JP2013227254A - Purification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purification method capable of efficiently purifying an acidic protein with excellent precision from a sample solution containing the acidic protein.SOLUTION: A purification method of purifying an acidic protein from a sample solution containing the acidic protein and contaminants consisting of substances other than the acidic protein includes: a first process of bringing the sample solution into contact with an adsorbent composed of fluoroapatite to adsorb the acidic protein with the adsorbent composed of fluoroapatite; and a second process of supplying an eluent to the adsorbent composed of fluoroapatite and fractionating a resultant eluate by a predetermined amount to purify the acidic protein in the fractionated eluate in each fraction. In the first process, the adsorbent is brought into contact with a Ca ion beforehand and/or substantially simultaneously when bringing the sample solution into contact with the adsorbent.

Description

  The present invention relates to a purification method for purifying an acidic protein from a sample solution.

  In recent years, acidic proteins such as antibodies contained in plasma (natural antibodies) and recombinant fusion proteins (artificial antibodies) having a constant (Fc) part produced by genetically engineered host cells As a purification method, an affinity chromatography method or an ion exchange chromatography method is used.

  In particular, the affinity chromatography method uses, as an adsorbent, a binding protein such as protein A or protein G immobilized on an individual support. Since the constant (Fc) part of the fusion protein has an excellent reversible interaction, the antibody and the recombinant fusion protein can be easily purified using such interaction, and thus are widely used ( For example, refer nonpatent literatures 1 and 2.).

  Further, when such an antibody and recombinant fusion protein are used for pharmacological purposes, it is necessary to remove contaminants contained in the sample solution together with the antibody and recombinant fusion protein with high accuracy.

  This contaminant includes aggregates of acidic proteins, inactive or partially inactive acidic proteins, and in the case of recombinant fusion proteins, DNA, RNA, proteins, etc. derived from host cells In the case of an antibody, proteins such as albumin, β globulin, and γ globulin contained in plasma are exemplified. Furthermore, when the affinity chromatography method is used for purification, binding proteins such as protein A and protein G released from the solid support and a complex of the binding protein and acidic protein are also included.

  In particular, aggregates of acidic proteins, as well as binding proteins and complexes of binding proteins and acidic proteins, are immunogenic and can be toxic if included in large amounts, thus It is necessary to remove it with accuracy.

  However, in reality, there is no known purification method that can efficiently remove such contaminants with excellent accuracy and purify acidic protein.

R. Vola et al. (1994), Cell Biophys. 24-25: 27-36 Aybay and Imir (2000), J. MoI. Immunol. Methods 233 (1-2): 77-81

  An object of the present invention is to provide a purification method capable of purifying acidic protein efficiently and with excellent accuracy from a sample solution containing acidic protein.

Such an object is achieved by the present inventions (1) to (10) below.
(1) A purification method for purifying the acidic protein from a sample solution containing the acidic protein and a contaminant comprising a substance other than the acidic protein,
A first step of bringing the sample solution into contact with an adsorbent composed of fluorapatite, and adsorbing the acidic protein to the adsorbent composed of fluorapatite;
The eluent is supplied to the adsorbent composed of the fluorapatite, and the resulting effluent is fractionated by a predetermined amount to purify the acidic protein in the effluent of each fractionated fraction. And a second step to
In the first step, before the sample solution is brought into contact with the adsorbent and / or almost simultaneously, the adsorbent is brought into contact with Ca ions.

  Thereby, acidic protein can be efficiently purified with excellent accuracy from a sample solution containing acidic protein.

  (2) In the first step, prior to bringing the sample liquid into contact with the adsorbent, the equilibration liquid containing the Ca ions is brought into contact with the adsorbent. .

  Thereby, when the sample solution is brought into contact with the adsorbent, a calcium site in which the surface of the phosphate group of the fluorapatite is covered with Ca ions can be reliably formed.

(3) The purification method according to (2), wherein the equilibration solution includes at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .

  Thus, the equilibration liquid contains a salt by containing a salt, so that the equilibration liquid contains Ca ions. Therefore, Ca ion can be made to contact an adsorbent by making this equilibration liquid contact an adsorbent.

  (4) The purification method according to any one of (1) to (3), wherein in the first step, the sample solution brought into contact with the adsorbent further contains the Ca ions.

  Thereby, when the sample solution is brought into contact with the adsorbent, a calcium site in which the surface of the phosphate group of the fluorapatite is covered with Ca ions can be reliably formed.

(5) The purification method according to (4), wherein the sample solution includes at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .

  Thus, a sample liquid will contain Ca ion because a sample liquid shall contain a salt. Therefore, Ca ion can be made to contact an adsorbent by making this sample liquid contact an adsorbent.

  (6) Prior to the second step, the purification according to any one of (1) to (5) above, wherein the adsorbent adsorbed with the acidic protein is washed using a washing solution containing the Ca ions. Method.

  Thus, contaminants that are not adsorbed by the adsorbent but remain in the column or the like can be reliably removed.

(7) The purification method according to (6), wherein the cleaning liquid contains at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .

  As a result, contaminants remaining in the column or the like can be reliably removed without eluting the acidic protein from the adsorbent.

  (8) The purification method according to any one of (1) to (7), wherein the eluate is a phosphate buffer having a concentration of 1 mM to 400 mM.

  Thereby, acidic protein can be reliably eluted from the adsorbent comprised with the fluorapatite which Ca ion contacted.

  (9) The purification method according to (8), wherein the eluate is a phosphate buffer having a pH of 4 to 12.

  Thereby, acidic protein can be reliably eluted from the adsorbent comprised with the fluorapatite which Ca ion contacted.

  (10) The purification method according to any one of (1) to (9), wherein the acidic protein has an immunoglobulin constant domain.

  The purification method of the present invention is suitably applied to the purification of acidic proteins having immunoglobulin constant domains.

  According to the purification method of the present invention, an acidic protein can be efficiently purified with excellent accuracy from a sample solution containing the acidic protein and a contaminant composed of a substance other than the acidic protein.

  In addition, since the fluorapatite chromatography process uses a material composed of fluorapatite as the adsorbent, the choice of eluent used to elute acidic protein from the adsorbent is widened, and the pH value of the eluate can be adjusted over a wide range. Therefore, it can be reliably set according to the conditions under which acidic protein is purified with high accuracy in the effluent of each fractionated fraction.

It is a longitudinal cross-sectional view which shows an example of the adsorption | suction apparatus used for the purification method of this invention.

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

  The “adsorbent” 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.

  “Affinity chromatography” refers to specific reversible interactions between biomolecules (eg, isoelectric point, hydrophobicity or size) rather than the general properties of molecules (eg, isoelectric point, hydrophobicity or size) to perform chromatographic separation. , The ability of protein A to bind to the Fc part of an IgG antibody). Specifically, affinity chromatography uses, for example, a solid support on which a binding protein such as protein A is immobilized as an adsorbent, and determines the difference in the adsorption force (binding force) of molecules to the adsorbent. Refers to chromatographic separation of molecules on the basis (see Ostrove (1990) Guide to Protein Purification, Methods in Enzymology 182: 357-379).

  “Anion exchange chromatography” refers to the exchange of anions contained in a sample solution with an ion exchange resin having a positively charged group such as a tertiary or quaternary amino group. Chromatography using

  “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, e.g., Charles A Hasmann and J. Donald Capra, Immunoglobulin: Structure and Function, Ed. William E. Paul, 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.

  “Acid protein” is a protein having an acidic isoelectric point (pI) that is negatively charged in a neutral pH range and generally containing a large amount of acidic amino acids such as aspartic acid and glutamic acid. Say.

  An “inactive protein” or “partially inactive protein” has the same amino acid sequence and molecular weight as an acidic protein, but has a binding ability to a target (such as an antigen) as compared to an acidic protein. It means that it has no or even very low.

  “Protein A” is a protein that was first discovered in the cell wall of Staphylococcus and has a property of specifically binding to the Fc region of IgG antibody. In the present invention, “protein A” refers to any protein that is the same or substantially similar to protein A derived from staphylococci, including commercially available and / or recombinant protein A. In the present invention, the biological activity of protein A for determining substantial similarity is the ability of IgG antibodies to bind to the Fc region.

  “Protein G” is a protein that was first discovered on the cell wall of Streptococcus and has the property of specifically binding to the Fc part of an IgG antibody. In the present invention, “protein G” refers to any protein that is the same or substantially similar to protein G derived from streptococci, and includes commercially available and / or recombinant protein G. In the present invention, the biological activity of protein G for determining substantial similarity is the ability of IgG antibodies to bind to the Fc region.

  “Protein LG” refers to a recombinant fusion protein that binds to an IgG antibody, and includes a part of both protein G and protein L. “Protein L” is a protein initially isolated from the cell wall of Peptostreptococcus. Protein LG contains an IgG binding domain derived from both proteins L and G (see, eg, Vola et al. (1994), Cell Biophys. 24-25: 27-36). In the present invention, “protein LG” refers to any protein that is the same or substantially similar to protein LG, and includes commercially available and / or recombinant protein LG. For the purposes of the present invention, the biological activity of protein LG to determine substantial similarity is the ability of an IgG antibody to bind to the Fc region.

  “Recombinant fusion protein” refers to any protein comprising part or all of two or more proteins that are not fused in their native state, such as, for example, the Fc of an antibody. Human receptor activator of NF-κB (huRANK: Fc) fused to the region, intimal endothelial cell kinase-δ (TEKδ: Fc) fused to the Fc portion of the antibody, and tumor necrosis factor fused to the Fc portion of the antibody A receptor (TNFR: Fc), but is not limited thereto.

  “Purify acidic protein” refers to the amount of extraneous or inconvenient factors that may be present in a sample solution containing acidic protein, particularly the amount of biopolymers such as proteins and DNA that differ from the acidic protein to be purified. It means to reduce. Also, the presence of a protein different from the acidic protein to be purified can be identified by any suitable method, including gel electrophoresis and staining and / or ELISA assays. Furthermore, the presence of DNA can be identified by any suitable method, including gel electrophoresis and staining and / or assays using polymerase chain reaction.

  “Substantially similar” means that the amino acid sequences of the proteins are at least 80%, preferably at least 90% identical to each other and retain the biological activity of the unaltered protein or are active in the desired manner. When changed, the proteins are said to be substantially similar. Included in amino acids considered identical for purposes of determining whether proteins are substantially similar are conservative substitutions that may not affect biological activity, including: That is, Ala is Ser, Val is Ile, Asp is Glu, Thr is Ser, Ala is Gly, Ala is Thr, Ser is Asn, Ala is Val, Ser is Gly, and Tyr is Phe. , Ala is Pro, Lys is Arg, Asp is Asn, Leu is Ile, Leu is Val, Ala is Glu, Asp is Gly, and vice versa. (See, eg, Neuroth et al., The Proteins, Academic Press, New York (1979)).

  “TNFR” refers to a protein comprising an amino acid sequence that is identical or substantially similar to the sequence of a natural mammalian tumor necrosis factor receptor (TNFR). Biological activity to determine substantial similarity includes binding tumor necrosis factor (TNF), transmitting a biological signal initiated by TNF binding to the cell, or natural (ie non-recombinant). By means of the ability to cross-react with anti-TNFR antibodies raised against source-derived TNFR. The TNFR can be any mammalian TNFR, including murine or human TNFR. Such TNFRs are described in US Pat. No. 5,395,760 and US Pat. No. 5,610,279. A particularly preferred TNFR is that described in US Pat. No. 5,395,760, which is a glycosylated form and has an apparent molecular weight of about 80 kilodaltons by SDS-PAGE.

  “TNFR: Fc” refers to a recombinant fusion protein comprising all or part of the extracellular domain of TNFR fused to the Fc region of an antibody. Such extracellular domains include amino acid sequences substantially similar to amino acid sequences 1-163, 1-185 or 1-235 of FIG. 2A of US Pat. No. 5,395,760, which include It is not limited.

Next, an example of an adsorption device (separation device) used in the purification method of the present invention will be described.
In the following, as the adsorption device, the type of adsorbent provided in this device is different, that is, an immobilization member in which a binding protein such as protein A is immobilized on a solid support, an anion exchange resin, Prepare three adsorbents each composed of fluorapatite. Using these adsorbers, the sample liquid containing the purified protein is subjected to affinity chromatography, anion exchange chromatography, and fluorapatite chromatography in this order. A case where purified protein is purified will be described as a representative.

<Adsorption device>
FIG. 1 is a longitudinal sectional view showing an example of an adsorption device used in the purification method of 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, when the acidic protein to be purified is purified (separated), the inflow side is a side for supplying a liquid such as a sample solution (liquid containing acidic protein) or an eluate into the adsorption device. On the other hand, the outflow side means the side opposite to the inflow side, that is, the side from which the sample liquid flows out from the adsorption device as the outflow liquid.

  In the following description, the configuration of the adsorption device 1 other than the adsorbent 3 will be described, and then the adsorbent 3 will be described.

  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. The adsorbent 3 is filled in at least a part of the adsorbent filling space 20 (almost full in this embodiment).

  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.

  Of the three adsorbers, one adsorber 1 (first adsorber 1A) is used in an affinity chromatography process to be described later, and the adsorbent 3 is a binding protein such as protein A on a solid support. The second adsorbing device 1 (second adsorbing device 1B) is used in the anion exchange chromatography process described later, and the adsorbent 3 is an anion exchange resin. Further, the three adsorbing devices 1 (third adsorbing device 1C) are used in a fluorapatite chromatography process to be described later, and the adsorbent 3 is composed of fluorapatite.

The immobilization member is obtained by immobilizing a binding protein on a solid support.
Since this immobilization member exhibits excellent adsorption properties for various substances having reversible interaction (affinity) with the binding protein (for example, acidic protein having immunoglobulin constant domain), It is suitably used as an adsorbent that adsorbs various substances having reversible interactions.

  Although it does not specifically limit as binding protein, For example, it is a protein provided with the characteristic which couple | bonds with an immunoglobulin constant domain, For example, protein G, protein LG, protein A etc. are mentioned, Among these, These can be used alone or in combination of two or more.

  In addition, examples of the constituent material of the solid support include agarose, sepharose, silica, collodion charcoal, and sand, and one or more of these can be used in combination.

  Anion exchange resins are those in which a positively charged group such as a tertiary or quaternary amino group is introduced into a polyethylene or polystyrene main chain.

  Since the anion exchange resin having such a configuration has a positively charged group, the anion exchange resin exhibits an adsorption ability based on electrostatic interaction as an anion exchanger. For this reason, an anion exchange resin exhibits an excellent adsorption capacity for various substances having a negative charge (negatively charged).

  Here, although acidic protein is also a negatively charged substance, the condition of the sample solution is that acidic protein does not bind to the anion exchange resin, but among the substances other than acidic protein contained in the sample solution, it is better than acidic protein. The condition is set such that a substance having a large negative charge (for example, DNA or the like) binds. When the sample solution is brought into contact with the anion exchange resin under these conditions, the acidic protein does not bind to the anion exchange resin, and a substance having a negative charge larger than that of the acidic protein binds to the anion exchange resin. Therefore, in the anion exchange chromatography process to be described later, by collecting the flow-through liquid and the washing liquid as a sample liquid that has been subjected to the anion exchange chromatography process, the sample liquid contains acidic protein, A substance having a negative charge larger than the acidic protein contained in the sample solution is adsorbed and removed by the anion exchange resin.

_{Fluoroapatite} is Ca ₁₀ (PO ₄ ) ₆ (OH) _2-2x F _2X [wherein x is 0 <x ≦ 1. And at least a part of the hydroxyl group of hydroxyapatite is substituted with a fluorine atom.

  As the fluorapatite having such a configuration, one having a Ca / P ratio of 1.0 to 2.0 is used, and Ca ions (calcium sites) and phosphate groups (phosphate sites) are regularly arranged at high density. In general, it has an adsorptive capacity based on electrostatic interaction as an amphoteric ion exchanger. For this reason, the particle | grains comprised with the fluorapatite exhibit the outstanding adsorption capacity with respect to the various substances which have a positive or negative charge.

  In the present invention, Ca ions are brought into contact with the adsorbent 3 before and / or substantially at the same time as the sample solution containing the purified protein is brought into contact with the adsorbent 3 composed of fluorapatite having such adsorption characteristics. That is, at least one of the process of bringing Ca ions into contact with the adsorbent 3 and the process of bringing Ca ions into contact with the adsorbent 3 is performed almost simultaneously with the process of bringing the sample liquid into contact with the adsorbent 3. Thus, when Ca ions are brought into contact with the adsorbent 3, among the Ca ions (calcium sites) and phosphate groups (phosphate sites) of the fluorapatite, Ca ions are selectively produced with respect to the phosphate groups. Adsorb. Therefore, the surface of the phosphate group is covered with Ca ions adsorbed on the phosphate group (phosphate site), and as a result, a second calcium site is formed.

  Therefore, particles composed of fluorapatite in contact with Ca ions do not have the ability to adsorb various substances having a positive charge and exhibit better adsorption ability to various substances having a negative charge. Become. That is, such particles exhibit an adsorption ability to selectively adsorb various substances having negative charges. Furthermore, a large amount of various substances having negative charges can be adsorbed. Therefore, the amount of acidic protein that can be purified by one treatment can be improved, and acidic protein can be purified more efficiently.

  Using the adsorption characteristics of fluorapatite contacted with Ca ions as described above, the sample solution is contacted with fluorapatite under the condition that acidic protein having a negative charge contained in the sample solution binds to fluorapatite. Let As a result, among the various substances contained in the sample solution, various substances having a positive charge and various substances having a negative charge smaller than the purified protein to be purified do not bind to fluorapatite, but have a negative charge almost equal to that of acidic protein. And various substances having a negative charge larger than that of acidic protein bind to fluorapatite. Therefore, in the fluorapatite chromatography process described later, after the acidic protein contained in the sample liquid is adsorbed to the adsorbent 3, the eluate is supplied to the adsorbent 3, and the resulting effluent is supplied in predetermined amounts. By fractionating, the effluent of each fraction obtained by this fractionation contains acidic protein, and contaminants other than acidic protein are adsorbed and removed by fluorapatite. Acidic protein is purified.

  In addition, since fluorapatite is superior in acid resistance and alkali resistance as compared to hydroxyapatite, the range of selection of the eluate used when obtaining the effluent of each fraction in the fluorapatite chromatography treatment is widened. That is, the pH value of the eluate can be set in a wide range. Therefore, it can be reliably set according to the conditions under which the acidic protein is purified with high accuracy in the effluent of the target fraction.

  These fluorapatites can be synthesized by a known wet synthesis method, dry synthesis method, or the like. In this case, the fluorapatite may contain substances remaining in the synthesis (such as raw materials) or secondary reaction products generated in the course of the synthesis.

  The form (shape) of the adsorbent 3 filled in the three adsorbing apparatuses 1 (the first adsorbing apparatus 1A, the second adsorbing apparatus 1B, and the third adsorbing apparatus 1C) as described above is shown in FIG. As shown, it is preferably in the form of particles (granular), but in addition, for example, a pellet (small block), a block (for example, a porous body in which adjacent pores communicate with each other, a honeycomb shape, Monolith) or the like. In addition, by making the adsorbent 3 particulate, the surface area can be increased, and the separation characteristics of acidic protein and contaminants can be improved.

  The average particle size of the granular adsorbent 3 is not particularly limited, but is preferably about 0.5 to 150 μm, 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, from such an adsorption device (separation device) 1 (first adsorption device 1A, second adsorption device 1B, and third adsorption device 1C), an acidic protein and a substance other than the acidic protein are used. A purification method for purifying an acidic protein from a sample solution containing contaminants will be described.

<Purification method>
The purification method of the present embodiment includes an affinity step in which at least a part of contaminants is removed from the sample solution by subjecting the sample solution to affinity chromatography, and an anion exchange chromatography on the sample solution that has been subjected to the affinity step. An anion exchange process that removes at least a part of the contaminants from the sample liquid that has been subjected to the affinity process, and a sample liquid that has been subjected to the anion exchange process are subjected to fluorapatite chromatography. And a fluorapatite process for purifying the acidic protein from the sample solution that has been subjected to the anion exchange process.

Hereinafter, this purification method will be described in detail.
[1] Preparation Step First, a sample solution containing an acidic protein to be purified and a contaminant that is a substance other than the acidic protein is prepared.

  An acidic protein is negatively charged in a neutral pH region, and in this embodiment, an acidic protein is a protein or protein complex having an immunoglobulin constant domain (for example, the Fc part of an antibody). Such an acidic protein exhibits the property of adsorbing to the immobilization member based on the reversible interaction (affinity) of the immunoglobulin constant domain with the binding protein in the affinity chromatography treatment [2].

  Acidic proteins having immunoglobulin constant domains are produced, for example, by living host cells that have been genetically engineered to produce such acidic proteins.

  In this case, methods for genetically engineering cells to produce this acidic protein are well known in the art (see, eg, Ausubel et al. (1990), Current Protocols in Molecular Biology (Willey, See New York.)

  The acidic protein may have one or more immunoglobulin constant domains, may have one or more immunoglobulin variable domains, and does not have immunoglobulin variable domains. It may be.

  Furthermore, the acidic protein may be a natural protein (natural antibody) or a recombinant fusion protein. Examples of the recombinant fusion protein include those having the Fc part of an antibody, 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 protein include flt3 ligand (see International Patent Application WO94 / 28391), CD40 ligand (see US Pat. No. 6,087,329), erythropoietin, thrombopoietin, calcitonin, Fas ligand, and reception of NF-κB. Ligand for body activator (RANKL), tumor necrosis factor (TNF) -related apoptosis-inducing ligand (TRAIL, see international patent application WO97 / 01633), thymic stroma-derived lymphopoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor ( GM-CSF, Australian patent No. 588819), mast cell growth factor, stem cell growth factor, epidermal growth factor, RANTES, growth hormone, insulin, insulinotropin, insulin-like increase Factors, 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 can be mentioned.

  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 proteins or a protein substantially similar to such a receptor. The thing containing 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, the acidic protein may be an antibody (natural antibody) or a part thereof, and a chimeric antibody, for example, an antibody in which a human immunoglobulin constant domain is bound to one or more murine immunoglobulin variable domains, or a fragment 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.

  Contaminants collectively refer to foreign or inconvenient molecules other than the acidic protein to be purified contained in the sample solution, for example, DNA, RNA, protein (basic protein), protein aggregates. Such biopolymers are mentioned.

  Specifically, as a contaminant, when an acidic protein is produced by a genetically engineered host cell, in addition to DNA, RNA and protein derived from the host cell, medium components, and the aggregation of the acidic protein In the case where the acidic protein is a natural antibody, proteins such as albumin, β globulin, and α globulin contained in plasma are exemplified. Furthermore, a binding protein such as protein A that has been detached from the adsorbent (immobilization member) 3 provided in the first adsorption device 1A used in the affinity chromatography process [2] and leached into the sample solution, Also included are complexes with acidic proteins.

[2] Affinity chromatography treatment (affinity process)
[2-1] Supplying Step to First Adsorption Device Next, the obtained sample solution is first supplied to the first adsorption device 1A among the three adsorption devices 1. That is, it is supplied to the adsorbent 3 through the inflow pipe 24 and the filter member 4 of the first adsorption device 1A.

  As a result, the sample liquid passes through the column 2 (first adsorption device 1A), and the sample liquid comes into contact with the adsorbent 3 composed of the immobilizing member in which the binding protein is immobilized on the solid support. To do.

  At this time, among the substances contained in the sample liquid, those having a high reversible interaction (affinity) with respect to the solid support (particularly, binding protein) are adsorbed (held) in the column 2, and the solid support is obtained. A substance having a low reversible interaction (affinity) with respect to (particularly binding protein) flows out from the column 2 through the filter member 5 and the outflow pipe 25 as an effluent.

  That is, acidic proteins having immunoglobulin constant domains and contaminants with high affinity to the solid support are adsorbed in the column 2, and contaminants with low affinity to the solid support. Flows out of the column 2.

  Before and after the sample solution is supplied to the first adsorption device 1A, a solution that does not interfere with the binding of the immunoglobulin constant domain of the acidic protein to the binding protein is supplied to the first adsorption device 1A. Thus, the adsorbent 3 may be washed.

  Moreover, as a washing | cleaning liquid which wash | cleans the adsorption agent 3, what contains a buffer solution and / or salts can be used.

  Examples of the buffer solution include a phosphate buffer solution, a Tris buffer solution, an acetate buffer solution, and a citrate buffer solution, and one or more of them can be used in combination.

  Examples of the salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salt, and magnesium salt, and one or more of these are used in combination. I can.

  In addition, as this solution, specifically, for example, a solution in which Tris is added at a concentration of about 5 to 100 mM and sodium chloride is added at a concentration of about 50 to 250 mM.

[2-2] Fractionation step by first adsorption device Next, an eluate (first solution) for eluting acidic protein from the inflow pipe 24 of the first adsorption device 1A into the column 2 with respect to the adsorbent 3. 1 eluate). Then, the sample liquid (outflow liquid) flowing out from the column 2 through the outflow pipe 25 is recovered.

  As a result, the acidic protein adsorbed on the adsorbent 3 is detached from the adsorbent 3 by the action of the eluent, so that it is recovered in a state of being eluted into the sample liquid (sample liquid subjected to the affinity step).

  As the eluate, one having a function of inhibiting the binding to the immunoglobulin constant domain of the acidic protein by the binding protein is used. For example, a chaotropic agent that is a substance that increases or decreases the pH, for example, guanidinium, and What contains at least 1 sort (s) of salts is mentioned.

The eluate may contain acetic acid, glycine or citric acid.
In addition, the separation (elution) of acidic protein can be more reliably performed, for example, by lowering the pH of the eluate.

  Specifically, for example, by reducing the pH to about 4.5 or less, more preferably about 2.5 to about 3.5 using a solution containing citrate or acetate, the acidic protein is reduced. It can be detached from the adsorbent 3.

  When the acidic protein is released from the adsorbent 3 due to an increase in pH, the pH of the solution is preferably set to about 8.5 or more.

  The flow rate of the eluate is preferably about 50 to 400 cm / hour, more preferably about 100 to 360 cm / hour.

  By the operation as described above, the sample solution that has been subjected to the affinity chromatography treatment is recovered in a state where the acidic protein is first purified (at least a part of the contaminants is removed).

  By performing this step, in the collected sample solution, in addition to acidic protein and substances other than acidic protein, binding protein separated from the solid support, and this binding protein and acidic protein This complex is included as a contaminant.

  In addition, you may make it perform pH adjustment which adjusts the pH to about 2.5-4.5 with respect to the collect | recovered sample liquid (effluent) as needed. Thereby, even when a virus such as a vector virus is mixed in the contaminant, the virus can be reliably inactivated.

  Furthermore, you may make it filter with respect to the collect | recovered sample liquid. Although it does not specifically limit as a filter (filter membrane) used for this filtration, It is preferable that it is about 0.1-0.3 micrometer, and it is more preferable that it is about 0.2 micrometer. This ensures that contaminants (specifically DNA, etc.) that are larger in size than acidic proteins are reliably removed from the sample solution without trapping the acidic protein contained in the sample solution. can do.

[3] Anion exchange chromatography (anion exchange process)
[3-1] Supplying process to the second adsorption device Next, the sample liquid collected from the first adsorption apparatus 1A (sample liquid subjected to the affinity process) is supplied to the second adsorption apparatus 1B. . That is, the sample liquid collected from the first adsorption device 1A is supplied to the adsorbent 3 through the inflow pipe 24 and the filter member 4 of the second adsorption device 1B.

  As a result, the sample liquid passes through the column 2 (second adsorption device 1B), and the sample liquid comes into contact with the adsorbent 3 composed of the anion exchange resin.

  At this time, the pH of the buffer solution and the salt concentration contained in the sample solution collected from the first adsorption device 1A are set as follows.

  That is, the adsorbing ability to the adsorbent 3 composed of an anion exchange resin does not have to negative acidic protein but has a negative charge larger than that of acidic protein among contaminants (for example, DNA Etc.).

  In other words, in the anion exchange chromatography process [3], acidic protein does not adsorb (bond) to the adsorbent 3 composed of an anion exchange resin, but a substance other than acidic protein contained in the sample solution ( A substance (for example, DNA) having a negative charge larger than that of acidic protein among the contaminants) is performed under the condition of adsorbing (binding) to the adsorbent 3 composed of an anion exchange resin.

  Buffers or salts include, for example, sodium, potassium, ammonium, magnesium, calcium, chloride, fluoride, acetate, phosphate and / or citrate, and / or Tris buffer. Among them, Tris buffer or sodium phosphate buffer is preferable.

  Moreover, when using a sodium phosphate buffer, the density | concentration is although it does not specifically limit, Preferably it is 1-600 mM, More preferably, it sets to about 5-400 mM.

  Further, the pH of the phosphate buffer (sodium phosphate buffer) is preferably about 4 to 12, more preferably about 5.5 to 8.5.

[3-2] Fractionation step by the second adsorption device Next, a supply solution (washing solution) for causing acidic protein to flow out is supplied from the inflow pipe 24 of the second adsorption device 1B into the column 2. Then, the effluent that flows out from the column 2 through the effluent pipe 25 is recovered as a sample solution that has been subjected to the anion exchange step.

  At this time, at least a part of the contaminants (contaminants having a negative charge larger than that of the acidic protein) is adsorbed on the adsorbent 3 composed of the anion exchange resin, and the acidic protein is not adsorbed. Acidic proteins are included in the effluent and at least some contaminants are removed from the effluent.

  By adopting such a configuration, the negatively charged acidic protein is collected in the flow-through liquid and the washing liquid, and the adsorbent in which the contaminant having a negative charge larger than the acidic protein in the sample liquid is composed of an anion exchange resin. 3 is held. Therefore, since the elution step for eluting the acidic protein adsorbed on the adsorbent 3 is omitted, the purification method of the present embodiment for purifying the acidic protein can be simplified.

  Here, the supply liquid supplied into the column 2 is not mixed with the adsorbent 3 composed of the anion exchange resin, although the acidic protein does not bind to the sample liquid supplied in the step [3-1]. Among the substances, those having a negative charge larger than that of acidic protein are bound to the adsorbent 3 composed of an anion exchange resin.

  As such a supply solution, one having the same concentration and pH as the buffer solution or the salt described in the step [3-1] is used.

  The anion exchange chromatography process was performed by collecting the effluent, that is, the flow-through liquid and the washing liquid flowing out from the column 2 as the sample liquid subjected to the anion exchange step. The sample solution is recovered in a state where the acidic protein is second purified (and at least a part of the contaminants is removed).

[4] Fluoroapatite chromatography treatment (fluorapatite process)
In the fluorapatite chromatography treatment, the purification method of the present invention is applied.

  That is, in the fluorapatite chromatography, the sample solution (sample solution subjected to the anion exchange step) is brought into contact with the adsorbent 3 composed of fluorapatite, and the acidic protein is adsorbed to the adsorbent 3 composed of fluorapatite. The first step of adsorption and the adsorbent 3 composed of fluorapatite are supplied with an eluate, and the resulting effluent is fractionated by a predetermined amount so that the effluent of each fractionated fraction is obtained. And a second step of purifying the acidic protein. In the first step, before the sample solution is brought into contact with the adsorbent 3, and / or almost simultaneously, the adsorbent 3 is subjected to Ca ions. Is configured to contact.

  In the following, a case will be described in which Ca ions are brought into contact with the adsorbent 3 both before and almost simultaneously with the sample solution.

[4-1] Step of contacting Ca ions with the adsorbent First, the sample liquid recovered from the second adsorbing apparatus 1B (the sample liquid subjected to the anion exchange step) is brought into contact with the adsorbent 3. Prior to this, Ca ions are brought into contact with the adsorbent 3.

  That is, prior to supplying the sample liquid recovered from the second adsorption device 1B (the sample liquid subjected to the anion exchange step) to the third adsorption device 1C, the equilibration liquid containing Ca ions. Is supplied to the third adsorption device 1C. As a result, the sample liquid passes through the column 2 (third adsorption device 1C), and the equilibration liquid comes into contact with the adsorbent 3 made of fluorapatite, so that Ca ions come into contact with the adsorbent 3. It will be.

  Thus, when Ca ions are brought into contact with the adsorbent 3, among the Ca ions (calcium sites) and phosphate groups (phosphate sites) of the fluorapatite, Ca ions are selectively produced with respect to the phosphate groups. Adsorb. Therefore, the second calcium site whose surface of the phosphate group is covered is formed by the Ca ions adsorbed on the phosphate group (phosphate site).

  Therefore, the adsorbent 3 composed of fluorapatite in contact with Ca ions does not have the ability to adsorb various substances having a positive charge, and exhibits better adsorption ability to various substances having a negative charge. Therefore, the adsorbent 3 exhibits an adsorbing ability to selectively adsorb various substances having negative charges (for example, acidic protein).

The equilibration liquid is not particularly limited as long as it contains Ca ions, and examples thereof include an aqueous solution containing at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ . Thus, the equilibration liquid contains Ca ions by including the salt as described above. Therefore, by bringing the equilibration liquid into contact with the adsorbent 3, the Ca ions can be brought into contact with the adsorbent 3.

  In this case, the concentration of the salt as described above is not particularly limited, but is preferably set to about 1 to 20 mM, more preferably about 2 to 5 mM. By setting the salt concentration within such a range, the Ca ions dissociated from the salt in the equilibration solution can be reliably brought into contact with the adsorbent 3.

  Further, the equilibration solution may be a buffer solution such as a Hepes buffer solution or a Tris buffer solution. In this case, the concentration of the buffer solution is preferably 10 to 200 mM, more preferably 10 to 50 mM, and even more preferably 10 mM.

  The pH of the equilibration liquid is preferably about 6.1 to 8.1, more preferably about 7.2.

[4-2] Supply process to the third adsorption device (first process)
Next, the sample liquid (sample liquid subjected to the anion exchange process) collected from the second adsorption apparatus 1B is supplied to the third adsorption apparatus 1C including the adsorbent 3 that has been contacted with Ca ions. To do. That is, the sample liquid collected from the second adsorption device 1B is supplied to the adsorbent 3 that has been contacted with Ca ions through the inflow pipe 24 and the filter member 4 of the third adsorption device 1C.

At this time, in this embodiment, at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ is added to the sample liquid collected from the second adsorption device 1B in advance. As a result, the sample solution contains the salt as described above.

  Therefore, Ca ions come into contact with the adsorbent 3 almost simultaneously with the passage of the sample liquid through the column 2 (third adsorbing device 1C) and the adsorbent 3 made of fluorapatite. It will be.

  As described above, since Ca ions are brought into contact with the adsorbent 3 almost simultaneously with the adsorbent 3 in contact with the sample liquid, Ca ions coexist in the sample liquid. (In particular, it is possible to accurately suppress or prevent Ca ions from eluting from the second calcium site formed in the step [4-1]). Thereby, the reduction | decrease of Ca ion (calcium site) which fluorapatite has by contacting sample liquid with the adsorbent 3 can be suppressed or prevented exactly.

  Therefore, the adsorbent 3 more reliably exhibits the adsorbing ability for selectively adsorbing various negatively charged substances (for example, acidic proteins) described in the step [4-1].

  Therefore, when the sample liquid is brought into contact with the adsorbent 3 having such adsorption characteristics under the condition that the acidic protein contained in the sample liquid binds to the adsorbent 3 (fluorapatite in contact with Ca ions). Of the various substances contained in the sample solution, various substances having a positive charge and various substances having a negative charge smaller than the purified protein to be purified do not bind to the adsorbent 3. On the other hand, the acidic protein, various substances having a negative charge substantially equal to the acidic protein, and various substances having a negative charge larger than that of the acidic protein bind to the adsorbent 3.

  Therefore, various substances (contaminants) having positive charges that are not bound to the adsorbent 3 flow out from the column 2 through the filter member 5 and the outflow pipe 25. In contrast, acidic protein or the like bound to the adsorbent 3 is held in the column 2.

  Moreover, although the density | concentration of the above salts is not specifically limited, Preferably it is 1-20 mM, More preferably, it sets to about 2-5 mM. By setting the salt concentration within such a range, the Ca ions dissociated from the salt in the sample solution can be reliably brought into contact with the adsorbent 3.

  Furthermore, the pH of the sample solution is preferably about 6.1 to 8.1, and more preferably about 7.2.

[4-3] Adsorbent Cleaning Step Next, the adsorbent 3 to which acidic protein or the like is bound (adsorbed) is cleaned by supplying a cleaning liquid containing Ca ions to the third adsorption device 1C.

  As a result, various substances (contaminants) having a positive charge that are not adsorbed on the adsorbent 3 but remain in the column 2 can be reliably obtained from the column 2 through the filter member 5 and the outflow pipe 25. Can be drained into.

As this cleaning liquid, the same liquid as the equilibration liquid described in the above step [4-1] can be used. That is, as the cleaning liquid, an aqueous solution containing at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ can be used. As a result, various substances (contaminants) having a positive charge remaining in the column 2 can be removed from the column 2 without dissociating (eluting) the acidic protein bound to the adsorbent 3 from the adsorbent 3. It can be surely drained.

  This step [4-3] can be omitted depending on the type of eluate used in the next step [4-4].

[4-4] Fractionation step by the third adsorption device (second step)
Next, an eluate for causing acidic protein to flow out is supplied from the inflow pipe 24 of the third adsorption device 1C into the column 2, and the effluent flowing out from the column 2 through the outflow pipe 25 is supplied to the column 2. Fractionate (collect) quantitatively.

  Thereby, the acidic protein and the contaminant adsorbed on the adsorbent 3 are recovered (separated) in a state of being eluted in each fraction in accordance with the difference in adsorbing force with respect to the adsorbent 3 possessed by each.

  In other words, the adsorbent 3 contains acidic proteins and contaminants (acidic proteins, various substances having a negative charge substantially equal to acidic proteins and various substances having a negative charge larger than that of acidic proteins). ) Because of the specific adsorption by force, acidic protein and contaminants are separated and purified according to the difference in adsorption force.

  Here, when the fluorapatite contacted with Ca ions is used as the adsorbent 3, the fluorapatite contacted with Ca ions is, as described above, the Ca ions (calcium sites) originally possessed by the fluorapatite and the phosphate group. It has the structure which has the 2nd calcium site by which the surface of the phosphate group was covered by Ca ion adsorbed to (phosphate site). Therefore, the adsorbent 3 exhibits excellent adsorbing ability with respect to various negatively charged substances, and the various negatively charged substances are adsorbed with their own specific adsorbing power. By appropriately setting these conditions, it becomes possible to remove the contaminants contained in the sample liquid with high accuracy.

  The eluent supplied into the column 2 is not particularly limited, but a phosphate buffer containing sodium phosphate, potassium phosphate and lithium phosphate is preferably used.

  The pH of the phosphate buffer is not particularly limited, but is preferably set to about 4 to 12, more preferably about 5.5 to 8.5. Thereby, an acidic protein can be eluted reliably and the improvement of the purification rate can be aimed at. Further, since the adsorbent 3 is made of fluorapatite in contact with Ca ions, the adsorbent 3 has excellent acid resistance, so even if a phosphate buffer solution in such a pH range is used. Further, alteration (dissolution, etc.) of the adsorbent 3 can be suitably prevented, and a change in separation ability in the third adsorption device 1C can also be reliably prevented.

  The temperature of the phosphate buffer is not particularly limited, but is preferably about 30 to 50 ° C, more preferably about 35 to 45 ° C. Thereby, alteration (denaturation) of the acidic protein to be separated can be prevented.

  Therefore, the yield (recovery rate) of the target acidic protein can be improved by using a phosphate buffer solution in such a pH range and temperature range as the eluent.

  Further, the concentration of the phosphate buffer is preferably 500 mM or less, and more preferably 400 mM or less. By performing separation of acidic protein using a phosphate buffer solution having such a concentration, adverse effects on the acidic protein due to metal ions in the phosphate buffer solution can be prevented.

  Specifically, it is more preferable to use a phosphate buffer having a concentration of about 1 to 400 mM. Further, it is preferable that the phosphate buffer is changed continuously or stepwise during the separation operation of the acidic protein. Thereby, the efficiency of separation operation of acidic protein can be achieved.

The phosphate buffer may further contain a salt such as NaCl, KCl, MgCl, NH ₄ Cl. Thereby, the yield of the target acidic protein can be improved more.

  Although the density | concentration of such a salt is not specifically limited, For example, Preferably it is about 30 mM-about 1.0M, More preferably, it sets to about 300-500 mM.

  Further, the phosphate buffer may contain hepes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), tris- (Tris-hydroxymethylammonium), and the like. In this case, the concentration of these in the phosphate buffer is preferably 10 to 200 mM, more preferably 10 to 50 mM, and even more preferably 10 mM.

The flow rate of the phosphate buffer is preferably about 0.1 to 10 mL / min, more preferably about 1 to 5 mL / min. By separating acidic protein at such a flow rate, the target acidic protein can be reliably separated without requiring a long time for the separation operation, that is, a high-purity acidic protein can be obtained.
By the above operation, acidic protein is recovered in a predetermined fraction.

  In the present embodiment, the case has been described where Ca ions are brought into contact with the adsorbent 3 before and almost simultaneously with the adsorbent 3. However, the present invention is not limited to this case. It may be either before or almost simultaneously. However, when the sample liquid is brought into contact with the adsorbent 3 by contacting the adsorbent 3 with Ca ions both before and almost simultaneously with the sample liquid as in the present embodiment, fluorine is used. The 2nd calcium site by which the surface of the phosphate group which apatite has was covered with Ca ion can be formed more certainly.

  Furthermore, in the present embodiment, for the purpose of purifying an acidic protein having an immunoglobulin constant domain, the sample solution is subjected to affinity chromatography, anion exchange chromatography, and fluorapatite chromatography in this order in three steps. However, when the acidic protein does not have an immunoglobulin constant domain, the affinity chromatography treatment and the anion exchange chromatography treatment can be omitted. In addition, although it does not specifically limit as acidic protein which does not have immunoglobulin constant domain, For example, BSA, HSA, fibrinogen, pepsinogen, alpha-globulin, beta-globulin etc. are mentioned, Of these, 1 type or 2 types A combination of the above can be used.

  In addition to the above three-step process, the sample solution is subjected to other processes such as a hydrophobic interaction chromatography process, a metal affinity chromatography process, a volume exclusion chromatography process, and an ultrafiltration process. You may do it.

  Although the purification method of the present invention has been described above, the present invention is not limited to this.

  For example, in the present invention, for any purpose, the previous step of the step [4-1], the intermediate step existing between the steps [4-1] to [4-4], or the step [4-4] You may make it add the post process.

Next, specific examples of the present invention will be described.
・ Measurement of adsorption amount of acidic protein (BSA) (Example)
-1- First, a φ4.0 × 100 mm stainless steel column (adsorber) packed with ceramic fluoroapatite Type II, 40 μm (Bio-rad Laboratories, Inc.) as an adsorbent was prepared.

  -2- Next, a sample solution in which 10 mg / mL BSA is dissolved in 1 mM phosphate buffer solution (pH 5.0) is prepared, and then this sample solution is supplied to the adsorption device at a flow rate of 1 mL / min (apply). did.

  -3- Next, the amount of BSA dynamically adsorbed on the adsorbent was measured using a chromatographic apparatus (manufactured by Bio-rad, "DuoFlow").

The measurement of the dynamic adsorption amount of BSA by the above steps 1- to 3- is carried out by setting the concentration of CaCl _{2 in} the sample solution (phosphate buffer solution) to 0 mM, 0.1 mM and 0.5 mM. I went instead.

Furthermore, the measurement of the dynamic adsorption amount of BSA at the CaCl ₂ concentration was repeated 20 times.

(Comparative example)
As the adsorbent provided in the adsorber, the dynamics of BSA were the same as in the above example except that ceramic hydroxyapatite Type II, 40 μm (Bio-rad Laboratories, Inc.) was used instead of ceramic fluoroapatite. The measurement of the amount of adsorption was repeated 20 times.

  Table 1 shows the measured values obtained in the first and twentieth measurements of the measured dynamic adsorption amount of BSA in the above Examples and Comparative Examples.

As described above, as is clear from the first measurement values of the examples and comparative examples, both the case where ceramic fluoroapatite is used as the adsorbent provided in the adsorption device and the case where ceramic hydroxyapatite is used are used. As a result, as the concentration of CaCl ₂ in the sample solution increased, the adsorption amount of BSA (acidic protein) was improved.

  However, as is apparent from the measured values in the 20th time of the examples and comparative examples, when ceramic fluoroapatite is used as the adsorbent provided in the adsorption device, the amount of adsorption of BSA is also increased by using the adsorbent multiple times. Although no change was observed, when ceramic hydroxyapatite was used, the amount of adsorbed BSA was greatly reduced by using the adsorbent multiple times.

  This is because the adsorption apparatus of the example uses ceramic fluoroapatite as the adsorbent, and the adsorption of BSA to the adsorbent using a low pH range sample solution such as pH 5.0 is repeated several times. Even if it is carried out, it is considered that dissolution of the adsorbent (ceramic fluoroapatite) is accurately prevented.

  In contrast, in the adsorption device of the comparative example, ceramic hydroxyapatite as an adsorbent is adsorbed by repeatedly adsorbing BSA to the adsorbent using an eluent in a low pH region (pH 5.0). It is inferred that the amount of BSA adsorbed at the 20th time decreased due to the dissolution of the agent.

  From the above, compared with the case of using ceramic hydroxyapatite as the adsorbent, when using ceramic fluoroapatite as the adsorbent, when adsorbing acidic proteins in the low pH region (acidic conditions) It was found that the adsorption can be stably (long-term) by adding Ca ions.

DESCRIPTION OF SYMBOLS 1 Adsorber 1A 1st adsorber 1B 2nd adsorber 1C 3rd 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 (10)

A purification method for purifying the acidic protein from a sample solution containing the acidic protein and a contaminant comprising a substance other than the acidic protein,
A first step of bringing the sample solution into contact with an adsorbent composed of fluorapatite, and adsorbing the acidic protein to the adsorbent composed of fluorapatite;
The eluent is supplied to the adsorbent composed of the fluorapatite, and the resulting effluent is fractionated by a predetermined amount to purify the acidic protein in the effluent of each fractionated fraction. And a second step to
In the first step, before the sample solution is brought into contact with the adsorbent and / or almost simultaneously, the adsorbent is brought into contact with Ca ions.   The purification method according to claim 1, wherein, in the first step, the equilibration liquid containing the Ca ions is brought into contact with the adsorbent prior to contacting the sample liquid with the adsorbent. The purification method according to claim 2, wherein the equilibration liquid contains at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .   The purification method according to any one of claims 1 to 3, wherein, in the first step, the sample liquid brought into contact with the adsorbent further contains the Ca ions. The purification method according to claim 4, wherein the sample solution contains at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .   The purification method according to any one of claims 1 to 5, wherein, prior to the second step, the adsorbent adsorbed with the acidic protein is washed with a washing solution containing the Ca ions. The purification method according to claim 6, wherein the cleaning liquid contains at least one of CaCl ₂ , Ca (NO ₃ ) ₂ , CaSO _4, and CaCO ₃ .   The purification method according to any one of claims 1 to 7, wherein the eluate is a phosphate buffer having a concentration of 1 mM to 400 mM.   The purification method according to claim 8, wherein the eluate is a phosphate buffer having a pH of 4 to 12.   The purification method according to claim 1, wherein the acidic protein has an immunoglobulin constant domain.

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