WO2018074559A1 - Complex of medical protein and polyamino acid, stabilization method and use thereof - Google Patents

Abstract

The purpose of the present invention is to provide a method whereby an easily denaturing medical protein can be more stabilized and easily concentrated, stored, etc. and, at the time of dosing, the protein can be easily converted into the original form. Compared with a complex of a medical protein and a polyamino acid with a random structure, a complex of the medical protein and the polyamino acid with an α-helix structure can more stabilize the protein, shows excellent stability during transportation and storage and is easy to handle in use.

Description

Complex of medical protein and polyamino acid, stabilization method and use thereof

The present invention relates to a complex with a medical protein using an α-helix structure of a polyamino acid, a stabilization method and its use.

Due to advances in genetic recombination technology, various protein drugs have been developed since the 1980s. In particular, the progress of antibodies known as molecular targeted drugs has been remarkable and has contributed to the treatment of intractable diseases such as cancer and rheumatoid arthritis that have been difficult to treat.
Since oral administration of protein pharmaceuticals is difficult unlike pharmaceuticals with low molecular weight compounds, they are often administered into the body by injection. Of these, subcutaneous injection is expected to be a new administration method because it is less painful and simple and can be self-administered (patient self-injection). However, since subcutaneous injection restricts the dose to 1.5 mL or less, it is usually necessary to prepare a high concentration protein solution of 100 mg / mL or more.

In order to increase the concentration of protein pharmaceuticals, a method of re-dissolving the powder formulation is widely used. Although only a small amount of a lyophilized protein powder is dissolved, there are some difficult points in practice. First, it must be taken into account that the protein is unstable. When protein in a powder state is dissolved, physicochemical stress such as shear stress or surface tension is applied, which may irreversibly denature. Furthermore, the aggregation of the denatured protein tends to occur as the concentration of the protein solution to be prepared increases. Aggregates not only reduce the effectiveness of protein drugs, but also adversely affect safety such as causing undesirable immune reactions. In addition to protein instability, the time required for dissolution is also a realistic issue. When preparing a high-concentration salt solution, it can be dissolved by stirring with a stirrer or vortex, but the protein is unstable and must be handled with care to avoid denaturation. In the actual formulation, after adding a solvent such as saline to the powder formulation in the vial, dissolve it by gently shaking the vial so that it does not foam. Therefore, it may take several tens of minutes to several hours to dissolve all the protein powder.

Another approach is protein enrichment. That is, it is a method of preparing a high concentration protein solution by selectively removing the solvent from the low concentration protein solution and reducing the amount of the solvent. Typical concentration methods include ultrafiltration, chromatography, and evaporation. Examples of methods for re-dissolving in powder form include freeze drying and spray drying.
However, since the number of operation steps increases, equipment and time costs remain as problems. In recent years, new concentration methods that do not require devices such as liquid-liquid phase separation, gelation, and crystallization have been developed, but problems such as irreversible denaturation of proteins accompanying processing remain. Thus, in order to obtain a high concentration protein solution, 1) development of a method that does not require denaturation of the protein, 2) a simple and rapid process, and 3) no investment such as an apparatus is expected. Furthermore, considering that the types of protein drugs will increase in the future, regardless of antibodies, enzymes, hormones, etc., 4) a general-purpose method that can be used for various proteins is desirable.
The present applicant has disclosed an aqueous suspension containing a protein / polyamino acid complex in Patent Document 1. The aqueous suspension containing the protein / polyamino acid complex can be concentrated by removing the solvent, and the protein can be dissociated by adding a low concentration electrolyte and used as a pharmaceutical product.

WO2015 / 06491

A number of methods for stabilizing medical proteins are provided, but there are further requirements. There is a demand for a method that can stabilize medical proteins that are easily denatured, can be concentrated and stored easily, can be easily adjusted, and can be administered with weak force.

The present inventors provide a complex that can stabilize a medical protein and increase the storage stability of the medical protein, and can administer the complex directly into a living body when necessary. The present invention seeks to provide a complex of α-helix structure of polyamino acid and medical protein, and use thereof.

That is, the present invention provides the following.
(1) A complex of a medical protein and an α-helix structure of a polyamino acid.
(2) An injection containing an aqueous suspension containing the complex according to (1) above or a concentrate thereof.
(3) The complex, aqueous suspension, concentrate or injection of the complex according to (1) or (2), wherein the polyamino acid is polylysine, polyglutamic acid or a water-soluble salt thereof.
(4) The aqueous suspension according to (2) or (3), wherein the pH of the aqueous suspension is 3.5 to 4.5.
(5) A method of forming a complex of an antibody protein and an α-helical structure of a polyamino acid having a molecular weight of 1.5 kDa to 5.5 kDa, and stabilizing the antibody protein from the complex with a random structure of the polyamino acid.
(6) A method of forming a complex of an antibody protein and an α-helix structure of a polyamino acid having a molecular weight of more than 5.5 kDa, and stabilizing the antibody protein from the complex with a random structure of the polyamino acid.
(7) The method for stabilizing a protein according to (5) or (6), wherein the polyamino acid is polyglutamic acid.
(8) The antibody protein is formed into a complex with polyglutamic acid at pH 3.5 to 4.5, an electrolyte (salt) is added to the complex, and the pH is increased to make the antibody protein complex. A method of dissociation from the body.

When a complex of a protein for medical use and an α-helical structure of polyamino acid is formed, the protein can be more stabilized than a complex of a random structure of polyamino acid obtained in the past, and stable during transportation and storage. Excellent in handling and handling at the time of use.

FIG. 1 is a schematic diagram for explaining an α-helix structure of polyglutamic acid at pH 4 and a random structure at pH 7. FIG. 2A is a graph showing the results of Table 1. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 2B is a graph showing the results of Table 1. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. FIG. 3A is a graph showing the results of Table 3. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 3B is a graph showing the results of Table 3. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. FIG. 4A is a graph showing the results of Table 6. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 4B is a graph showing the results of Table 6. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of human IgG2 monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. It is a graph which shows the result of the residual rate before and behind the shaking stress of Table 7. It is a graph which shows the result of the residual rate before and behind the heat stress of Table 8. FIG. 7A is a graph showing the results of Table 9. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 7B is a graph showing the results of Table 9. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. FIG. 8A is a graph showing the results of Table 10. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 8B is a graph showing the results of Table 10. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. FIG. 9A is a graph showing the results of Table 11. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 9B is a graph showing the results of Table 11. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. The vertical axis represents the measured recovery rate of the complex. FIG. 10A is a graph showing the results of Table 12. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. A vertical axis | shaft shows the formation rate of the measured composite_body | complex. FIG. 10B is a graph showing the results of Table 12. The horizontal axis represents the concentration indicating the parts by mass of poly-L-glutamic acid per 1 part by mass of the anti-TNFα monoclonal antibody. The vertical axis represents the measured recovery rate of the complex.

[1. Complex of medical protein and α-helix structure polyamino acid)
Alpha helix is one of the common motifs of protein secondary structure and has a right-handed helix shape resembling a spring. Short polypeptide chains in solution are less likely to take an α-helix structure because the entropy required to form the helix is not compensated by the stability of the helix binding. Different amino acid sequences show different tendencies for α-helix formation. Methionine, alanine, leucine, glutamic acid, and lysine have a strong tendency to make a helix, but proline, glycine, tyrosine, and serine are difficult to make a helix.
The present inventors have obtained the following two new findings and made the present invention. First, polyglutamic acid having a chain length of a certain length or more has an α-helix structure at a low pH, and the binding when forming a complex with an antibody protein becomes stronger and can be further stabilized. Secondly, polyglutamic acid has a random structure when the pH is high, so that the binding force with the antibody protein is weakened and the protein is easily dissociated from the complex. The complex of the protein for medical use and the α-helix structure of polyamino acid does not require that all proteins in the aqueous solution form a complex with the α-helix structure. If a complex with the structure is formed, it can be stabilized more than a complex with the random structure, so that a part of the protein in the aqueous solution only needs to form a complex with the α helix structure.
FIG. 1 is a schematic diagram illustrating an α-helix structure of polyglutamic acid at pH 4 and a random structure at pH 7. In a random structure, a complex is formed by electrostatic interaction. It is thought that the hydrophobic interaction other than the electrostatic interaction works further in the complex with the α helix structure.

When a protein for medical use forms a complex with the α-helix structure of a polyamino acid, a stable complex with a higher binding force to the protein is obtained than a complex with a random structure of a polyamino acid. Spread. In general, additives such as excipients, surfactants, and stabilizers are used to stabilize protein drugs. When these are used, the type, combination, and amount added are optimized for each protein drug. In order to do so, it is necessary to make a complicated and enormous study. The aqueous suspension of the present invention can be easily stabilized by forming a complex with a polyamino acid, and it is not necessary to add an additive that has been necessary in the past, and its usefulness can be enhanced. . Further, it does not require a complicated dissolving operation required for a freeze-dried preparation, and can be administered as it is, or can be administered as an aqueous liquid after being dissolved by adding an inorganic salt typified by sodium chloride at the time of use. Furthermore, even if the protein concentration is high, the aqueous suspension of the complex has the characteristic that the viscosity is low, so that the amount of waste remaining in the container at the time of use can be reduced, and a syringe can be used. At the time of administration, it can be administered with a weaker force than an aqueous protein solution having the same concentration.

The medical protein to be used is not limited. The molecular weight is preferably 0.5 kDa to 1000 kDa, and an antibody protein having high utility as a pharmaceutical preparation such as adalimumab known as an active ingredient of Humira (registered trademark) or denosumab known as an active ingredient of Lanmark (registered trademark) should be used. preferable.
(antibody)
For example, muromonab-CD3, rasutuzumab, rituximab, rituximab, cetuximab, rituximab, rituximab, rituximab, rituximab, rituximab, rituximab, rituximab, rituximab, rituximab Golimumab, force nakinumab, denosumab, mogum lizumab, cello lizumab pegol, offatumumab, pertuzumab, 卜 rasutuzumab emtansine, brentuximab vedotin, natalizumab, nipolumab, alemtuzumab edema , Abciximab, siltuximab, daclizumab, efalizumab, obinutuzumab, vedolizumab, pembu There are lolizumab, ixekizumab, diridabumab, ipilimumab, berimumab, laxibakumab, ramsirmafu.
(Fusion protein)
For example, there are etanercept, avatarcept, romiplostim, and aflibercept.
Examples of other medical proteins include enzymes, blood coagulation / fibrinolytic factors, serum proteins, hormones, vaccines, interferons, erythropoietins, and cytokines.

(Polyamino acid)
The complex of the present invention can be produced by adjusting the pH of a protein and a polyamino acid in an aqueous solution and combining the protein with a protein under the condition that the polyamino acid takes an α structure. The aqueous suspension containing the complex can contain water, a buffer, a protein, a polyamino acid, and a pH adjuster in an appropriate combination. However, it is preferable not to contain additives other than the components described here. Moreover, protein can be concentrated by concentrating the aqueous suspension containing the complex. A protein can be obtained by adding a low-concentration electrolyte to an aqueous suspension containing the complex to dissociate the complex.
Since a complex is mainly formed through electrostatic interaction, a protein and a polyamino acid having an opposite charge are combined to form a complex.
Examples of anionic polyamino acids used for cationic proteins are polyglutamic acid (MW: 750 to 5000, pI: 2.81 to 3.46), polyglutamic acid (MW: 3000 to 15000, pI: 2.36 to 3). .00), polyglutamic acid (MW: 15000 to 50000, pI: 1.85 to 2.36), polyglutamic acid (MW: 50,000 to 100,000, pI: 1.56 to 1.85), polyaspartic acid (MW: 2000-11000, pI: 2.06-2.75), polyaspartic acid (MW: 5000-15000, pI: 1.93-2.39) and at least one selected from the group consisting of these water-soluble salts Two anionic polyamino acids, which can form a complex with a cationic protein.
Examples of cationic polyamino acids used for anionic proteins are polylysine (MW: 1000 to 5000, pI: 10.85 to 11.58), polylysine (MW: 4000 to 15000, pI: 11.49 to 12.06). ), Polylysine (MW: 15000-30000, pI: 12.06-12.37), polylysine (MW: 30000-, pI: 12.37-), polyarginine (MW: 5000-15000, pI: 13.49) To 13.97), polyarginine (MW: 15000 to 70000, pI: 13.98 to 14.00), polyarginine (MW: 70000 to, pI: 14.00), polyhistidine (MW: 5000 to 25000, pI: 7.74-8.30) and at least selected from the group consisting of these water-soluble salts One of a cationic polyamino acid, complexes of these with anionic protein can be formed.

Examples of the electrolyte include NaCl, KCl, CaCl ₂ , MgCl _{2, and the} like. Preferably, NaCl having the highest biocompatibility is selected. The electrolyte concentration is not limited, but it is 5% by mass or less, and a sufficient amount for dissolving the complex can be used.

[Buffer]
The buffer used in the present invention refers to a solution having a buffering action against the hydrogen ion concentration, and the buffer is adjusted so that the pH does not change greatly even if a small amount of acid or base is added or the concentration slightly changes. The aqueous solution. The buffer solution is not particularly limited as long as it is usually used chemically, but a biologically used buffer solution is preferable. More preferable forms of the buffer include phosphate buffer, citrate buffer, citrate-phosphate buffer, tartrate buffer, acetate buffer, MOPS buffer (3- (N-morpholino) propanesulfonic acid) buffer. , MOPS-HaOH buffer, PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid)) buffer, TrisHCl buffer (trishydroxymethylaminomethane-HCl buffer), MES (2-Morpholinoethanesulfonic acid, monohydrate) Buffer, HEPES (4- (2-Hydroxyethyl) -1-piperazine etheric acid) buffer, HEPES-NaOH buffer, Glycine N OH buffer, glycine-hydrochloric acid buffer, glycylglycine-NaOH buffer, glycylglycine-KOH buffer, tris-borate buffer, boric acid-NaOH buffer, borate buffer, TES buffer (N -Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer solution), imidazole buffer solution, and other biological samples that can be administered to a living body can be used. The formulation of the buffer into the aqueous suspension containing the complex can be administered without previously dissociating the complex, and is suitable for an administration method in which the protein is dissociated in the body after administration.

It has been found that the complex of the present invention, the aqueous suspension containing the complex or its concentrate, and the injection containing these have no general toxicity. Formulated for any route of administration, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if necessary, for local treatment, intralesional administration. Parenteral injection includes intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or subcutaneous administration. Preferably, it is administered by injection, most preferably by intravenous or subcutaneous injection. Including topical, particularly transdermal, transmucosal, rectal, oral administration, or local administration, eg, through a force tape placed near the desired site. Depending on the route of administration, pharmaceutically acceptable excipients or diluents may be included. Examples of such excipients include water and pharmaceutically acceptable organic solvents.
A complex of a medical protein and an α-helix structure of a polyamino acid can be formed at a low pH, and is more stable than a complex of a medical protein and a random structure of a polyamino acid. Therefore, it can be stored as a complex for a long period of time and stored at room temperature until administration, and when administered into the body, it becomes an environment of pH 7, and the α-helix structure of the complex becomes a random structure and the protein is easily dissociated. It can be effectively used as a protein for use.

2. Production method of the complex The polyamino acid has an α-helix structure at a relatively low pH. For example, the complex can be produced by contacting the polyamino acid and the protein in a buffer solution of pH 3.5 to 4.5. . The complex of the α-helix of the produced polyamino acid and the protein is more stable than the complex of the same polyamino acid and a random structure. If necessary, an electrolyte (salt) is added to the aqueous suspension containing the complex and allowed to dissociate as it is or at a higher pH to obtain a protein.
[Precipitation-re-dissolution method]
A polyamino acid having an opposite charge is added to the protein stock solution (Step 1) under the condition of α-helix structure to form a precipitating complex (Step 2). Next, this complex is precipitated (Step 3), and the protein solution can be concentrated by removing the supernatant from the precipitate (Step 4). Thereafter, by administering the complex as it is in the living body, the pH of the polyamino acid becomes a random structure, the protein is easily dissociated, and the original native protein can be obtained. The complex can be redissolved in advance by adding a salt at a concentration necessary for dissociation, and then administered in vivo (Step 5). As a specific condition, it is preferable to set the final concentration of salt at the time of re-dissolution to 150 mM, which is equivalent to physiological saline.

Hereinafter, the present invention will be described using specific examples, but the present invention is not limited to these examples.
The method for measuring and calculating the formation rate and the recovery rate in the examples is not limited. For example, the formation rate of the protein / poly-L-glutamic acid complex in the aqueous suspension is determined by precipitating the complex, When measured by a size exclusion chromatograph (hereinafter sometimes referred to as SEC) method and the amount of protein is 0%, it is assumed that all proteins are in a complex, and the formation rate is 100%. The recovery rate is determined by measuring the solution from which the complex is dissociated by SEC, taking into consideration the formation rate, and if the protein concentration is the same as the concentration of the chemical solution before complex formation, The value obtained as the recovery rate of 100% can be assumed to be formed and dissociated.
In the examples described below, when both the formation rate and the recovery rate are high, the aqueous suspension of the present invention can be used as it is as a protein preparation, and either the formation rate or the recovery rate is excellent Can be used as a protein preparation with improved conditions. When both the formation rate and the recovery rate are small, it is useful for studying the mechanism of the complex formation of the present invention by examining the conditions.
In the table below, as a guideline, vertical lines are drawn in the column of example numbers so that the results with excellent formation rate and recovery rate can be distinguished from the other results.

1. [Preparation of aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex targeting RANKL (receptor activator for nuclear factor-κB ligand)]
(Test Example 1)
[Formation of complex]
A human IgG2 monoclonal antibody was prepared to a concentration of 1.0 mg / mL in 10 mM phosphate buffer (pH 4.0), and 0.04 to 0.14 relative to 1 part by mass of the human IgG2 monoclonal antibody. A part by weight of poly-L-glutamic acid (E1: MW: 1.5 kDa-5.5 kDa) was added to obtain an aqueous suspension containing a human IgG2 monoclonal antibody / poly-L-glutamic acid complex. This aqueous suspension was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected, and the protein content was measured by size exclusion chromatography (SEC) (measuring instrument Shimadzu LC-20A). In addition, a solution prepared by adjusting the concentration of human IgG2 monoclonal antibody to a concentration of 1.0 mg / mL in 10 mM phosphate buffer (pH 4.0) was prepared as a control solution (Example 1-1), and size exclusion was performed. The protein content was measured by a chromatographic method (SEC), and the protein content in the supernatant of the aqueous suspension was subtracted to obtain the complex formation rate.
[Dissociation of complex]
To the resulting complex, an isotonic physiological buffer saline solution (PBS) (trade name Dulbecco's PBS (-) powder “Nissui”) is added to dissociate the antibody protein from the complex, and size exclusion chromatography. The protein content was measured by the graph method (SEC). A solution prepared by adjusting the concentration of human IgG2 monoclonal antibody to a concentration of 1.0 mg / mL in PBS (pH 7.0) was prepared as a control solution (Example 1-1), and the protein content was measured by SEC. The recovery rate was calculated as a relative value with the protein concentration of the control solution as 100%. The results are shown in Table 1, FIGS. 2A and 2B. Similar curves were obtained for the formation rate and the recovery rate.

(Test Example 2)
A human IgG2 monoclonal antibody was used to form a complex with α-helix at pH 4 in the same manner as in Test Example 1, and the formation rate was measured. The resulting complex was dissociated with a sodium chloride-10 mM phosphate buffer (pH 4.0) prepared to a final concentration of 150 mM, and the recovery rate was measured in the same manner as in Test Example 1. The results are shown in Table 2.

(Test Example 3)
In the same manner as in Test Example 1 except that 0.04 to 0.13 parts by mass of poly-L-glutamic acid (E4: MW: 50 kDa-100 kDa) was added to 1 part by mass of the human IgG2 monoclonal antibody, α at pH 4 A complex with a helix was formed, and the formation rate was measured. A physiological buffer salt solution (trade name Dulbecco PBS (-) powder “Nissui”) that is isotonic is added to the resulting complex, and poly-L-glutamic acid (E4: MW: 50 kDa-100 kDa) is used as a random structure. The antibody protein was dissociated from the complex, and the recovery rate was measured in the same manner as in Test Example 1. The results are shown in Table 3, FIGS. 3A and 3B. The complex formation rate with polyglutamic acid having a high molecular weight was high, and the recovery rate was also high.

(Test Example 4)
In the same manner as in Test Example 1 except that 0.04 to 0.13 parts by mass of poly-L-glutamic acid (E4: MW: 50 kDa-100 kDa) was added to 1 part by mass of the human IgG2 monoclonal antibody, α at pH 4 A complex with a helix was formed, and the formation rate was measured. The resulting complex was dissociated with a sodium chloride-10 mM phosphate buffer (pH 4.0) prepared to a final concentration of 150 mM, and the recovery rate was measured in the same manner as in Test Example 1. The results are shown in Table 4. The complex formation rate with polyglutamic acid having a high molecular weight was high, but the recovery rate was low.

From the results of Test Example 4, it is understood that polyglutamic acid has an α-helix structure near pH 4, and a more stable complex is formed that cannot be dissociated when the side chain of polyglutamic acid becomes long. This effect cannot be explained by hydrophobic interactions alone. In addition, the antibody protein complex bound to the α-helix structure polyglutamic acid is more stable than the complex with the conventional random structure, and the recovery rate is high if the polyglutamic acid is dissociated into a random structure at pH 7. Thus, it can be seen that the antibody protein complex bound to the polyglutamic acid having an α-helix structure is dissociated when administered in vivo.

(Test Example 5)
The formation of the poly-L-glutamic acid (E1: MW: 1.5 kDa-5.5 kDa) complex of Test Example 1 was carried out in the same manner as in Test Example 1, except that a 10 mM phosphate buffer (pH 7.0) was used. When the protein was dissociated from the obtained complex, an isotonic physiological buffer saline solution (trade name Dulbecco PBS (-) powder “Nissui”) was added. The results measured in the same manner as in Test Example 1 are shown in Table 5. In the formation of a poly-L-glutamic acid (E1: MW: 1.5 kDa-5.5 kDa) complex, the complex formation rate was low and the recovery rate was low at pH 7.0.

(Test Example 6)
Other than adding 0.01-0.04 parts by mass of poly-L-glutamic acid (E4: MW: 50 kDa-100 kDa) to 1 part by mass of human IgG2 monoclonal antibody to make a 10 mM phosphate buffer (pH 7.0) Formed a composite as in Test Example 1. A physiological buffer saline solution (trade name Dulbecco PBS (−) powder “Nissui”) that is isotonic is added to the obtained complex, and the antibody protein is dissociated from the complex at pH 7 to obtain Test Example 1 Similarly, the recovery rate was measured. The results are shown in Table 6, FIG. 4A and FIG. 4B. The complex with E4 formed at pH 7 slightly decreased in the formation rate and the recovery rate.

2. [Tolerance of shaking stress of aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex targeting RANKL (receptor activator for nuclear factor-κB ligand)]
(Test Example 7)
Filled with aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex of Example 1-7 (E1 / pH4), Example 3-5 (E4 / pH4) and Example 6-5 (E4 / pH7) The polypropylene disposable tube was placed on a shaker (Bioshaker VR-36) and shaken at 500 rpm at room temperature for 27 days. In each buffer, control solutions (Examples 1-1 and 6-1) prepared to have a concentration of human IgG2 monoclonal antibody at a concentration of 1.0 mg / mL and adjusted to pH 4 and pH 7 were shaken at the same time. . In the table, the formulation dilution (Example 7-1) was diluted with a brand name Lanmark formulation having a pH of 5 and used as a control solution.

[Measurement of shaking residual rate]
The aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex and the control solution before and after shaking were centrifuged at 14000 rpm for 15 minutes, and the supernatant was collected and suspended by SEC. And the protein content contained in the control solution was measured. Table 7 and FIG. 5 show the measurement results of the protein content after shaking with the protein content before shaking as 100%.

From the results of Table 7, it can be seen that the stability against shaking stress is very high when the antibody protein is a complex with an α-helix structure. It can be seen that the complex formed at pH 4 is more stable than the complex formed at pH 7.

3. [Heat resistance of aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex targeting RANKL (receptor activator for nuclear factor-κB ligand)]
(Test Example 8)
Separately prepared in the same manner as in Test Example 7, the human IgG2 monoclonal antibody poly-L- of Example 1-7 (E1 / pH4), Example 3-5 (E4 / pH4), and Example 6-5 (E4 / pH7) It was placed in a polypropylene disposable tube filled with an aqueous suspension containing a glutamic acid complex and kept at 60 ° C. for 7 days. In addition, in each buffer, control solutions (Examples 1-1 and 6-1) prepared by adjusting the pH of the human IgG2 monoclonal antibody so as to have a concentration of 1.0 mg / mL to pH 4 and pH 7 were similarly used. It was kept at 60 ° C. for 7 days. In the table, the formulation dilution (Example 7-1) was diluted with a brand name Lanmark formulation having a pH of 5 and used as a control solution.

[Measurement of residual heating rate]
The aqueous suspension containing human IgG2 monoclonal antibody / poly-L-glutamic acid complex and the control solution before and after heating were centrifuged at 14000 rpm for 15 minutes, the supernatant was collected, and the suspension and The protein content contained in the control solution was measured. Table 8 and FIG. 6 show the measurement results of the protein content after heating, where the protein content before heating is 100%.

From the results in Table 8, it can be seen that the stability against heat stress is very high when the antibody protein is a complex. The stability of the complex was not significantly different between the complex formed at pH 4 and the complex formed at pH 7.

4). [Preparation of aqueous suspension containing anti-TNFα monoclonal antibody / poly-L-glutamic acid complex]
(Test Example 9)
[Formation of complex]
In 10 mM acetate buffer (pH 4.0), an anti-TNFα monoclonal antibody was prepared to a concentration of 1 mg / mL, and 0.01-1.0 parts by mass of poly-polyamide was added to 1 part by mass of the anti-TNFα monoclonal antibody. L-glutamic acid (E1: MW: 1.5 kDa-5.5 kDa) was added to obtain an aqueous suspension containing an anti-TNFα monoclonal antibody / poly-L-glutamic acid complex. This aqueous suspension was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected, and the protein content was measured by size exclusion chromatography (SEC) (measuring instrument Shimadzu LC-20A). In addition, a solution prepared by adjusting the anti-TNFα monoclonal antibody to a concentration of 1.0 mg / mL in 10 mM acetate buffer (pH 4.0) was prepared as a control solution, and the protein content was measured by SEC. The protein content in the supernatant of the suspension was subtracted to obtain the complex formation rate.

[Dissociation of complex]
An isotonic physiological buffer salt solution (trade name Dulbecco PBS (-) powder “Nissui”) is added to the resulting complex, the antibody protein is dissociated from the complex, and the protein content is measured by SEC. The recovery rate was calculated as a relative value with the protein concentration of the control solution as 100%. The results are shown in Table 9 and FIGS. 7A and 7B. Similar curves were obtained for the formation rate and the recovery rate.

(Test Example 10)
In the same manner as in Test Example 9 with respect to 1 part by mass of the anti-TNFα monoclonal antibody, a complex with α-helix was formed at pH 4 and the formation rate was measured. The obtained complex was dissociated with a sodium chloride-acetate buffer solution (pH 4.0) prepared to a final concentration of 150 mM, and the recovery rate was measured in the same manner as in Test Example 9. The results are shown in Table 10 and FIGS. 8A and 8B. The complex formation rate was high, but the recovery rate was low.

From the results shown in Table 10, the complex bound to the α-helix of polyglutamic acid at pH 4 has a hydrophobic interaction other than electrostatic interaction, so it does not dissociate with salt alone, and the recovery rate is poor. I understand that.

(Test Example 11)
In the same manner as in Test Example 9, except that 0.01 to 1.4 parts by mass of poly-L-glutamic acid (E4: MW: 50 kDa to 100 kDa) was added to 1 part by mass of the anti-TNFα monoclonal antibody. A complex with Lix was formed and the formation rate was measured. A physiological buffer salt solution (trade name Dulbecco PBS (-) powder “Nissui”) that is isotonic is added to the resulting complex, and poly-L-glutamic acid (E4: MW: 50 kDa-100 kDa) is used as a random structure. The antibody protein was dissociated from the complex, and the recovery rate was measured in the same manner as in Test Example 9. The results are shown in Table 11, FIG. 9A and FIG. 9B. The complex formation rate was high, and the recovery rate increased as the mass ratio increased.

(Test Example 12)
In the same manner as in Test Example 11 with respect to 1 part by mass of the anti-TNFα monoclonal antibody, a complex with α-helix was formed at pH 4 and the formation rate was measured. The obtained complex was dissociated with a sodium chloride-acetate buffer (pH 4.0) prepared to a final concentration of 150 mM, and the recovery rate was measured in the same manner as in Test Example 9. The results are shown in Table 12, FIG. 10A and FIG. 10B. The complex formation rate was high, but the recovery rate was low.

From the results shown in Table 12, the complex bound to the α-helix of polyglutamic acid (E4) at pH 4 does not dissociate and recovers due to further hydrophobic interaction other than electrostatic interaction. You can see that the rate is bad.
In this experiment, as with human IgG2 monoclonal antibody, protein can be recovered from the complex by making it a neutral solution containing salt, so the complex of the protein bound to the α-helix of polyglutamic acid is It was found to dissociate when administered to a living body.

By using the complex of the present invention and a method using the same, the protein solution can be rapidly and easily concentrated. Furthermore, the complex with the α helix structure obtained in the present invention is more stable than the complex with a normal random structure. Until now, protein aggregates and precipitates have been hated in the medical field, and the possibility of handling them as a preparation has not been studied. However, controlled aggregation and precipitation are also effective for protein concentration and stabilization, and are highly useful as protein preparations.

Claims (8)

Complex of protein for medical use and α-helix structure of polyamino acid. An injection containing an aqueous suspension containing the complex according to claim 1 or a concentrate thereof. The complex, aqueous suspension, concentrate or injection of claim 1, wherein the polyamino acid is polylysine, polyglutamic acid or a water-soluble salt thereof. The aqueous suspension according to claim 2 or 3, wherein the pH of the aqueous suspension is 3.5 to 4.5. A method in which a complex of an antibody protein and an α-helix structure of a polyamino acid having a molecular weight of 1.5 kDa to 5.5 kDa is formed, and the antibody protein is stabilized from the complex of the random structure of the polyamino acid. A method of stabilizing an antibody protein from a complex with a random structure of a polyamino acid by forming a complex of the antibody protein and an α-helix structure of a polyamino acid having a molecular weight of more than 5.5 kDa. The method for stabilizing a protein according to claim 5 or 6, wherein the polyamino acid is polyglutamic acid. The antibody protein is formed into a complex with polyglutamic acid at pH 3.5 to 4.5, an electrolyte (salt) is added to the complex, and the pH is increased to dissociate the antibody protein from the complex. How to make.

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