 Purification of antibody using Fc-binding protein-immobilized gel (1) Escherichia coli was transformed with plasmid DNA containing a polynucleotide encoding an Fc-binding protein consisting of the amino acid sequence of SEQ ID NO: 1 to transform the transformant. Obtained. The obtained Fc binding protein was obtained by culturing the obtained transformant and further purifying the Fc binding protein from the obtained bacterial cells.
(2) After introducing an amino group by sequentially reacting 1,6-hexanediol diglycidyl ether and ethylenediamine with a hydroxyl group in a vinyl polymer gel for separation agent (Toyopearl, manufactured by Tosoh Corporation), 3-maleimidopropionic acid N- Succinimidyl was reacted to obtain Toyopearl activated with a maleimide group. The obtained activated Toyopearl was reacted with the Fc-binding protein prepared in (1) to obtain an Fc-binding protein-immobilized gel.
(3) A Tricorn 5/20 column (manufactured by GE Healthcare) was filled with 0.1 mL of an Fc-binding protein-immobilized gel and placed in a liquid chromatograph.
(4) CHO cell culture supernatant (hereinafter simply referred to as “the CHO cell containing the antibody” obtained by culturing CHO cells (ATCC No. CRL-12445) producing anti-IL-8 antibody and clarifying the culture supernatant). 1 mL (referred to as “CHO cell culture supernatant”) was added to a column packed with an Fc-binding protein-immobilized gel previously washed and equilibrated with PBS (Phosphate Buffered Saline).
(5) After washing the column by passing PBS for 10 minutes (50 CV (Column Volume)), 50 mM glycine-hydrochloric acid buffer (pH 3.0) was passed for 3 minutes (15 CV) to adsorb to the gel. The antibody was eluted and collected.
(6) The cultured cell-derived protein (HCP) contained in the recovered antibody solution was quantified according to the attached protocol by ELISA using CHO Host Cell Protein 3rd Generation (manufactured by CYGNUS Technologies).

  As a result, the recovered antibody solution contained 6998 ng of HCP.

Example 2 Examination of Second Washing in Antibody Purification Using Fc-Binding Protein Immobilized Gel Antibody purification using an Fc-binding protein-immobilized gel as shown in FIG.
(1) After washing and equilibrating the column filled with the Fc-binding protein-immobilized gel prepared in Example 1 (3) with PBS, 1 mL of the CHO cell culture supernatant obtained in Example 1 (4) Was added.
(2) The first washing was performed by passing PBS through the column for 10 minutes (50 CV), then the second washing was performed by passing the washing solution through the column for 3 minutes (15 CV), and finally 50 mM. The glycine-hydrochloric acid buffer (pH 3.0) was passed through a column for 3 minutes (15 CV) to elute and collect the antibody adsorbed on the gel.
(3) HCP in the antibody solution recovered by the method described in Example 1 (6) was quantified.

  First, the pH of the cleaning solution used in the second cleaning was examined. As a result, the amount of HCP in the antibody solution decreased by washing with a buffer solution of pH 8 or higher compared to when the second washing was not performed (FIG. 2). Further, when sodium chloride was added to the buffer solution, the amount of HCP in the antibody solution was further reduced (FIG. 2).

  Therefore, the concentration of sodium chloride added to the buffer was examined. As a result of the second washing using the washing solution in which various concentrations of sodium chloride were added to the borate buffer (pH 9), when sodium chloride was added in a final concentration of 0.2 M or more, the buffer without addition of sodium chloride The amount of HCP in the antibody solution was reduced compared to when the second washing was performed with the solution. In particular, when the second washing was performed with a buffer solution to which sodium chloride was added at a final concentration of 0.4 M to 0.5 M, the amount of HCP in the antibody solution was further reduced (FIG. 3).

  Next, antibody purification from the CHO cell culture supernatant was examined using a washing solution in which an organic solvent was further added to a borate buffer solution (pH 9) containing 0.5 M sodium chloride at a final concentration. As a result, the amount of HCP in the antibody solution was further reduced by performing the second washing with a washing liquid to which a water-soluble organic solvent such as isopropanol or acetonitrile was further added as compared with the case where no water-soluble organic solvent was added (FIG. 4). . In addition, when isopropanol was added in the water-soluble organic solvent and examined, the amount of HCP in the antibody solution decreased as the amount of isopropanol added increased, and when 5% (v / v) or more was added, water-soluble organic solvent was added. Compared to when no solvent was added, the amount of HCP in the antibody solution was about one-third or less (FIG. 4).

  Next, antibody purification from the CHO cell culture supernatant using a washing solution in which urea was further added to a borate buffer solution (pH 9) added with 0.5 M sodium chloride at a final concentration was examined. As a result, when the second washing was performed with a washing solution to which urea was added at a final concentration of 0.5 M or more, the amount of HCP in the antibody solution was further reduced as compared with the case where urea was not added (FIG. 5). On the other hand, no effect was observed when the amount of urea added was 0.1 M in the final concentration (FIG. 5).

  FIG. 6 summarizes the results of FIGS. In addition, when a second wash is performed using a wash solution in which isopropanol and urea, which are water-soluble organic solvents, are added to a borate buffer solution (pH 9) containing 0.5 M sodium chloride at a final concentration, only isopropanol or urea is obtained. The amount of HCP in the antibody solution was further reduced compared to when the second washing was performed using the added washing solution (FIG. 6).

Example 3 Antibody Purification by Protein A-Immobilized Agarose Gel (1) 0.1 mL of MabSelect SuRe (manufactured by GE Healthcare), a protein A-immobilized agarose gel, was added to a Tricorn 5/20 column (manufactured by GE Healthcare). Packed and placed in a liquid chromatograph.
(2) 1 mL of the CHO cell culture supernatant obtained in Example 1 (4) was added to a column packed with protein A-immobilized agarose gel that had been washed and equilibrated in advance with PBS.
(3) Antibody purification and HCP quantification were carried out by the methods described in Example 1 (5) and (6).

  As a result, the recovered antibody solution contained 558 ng of HCP.

Reference Example 1 Examination of Second Washing in Antibody Purification by Protein A-Immobilized Agarose Gel Antibody purification by protein A-immobilized agarose gel according to the protocol with the second washing shown in FIG. 1 was examined.
(1) After washing and equilibrating the column filled with the protein A-immobilized agarose gel prepared in Example 3 (1) with PBS, 1 mL of the CHO cell culture supernatant obtained in Example 1 (4) was used. Added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). As the washing solution, a 50 mM citrate buffer (pH 4.4) or a Tris buffer (pH 9) containing 10% (v / v) isopropanol and 1.0 M (final concentration) urea was used.

  The results are shown in FIG. By performing the second washing with the washing liquid, the amount of HCP in the antibody solution was reduced as compared with the case where the second washing was not performed. However, when citrate buffer was used as the washing solution, it was confirmed that a part of the antibody adsorbed on the gel leaked into the washing solution, and the antibody recovery rate in the antibody solution decreased. When the buffer solution containing only isopropanol and urea was used as the washing solution, the decrease rate of the HCP amount was only about 15%.

Example 4 Antibody Purification with Protein A-Immobilized Vinyl Polymer Gel (1) 0.1 mL of TOYOPEARL AF-rProtein A-650F (Tosoh Corp.), which is a protein A-immobilized vinyl polymer gel, was added to a Tricorn 5/20 column (GE Health). And manufactured in a liquid chromatograph.
(2) 1 mL of the CHO cell culture supernatant obtained in Example 1 (4) was added to a column packed with a protein A-immobilized vinyl polymer gel that had been previously washed and equilibrated with PBS.
(3) Antibody purification and HCP quantification were carried out by the methods described in Example 1 (5) and (6).

  As a result, the collected antibody solution contained 1001 ng of HCP.

Example 5 Examination of Second Washing in Antibody Purification with Protein A-Immobilized Vinyl Polymer Gel Antibody purification with protein A-immobilized vinyl polymer gel was examined according to the protocol with the second washing shown in FIG.
(1) After washing and equilibrating the column filled with the protein A-immobilized vinyl polymer gel prepared in Example 4 (1) with PBS, 1 mL of the CHO cell culture supernatant obtained in Example 1 (4) Was added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was Tris buffer (pH 9) to which 10% (v / v) isopropanol and 1.0 M urea were added, or 10% (v / v) isopropanol and 0.5 M (final concentration) sodium chloride. A borate buffer solution (pH 9) to which was added was used.

  The results are shown in FIG. Compared with the case where the second washing was not performed by performing the second washing with the washing solution, the amount of HCP in the antibody solution was reduced, and a buffer solution containing sodium chloride and isopropanol was used as the washing solution. In particular, the amount of HCP was reduced (632 ng when using a Tris buffer added with isopropanol and urea, and 102 ng when using a borate buffer added with sodium chloride and isopropanol).

Example 6 Examination of addition of washing components in antibody purification using Fc-binding protein-immobilized gel In Examples 2 and 5 and Reference Example 1, the second washing was added to purify the antibody. Instead of the second washing, components to be added to the washing solution used in the second washing were previously added to the solution to be purified to purify the antibody. The protocol is shown in FIG.
(1) After each component was added to 1 mL of the CHO cell culture supernatant obtained in Example 1 (4), the column was packed with an Fc-binding protein-immobilized gel previously washed and equilibrated with PBS (Example 1 ( 3)) or added to a column packed with protein A-immobilized vinyl polymer gel (prepared in Example 4 (1)). The component to be added is sodium chloride (0.5 M for CHO cell culture supernatant), isopropanol (10% (v / v)), or sodium chloride (0.5 M for CHO cell culture supernatant) + isopropanol ( 10% (v / v)).
(2) After washing by passing PBS through the column for 10 minutes (50 CV), 50 mM glycine-hydrochloric acid buffer (pH 3.0) was passed through the column for 3 minutes (15 CV) to adsorb to the gel. The antibody was eluted and collected.
(3) HCP was quantified by the method described in Example 1 (6).

  FIG. 10 shows the results when using the Fc-binding protein-immobilized gel, and FIG. 11 shows the results when using the protein A-immobilized vinyl polymer gel. In either case, the amount of HCP in the antibody solution is greatly reduced by adding sodium chloride to the solution to be purified (CHO cell culture supernatant) before purification, compared with the case of purifying the solution to be purified as it is. did. When the Fc-binding protein-immobilized gel was used, the amount of HCP in the antibody solution was further reduced by further adding isopropanol to the solution to be purified (FIG. 10).

Example 7 Antibody Purification from Solution Containing High Concentration of Antibody Using Fc Binding Protein Immobilized Gel (Part 1)
The antibody contained in the CHO cell culture supernatant obtained in Example 1 (4) is about 0.08 mg / mL, but due to the recent progress in antibody production technology, the antibody concentration contained in the culture supernatant is 1 mg / mL. Examples of more than mL, and further 10 mg / mL or more have been reported. Therefore, it was examined whether the antibody purification method of the present invention can be applied to a solution containing a high concentration of antibody.
(1) Human gamma globulin (manufactured by Chemical and Serum Therapy Laboratories) was added to the CHO cell supernatant to a final concentration of 2 mg / mL to prepare a CHO cell culture supernatant containing a high concentration of antibody.
(2) The column packed with the Fc-binding protein-immobilized gel prepared in Example 1 (3) was washed and equilibrated with PBS, and then the CHO cell culture supernatant containing the antibody prepared in (1) at a high concentration 1 mL was added.
(3) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was phosphate buffer (pH 7) to which 10% (v / v) isopropanol and 0.5M (final concentration) sodium chloride were added, or 10% (v / v) isopropanol and 0.5M (pH). A borate buffer solution (pH 9) to which sodium chloride (final concentration) was added was used.

  The results are shown in FIG. By performing the second wash using a neutral or basic buffer to which isopropanol and sodium chloride have been added, the amount of HCP in the antibody solution is reduced to 1/10 compared to the case where the second wash is not performed. It decreased to the following.

Example 8 Antibody Purification from Solution Containing High Concentration of Antibody Using Fc Binding Protein Immobilized Gel (Part 2)
In Example 6, it has been confirmed that the amount of HCP in the antibody solution decreases when the components to be added to the washing solution are added to the solution to be purified in advance and purification is performed without performing the second washing. Therefore, it was confirmed whether the amount of HCP was further reduced by adding the component added to the cleaning solution to the solution to be purified and further performing the second cleaning.
(1) Fc-binding protein-immobilized gel prepared by adding 0.5 M sodium chloride to 1 mL of CHO cell culture supernatant containing a high concentration of the antibody prepared in Example 7 (1), and then washing and equilibrating in advance with PBS Was added to the column (produced in Example 1 (3)).
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was phosphate buffer (pH 7) to which 10% (v / v) isopropanol and 0.5M (final concentration) sodium chloride were added, 10% (v / v) isopropanol and 0.5M (final). Tris buffer (pH 8) to which 10% (v / v) isopropanol and 0.5 M (final concentration) sodium chloride were added (pH 9), 10% (v) Borate buffer (pH 9) with ethanol / v) and 0.5M (final concentration) sodium chloride, or 20% (v / v) ethanol and 0.5M (final concentration) sodium chloride. The borate buffer solution (pH 9) was used.

  The results are shown in FIG. By performing the second washing with a buffer solution to which a water-soluble organic solvent such as isopropanol or ethanol and sodium chloride are added, the amount of HCP in the antibody solution is 7 minutes compared to the case where the second washing is not performed. It decreased to 1 or less. It can also be seen that the amount of HCP in the antibody solution is reduced compared to the case where the solution to be purified is directly added to the column (Example 7). That is, it can be seen that the amount of HCP in the antibody solution is further reduced by performing the second washing and adding the components added to the washing solution used for the second washing to the solution to be purified.

  In addition, it can be said that it is preferable that the pH of a washing | cleaning liquid is high (basic side) at the point which the amount of HCP in an antibody solution reduces. It can also be seen that the amount of HCP in the antibody solution decreases with increasing amount of ethanol, as with isopropanol.

Example 9 Antibody Purification from Solution Containing High Concentration of Antibody Using Protein A-Immobilized Agarose Gel (Part 1)
(1) After washing and equilibrating the column filled with the protein A-immobilized agarose gel prepared in Example 3 (1) with PBS, CHO cell culture containing the antibody prepared in Example 7 (1) at a high concentration 1 mL of supernatant was added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was Tris buffer (pH 9) with 10% (v / v) isopropanol and 1.0M (final concentration) urea added, 10% (v / v) isopropanol and 0.5M (final concentration). A phosphate buffer solution (pH 7) to which sodium chloride was added, or a borate buffer solution (pH 9) to which 10% (v / v) isopropanol and 0.5 M (final concentration) sodium chloride were added was used.

  The results are shown in FIG. When the second wash was performed using a buffer solution to which only isopropanol and urea were added without adding sodium chloride, the rate of decrease in the amount of HCP in the antibody solution was 25 compared to the case where the second wash was not performed. %. On the other hand, when the second washing was performed with a buffer solution containing sodium chloride and isopropanol, the amount of HCP in the antibody solution decreased to 1/20 or less.

Example 10 Antibody Purification from a Solution Containing High Concentration of Antibody on Protein A-Immobilized Agarose Gel (Part 2)
(1) After adding sodium chloride to 0.5 M to 1 mL of the CHO cell culture supernatant containing a high concentration of the antibody prepared in Example 7 (1), the protein A was immobilized by washing and equilibrating in advance with PBS. It added to the column (made by Example 3 (1)) filled with the agarose gel.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was phosphate buffer (pH 7) to which 10% (v / v) isopropanol and 0.5M (final concentration) sodium chloride were added, or 10% (v / v) isopropanol and 0.5M (pH). A borate buffer solution (pH 9) to which sodium chloride (final concentration) was added was used.

  The amount of HCP in the antibody solution was decreased by performing the second washing with a buffer solution to which sodium chloride and isopropanol had been added, compared to the case without the second washing (without the second washing: 443 ng, When using phosphate buffer: 107 ng, when using borate buffer: 109 ng).

Example 11 Antibody Purification from Solution Containing High Concentration of Antibody with Protein A-Immobilized Vinyl Polymer Gel (Part 1)
(1) The column filled with the protein A-immobilized vinyl polymer gel prepared in Example 4 (1) was washed with PBS and equilibrated, and then CHO cells containing the antibody prepared in Example 7 (1) at a high concentration. 1 mL of culture supernatant was added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was phosphate buffer (pH 7) to which 10% (v / v) isopropanol and 0.5M (final concentration) sodium chloride were added, or 10% (v / v) isopropanol and 0.5M (pH). A borate buffer solution (pH 9) to which sodium chloride (final concentration) was added was used.

  By performing the second washing with a buffer solution to which sodium chloride and isopropanol had been added, the amount of HCP in the antibody solution was reduced as compared to the case without the second washing (without the second washing: 1157 ng, When using phosphate buffer: 81 ng, when using borate buffer: 85 ng).

Example 12 Antibody Purification from a Solution Containing High Concentration of Antibody with Protein A-Immobilized Vinyl Polymer Gel (Part 2)
(1) After adding sodium chloride to a final concentration of 0.5 M to 1 mL of a CHO cell culture supernatant containing a high concentration of the antibody prepared in Example 7 (1), protein A was washed and equilibrated in advance with PBS. It added to the column (made by Example 4 (1)) filled with the fixed vinyl polymer gel.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was phosphate buffer (pH 7) to which 10% (v / v) isopropanol and 0.5M (final concentration) sodium chloride were added, or 10% (v / v) isopropanol and 0.5M (pH). A borate buffer solution (pH 9) to which sodium chloride (final concentration) was added was used.

  The amount of HCP in the antibody solution was reduced by performing the second washing with a buffer solution containing sodium chloride and isopropanol compared to the case without the second washing (without the second washing: 518 ng, When using phosphate buffer: 91 ng, when using borate buffer: 69 ng).

Example 13 Antibody Purification from Solution Containing High Concentration of Antibody Using Fc Binding Protein Immobilized Gel (Part 3)
(1) The column packed with the Fc-binding protein-immobilized gel prepared in Example 1 (3) was washed and equilibrated with PBS, and then the CHO cells containing the antibody prepared in Example 7 (1) at a high concentration 1 mL of culture supernatant was added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution was a buffer solution (pH 7, pH 9 or pH 10) containing 0.1% (v / v) Tween 20 and 0.5M (final concentration) sodium chloride, 0.5% (v / v). Tween 20 and 0.5 M (final concentration) sodium chloride buffer (pH 7, pH 9 or pH 10), or 0.5% (v / v) Tween 80 and 0.5 M (final concentration) sodium chloride. The added buffer (pH 7, pH 9 or pH 10) was used.

  The results are shown in FIG. The amount of HCP in the antibody solution is obtained by performing the second washing with a neutral or basic buffer solution to which a nonionic surfactant and sodium chloride are added, as compared with the case where the second washing is not performed. Decreased from 60% to 80%.

Example 14 Antibody Purification from Solution Containing High Concentration of Antibody Using Fc Binding Protein Immobilized Gel (Part 4)
(1) After the column packed with the Fc-binding protein-immobilized gel prepared in Example 1 (1) was washed and equilibrated with PBS, the CHO cells containing the antibody prepared in Example 7 (1) at a high concentration 1 mL of culture supernatant was added.
(2) The antibody was purified by the method described in Example 2 (2), and HCP was quantified by the method described in Example 1 (6). The washing solution is a buffer solution (pH 10) to which 0.5 M (final concentration) L-valine and 0.5 M sodium chloride are added, 0.2 M (final concentration) L-leucine and 0.5 M sodium chloride. Added buffer (pH 10), 0.4 M (final concentration) L-leucine and 0.5 M sodium chloride added buffer (pH 10), 0.2 M (final concentration) L-isoleucine and 0.5 M Buffer (pH 10) to which sodium chloride was added, buffer solution (pH 10) to which 0.4 M (final concentration) L-isoleucine and 0.5 M sodium chloride were added, 0.05 M (final concentration) glycine and 0 A buffer solution (pH 10) added with 5 M sodium chloride, or a buffer solution (pH 10) added with 0.2 M (final concentration) glycine and 0.5 M sodium chloride was used.

  The results are shown in FIG. Performing the second wash with a basic buffer to which hydrophobic amino acids (valine, leucine, isoleucine) and sodium chloride have been added. Compared with the case of washing with an acid buffer (pH 10)), the amount of HCP in the antibody solution decreased. On the other hand, when the second washing is performed using a basic buffer solution to which glycine and sodium chloride are added, compared with the case of washing with a buffer solution to which no amino acid has been added (borate buffer solution (pH 10)), in the antibody solution. The amount of HCP increased.

  The purification method of the present invention can improve the purification purity of the antibody as compared with the conventional antibody purification method using the carrier for affinity chromatography. Since the purification process load of the latter stage can be greatly reduced by the purification method of the present invention, high-purity antibodies can be produced at low cost as a result.

Claims (7)

A step of equilibrating a column packed with a carrier for affinity chromatography, a step of adding a solution containing an antibody to the equilibrated column, a step of washing impurities contained in the solution containing the antibody, and an eluent Elution of the antibody using a method for purifying an antibody,
The step of washing the contaminants is a first washing step of washing with the solution used for equilibration of the column, and a neutral or basic buffer containing sodium chloride having a final concentration of 0.2 M or more. The said purification method which consists of a 2nd washing | cleaning process. The purification method according to claim 1, wherein the step of adding the solution containing the antibody to the equilibrated column is performed after adding sodium chloride so that the concentration of the solution becomes 0.2 M or more with respect to the solution containing the antibody. The buffer used in the second washing step further contains any one or more of a water-soluble organic solvent, a nonionic surfactant, a hydrophobic amino acid, and urea having a final concentration of 0.3 M or more. 2. The purification method according to 2. The purification method according to claim 3, wherein the water-soluble organic solvent is any one of isopropanol, acetonitrile, and ethanol. The purification method according to claim 3, wherein the hydrophobic amino acid is any one of valine, leucine and isoleucine. Equilibrating a column packed with a carrier for affinity chromatography;
Adding sodium chloride so as to be 0.2 M or more with respect to the solution containing the antibody;
Adding a solution containing the antibody to the equilibrated column;
Washing impurities contained in the solution containing the antibody with the solution used for equilibration of the column;
Elution of the antibody with an eluate.
Antibody purification method. The purification method according to any one of claims 1 to 6, wherein the carrier for affinity chromatography is a carrier on which Fc-binding protein or protein A is immobilized.

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