JP2016003192A - Buffer solution and purification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a buffer solution which, in purifying purification protein to be purified from in a sample solution by using adsorbent comprising a calcium phosphate-based compound, can suppress decomposition/degradation of the adsorbent over a long period of time and is used as elute; and a purification method for purifying purification protein from in the sample solution in which the buffer solution is used as elute.SOLUTION: The buffer solution is used as elute in selectively eluting purification protein to be purified which has been adsorbed on a carrier at least whose surface is constituted by a calcium phosphate-based compound, from the carrier. The buffer solution contains at least one of phosphoric acid and salt thereof and at least one of a compound having a carboxyl group and salt thereof.

Description

  The present invention relates to a buffer solution used as an eluent when purifying a purified protein to be purified from a sample solution, and a purification method for purifying a purified protein from a sample solution using such a buffer solution as an eluent. It is.

  In recent years, as a purification method for purifying proteins such as antibodies (natural antibodies) contained in plasma and recombinant fusion proteins (artificial antibodies) produced by living genetically engineered host cells from a sample solution A hydroxyapatite method using a calcium phosphate compound such as hydroxyapatite or fluorapatite as an adsorbent (chromatographic carrier) has been proposed (for example, see Patent Document 1).

  In this hydroxyapatite method, generally, out of contaminants including a protein to be purified (purified protein) adsorbed on an adsorbent and other proteins (contaminated protein), only the purified protein is used as an effluent. An elution method is used in which the effluent is recovered while leaving the contaminants adsorbed on the adsorbent.

  In this elution method, for example, a buffer solution in which an eluent such as sodium chloride is added to a low-concentration phosphate buffer is used as the eluent. However, when a buffer solution containing sodium chloride (eluent) is used, the sodium ion of sodium chloride and the hydrogen ion of the phosphate group of the calcium phosphate compound are ion-exchanged, resulting in a rapid elution pH. To drop. For this reason, the adsorbent composed of the calcium phosphate compound is dissolved, so that the adsorbent is altered and deteriorated. As a result, there is a problem that the function as the adsorbent (carrier) is rapidly reduced.

  In order to suppress the decrease in pH, it is conceivable to increase the concentration of phosphoric acid in the eluate. In this case, contaminants will be eluted in addition to the purified protein. It is not preferable for improving the rate. Although a method using a good buffer such as MES buffer or HEPES buffer is also conceivable, the good buffer is expensive and expensive, so it is suitable for a laboratory level with a small scale, but an industrial level with a large scale. Not suitable for.

Special Table 2009-35464

  It is an object of the present invention to suppress alteration and deterioration of an adsorbent over a long period of time when purifying a purified protein to be purified from a sample solution using an adsorbent composed of a calcium phosphate compound. Another object of the present invention is to provide a buffer used as an eluate and a purification method for purifying a purified protein from a sample solution using the buffer as an eluent.

Such an object is achieved by the present inventions (1) to (7) below.
(1) A buffer used as an eluent when selectively purifying a purified protein to be purified, adsorbed on a carrier composed of a calcium phosphate compound at least on its surface, from the carrier,
A buffer comprising at least one of phosphoric acid and a salt thereof and at least one of a compound having a carboxy group and a salt thereof.

  By using this buffer solution as an eluent, when the purified protein to be purified from the sample solution is purified using an adsorbent composed of a calcium phosphate compound, this adsorbent is altered and deteriorated over a long period of time. Can be suppressed

  (2) The buffer solution according to (1), wherein the pH of the buffer solution is 5.5 or more and less than 8.0.

  Thereby, quality change (denaturation | denaturation) of purified protein can be prevented, and it can prevent that the characteristic changes. In addition, alteration of the carrier (dissolution, etc.) can be suitably prevented, and changes in the separation capacity in the adsorption apparatus can be prevented over a long period of time, so that the target protein can be purified with high accuracy over a long period of time. can do.

  (3) The buffer solution according to (1) or (2), wherein the total content of the phosphoric acid and the salt thereof is 1.0 mM or more and 400 mM or less.

  Thereby, it can suppress or prevent more accurately that the foreign substance adsorb | sucked to a support | carrier is eluted with a refined protein.

  (4) The buffer according to any one of (1) to (3), wherein the total content of the compound having a carboxy group and a salt thereof is 0.1 M or more and 5 M or less.

  Thereby, it can suppress or prevent more accurately that the foreign substance adsorb | sucked to a support | carrier is eluted with a refined protein.

  (5) The buffer solution according to any one of (1) to (4), wherein the compound having a carboxy group is an aliphatic carboxylic acid having two or more carbon atoms.

(6) The purified protein is a natural antibody having an immunoglobulin constant domain,
Or the buffer solution in any one of said (1) thru | or (5) which is a recombinant fusion protein.

  The buffer and the purification method of the present invention are suitably applied to the purification of natural antibodies or recombinant fusion proteins.

(7) A separation method for separating the purified protein from a sample solution containing a plurality of types of proteins including the purified protein,
A supply step of supplying the sample solution into an apparatus in which the carrier is filled in at least a part of a filling space;
The buffer solution according to any one of the above (1) to (6) is supplied into the device, and the effluent flowing out from the device is fractionated by a predetermined amount. And a fractionation step of purifying the purified protein in the fraction effluent.

  As a result, the purified protein can be purified from the sample solution while suppressing alteration and deterioration of the adsorbent over a long period of time.

  According to the buffer solution of the present invention, when the purified protein to be purified from the sample solution is purified using the adsorbent composed of the calcium phosphate compound, the alteration / deterioration of the adsorbent is suppressed over a long period of time. be able to.

  Therefore, according to the purification method of the present invention using such a buffer solution, the target protein can be purified with high accuracy over a long period of time.

It is a longitudinal cross-sectional view which shows an example of the adsorption | suction apparatus used by this invention. It is a figure which shows the change of pH accompanying supply of the eluate in Example 1A and Comparative Example 1A. It is a figure which shows the change of pH accompanying supply of the eluate in Example 2A, 3A and Comparative Example 2A. 4 is an absorbance curve measured when proteins contained in a 4STD sample were separated by an adsorption device using acetic acid (Example 1B) or sodium chloride (Reference Example 1B) as an eluent. It is an absorbance curve measured when BSA contained in a BSA sample was separated by an adsorption device using acetate (Example 2B) or sodium chloride (Reference Example 2B) as an eluent. It is an absorbance curve measured when IgG contained in an IgG sample was separated by an adsorption device using acetate (Example 3B) or sodium chloride (Reference Example 3B) as an eluent. It is an absorbance curve measured when DNA contained in a DNA sample was separated by an adsorption device using acetate (Example 4B) or sodium chloride (Reference Example 4B) as an eluent.

Hereinafter, preferred embodiments of the buffer and the purification method of the present invention will be described in detail.
Prior to describing the buffer solution and the purification method of the present invention, the terms used in the description are defined below in describing the preferred embodiment.

  “Antibody” refers to a protein or protein complex, each containing at least one immunoglobulin variable domain and at least one immunoglobulin constant domain. Also, the “antibody” may be a single chain antibody, a dimeric antibody, or any higher protein complex, including but not limited to heterodimeric antibodies. is not.

“Immunoglobulin constant domain” refers to an immunoglobulin domain that is identical or substantially similar to a C _L , C _H 1, C _H 2, C _H 3 or C _H 4 domain of human or animal origin, This also includes the Fc part. (See, for example, Charles A Hasmann and J. Donald Capra, Immunoglobulin: Structure and Function, William E. Paul, Ed., Fundamental Immunology, 2nd edition, 209, 210-218 (1989)).

An “immunoglobulin variable domain” refers to an immunoglobulin domain that is the same or substantially similar to a _VL or _VH domain from a human or animal origin. In the present invention, the biological activity of an immunoglobulin variable domain for determining substantial similarity is antigen binding.

  “Adsorbent (carrier)” is composed of at least one kind of molecule immobilized on a solid support or at least one kind of molecule that is itself a solid, and is used for performing various types of chromatography. In the present invention, at least the surface is composed of a calcium phosphate-based compound.

  Next, an example of an adsorption apparatus used in the purification method of the present invention, that is, an adsorption apparatus (separation apparatus) used for protein purification using the buffer solution of the present invention as an eluent will be described.

<Adsorption device>
FIG. 1 is a longitudinal sectional view showing an example of an adsorption device used in the present invention. In the following description, the upper side in FIG. 1 is referred to as “inflow side” and the lower side is referred to as “outflow side”.

  Here, the inflow side means that when purifying (separating) the purified protein (target substance) to be purified, the sample solution (liquid containing the purified protein) and a liquid such as a buffer solution as an eluent are adsorbed by the adsorption device. The outflow side is the side opposite to the inflow side, that is, the side from which the liquid flows out from the adsorbing device as the outflow liquid.

  The adsorption device 1 shown in FIG. 1 has a column 2, an adsorbent (filler) 3, and two filter members 4 and 5.

  The column 2 includes a column main body 21 and caps (lid bodies) 22 and 23 attached to the inflow side end portion and the outflow side end portion of the column main body 21, respectively.

  The column main body 21 is composed of, for example, a cylindrical member. Examples of the constituent material of each part (each member) constituting the column 2 including the column main body 21 include various glass materials, various resin materials, various metal materials, various ceramic materials, and the like.

  In the column body 21, caps 22, 23 are screwed into the inflow side end and the outflow side end, respectively, in a state where the filter members 4, 5 are arranged so as to block the inflow side opening and the outflow side opening, respectively. It is attached by the match.

  In the column 2 having such a configuration, an adsorbent filling space 20 is defined by the column main body 21 and the filter members 4 and 5. At least a part of the adsorbent filling space 20 (almost full in this embodiment) is filled with the adsorbent (carrier) 3.

  The volume of the adsorbent filling space 20 is appropriately set according to the volume of the sample solution, and is not particularly limited, but is preferably about 0.05 to 100 mL, more preferably about 0.1 to 100 mL with respect to 1 mL of the sample solution. More preferably, about 0.5 to 50 mL.

  Further, the column 2 is configured such that liquid tightness between the column main body 21 and the caps 22 and 23 is ensured with the caps 22 and 23 being mounted.

  An inflow pipe 24 and an outflow pipe 25 are fixed (fixed) in a liquid-tight manner at substantially the centers of the caps 22 and 23, respectively. A sample liquid (liquid) is supplied to the adsorbent 3 through the inflow pipe 24 and the filter member 4. The sample solution supplied to the adsorbent 3 passes between the adsorbents 3 (gap) and flows out of the column 2 through the filter member 5 and the outflow pipe 25.

  Each of the filter members 4 and 5 has a function of preventing the adsorbent 3 from flowing out of the adsorbent filling space 20. These filter members 4 and 5 are, for example, glass nonwoven fabrics, foams (communication holes) made of a synthetic resin such as a glass sintered body, metal or polyurethane, polyvinyl alcohol, polypropylene, polyether polyamide, polyethylene terephthalate, and polybutylene terephthalate. Sponge-like porous body), woven fabric, mesh or the like.

  Moreover, the adsorption | suction apparatus 1 is provided with the thing by which the surface was comprised with the calcium-phosphate type compound at least as the adsorbent 3. FIG.

  Since the adsorbent 3 specifically adsorbs with the specific adsorption (supporting) force of the target purified protein contained in the sample solution, the purified protein and the Contaminants (impurities) such as other contaminating proteins different from the purified protein are separated and purified.

As the calcium phosphate-based compound is not particularly limited, for example, hydroxyapatite _{(Ca 10 (PO 4) 6} (OH) 2), TCP (Ca 3 (PO 4) 2), Ca 2 P 2 O 7, Ca (PO ₃ ) ₂ , DCPD (CaHPO ₄ .2H ₂ O), Ca ₄ O (PO ₄ ) _2, or some of these substituted with other atoms or atomic groups, etc. Species or a combination of two or more can be used.

  Among these, the calcium phosphate compound preferably has hydroxyapatite as a main component. In particular, since hydroxyapatite is close to a component constituting a living body, it is possible to suitably prevent the purified protein from being altered (denatured) when adsorbed / separated purified protein. Furthermore, the hydroxyapatite is preferably one in which at least a part of the hydroxyl group it has is substituted with a fluorine atom. Hydroxyapatite substituted with a fluorine atom (hereinafter referred to as “fluorapatite”) has a fluorine atom (fluorine ion) present in the crystal structure, and thus the calcium atom (calcium ion) is released from the fluorine apatite. It can prevent more reliably. Moreover, the strength of the adsorbent 3 is further improved by forming at least the vicinity of the surface of the adsorbent 3 with such fluorapatite. Hereinafter, hydroxyapatite and hydroxyapatite substituted with fluorine atoms may be collectively referred to as “apatite”.

  Moreover, the adsorbent 3 which has high intensity | strength can be obtained by comprising at least the surface vicinity with a calcium-phosphate type compound. For this reason, it is possible to prevent the adsorbent 3 from being easily deformed / collapsed by its own weight for a long time. Therefore, it is possible to prevent the adsorbent 3 filled in the lower part of the adsorbent filling space 20 from collapsing and causing the adsorber 1 to be clogged. As a result, a large amount of sample solution can be processed, that is, the purified protein to be purified can be reliably separated from the sample solution.

  The form (shape) of the adsorbent 3 as described above is preferably particulate (granular) as shown in FIG. 1, but in addition, for example, pellet (small block), block (for example, Alternatively, a porous body in which adjacent pores communicate with each other, a honeycomb shape, a monolith, or the like can be used. In addition, by making the adsorbent 3 particulate, the surface area thereof can be increased, and the protein separation characteristics can be improved.

  The average particle diameter of the particulate adsorbent 3 is not particularly limited, but is preferably about 0.5 to 150 μm, and more preferably about 10 to 80 μm. By using the adsorbent 3 having such an average particle diameter, it is possible to ensure a sufficient surface area of the adsorbent 3 while reliably preventing the filter member 5 from being clogged.

  Next, a purification method (purification method of the present invention) for purifying a target protein contained in a sample solution using such an adsorption device (separation device) 1 will be described.

<Purification method>
In the purification method of the present embodiment, the purified protein to be purified is contained in the adsorption device 1 in which at least a part of the filling space is filled with a filler (carrier) whose surface is composed of a calcium phosphate compound. By supplying a sample solution and supplying the buffer solution of the present invention into the adsorption device 1 and fractionating the effluent flowing out of the adsorption device 1 by a predetermined amount. In the effluent of each fractionated fraction, there is a fractionation step (second step) for separating the purified protein.

Hereinafter, this purification method will be described in detail.
In the following, a case where the target purified protein is purified from a sample solution containing an immunoglobulin constant domain and containing a natural antibody or a recombinant fusion protein will be described as a representative.

[1] Supplying step First, after preparing a sample solution containing the purified protein to be purified, this sample solution is contacted by supplying it to the adsorbent (filler) 3.

  [1-1] First, a sample solution containing a purified protein (target substance) to be purified is prepared.

  This purified protein is a protein having an immunoglobulin constant domain (eg, the Fc portion of an antibody) or a complex of the protein, and is produced, for example, by a host cell that has been genetically engineered to produce such a protein. Is done.

  In this case, methods for genetically engineering cells to produce purified protein are well known in the art (eg, Ausubel et al. Ed. (1990), Current Protocols in Molecular Biology (Willey, NY)). )reference.).

  The purified protein only needs to have one or more immunoglobulin constant domains, may have one or more immunoglobulin variable domains, and does not have immunoglobulin variable domains. There may be.

  Further, the purified protein may be a natural protein (natural antibody) or a recombinant fusion protein, and examples of the recombinant fusion protein include those having the Fc part of an antibody, which are non-antibody proteins. Also good.

  Specific examples of recombinant fusion proteins include those comprising one or more immunoglobulin constant domains (eg, the Fc portion of an antibody) and a protein that is the same or substantially similar to any of the following proteins.

  Examples of the purified protein include flt3 ligand (see International Patent Application WO94 / 28391), CD40 ligand (see US Pat. No. 6,087,329), erythropoietin, thrombopoietin, calcitonin, Fas ligand, NF-κB. Ligand for receptor activator (RANKL), tumor necrosis factor (TNF) -related apoptosis-inducing ligand (TRAIL, see International Patent Application WO 97/01633), thymic stroma-derived lymphopoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (See GM-CSF, Australian Patent No. 588819), mast cell growth factor, stem cell growth factor, epidermal growth factor, RANTES, growth hormone, insulin, insulinotropin, insulin Growth factor, parathyroid hormone, interferon, nerve growth factor, glucagon, interleukins 1-18, colony stimulating factor, lymphotoxin-β, tumor necrosis factor (TNF), leukemia inhibitory factor, oncostatin-M, and cell surface molecule ELK And various ligands for Hek (for example, ligands for eph-related kinases or LERKS) and the like.

  Also, specific examples of recombinant fusion proteins include one or more immunoglobulin constant domains (eg, the Fc portion of an antibody) and a receptor for any of the purified proteins or substantially similar to such a receptor. The thing containing protein is mentioned.

  The receptors include both forms of TNFR (referred to as p55 and p75), type I and type II interleukin-1 receptors (EP Patent No. 0 460 846, US Pat. No. 4,968,607 and US Pat. No. 5,767,064), interleukin-2 receptor, interleukin-4 receptor (see EP Patent No. 0 367 566 and US Pat. No. 5,856,296), interleukin-15. Receptor, interleukin-17 receptor, interleukin-18 receptor, granulocyte macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptor for oncostatin-M and leukemia inhibitory factor, NF-κB Receptor activator (see RANK, US Pat. No. 6,271,349), TRAIL Receptors (including the TRAIL receptors 1, 2, 3 and 4), and receptors that comprise death domains, e.g., Fas or Apoptosis inducing receptor (AIR), and the like.

  In addition, specific examples of recombinant fusion proteins include one or more immunoglobulin constant domains (eg, the Fc portion of an antibody) and a differentiation antigen (referred to as a CD protein) or a differentiation antigen or a protein that is substantially similar to a differentiation antigen. The thing containing is mentioned.

  The differentiation antigen is shown in Leukocyte Type VI (Proceedings of the VITH International Workshop and Conference, Kishimoto, Kikutani et al., Ed., Japan, Kobe, 1996). CD40, and ligands for them (CD27 ligand, CD30 ligand, etc.).

  Some CD antigens are members of the TNF receptor family, including 41BB ligand and OX40. The ligand is a member of the TNF family similar to the 41BB ligand and the OX40 ligand.

  Examples include recombinant fusion proteins comprising at least one immunoglobulin constant domain and all or part of a protein substantially similar to any of the following proteins or their ligands or any of these: For example, members of the metalloproteinase-disintegrin family, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein AI, globin, IL -2 antagonists, α-1 antitrypsin, TNF-α converting enzyme, ligands for any of the enzymes, and numerous other enzymes and their ligands.

  Furthermore, purified proteins may include antibodies (natural antibodies) or portions thereof, and chimeric antibodies, such as antibodies in which human immunoglobulin constant domains are linked to one or more murine immunoglobulin variable domains, or fragments thereof. Alternatively, it may be a conjugate containing an antibody and a cytotoxic substance or a luminescent substance. Cytotoxic or luminescent substances include maytansine derivatives (eg DM1); enterotoxins (eg staphylococcal enterotoxins); iodine isotopes (eg I-125); technium isotopes (eg Tc-99m ); Cyanine fluorescent dyes (eg, Cy5.5.18); and ribosome inactivating proteins (eg, bouganin, gelonin, or saporin-S6).

  Examples of the antibody, antibody / cytotoxin conjugate, or antibody / luminophore conjugate include those that recognize any one or a combination of the above proteins and / or the following antigens. , CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α IL-1β, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor Subunit, PDGF-β, VEGF, TGF, TGF-β2, TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigen C In serum of patients with A125, tumor antigen MUCI, PEM antigen, LCG (gene product expressed in association with lung cancer), HER-2, tumor-associated glycoprotein TAG-72, SK-1 antigen, colon cancer and / or pancreatic cancer Tumor-associated epitopes present in high concentrations in breast, colon, squamous cells, prostate, pancreas, lung and / or kidney cancer cells, and / or cancer-associated epitopes or proteins expressed in melanoma, glioma or neuroblast, Tumor necrosis core, integrin α4β7, integrin VLA-4, B2 integrin, TRAIL receptors 1, 2, 3 and 4, RANK, RANK ligand, TNF-α, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), cells Interadhesion molecule-3 (ICAM-3), leucointegrin adhesin, platelet sugar tamper GpIIb / IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (factor VIIa-tissue factor inhibitor), MHC I, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), tumor necrosis factor (TNF) CTLA-4 (which is a cytotoxic T lymphocyte-related antigen), Fc-γ-1 receptor, HLA-DR 10β, HLA-DR antigen, L-selectin, IFN-γ, respiratory rash virus, human immunity Examples include deficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutans and Staphylococcus aures.

  Such a purified protein-containing sample solution contains impurities (contaminants) other than this purified protein. Examples of such impurities include DNA, RNA, and immunoglobulin constant domains. Examples thereof include biopolymers such as proteins (contaminating proteins) other than proteins (purified proteins).

  More specifically, when the purified protein is produced by a genetically engineered host cell, the contaminant is a protein that does not have DNA, RNA, or immunoglobulin constant domain derived from the host cell (contaminant protein). In addition, when the protein is a natural antibody, a protein having no immunoglobulin constant domain (contaminating protein) such as albumin, β globulin, and α globulin contained in plasma is included.

  In addition, the sample solution prepared in the present embodiment may be any sample solution as long as it contains impurities in addition to the purified protein to be purified, and may be obtained by removing a part of the impurities in advance.

  Examples of the method for removing contaminants from the sample solution in advance include affinity chromatography using, as an adsorbent, a material having an immobilizing member in which a binding protein such as protein A is immobilized on a solid support. It is done.

  In this embodiment, even if the method for removing contaminants as described above is applied to the sample solution, not all of the contaminants are removed from the sample solution, and at least some of the contaminants are It will remain in the sample solution.

  [1-2] Next, the obtained sample solution is supplied to the adsorbent 3 through the inflow pipe 24 and the filter member 4, and is passed through the column 2 (adsorption device 1). Make contact.

  As a result, a purified protein (target substance to be purified) that adsorbs to the adsorbent 3 and a contaminant protein other than this protein that has a relatively high adsorbing capacity to the adsorbent 3 are contained in the column 2. Retained. Then, contaminating proteins having low adsorbability with respect to the adsorbent 3 and contaminants other than proteins flow out from the column 2 through the filter member 5 and the outflow pipe 25.

[2] Fractionation step Next, an eluate (washing solution) for flowing the purified protein is supplied from the inflow pipe 24 of the adsorption device 1 into the column 2. Then, the effluent flowing out from the column 2 through the effluent pipe 25 is fractionated (collected) by a predetermined amount. As a result, the target purified protein adsorbed on the adsorbent 3 and other contaminants (contaminating proteins, etc.) are separated in each fraction according to the difference in adsorbing force with respect to the adsorbent 3 possessed by each. It is recovered (separated) in the state of elution.

  Therefore, the purified protein is purified by collecting the fraction from which the target purified protein is eluted as a fraction.

  Here, as an eluent supplied into the column 2, for example, when a phosphate buffer solution to which an eluent such as sodium chloride is added is used, sodium ions of sodium chloride and a calcium phosphate system constituting the adsorbent 3 Ion exchange with hydrogen ions of the phosphate group of the compound. As a result, as described above, the pH of the eluate is drastically lowered, so that the adsorbent 3 is altered and deteriorated due to dissolution of the calcium phosphate compound, and as a result, the function as the adsorbent (carrier) 3 is achieved. There was a problem that it declined early.

  On the other hand, in the present invention, a buffer solution containing at least one of phosphoric acid and a salt thereof and at least one of a compound having a carboxy group and a salt thereof as an eluent supplied into the column 2 Is used. That is, in the present invention, a buffer solution containing at least one of a compound having a carboxy group and a salt thereof is used as an eluent in place of sodium chloride.

  Thus, by using an eluate (buffer solution) containing at least one of a compound having a carboxy group and a salt thereof, the pH of the eluate can be reduced by ion exchange as in the case of containing sodium chloride. In this case, since the carboxy group of the compound having a carboxy group (and its salt) has a buffering capacity in the region of low pH (about 4.0 to 6.0), the eluate The decrease in pH can be suppressed. For this reason, the adsorbent (carrier) 3 can be maintained over a long period of time because the alteration / deterioration of the adsorbent 3 due to the dissolution of the adsorbent 3 having at least the surface composed of the calcium phosphate compound can be dissolved. Can be used repeatedly.

  Although it does not specifically limit as a compound which has a carboxy group, For example, amino acids other than aliphatic carboxylic acid and aromatic carboxylic acid are mentioned.

  Moreover, examples of the aromatic carboxylic acid include phthalic acid, trimellitic acid, pyromellitic acid, benzophenone tetracarboxylic acid, and the like, and one or more of these can be used in combination.

  The aliphatic carboxylic acid is not particularly limited, for example, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, fumaric acid, Maleic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, sebacic acid, dodecanedioic acid, pimelic acid and the like can be mentioned, and one or more of these can be used in combination.

  Among these, the compound having a carboxy group is preferably an aliphatic carboxylic acid (linear saturated monocarboxylic acid) having two or more carbon atoms.

  Furthermore, the aliphatic carboxylic acid having two or more carbons is preferably acetic acid. Here, the salt of acetic acid (acetate) is a monovalent salt having one carboxy group. Since this acetate salt is presumed to function as an eluent having an elution mechanism substantially similar to that of sodium chloride, contaminants adsorbed on the adsorbent 3 are eluted together with the purified protein even if contained in a high concentration in the buffer solution. This can be suppressed or prevented more accurately.

  In addition, as a salt of the compound which has a carboxy group, the sodium salt, potassium salt, ammonium salt, etc. of this compound are mentioned, for example.

  The total content of the compound having a carboxy group and its salt is within a range where the compound having a carboxy group and its salt function as an eluent, that is, the purified protein to be purified is selectively eluted from the adsorbent 3. For example, it is preferably set to 0.1M or more and 5M or less, more preferably 1M or more and 2M or less. Thereby, it can suppress or prevent more accurately that the foreign material adsorbed to the adsorbent 3 is eluted together with the purified protein.

  Moreover, sodium phosphate, potassium phosphate, ammonium phosphate etc. are mentioned as phosphate among phosphoric acid and its salt.

  Furthermore, although total content of phosphoric acid and its salt is not specifically limited, For example, Preferably it sets to 1.0 mM or more and 400 mM or less, More preferably, it sets to 10 mM or more and 100 mM or less. Thereby, it can suppress or prevent more accurately that the foreign material adsorbed to the adsorbent 3 is eluted together with the purified protein.

  The pH of the eluate, that is, a buffer containing at least one of phosphoric acid and a salt thereof and at least one of a compound having a carboxy group and a salt thereof is preferably 5. It is set to 5 or more and less than 8.0, more preferably 6.5 or more and 7.5 or less. Thereby, alteration (denaturation) of the purified protein to be purified can be prevented, and changes in its characteristics can be prevented. In addition, alteration (dissolution, etc.) of the adsorbent (carrier) 3 can be suitably prevented, and a change in the separation ability in the adsorption device 1 can be prevented over a long period of time. It can be purified with high accuracy.

  Furthermore, the temperature of the eluate (buffer solution) is not particularly limited, but is preferably about 30 to 50 ° C, and more preferably about 35 to 45 ° C. Thereby, alteration (denaturation | denaturation) of purified protein can be prevented.

  Furthermore, the flow rate of the eluate is preferably about 0.1 to 10 mL / min, and more preferably about 1 to 5 mL / min. By purifying the purified protein at such a flow rate, it is possible to reliably separate the purified protein and the contaminants without requiring a long time for the separation operation (purification operation), that is, to purify the purified protein with high purity. Can be purified.

  By the operation as described above, the target purified protein is recovered in a predetermined fraction. Further, contaminants are collected in a fraction different from the fraction from which the target purified protein is collected.

  The buffer solution and purification method of the present invention have been described above, but the present invention is not limited to this.

  For example, in the present invention, a pre-process of the process [1], an intermediate process existing between the processes [1] and [2], or a post-process of the process [2] is added for an arbitrary purpose. May be.

  Furthermore, as a method for removing a part of the contaminants mentioned in the above step [1-1] and roughly purifying the purified protein, instead of using the affinity chromatography method, an anion exchange resin or An ion exchange chromatography method including a cation exchange resin may be used.

  In the above embodiment, the case where a natural antibody or a recombinant fusion protein having an immunoglobulin constant domain is purified as a purified protein has been described. However, the present invention is not limited to such a protein, and fibrinogen, pepsinogen and the like. According to the present invention, a protein having no immunoglobulin constant domain can be purified as a purified protein.

Next, specific examples of the present invention will be described.
-Effect of pH due to contact of eluate on hydroxyapatite (Example 1A)
-1- First, as an adsorber, a column (size φ4.0 mm × 100 mm) in which about 0.8 g of hydroxyapatite beads (CHT Type I, average particle size 40 μm, manufactured by HOYA Technosurgical) was packed as an adsorbent was prepared. .

  -2- Next, 15 CV of the equilibration solution is supplied at a flow rate of 0.5 mL / min, then 10 CV of the eluate is supplied at a flow rate of 0.5 mL / min, and then the washing solution is supplied at a flow rate of 0.5 mL / min. Then, 6.4 CV was supplied, and the pH of the effluent flowing out from the column was measured.

The equilibration solution is a phosphate buffer (pH 6.5) containing 10 mM sodium phosphate, and the eluate is a phosphate buffer (pH 6) containing 10 mM sodium phosphate and 1 M CH ₃ COOH. 5) and a phosphate buffer solution (pH 6.5) containing 400 mM sodium phosphate was used as the washing solution.

(Comparative Example 1A)
The pH of the effluent flowing out of the column was adjusted in the same manner as in Example 1A, except that a phosphate buffer (pH 6.5) containing 10 mM sodium phosphate and 1 M NaCl was used as the eluent. It was measured.

  As a result, as shown in FIG. 2, Comparative Example 1A showed a result that a decrease in pH was significantly observed with the supply of the eluate, whereas Example 1A was accompanied with the supply of the eluate. The results showed that the decrease in pH was suppressed as compared with Comparative Example 1A. Moreover, in Example 1A, after pH fell, the result returned to the original pH vicinity by supply of a little eluate compared with Comparative Example 1A was shown.

(Example 2A)
-1- First, as an adsorber, a column (size 4 mm ID × 100 mm) in which about 0.7 g of hydroxyapatite beads (CHT Type I, average particle size 40 μm, manufactured by HOYA Technosurgical) is filled as an adsorbent is prepared. did.

  -2- Next, the eluate was supplied at 10 CV at a flow rate of 1.0 mL / min, and the pH of the effluent flowing out of the column was measured.

  In addition, the buffer solution (pH 6.5) containing 5 mM MES (2-morpholino ethanesulfonic acid), 800 mM NaCl, and 400 mM histidine was used for the eluate.

(Example 3A)
In the same manner as in Example 2A, except that a buffer solution (pH 6.5) containing 5 mM MES (2-morpholinoethanesulfonic acid), 800 mM NaCl, and 100 mM histidine was used as an eluent. The pH of the effluent flowing out from was measured.

(Comparative Example 2A)
The effluent flowing out from the column in the same manner as in Example 2A, except that a buffer solution (pH 6.5) containing 5 mM MES (2-morpholinoethanesulfonic acid) and 800 mM NaCl was used as the eluent. The pH of the liquid was measured.

  As a result, as shown in FIG. 3, Comparative Example 2A showed a result in which a decrease in pH was remarkably observed with the supply of the eluate, whereas in Examples 2A and 3A, the supply of the eluate was shown. As a result, the decrease in pH was suppressed as compared with Comparative Example 2A, and this tendency was particularly noticeable in Example 2A containing 400 mM histidine.

・ Purification by eluate (buffer) using hydroxyapatite such as protein [4STD purification]
(Example 1B)
-1- First, as an adsorber, a column (size φ4 mm × 100 mm) in which about 0.8 g of hydroxyapatite beads (CHT Type I, average particle diameter of 40 μm, manufactured by HOYA Technosurgical) was packed as an adsorbent was prepared.

  -2- Next, the column was washed by supplying 10 mM NaPB (pH 6.5) at a flow rate of 1 mL / min for 10 minutes, and a phosphate buffer solution containing 10 mM sodium phosphate ( Equilibrated at pH 6.5).

  -3- Next, 1 mM phosphoric acid is added to the column obtained in the step 2-2 so that myoglobin is 5 mg / mL, ovalbumin 10 mg / mL, α-chymotrypsinogen A 5 mg / mL, and cytochrome C 5 mg / mL. 50 μL of sample (4STD sample) dissolved in sodium buffer (pH 6.5) was supplied.

  -4- Next, a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate is supplied at a flow rate of 1 mL / min for 3 minutes, and then the eluate A and the eluate B are mixed at a volume ratio thereof. The eluate B is supplied at a flow rate of 1 mL / min for 15 minutes so that the eluate B continuously changes from 0% to 100%, and then a phosphate buffer solution (pH 6.5) containing 400 mM sodium phosphate is added at 1 mL / min. Supplying for 7 minutes at a flow rate of min, the effluent flowing out from the column was fractionated by 1 mL.

The eluent A is a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate, and the eluent B is a phosphate buffer solution (containing 10 mM sodium phosphate and 1M CH ₃ COOH). Each of pH 6.5) was used.

(Reference Example 1B)
Reference Example 1B was carried out in the same manner as in Example 1B, except that a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate and 1M NaCl was used as eluent B in Step -4-. A 4STD sample was purified using an adsorber.

  FIG. 4 shows the absorbance at 280 nm of the effluents measured in Step -4- in Example 1B and Reference Example 1B, respectively.

  As is clear from FIG. 4, in both Example 1B and Reference Example 1B, proteins were eluted in the effluent in the order of basic protein, neutral protein, and acidic protein, and acetic acid and its salts. Was found to have almost the same elution mechanism as sodium chloride.

[Purification of bovine serum albumin (BSA)]
(Example 2B)
A sample (BSA sample) dissolved in 1 mM sodium phosphate buffer (pH 6.5) so that bovine serum albumin is 10 mg / mL in the column obtained in the step -2- in the step -3- The BSA sample in Reference Example 1B was purified using an adsorption device in the same manner as in Example 1B except that 50 μL was supplied.

(Reference Example 2B)
Reference Example 2B was carried out in the same manner as in Example 2B, except that a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate and 1M NaCl was used as eluent B in Step -4-. The BSA sample was purified using an adsorber.

  FIG. 5 shows the absorbances at 280 nm of the effluents measured in Example 4B and Reference Example 2B in step -4-.

  As is apparent from FIG. 5, in both Example 2B and Reference Example 2B, BSA was not eluted in the effluent by elution with Eluent A and Eluent B, but phosphorous containing 400 mM sodium phosphate. Elution into the effluent was first made by supplying an acid buffer solution (pH 6.5), and it was found that acetic acid and its salt have almost the same elution mechanism as sodium chloride.

[Purification of human immunoglobulin (antibody; IgG)]
(Example 3B)
In the step -3-, 50 μL of a sample (IgG sample) dissolved in 1 mM sodium phosphate buffer (pH 6.5) was added to the column obtained in the step 2-2 so as to be IgG 2 mg / mL. A DNA sample in Reference Example 3B was purified using an adsorption device in the same manner as in Example 1B except that it was supplied.

(Reference Example 3B)
Reference Example 3B was carried out in the same manner as in Example 3B, except that a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate and 1M NaCl was used as eluent B in Step-4. IgG samples were purified using an adsorber.

  FIG. 6 shows the absorbance at 280 nm of the effluent measured in Example 4B and Reference Example 3B in Step -4-.

  As apparent from FIG. 6, in both Example 3B and Reference Example 3B, IgG was eluted in the effluent by elution with eluent A and eluent B, and acetic acid and its salt were sodium chloride. It was found that it has almost the same elution mechanism.

[Purification of salmon DNA]
(Example 4B)
In the step-3-, 50 μL of a sample (DNA sample) dissolved in 1 mM sodium phosphate buffer (pH 6.5) was added to the column obtained in the step-2- so that the salmon DNA was 1 mg / mL. A DNA sample in Reference Example 4B was purified using an adsorption device in the same manner as in Example 1B except that it was supplied.

(Reference Example 4B)
Reference Example 4B was carried out in the same manner as in Example 4B, except that a phosphate buffer solution (pH 6.5) containing 10 mM sodium phosphate and 1M NaCl was used as eluent B in Step -4-. The DNA sample was purified using an adsorption device.

  FIG. 7 shows the absorbance at 280 nm of the effluent measured in Example 4B and Reference Example 4B in the step -4-.

  As is clear from FIG. 7, in both Example 4B and Reference Example 4B, DNA was not eluted in the effluent by elution with eluate A and eluent B, but phosphorous containing 400 mM sodium phosphate. Elution into the effluent was first made by supplying an acid buffer solution (pH 6.5), and it was found that acetic acid and its salt have almost the same elution mechanism as sodium chloride.

DESCRIPTION OF SYMBOLS 1 Adsorber 2 Column 20 Adsorbent filling space 21 Column main body 22, 23 Cap 24 Inflow pipe 25 Outflow pipe 3 Adsorbent 4, 5 Filter member

Claims (7)

A buffer used as an eluent when selectively purifying a purified protein to be purified adsorbed on a carrier composed of a calcium phosphate compound at least on its surface;
A buffer comprising at least one of phosphoric acid and a salt thereof and at least one of a compound having a carboxy group and a salt thereof.   The buffer solution according to claim 1, wherein pH of the buffer solution is 5.5 or more and less than 8.0.   The buffer solution according to claim 1 or 2, wherein a total content of the phosphoric acid and a salt thereof is 1.0 mM or more and 400 mM or less.   The buffer solution according to any one of claims 1 to 3, wherein a total content of the compound having a carboxy group and a salt thereof is 0.1 M or more and 5 M or less.   The buffer solution according to any one of claims 1 to 4, wherein the compound having a carboxy group is an aliphatic carboxylic acid having two or more carbon atoms.   The buffer solution according to any one of claims 1 to 5, wherein the purified protein is a natural antibody or a recombinant fusion protein having an immunoglobulin constant domain. A separation method for separating the purified protein from a sample solution containing a plurality of types of proteins including the purified protein,
A supply step of supplying the sample solution into an apparatus in which the carrier is filled in at least a part of a filling space;
A buffer solution according to any one of claims 1 to 6 is supplied into the device, and the effluent flowing out of the device is fractionated by a predetermined amount, whereby each fractionated fraction is separated. And a fractionation step of purifying the purified protein in the effluent of the water.

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