HK1084340B - Stabilized preparation containing protein - Google Patents

Description

Stable protein-containing formulations

Technical Field

The present invention relates to stable protein-containing formulations. More particularly, the present invention relates to stable protein-containing formulations comprising poloxamers as surfactants.

Background

With the development of genetic engineering techniques, it has become possible to use physiologically active proteins such as antibodies, enzymes, hormones and cytokines as pharmaceutical products. In order to provide them in a stable high quality, it is necessary to establish preparation conditions and storage conditions capable of maintaining the structure and activity.

In general, a problem encountered during storage of proteins is deterioration phenomena, such as the formation of insoluble aggregates, which must be prevented.

For example, antibodies such as immunoglobulins, monoclonal antibodies and humanized antibodies are unstable proteins that are susceptible to physical or chemical changes such as clumping or clustering under the stress of filtration, concentration and heating during purification and formulation processing, and under the stress of heat, light and transportation during storage of stock solutions or formulations.

When the antibody is obtained by genetic engineering techniques, antibody-producing cells are cultured and purified in large quantities to obtain an antibody-containing solution, which is then cryopreserved and thawed before preparation of a preparation. However, the residual antibody content in the solution is reduced due to the formation of antibody dimers or insoluble particles or insoluble foreign matter during repeated freeze/thaw cycles or degradation of the antibody to form degradation products during long term storage.

In order to suppress the formation of such insoluble foreign substances and obtain a stable protein-containing preparation, the use of a surfactant is indispensable, and in particular, such surfactants as polysorbate 20 and 80 have been widely used. However, for Protein preparations which are easily oxidizable, antioxidants such as L-methionine have to be included in addition to polysorbate 80 (JPA No.2000-247903, J.pharm.Sci.90: 3(2001)), since polysorbate 80 tends to oxidize proteins (PDA J.pharm.Sci.technol.50: 3 (1996); Formulation, charaterization, and Stability of Protein drugs. plenum Press, New Yolk, (1996)), thereby reducing the biological activity of the antibody preparation. The addition of the antioxidant requires complicated operations such as strict measurement of the specification and content of the antioxidant.

Accordingly, it is desirable to provide a surfactant that can inhibit oxidation of proteins without adding an antioxidant and can inhibit formation of insoluble foreign matter in protein formulations. Lyophilized formulations are capable of inhibiting oxidation of proteins (e.g., JPA No.2000-247903), but there is a great need for convenient solution formulations that eliminate the reconstitution step and it would also be desirable to provide a protein-containing formulation that is stable even when present as a solution formulation.

It is an object of the present invention to find a surfactant which can inhibit oxidation of a protein without adding an antioxidant to maintain the biological activity of the protein and also inhibit formation of insoluble foreign matter in a protein preparation and to provide a stable protein-containing preparation containing the surfactant.

Disclosure of Invention

As a result of intensive studies to achieve the above object, we have found that the addition of poloxamer as a surfactant can maintain the biological activity of a protein without causing oxidation of the protein in the absence of an antioxidant, and can suppress the formation of insoluble foreign matter in a protein-containing preparation, and have completed the present invention.

Accordingly, the present invention provides:

(1) a protein formulation comprising a poloxamer as a surfactant;

(2) the protein preparation as defined in the above (1), wherein the poloxamer is poloxamer 188;

(3) the protein preparation as defined in the above (1) or (2), which is a solution preparation;

(4) a protein preparation as defined in any one of (1) to (3) above, wherein the protein is an immunoglobulin;

(5) the protein preparation as defined in the above (4), wherein the immunoglobulin is a humanized antibody;

(6) the protein preparation as defined in the above (4), wherein the immunoglobulin is an anti-tissue factor antibody;

(7) the protein preparation as defined in the above (6), wherein said anti-tissue factor antibody is a humanized anti-tissue factor antibody;

(8) a protein preparation as defined in any one of the above (1) to (9), which does not contain an antioxidant as an additive;

(9) a method for maintaining biological activity and inhibiting formation of insoluble foreign matter in a protein preparation by adding poloxamer as a surfactant without adding an antioxidant;

(10) the protein preparation as defined in any one of (1) to (3) above, wherein the protein is granulocyte colony stimulating factor; and

(11) a protein preparation as defined in any one of (1) to (3) above, wherein said protein is parathyroid hormone.

Brief description of the drawings

FIG. 1 shows the change of biological activity with time in anti-human tissue factor antibody solution formulations containing different surfactants.

FIG. 2 shows an anion exchange chromatogram of an anti-human tissue factor antibody solution formulation containing poloxamer 188 or polysorbate 80.

FIG. 3 shows the effect of addition of L-methionine to anti-human tissue factor antibodies on the loss of activity induced by polysorbate 80, compared to the effect of addition of poloxamer.

FIG. 4 shows a chromatogram showing the effect of polysorbate 80, polysorbate 20 or poloxamer 188 on granulocyte colony stimulating factor oxidation.

FIG. 5 shows a chromatogram showing the effect of polysorbate 80, polysorbate 20 or poloxamer 188 on parathyroid hormone oxidation.

Most preferred embodiments of the invention

The "protein-containing preparation" used in the present invention means a preparation containing a protein, preferably a physiologically active protein as an active ingredient, which is prepared for administration to animals such as humans, and includes lyophilized preparations and solution preparations.

Proteins useful in the formulations of the present invention include, but are not limited to, antibodies, enzymes, cytokines, and hormones. Specifically, they include, but are not limited to, hematopoietic factors such as granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), Erythropoietin (EPO), and thrombopoietin; cytokines such as interferon, IL-1 and IL-6; an immunoglobulin; a monoclonal antibody; a humanized antibody; tissue Plasminogen Activator (TPA); urokinase; a serum albumin; a coagulation factor VIII; leptin; insulin; stem cell growth factor (SCF). Among these proteins, hematopoietic factors such as G-CSF and EPO, parathyroid hormone (PTH) and immunoglobulins, particularly antibodies, are preferred. Among the antibodies, an anti-tissue factor antibody is particularly preferable.

The proteins used in the formulations of the present invention have substantially the same biological activity as those physiologically active proteins of mammals, particularly humans, including those derived from natural sources, preferably those obtained by genetic engineering. Genetically engineered proteins may have the same amino acid sequences as those of the native protein, or may comprise deletions, substitutions, or additions of one or more amino acids in the amino acid sequence but retain the biological activity. Physiologically active proteins also include proteins chemically modified with PEG or the like.

Particularly preferred is a protein having a sugar chain. The sugar chain may be derived from any source, but is preferably added to mammalian cells. Mammalian cells include, for example, Chinese Hamster Ovary (CHO) cells, BHK cells, COS cells, human-derived cells, and the like, with CHO cells being most preferred.

When the protein is EPO, EPO can be prepared by any method, such as it can be extracted from human urine, isolated and purified by various techniques, or produced by genetic engineering techniques (e.g., JPA No. SHO 61-12288) in Chinese Hamster Ovary (CHO) cells, BHK cells, COS cells, human-derived cells, etc., and then extracted and isolated and purified by various techniques. Chemically modified EPO with PEG or the like is also included (see International patent application publication No. WO90/12874). EPO without sugar chains and chemically modified with PEG or the like is also included. Also included are EPO analogs in which EPO is modified to increase the number of glycosylation site(s) in the N-linked sugar chain binding site or O-linked sugar chain binding site in the amino acid sequence of EPO (see, for example, JPA No. HEI 8-151398 and JPA No. HEI 8-506023). Further, the number of sugar chains can be increased by increasing the content of sialic acid or the like without changing the number of sugar chain-binding sites.

When the protein is G-CSF, then any high purity human G-CSF can be used. The G-CSF of the invention can be prepared by any method, e.g. they can be extracted from cultures of human tumor cell lines, isolated and purified by different techniques, or in bacterial cells such as e.coli; a yeast cell; cultured animal cells such as Chinese Hamster Ovary (CHO), C127 or COS cells are produced by genetic engineering techniques and then extracted and isolated and purified by various techniques. G-CSF is preferably produced by genetic recombination in e.coli, yeast or CHO cells, most preferably in CHO cells. Also included is G-CSF chemically modified with PEG or the like (see International patent application publication No. WO90/12874).

When the protein is an antibody, the antibody is not particularly limited as long as it binds to an antigen of interest, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, a chimeric antibody, a humanized antibody, a human antibody, or the like may be used as appropriate. The antibody may be polyclonal or monoclonal, but is preferably monoclonal because homogeneous antibodies can be stably produced. Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art.

The hybridoma producing the monoclonal antibody can be constructed basically by the following known techniques. A monoclonal antibody-producing cell (hybridoma) is selected by immunizing a host cell with a target antigen or a cell expressing the target antigen according to a standard immunization technique, fusing the resulting immune cell into a known parent cell by a standard cell fusion technique, and then screening the fused cell by a standard screening method. Construction of hybridomas can be carried out following Methods such as Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). If the antigen is low in immunogenicity, it may be conjugated to an immunogenic macromolecule such as albumin and used for immunization.

Recombinant antibodies can be used, which are produced by transforming a host with an appropriate vector containing the antibody gene cloned from the hybridoma using genetic engineering techniques (see, e.g., Carl, A.K. Borrexack, James, W.Larrick, THERAPEUTICMONOLONAL ANTIBODIES.MACMILLIAN PUBLISHERS, 1990). In particular, the cDNA sequence of the antibody variable region (V region) is synthesized from the mRNA of the hybridoma using reverse transcriptase. Once the DNA sequences encoding the V regions of the antibody of interest are obtained, they can be bound to DNA sequences encoding the constant regions (C regions) of the antibody of interest and integrated into an expression vector. Alternatively, the DNA sequence encoding the V region of the antibody may be incorporated into an expression vector comprising the DNA sequence of the C region of the antibody. They may be incorporated into expression vectors in such a way that they can be expressed under the control of regulatory regions such as enhancers and promoters. The host cell can then be transformed with this expression vector to express the antibody.

In the present invention, recombinant antibodies, i.e., antibodies that have been artificially modified to reduce antigenicity in humans or for other purposes, such as chimeric antibodies and humanized antibodies, can be used. These modified antibodies can be prepared by known methods. Chimeric antibodies, which are composed of antibody heavy and light chain variable regions and human antibody heavy and light chain constant regions obtained from a non-human mammal such as a mouse, can be obtained by ligating a DNA sequence encoding a mouse antibody variable region with a DNA sequence encoding a human antibody constant region, transforming a host with an expression vector comprising the linker sequence, and allowing it to produce the chimeric antibody.

Humanized antibodies, also called reshaped human antibodies, are obtained by grafting Complementarity Determining Regions (CDRs) of an antibody from a non-human mammal such as a mouse into complementarity determining regions of a human antibody, and typical genetic recombination techniques for preparing humanized antibodies are also known. Specifically, DNA sequences designed to link CRDs of mouse antibodies to Framework Regions (FRs) of human antibodies were synthesized by PCR from a plurality of oligonucleotides prepared to have terminal overlapping regions. The resulting DNA sequence is ligated to a DNA sequence encoding a human antibody constant region and then integrated into an expression vector, which is transformed into a host to allow it to produce a reshaped antibody (see European patent application publication No. EP239400, International patent application publication No. WO96/02576). The FRs of the human antibodies to which the CDRs are linked are selected in such a way that the complementarity determining regions form appropriate antigen binding sites. If necessary, the reshaped humanized antibody may have some amino acid changes in the framework regions of the variable regions so that the CDRs form the appropriate antigen binding site (Sato, K., et al, Cancer Res. (1993) 53.851-856).

Methods for obtaining human antibodies are also known. For example, a target human antibody having a binding activity to a target antigen can be obtained by immunizing human lymphocytes with the target antigen or a cell expressing the target antigen in vitro and fusing the immunized lymphocytes with human myeloma cells such as U266 (see JPB No. HEI 1-59878). The human antibody of interest can also be obtained by immunizing a transgenic animal having all human antibody gene components with an antigen (see International patent application publication Nos. WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, WO 96/33735). Methods for obtaining human antibodies by panning using human antibody libraries are also known. For example, phage binding to an antigen can be selected by expressing a human antibody variable region as a single chain antibody fragment (scFv) on the phage surface by phage display. The DNA sequence encoding the variable region of the human antibody that binds to the antigen is determined by analyzing the gene of the selected phage. On the basis of the determined DNA sequence of the scFv fragment binding to the antigen, a fully human antibody can be obtained by preparing an appropriate expression vector. Such processes are known from WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388.

When the antibody is prepared by transforming the primarily isolated antibody into a suitable host, the suitable host may be used in combination with an expression vector. Suitable eukaryotic cells for use as hosts include animal cells, plant cells, and fungal cells. Known animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa and Vero cells; (2) amphibian cells such as xenopus oocytes; or (3) insect cells such as sf9, sf21 and Tn 5. Known plant cells include cells of the genus Nicotiana such as Nicotiana tabacum, which can be used as callus cultures. Known fungal cells include yeasts, for example Saccharomyces species such as Saccharomyces serevisiae and filamentous fungi such as Aspergillus species such as Aspergillus niger (Aspergillus miger). Prokaryotic cells can be used as production systems using bacterial cells. Known bacterial cells include E.coli (E.coli) and Bacillus subtilis. The antibody can be obtained by transforming these cells with the antibody gene of interest and culturing the transformed cells in vitro.

The antibody contained in the stable preparation of the present invention includes, but is not limited to, an anti-IL-6 receptor antibody, an anti-HM 1.24 antigen monoclonal antibody, an anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody), an anti-tissue factor antibody, and the like.

Preferred reshaped humanized antibodies for use in the present invention include humanized anti-IL-6 receptor antibody (hPM-1) (see International patent application publication No. WO92-19759), humanized anti-HM 1.24 antigen monoclonal antibody (see International patent application publication No. WO98-14580), and humanized anti-parathyroid hormone-related peptide antibody (anti-PTHr P antibody) (see International patent application publication No. WO98-13388).

We prepared a human/mouse chimeric antibody composed of the variable region (V region) of a mouse monoclonal antibody against human tissue factor and the constant region (C region) of a human antibody, and the complementarity determining regions in the light chain (L chain) variable region and the heavy chain (H chain) variable region of a mouse monoclonal antibody against human tissue factor transferred into a human antibody, and reported that these would be expected to be excellent therapeutic agents for DIC, arterial thrombosis, and venous thrombosis (WO99/51743, WO 01/24626). Particularly preferred is a humanized anti-human tissue factor antibody described in WO99/51743, which is a recombinant antibody produced by CHO cells, comprising a combination of humanized H chain form i and humanized L chain form b 2. A number of anti-human tissue factor antibodies have been reported (WO99/51743, WO88/07543, WO96/40921, WO98/40408, WO 01/70984). They can be prepared by methods known to those skilled in the art, since their antigenic tissue factors are known (Ito T et al, J.biochem.114.691-696, (1993)). These anti-human tissue factor antibodies are also preferred antibodies for use in the present invention.

The antibodies comprised in the formulation of the invention may belong to any immunoglobulin class, preferably IgG, such as IgG1, IgG2, IgG3 and IgG 4.

As used herein, an "antibody-containing solution" may be a solution containing any antibody, whether biologically derived or recombinant, preferably a culture medium in which antibody-containing mammalian cells such as CHO cells are cultured, or a solution obtained by subjecting such a culture medium to a certain treatment such as partial purification (bulk solution), or a solution preparation prepared as described above for administration to an animal such as a human.

As used herein, "insoluble foreign substance" means an insoluble foreign substance that should not be contained in a solution preparation that is easily detected, and the solution preparation is clear when the solution preparation is placed in a container under an incandescent lamp and observed with the naked eye at a position where the light intensity is about 1000 lux, as defined in the section of detection of insoluble foreign substance for injection in the general test, method and apparatus in the japanese pharmacopoeia.

As used herein, "biological activity of an antibody" refers to the ability of an antibody to bind to its antigen, and can be determined using an antigen neutralization activity assay. When the antibody is a humanized anti-human tissue factor antibody, a formulation that retains the biological activity of the antibody means that the formulation retains 60% or more, preferably 70% or more, of the biological activity of the antibody stock after 6 months of accelerated testing at 25 ℃; still more preferably 80% or more, most preferably 90% or more.

In the present invention, the purity of the antibody preparation can be checked by gel filtration chromatography and anion exchange chromatography as described below.

In the protein-containing preparation of the present invention, by adding poloxamer as a surfactant, the high biological activity of the preparation can be maintained without oxidation of the protein and the formation of insoluble foreign matter can be suppressed without causing oxidation of the protein in the absence of an antioxidant.

Poloxamers are nonionic surfactants consisting of a series of block copolymers of ethylene oxide and 1, 2-propylene oxide of the general formula:

HO(C₂H₄O)_a(C₃H₆O)_b(C₂H₄O)_aH

poloxamers include poloxamers 124, 188, 237, 338 and 407 as described in USP (united states pharmacopeia), with poloxamer 188 being particularly preferred for the present invention. Poloxamer 188 described in USP is a compound of the above formula where a is 80, b is 27, with an average molecular weight of 7680-. In addition, EP (european pharmacopoeia) describes poloxamers 182, 184 and 331. Pluronics (trade marks for poloxamers available from BASF) such as Pluronics L35, L43, L44, L61, L62, L64, F68, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, P123 and F127 are also included in the scope of poloxamers of the present invention.

Poloxamers have previously been used in pharmaceutical formulations as emulsifiers for fat emulsions for intravenous injection or as solubilizers to maintain clarity of elixirs or syrups, but have not been used as stabilizers for protein-containing formulations.

The amount of poloxamer added depends on the type of poloxamer used and the type and concentration of protein, but the amount of poloxamer 188 is typically 0.001-100mg/mL, preferably 0.005-50mg/mL, more preferably 0.01-10 mg/mL.

In the protein-containing formulation of the present invention, poloxamer is used as a surfactant, eliminating the need for the addition of antioxidants such as L-methionine that is required when polysorbates are used.

The protein-containing formulation of the present invention may comprise an amino acid as a stabilizer. Amino acids include, but are not limited to, leucine, tryptophan, serine, glutamic acid, arginine, histidine, and lysine and their salts.

To inhibit dimer formation during freeze/thaw cycles, the formulations of the present invention may also contain sugar alcohols such as mannitol and sorbitol; and non-reducing oligosaccharides, such as non-reducing disaccharides such as sucrose and trehalose or non-reducing trisaccharides such as raffinose. Non-reducing oligosaccharides are particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

Preferably, the antibody-containing solution formulation of the present invention is substantially free of proteins such as human serum albumin or pure gelatin as a stabilizer.

The pH of the antibody preparation of the present invention is preferably 4 to 8, more preferably 5 to 7.5, however, the pH depends on the antibody contained and is not limited to these values.

The formulations of the invention may also contain isotonic agents, for example, polyethylene glycol; and sugars such as dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

The antibody-containing solution formulation of the present invention may further include diluents, solubilizers, excipients, pH adjusters, demulcents, buffers, sulfur-containing reducing agents, antioxidants, and the like, if necessary. For example, sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid (thioglycolic acid) and salts thereof, sodium thiosulfate, glutathione, and mercapto-containing compounds such as thioalkanoic acids having 1 to 7 carbon atoms. Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α -tocopherol, tocopheryl acetate, L-ascorbic acid or its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate or chelating agents such as disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate. Other commonly used additives may also be included, such as inorganic salts, for example, sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; and organic salts such as sodium citrate, potassium citrate, and sodium acetate.

The formulations of the invention may be prepared as follows: these components are dissolved in an aqueous buffer solution known in the art of solution formulation, such as a phosphate buffer (preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate system) and/or a citrate buffer (preferably sodium citrate buffer), to make a solution formulation. The concentration of the buffer is typically 1-500mM, preferably 5-100mM, more preferably 10-50 mM.

The antibody-containing solution preparations of the present invention are usually administered by parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular or intraperitoneal injection) or transdermal, transmucosal, nasal or pulmonary administration, and may also be administered orally.

The protein-containing formulation of the present invention may be a lyophilized formulation or a solution formulation, preferably a solution formulation. Solution formulations are usually supplied in sealed and sterilized plastic or glass containers of defined volume, such as vials, ampoules or syringes, or large volumes such as bottles. From a convenient point of view, pre-filled syringes are preferred.

The amount of antibody contained in the preparation of the present invention is generally 0.1 to 200mg/ml, preferably 1 to 120mg/ml, depending on the kind of disease to be treated, the severity of the disease and the age of the patient and other factors.

As shown in the following examples, the solution formulation of the present invention can maintain the biological activity of the antibody formulation at a high level and inhibit the formation of insoluble foreign substances by adding poloxamer as a surfactant without adding an antioxidant.

The following examples further illustrate the invention but do not limit the scope of the invention. Various changes and modifications may be made by those skilled in the art in light of this disclosure, and are also within the scope of the invention.

Examples

Sample (I)

1. Antibody sample

A humanized anti-human tissue factor antibody described in WO99/51743, which comprises a combination of humanized H chain form i and humanized L chain form b2, is used as the anti-human tissue factor antibody. The anti-human tissue factor antibodies used in the examples described below were recombinant antibodies produced by CHO cells and belonging to the IgG4 class.

2. Granulocyte colony stimulating factor (G-CSF)

The granulocyte colony stimulating factor used was produced by genetic engineering techniques using genetic recombination methods in Chinese Hamster Ovary (CHO) cells and extracted and isolated/purified.

3. Parathyroid hormone (PTH)

Parathyroid hormone, which has 1-84 residues, is prepared by the method described in WO 9014415.

Test method

(1) TF neutralization Activity assay

Tissue Factor (TF) is a factor VII receptor expressed on the cell surface and is localized as a substantial trigger of the coagulation response. Tissue factor activates factors IX and X by forming a complex with factor VII. Thus, the biological activity of humanized anti-human tissue factor antibodies can be determined by the methods described below using factor VIIa solutions and factor X solutions.

1. The following solutions were prepared.

1) A.b. (test buffer): TBS (pH7.6) contained 5mmol/L CaCl₂，0.1％BSA。

2) Mixed solution of factor VIIa & thromboborel S: factor VIIa and Thromborel S were diluted to 0.1PEU/mL and 0.42mg/mL with A.B. respectively.

3) Factor X solution: factor X was diluted with A.B. to 0.25 PEU/mL.

4) Mixed solution of Testzyme chromogenic substrate S-2222: a1.5 mg/mL solution of the chromogenic substrate S-2222 was mixed with water and an aqueous solution of 1, 5-dimethyl-1, 5-diaza-undecamethylene polybromide in a ratio of 1: 2.

2. The mixed solution of factor VIIa and Thromborel S was dispensed into the plate at 60. mu.L/well and allowed to stand at room temperature for 60 minutes.

3. Anti-human tissue factor antibody stock solution (standard solution) and sample solution diluted with factor X solution were dispensed at 40 μ L/well in plates and allowed to stand at room temperature for 30 minutes.

4. The reaction was stopped by adding 10. mu.L/well of 0.5mol/L EDTA solution, and then the mixed solution of Testzyme chromogenic substrate S-2222 was dispensed at 50. mu.L/well into the plate and allowed to stand at room temperature for 30 minutes.

5. The absorbance at 405nm to 655nm was measured.

6. The biological activity of each test sample was calculated as a percentage of the standard solution based on standard curve analysis.

Abbreviations

TBS: tris buffered saline

BSA: bovine serum albumin

EDTA: ethylenediaminetetraacetic acid

(2) Ion exchange column chromatography (IEC)

The analysis conditions were as follows

Column: DEAE-NPR (4.6mm I.D.. times.3.5 cm)

Mobile phase:

a: 50mmol/L Tris buffer, pH8.0

B: 50mmol/L Tris buffer +500mmol/L NaCl, pH8.0

Gradient:

0-5min solution B0%

5-40min solution B0 → 50%

Flow rate: 1.0mL/min

And (3) detection: UV absorption at 280nm

Sample loading: equivalent to 100. mu.g

(3) Analysis of insoluble foreign bodies

The solution preparation contained in the container was visually observed under an incandescent lamp at a position where the light intensity was about 1000 lux as defined in the section of detection of insoluble foreign matter with respect to an injection in general detection, method and apparatus in the japanese pharmacopoeia.

Example 1: effect of surfactant addition on biological Activity

The biological activity of anti-human tissue factor antibody solution formulations containing different surfactants was tested over time. Biological activity (TF-neutralizing activity) of a sample (pH6.0) containing 2.3mg/mL of acetate buffer of an anti-human tissue factor antibody and 0.5mg/mL of polysorbate 20 or 80 (both manufactured by company A) or poloxamer 188 (manufactured by company B) as a surfactant after storage at 5 ℃ for 14 days and storage at 40 ℃ for 14 days was tested. For comparison, the surfactant-free group was also tested in the same manner. The results are shown in FIG. 1.

In contrast to the formulation containing polysorbate 80, which showed a significant loss of biological activity of the anti-human tissue factor antibody, the formulation containing poloxamer 188 maintained similar activity to the formulation without surfactant. Loss of activity was also observed in formulations containing polysorbate 20, known in the art as injectable surfactants.

Example 2: effect of surfactants on biological Activity and purity

The effect of adding a surfactant on the bioactivity and purity of the anti-human tissue factor antibody solution preparation was tested. After accelerated testing at 25 ℃ for 6 months, samples (pH5.5) containing 10mg/mL of acetate buffer of anti-human tissue factor antibody and 0.5mg/mL of polysorbate 80 (manufactured by C) or poloxamer 188 (manufactured by D) as a surfactant were tested for biological activity by TF-neutralization activity assay, and their purity was determined by Ion Exchange Chromatography (IEC). For comparison, stock solutions of anti-human tissue factor antibodies (bulk) without accelerated testing were also tested in the same manner. The results are shown in FIG. 2.

The biological activity of the formulation containing poloxamer 188 as surfactant was 92% of that of the anti-human tissue factor antibody stock solution, while the biological activity of the formulation containing polysorbate 80 was 52%.

In ion exchange chromatography, the main peak in the anti-human tissue factor antibody solution formulation containing polysorbate 80 decreased, and a new peak immediately preceded the main peak (indicated by an arrow in fig. 2). The peak portion immediately preceding the main peak is due to a derivative containing some oxidized amino acid residues. However, the formulation containing poloxamer 188 maintained similar chromatograms as the stock solution of anti-human tissue factor antibody.

These results show that poloxamer 188 is superior to polysorbate 80 in both biological activity and purity.

Example 3: effect of L-methionine addition on loss of anti-hTF antibody Activity induced by polysorbate 80 compared to the Effect of poloxamer addition

The effect of the addition of L-methionine on the loss of activity of anti-human tissue factor antibodies induced by polysorbate 80 was tested and compared to a formulation containing poloxamer. Acetate buffer solution containing 10mg/mL anti-human tissue factor antibody and the preparation method thereof

1)0.5mg/mL of polysorbate 80,

2)0.5mg/mL polysorbate 80 and 5mg/mL L-methionine, or

3)0.5mg/mL of a sample of poloxamer 188 (pH 5.5).

After an accelerated test at 25 ℃ for 6 months, the biological activity of each sample was determined by the TF-neutralization activity test and compared with that of a stock solution (bulk) of anti-human tissue factor antibody.

The results are shown in FIG. 3. The loss of activity induced by polysorbate 80 was inhibited by the addition of L-methionine. The sample containing poloxamer 188 showed similar or higher biological activity than the sample containing polysorbate 80 and L-methionine.

Example 4: effect of surfactant on insoluble foreign matter

The effect of the addition of a surfactant on the formation of insoluble foreign bodies in the anti-human tissue factor antibody solution formulation was tested. Immediately after the preparation and after 24 months of storage at 5 ℃, a sample (ph6.0) containing 10mg/mL of an anti-human tissue factor antibody acetate buffer and 0.5mg/mL of polysorbate 80 (manufactured by company C) or poloxamer 188 (manufactured by company D) as a surfactant was tested for insoluble foreign matter. For comparison, the surfactant-free group was also tested in the same manner. The results are shown in table 1. Shown is the number of bottles in which insoluble foreign matter was observed among 5 sample bottles. Insoluble foreign matter was observed in the surfactant-free samples immediately after preparation. However, no formation of insoluble foreign bodies was observed in the sample containing poloxamer 188 and polysorbate 80.

TABLE 1

Surface active agent	Does not contain	Poloxamer 188	Polysorbate 80
				The first 5-24 months	5/55/5	0/50/5	0/50/5

(4) Experiment of protein stabilizing Effect of Poloxamers

Different surfactants were added to the above-mentioned granulocyte colony stimulating factor solution preparation and parathyroid hormone solution preparation to evaluate their effects on the oxidation of granulocyte colony stimulating factor and parathyroid hormone.

Example 5: effect of surfactants on granulocyte colony stimulating factor oxidation

The effect of the surfactant on the granulocyte colony stimulating factor solution formulation was tested. After 5 weeks of accelerated testing at 25 ℃, the content of oxidized derivatives of granulocyte colony stimulating factor in a sample (ph6.5) containing 0.25mg/mL of colony stimulating factor phosphate buffer and 0.05% polysorbate 80 (manufactured by company a), polysorbate 20 (manufactured by company a) or poloxamer 188 (manufactured by company B) as a surfactant was measured by Reverse Phase Chromatography (RPC).

The analysis conditions were as follows.

Column: DAISOPAK SP-300-5-C4-P (4.6mm I.D.. times.25 cm)

Mobile phase:

acetonitrile, water and trifluoroacetic acid (400: 600: 1)

Nitrile, water and trifluoroacetic acid (800: 200: 1)

Gradient:

0-25min solution B20 → 90%

25-40min solution B90 → 90%

40-41min solution B90 → 20%

41-60min solution B20%

Flow rate: 0.3mL/min

Loading: 10 μ L

Column temperature: 35 deg.C

Detection wavelength: UV absorption at 215nm

The results are shown in FIG. 4.

Reversed phase chromatography showed that oxidized derivatives (indicated by arrows in figure 4) appeared immediately before the main peak of the granulocyte colony stimulating factor solution formulations containing different surfactants. These peaks are due in part to derivatives of granulocyte colony stimulating factor that contain some oxidized amino acid residues.

The highest level of oxidized derivatives was found in the sample containing polysorbate 20, followed by polysorbate 80, and again poloxamer 188.

Example 6: effect of surfactants on the Oxidation of Parathyroid hormone

The effect of the surfactant on the parathyroid hormone solution formulation was determined. After accelerated testing at 40 ℃ for 2 weeks, the content of oxidized derivatives of parathyroid hormone in a sample (pH5.0) containing 0.25mg/mL of parathyroid hormone citrate buffer and 0.05% of polysorbate 80 (manufactured by A), polysorbate 20 (manufactured by A) or poloxamer 188 (manufactured by B) as a surfactant was measured by Reverse Phase Chromatography (RPC).

The analysis conditions were as follows.

Column: YMC-Pack ODS A-312(4.6mm I.D.. times.15 cm)

Mobile phase:

acetonitrile, water and trifluoroacetic acid in the ratio of 0 to 1000 to 1

Nitrile, water and trifluoroacetic acid (600: 400: 1)

Gradient:

0-40min solution B40 → 60%

40-42min solution B60 → 60%

42-42.5min solution B60 → 40%

42.5-60min solution B40%

Flow rate: 1.0mL/min

Loading: 10 μ L

Column temperature: 25 deg.C

Detection wavelength: UV absorption at 215nm

The results are shown in FIG. 5.

Reversed phase chromatography showed that oxidized derivatives (indicated by arrows in fig. 5) appeared immediately before the main peak of parathyroid hormone solution formulations containing different surfactants. These peaks are due in part to derivatives of parathyroid hormone which contain some oxidized amino acid residues.

The highest amount of oxidized derivative was present in the sample containing polysorbate 20, followed by polysorbate 80, and again poloxamer 188.

These results show that poloxamer 188 is superior to polysorbate in terms of antioxidant effect on the protein solution formulation.

Industrial applicability

The stable protein-containing formulations of the invention show neither loss of biological activity nor formation of insoluble foreign matter even after long-term storage. They are stable formulations in which the production of oxidized derivatives of proteins is effectively inhibited.

Claims (11)

1. A protein formulation comprising a poloxamer as a surfactant, wherein the protein is a hematopoietic factor, a parathyroid hormone or an antibody.

2. The protein formulation of claim 1, wherein the poloxamer is poloxamer 188.

3. The protein formulation of claim 1 or 2 which is a solution formulation.

4. The protein formulation of any one of claims 1 to 3, wherein the protein is an antibody.

5. The protein formulation of claim 4, wherein the antibody is a humanized antibody.

6. The protein formulation of claim 4, wherein said antibody is an anti-tissue factor antibody.

7. The protein formulation of claim 6, wherein said anti-tissue factor antibody is a humanized anti-tissue factor antibody.

8. A protein preparation according to any one of claims 1 to 7, which does not contain an antioxidant as an additive.

9. A protein preparation according to any one of claims 1 to 3, wherein the protein is granulocyte colony stimulating factor.

10. A protein formulation as claimed in any one of claims 1 to 3, wherein said protein is parathyroid hormone.

11. A method for maintaining biological activity in a protein preparation and inhibiting the formation of insoluble foreign matter by adding poloxamer as a surfactant without adding an antioxidant, wherein the protein is hematopoietic factor, parathyroid hormone or antibody.