Lyophilized Preparation

Abstract

[Problem] To provide a pharmaceutical composition containing a fusion protein comprising an antibody and a lysosomal enzyme as an active ingredient, which is stable enough to permit its distribution to the market.

[Solution] A lyophilized formulation containing; a fusion protein comprising an antibody and a lysosomal enzyme as an active ingredient, and further containing a neutral salt, a disaccharide, a nonionic surfactant, and a buffer. Such a lyophilized formulation includes, for example, as an active ingredient, a fusion protein comprising an anti-transferrin receptor antibody and human iduronate-2-sulfatase, and further containing sodium chloride as the neutral salt, sucrose as the disaccharide, poloxamer as the nonionic surfactant, and phosphate buffer as the buffer.

Claims

1. A lyophilized formulation comprising; a fusion protein including an antibody and a lysosomal enzyme, as an active ingredient, and further a neutral salt, a disaccharide, a nonionic surfactant, and a buffer.

2. The lyophilized formulation according to claim 1, wherein the neutral salt is sodium chloride.

3. The lyophilized formulation according to claim 1 or 2, wherein the disaccharide is selected from the group consisting of trehalose, sucrose, maltose, and lactose.

4. The lyophilized formulation according to any one of claims 1 to 3, wherein the nonionic surfactant is polysorbate or poloxamer.

5. The lyophilized formulation according to any one of claims 1 to 3, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and polyoxyethylene(160)polyoxypropylene(30)glycol.

6. The lyophilized formulation according to any one of claims 1 to 5, wherein the buffer is a phosphate buffer.

7. The lyophilized formulation according to claim 1 or 2, wherein the disaccharide is sucrose, the nonionic surfactant is polyoxyethylene(160)polyoxypropylene(30)glycol, and the buffer is a phosphate buffer.

8. The lyophilized formulation according to any one of claims 1 to 7, wherein the amounts of the neutral salt, the disaccharide, and the ionic surfactant are 0.015 to 2.5 (w/w), 2.5 to 200 (w/w), and 0.005 to 6 (w/w), respectively, relative to the amount of the fusion protein.

9. The lyophilized formulation according to any one of claims 1 to 7, wherein the amounts of the neutral salt, the disaccharide, and the ionic surfactant are 0.05 to 0.5 (w/w), 5 to 50 (w/w), and 0.02 to 0.2 (w/w), respectively, relative to the amount of the fusion protein.

10. The lyophilized formulation according to any one of claims 1 to 7, wherein the amounts of the neutral salt, the disaccharide, and the ionic surfactant is 0.1 to 0.25 (w/w), 10 to 25 (w/w), and 0.04 to 0.1 (w/w), respectively, relative to the amount of the fusion protein.

11. The lyophilized formulation according to any one of claims 1 to 10, wherein the pH is 5.5 to 7.5 when dissolved in pure water.

12. The lyophilized formulation according to any one of claims 1 to 11, wherein the fusion protein is a fusion protein in which human lysosomal enzyme is linked to the light chain or the heavy chain of the antibody on the C-terminal side or the N-terminal side thereof by a peptide bond.

13. The lyophilized formulation according to any one of claims 1 to 11, wherein the fusion protein is a fusion protein in which human lysosomal enzyme is linked to the heavy chain of the antibody on the C-terminal side thereof by a peptide bond.

14. The lyophilized formulation according to any one of claims 1 to 11, wherein the fusion protein is a fusion protein in which human lysosomal enzyme is linked to the light chain or the heavy chain of the antibody on the C-terminal side or the N-terminal side thereof via a linker including one or more of amino acids.

15. The lyophilized formulation according to any one of claims 1 to 11, wherein the fusion protein is a fusion protein in which human lysosomal enzyme is linked to the heavy chain of the antibody on the C-terminal side thereof via a linker including one or more of amino acids.

16. The lyophilized formulation according to claim 14 or 15, wherein the linker includes the amino acid sequence of Gly-Ser.

17. The lyophilized formulation according to any one of claims 1 to 16, wherein the lysosomal enzyme is a human lysosomal enzyme.

18. The lyophilized formulation according to any one of claims 1 to 17, wherein the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartyl glucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA: α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl-protein thioesterase 1, tripeptidyl-peptidase 1, hyaluronidase 1, CLN1, and CLN2.

19. The lyophilized formulation according to claim 17, wherein the human lysosomal enzyme is human iduronate-2-sulfatase.

20. The lyophilized formulation according to any one of claims 1 to 19, wherein the antibody is a human antibody or a humanized antibody.

21. The lyophilized formulation according to any one of claims 1 to 20, wherein the antibody recognizes a molecule present on the surface of a vascular endothelial cell as an antigen.

22. The lyophilized formulation according to claim 21, wherein the vascular endothelial cells are human vascular endothelial cells.

23. The lyophilized formulation according to claim 21 or 22, wherein the vascular endothelial cells are cerebral vascular endothelial cells.

24. The lyophilized formulation according to claim 23, wherein the molecule present on the surface of the cerebral vascular endothelial cell is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, OATP-F, organic anion transporter, MCT-8 and monocarboxylate transporter.

25. The lyophilized formulation according to claim 20, wherein the antibody is a humanized anti-human transferrin receptor (hTfR) antibody.

26. The lyophilized formulation according to claim 20, wherein the antibody is a humanized anti-hTfR antibody, wherein the human lysosomal enzyme is human iduronate-2-sulfatase, wherein the fusion protein includes the humanized anti-hTfR antibody and the human iduronate-2-sulfatase, and wherein the fusion protein is selected from the group consisting of (a) to (c) below; (a) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:2, and wherein the heavy chain of the humanized anti-hTfR antibody having the amino acid sequence set forth as SEQ ID NO:8 is linked, on the C-terminal side thereof; to the human iduronate-2-sulfatase via a linker sequence, (b) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:4, and wherein the heavy chain of the humanized anti-hTfR antibody having the amino acid sequence set forth as SEQ ID NO:9 is linked, on the C-terminal side thereof, to the human iduronate-2-sulfatase via a linker sequence, (c) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:6, and wherein the heavy chain of the humanized anti-hTfR antibody having the amino acid sequence set forth as SEQ ID NO:10 is linked, on the C-terminal side thereof; to the human iduronate-2-sulfatase via a linker sequence.

27. The lyophilized formulation according to claim 20, wherein the antibody is a humanized anti-hTfR antibody, wherein the human lysosomal enzyme is human iduronate-2-sulfatase, wherein the fusion protein includes the humanized anti-hTfR antibody and the human iduronate-2-sulfatase, and wherein the fusion protein is selected from the group consisting of (a) to (c) below; (a) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:2, and wherein the heavy chain of the humanized anti-hTfR antibody is linked, on the C-terminal side thereof and via a linker sequence of Gly-Ser, to the human iduronate-2-sulfatase, and the whole linked heavy chain has the amino acid sequence set forth as SEQ ID NO:13, (b) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:4, and wherein the heavy chain of the humanized anti-hTfR antibody is linked, on the C-terminal side thereof and via a linker sequence of Gly-Ser, to the human iduronate-2-sulfatase, and the whole linked heavy chain has the amino acid sequence set forth as SEQ ID NO:15, and (c) the fusion protein, wherein the light chain of the humanized anti-hTfR antibody has the amino acid sequence set forth as SEQ ID NO:6, and wherein the heavy chain of the humanized anti-hTfR antibody is linked, on the C-terminal side thereof and via a linker sequence of Gly-Ser, to the human iduronate-2-sulfatase, and the whole linked heavy chain has the amino acid sequence set forth as SEQ ID NO:17.

28. The lyophilized formulation according to any one of claims 1 to 27, sealed in a container formed of a borosilicate glass or a hydrophobic resin.

29. The lyophilized formulation according to claim 28, wherein the container is formed by a cycloolefin copolymer, a ring-opened polymer of cycloolefin, or a hydrogenated ring-opened polymer of cycloolefin.

Description

EXAMPLES

[0165] While the present invention will be described in further detail below referring to examples, it is not intended that the present invention be limited to the examples.

[Example 1] Construction of Expression Vector for hI2S-Humanized Anti-hTfR Antibody Fusion Protein

[0166] An expression vector for hI2S-humanized anti-hTfR antibody fusion protein was constructed using genes encoding three types of humanized anti-hTfR antibodies (Nos. 1 to 3). The antibody No. 1 comprises a light chain having the amino acid sequence set forth as SEQ ID NO:2 and a heavy chain having the amino acid sequence set forth as SEQ ID NO:8, the antibody No. 2 comprises a light chain having the amino acid sequence set forth as SEQ ID NO:4 and a heavy chain having the amino acid sequence set forth as SEQ ID NO:9, the antibody No.3 comprises a light chain having the amino acid sequence set forth as SEQ ID NO:6 and a heavy chain having the amino acid sequence set forth as SEQ ID NO:10, respectively.

[0167] A pEF/myc/nuc vector (Invitrogen Inc.) was digested with KpnI and NcoI to cut out the region containing the EF-1α promoter and its first intron, and the region was blunt-ended with T4 DNA polymerase. A pCI-neo (Invitrogen Inc.) was digested with BglII and EcoRI to cut out the region containing the enhancer/promoter and intron of CMV, and then the region was blunt-ended with T4 DNA polymerase. The above region containing the EF-1α promoter and its first intron was inserted into this to construct a pE-neo vector. The pE-neo vector was digested with SfiI and BstXI and a region of approximately 1 kbp containing the neomycin resistance gene was cut out. Amplification of hygromycin gene was carried out by PCR reaction using primers Hyg-Sfi5′ (SEQ ID NO:11) and Hyg-BstX3′ (SEQ ID NO:12) and using pcDNA 3.1/Hygro(+)(Invitrogen Inc.) as a template. The amplified hygromycin gene was digested with SfiI and BstXI and inserted into the pE-neo vector from which the above neomycin resistance gene has been cut out to construct a pE-hygr vector.

[0168] A DNA fragment (SEQ ID NO:3) containing the gene encoding the full length of the light chain of the humanized anti-hTfR antibody No.1 having the amino acid sequence set forth as SEQ ID NO:2 was synthesized. A MluI sequence was introduced on the 5′ side of this DNA fragment and a NotI sequence on the 3′ side thereof. This DNA fragment was digested with MluI and NotI and incorporated between MluI and NotI of the pE-neo vector. The obtained vector was designated pE-hygr(LC1) which is a vector for expressing the light chain of humanized anti-hTfR antibody No. 1.

[0169] A DNA fragment (SEQ ID NO:5) containing a gene encoding the full length of the light chain of humanized anti-hTfR antibody No. 2 having the amino acid sequence set forth as SEQ ID NO:4 was synthesized. The MluI sequence was introduced on the 5′ side of this DNA fragment and the NotI sequence on the 3′ side thereof. This DNA fragment was digested with MluI and NotI and incorporated between MluI and NotI of the pE-neo vector. The resulting vector was designated pE-hygr(LC2) which is a vector for expressing the light chain of humanized anti-hTfR antibody No. 2.

[0170] A DNA fragment (SEQ ID NO:7) containing a gene encoding the full length of the light chain of humanized anti-hTfR antibody No. 3 having the amino acid sequence set forth as SEQ ID NO:6 was synthesized. The MluI sequence was introduced on the 5′ side of this DNA fragment and the Noll sequence on the 3′ side thereof. This DNA fragment was digested with MluI and NotI and incorporated between MluI and Noll of the pE-neo vector. The obtained vector was defined as pE-hygr(LC3) which is a vector for expressing the light chain of humanized anti-hTfR antibody No. 3.

[0171] A DNA fragment was artificially synthesized, having a nucleotide sequence set forth as SEQ ID NO:14 containing a gene encoding a protein in which hI2S having an amino acid sequence set forth as SEQ ID NO:1 is linked to the C-terminal side of the heavy chain of the humanized anti-hTfR antibody No. 1 having an amino acid sequence set forth as SEQ ID NO:8 via a linker having an amino acid sequence set forth as (Gly-Ser). This DNA fragment encodes a protein having the amino acid sequence set forth as SEQ ID NO:13, in which a heavy chain of humanized anti-hTfR antibody No. 1 binds to hI2S. This DNA fragment was digested with MluI and NotI and inserted between MluI and NotI of the pE-neo vector to construct pE-neo (HC-I2S-1).

[0172] A DNA fragment was artificially synthesized, having a nucleotide sequence set forth as SEQ ID NO:16 containing a gene encoding a protein in which hI2S having an amino acid sequence set forth as SEQ ID NO:1 is linked to the C-terminal side of the heavy chain of the humanized anti-hTfR antibody No. 2 having an amino acid sequence set forth as SEQ ID NO:9 via a linker having an amino acid sequence set forth as (Gly-Ser). This DNA fragment encodes a protein having the amino acid sequence set forth as SEQ ID NO:15, in which a heavy chain of humanized anti-hTfR antibody No. 2 binds to hI2S. This DNA fragment was digested with MluI and NotI and inserted between MluI and NotI of the pE-neo vector to construct pE-neo (HC-I2S-2).

[0173] A DNA fragment was artificially synthesized, having a nucleotide sequence set forth as SEQ ID NO:18 containing a gene encoding a protein in which hI2S having an amino acid sequence set forth as SEQ ID NO:1 is linked to the C-terminal side of the heavy chain of the humanized anti-hTfR antibody No. 3 having an amino acid sequence set forth as SEQ ID NO:10 via a linker having an amino acid sequence set forth as (Gly-Ser). This DNA fragment encodes a protein having the amino acid sequence set forth as SEQ ID NO:17, in which a heavy chain of humanized anti-hTfR antibody No. 3 binds to hI2S. This DNA fragment was digested with MluI and NotI and inserted between MluI and Noll of the pE-neo vector to construct pE-neo (HC-I2S-3).

[Example 2] Preparation of a High Expression Cell Lines of hI2S-Humanized Anti-hTfR Antibody Fusion Proteins

[0174] CHO cells (CHO-K1 obtained from American Type Culture Collection) were transformed with combinations of pE-hygr (LC1) and pE-neo (HC-I2S-1) constructed in Example 1, pE-hygr (LC2) and pE-neo (HC-I2S-2) constructed in Example 1 and pE-hygr (LC3) and pE-neo (HC-I2S-3) constructed in Example 1, respectively, using the GenePulser (Bio-Rad Inc.). Transformation of cells was in brief carried out by the following method.

[0175] 5×10.sup.5 CHO-KI cells were seeded in a 3.5 cm culture dish to which CD OptiCHO™ medium (Thermo Fisher Scientific Inc.) was added and cultured overnight at 37° C. under 5% CO.sub.2. After the culture, the cells were suspended in Opti-MEM™ I medium (Thermo Fisher Scientific Inc.) to a density of 5×10.sup.6 cells/mL. 100 μL of the cell suspension was collected, and thereto 5 μL each of the pE-hygr (LC1) and pE-neo (HC-I2S-1) plasmid DNA solutions both having been diluted to 100 μg/mL with CD OptiCHO™ medium was added. Electroporation was performed using GenePulser (Bio-Rad Inc.) to introduce the plasmids into the cells. After overnight culture under the condition of 37° C., 5% CO.sub.2, the cells were selectively cultured in CD OptiCHO™ medium supplemented with 0.5 mg/mL of hygromycin and 0.8 mg/mL of G418. For the combination of pE-hygr (LC2) and pE-neo (HC-I2S-2) and the combination of pE-hygr (LC3) and pE-neo (HC-I2S-3), the transformations of the cells were conducted by the same method.

[0176] Then, the cells selected above through the selection culture were seeded on 96-well plates so that not more than one cell might be seeded per well by limiting dilution. The cells then were cultured for about 10 days so that monoclonal colonies formed. Respective culture supernatants of the wells in which monoclonal colony formed were collected, the amount of the humanized antibody contained in culture supernatants was determined by ELISA, and the hI2S-humanized anti-hTfR antibody fusion protein high-expressing cell lines were selected.

[0177] The ELISA above was conducted as follows in general. To each well of 96-well microtiter plates (Nunc Inc.) was added 100 μL of a goat anti-human IgG polyclonal antibody solution diluted with 0.05 M sodium bicarbonate buffer (pH 9.6) to 4 μg/mL, and the plate was left to stand for at least one hour at room temperature so as to allow the antibody to be adsorbed by the plates. Then, after each well was washed three times with a phosphate-buffered saline (pH 7.4) supplemented with 0.05% Tween20 (PBS-T), 200 μL of Starting Block (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.) was added to each well, and the plates were left to stand for 30 minutes at room temperature. After each well was washed with PBS-T three times, the culture supernatant or the human IgG reference standard product which had been diluted with a phosphate buffer saline (pH 7.4) supplemented with 0.5% BSA and 0.05% Tween20 (PBS-BT) to appropriate concentrations, was added to each well, in the amount of 100 μL, and the plates were left to stand for at least one hour at room temperature. After the plates were washed three times with PBS-T, 100 μL, of HRP-labeled anti-human IgG polyclonal antibody solution which had been diluted with PBS-BT, was added to each well, and the plates were left to stand for at least one hour at room temperature. After the wells were washed three times with PBS-T, citrate-phosphate buffer (pH 5.0) containing 0.4 mg/mL o-phenylenediamine was added to each well, in the amount of 100 μL, and the wells were left to stand for 8 to 20 minutes at room temperature. Then, 1 mol/L sulfuric acid was added to each well in the amount of 100 μL to terminate the reaction, and the absorbance for each well was measured at 490 nm using a 96-well plate reader. The cells corresponding to the wells which exhibited the higher measurements were regarded as a high-expressing cell line for hI2S-humanized anti-hTfR antibody fusion protein.

[0178] A high-expressing cell line of a hI2S-humanized anti-hTfR antibody fusion protein obtained by transformation with combination of pE-hygr(LC1) and pE-neo(HC-I2S-1) was designated as a hI2S-anti-hTfR antibody expressing strain 1. The fusion protein of hI2S and humanized anti-hTfR antibody expressed by this cell line was designated as I2S-anti-hTfR antibody 1.

[0179] A high-expressing cell line of a hI2S-humanized anti-hTfR antibody fusion protein obtained by transformation with combination of pE-hygr(LC2) and pE-neo(HC-I2S-2) was designated as a hI2S-anti-hTfR antibody expressing strain 2. The fusion protein of hI2S and humanized anti-hTfR antibody expressed by this cell line was designated as I2S-anti-hTfR antibody 2.

[0180] A high-expressing cell line of a hI2S-humanized anti-hTfR antibody fusion protein obtained by transformation with combination of pE-hygr(LC3) and pE-neo(HC-I2S-3) was designated as a hI2S-anti-hTfR antibody expressing strain 3. The fusion protein of hI2S and humanized anti-hTfR antibody expressed by this cell line was designated as I2S-anti-hTfR antibody 3.

[0181] The sequence numbers of amino acid sequences of the light and heavy chains of humanized antibodies, the amino acid sequences of the variable regions contained in those light and heavy chains, and the amino acid sequences of CDR 1 to 3 contained in those variable regions, which are contained in I2S-anti-hTfR antibody 1, I2S-anti-hTfR antibody 2, or I2S-anti-hTfR antibody 3, are summarized in Table 1.

TABLE-US-00001 TABLE 1 SEQ ID NOS of the light chain and heavy chain amino acid sequences contained in the fusion protein Light chain Heavy chain Full Variable Full Variable Name of fusion protein length region CDR1 CDR2 CDR3 length region CDR1 CDR2 CDR3 I2S-anti-hTfR antibody 1 2 23 29, 30 31, 32 33 8 24 34, 35 36, 37 33, 39 I2S-anti-hTfR antibody 2 4 25 40, 41 42, 43 44 9 26 45, 46 47, 48 49, 50 I2S-anti-hTfR antibody 3 6 27 51, 52 53, 54 55 10 28 56, 57 58, 59 60, 61

[Example 3] Culture of hI2S-Anti-hTfR Antibody Expressing Strain

[0182] The hI2S-anti-hTfR antibodies were produced by the method described below. The hI2S-anti-hTfR antibody expressing strain 3 obtained in Example 2 was suspended in about 200 L of serum-free medium (EX-CELL Advanced CHO Fed-batch Medium, Sigma Aldrich Inc.) containing 4 mM L-alanyl-L-glutamine, 100 μmon hypoxanthine and 16 μmol/L thymidine to the density of about 2×10.sup.5 cells/mL. 140 L of this cell suspension was transferred to a culture tank. The cells were cultured for about 11 days at a temperature range of 34 to 37° C., while the medium was stirred with an impeller at a rate of 89 rpm, and the dissolved oxygen saturation of the medium was kept at about 40%. During the culture period, cell number, cell viability, and glucose and lactate concentrations of the medium were monitored. When the glucose concentration of the medium became less than 15 mmol/L, the glucose solution was immediately added to the medium so that the glucose concentration became 37.89 mmol/L. After completion of the culture, the medium was collected. The recovered medium was filtered with Millistak+HC Pod Filter grade D0HC (Merck Inc.) and further filtered with Millistak+HCgrade X0HC (Merck Inc.) to obtain a culture supernatant containing I2S-anti-hTfR antibody 3. The culture supernatant was subjected to ultrafiltration using a Pellicon™ 3 Cassette w/Ultracel PLCTK Membrane (pore size: 30 kDa, membrane area: 1.14 m.sup.2, Merck Inc.) and concentrated until the liquid volume was about 1/17. The concentrate was then filtered using OpticapXL600 (0.22 μm, Merck Inc.). The obtained solution was used as a concentrated culture supernatant.

[Example 4] Inactivation of the Virus

[0183] To the concentrated culture supernatant obtained in Example 3, tri-n-butyl phosphate (TNBP) and polysorbate 80 were added so that the final concentrations were 0.3% (v/v) and 1% (w/v), respectively, and gently stirred at room temperature for 4 hours. This process is conducted for inactivating the virus contaminating the culture supernatant. However, insofar as the culture is carried out using a serum-free medium not containing biological components, there is little possibility that viruses harmful to the human body are contaminated in the culture supernatant.

[Example 5] Purification of hI2S-anti-hTfR Antibodies

[0184] The concentrated culture supernatant after the virus inactivation was filtrated by a Millipak-200 Filter Unit (pore size: 0.22 μm, Merck Inc.) after adding thereto 20 mM Tris-HCl buffer (pH 7.0) containing 0.5 volume of 140 mM NaCl. The solution after filtration was loaded onto a MabSelect SuRe LX column (column volume: about 3.2 L, bed height: about 20 cm, GE Healthcare Inc.), which was a protein A affinity column, and equilibrated with 4 column volumes of 20 mM Tris-HCl buffer (pH 7.0) containing 140 mM NaCl, at a constant flow rate of 200 cm/hr to adsorb I2S-anti-hTfR antibody 3 to protein A.

[0185] Subsequently, the column was washed with 5 column volumes of 10 mM Tris-HCl buffer (pH 7.0) containing 500 mM NaCl and 450 mM arginine at the same flow rate. Then the column was further washed with 2.5 column volumes of 20 mM Tris-HCl buffer (pH 7.0) containing 140 mM NaCl at the same flow rate. Then I2S-anti-hTfR antibody 3 adsorbed to Protein A was eluted with 5 column volumes of 100 mM glycine buffer (pH 3.5) containing 140 mM NaCl. The eluate was immediately neutralized by receiving it in a container containing 1 M Tris-HCl buffer (pH 7.5) in advance.

[0186] To the above eluate from the Protein A affinity column, 200 mM phosphate buffer (pH 7.0), 10 mM MES buffer (pH 7.3) containing 4 M NaCl and 2 mM phosphate buffer, and 1 M Tris-HCl buffer solution (pH 8.0) were added in the order, and the concentrations of sodium phosphate and NaCl contained in the eluate were adjusted to 2 mM and 215 mM, respectively, and the pH of the eluate was adjusted to 7.3. The eluate was then filtered through Opticap XL 600 (pore size: 0.22 Merck Inc.). The solution after filtration was applied to a CHT Type II 40 μm column, a hydroxyapatite column (Column volume: about 3.2 L, bed height: about 20 cm, Bio-Rad Inc.), equilibrated with 4 column volumes of 10 mM MES buffer solution (pH 7.3) containing 215 mM NaCl and 2 mM sodium phosphate at a constant flow rate of 200 cm/hr to adsorb I2S-anti-hTfR antibody 3 to hydroxyapatite.

[0187] Subsequently, the column was washed with 5 column volumes of the same buffer at the same flow rate. Then I2S-anti-hTfR antibody 3 adsorbed on hydroxyapatite was eluted with 5 column volumes of 35 mM phosphate buffer (pH 7.3) containing 215 mM NaCl. Purification by the hydroxyapatite column was carried out twice using half volume of the eluate from the protein A affinity column.

[0188] To the above eluate from the hydroxyapatite column, dilute hydrochloric acid was added to adjust the pH to 6.5. Then, ultrafiltration was carried out using Pellicon™ 3 Cassette w/Ultracel PLCTK Membrane (pore size: 30 kDa, membrane area: 1.14 m.sup.2, Merck Inc.) to concentrate I2S-antihTfR antibody 3 in the solution at the concentration of about 2 mg/mL. The concentrate was then filtered using Opticap XL 600 (0.22 mm, Merck Inc.).

[0189] The above concentrated solution was applied to a Superdex 200 column, size exclusion column (column volume: about 12.6 L, bed height: 40 cm, GE Healthcare Inc.) equilibrated with 5 column volumes of 20 mM phosphate buffer (pH 6.5) containing 0.8 mg/mL NaCl and 75 mg/mL sucrose at a constant flow rate of 19 cm/hr, and the same buffer was supplied at the same flow rate. At this time, an absorbance photometer for continuously measuring the absorbance of the eluate was placed in the flow path of the eluate from the size exclusion column, and the absorbance at 280 nm was monitored. The fractions which corresponded to an absorption peak at 280 nm were collected as a fractions containing I2S-anti-hTfR antibody 3, which was designated as a purified product of I2S-anti-hTfR antibody. Purification on the size exclusion column was carried out twice using half volume of the eluate from the hydroxyapatite column.

[Example 6] Manufacture of Lyophilized Products Containing I2S-Anti-hTfR Antibodies

[0190] An aqueous solution, whose composition is shown in Table 2, was prepared using the purified I2S-anti-hTfR antibody obtained in Example 5. The aqueous solution was filtered using Vacuum Filter/Storage Bottle System, 0.22 μm (Corning Inc.). The solution after filtration was used as the test solution. Sodium hydroxide solution or dilute hydrochloric acid was added as needed to adjust the pH of the test solution to about 6.5.

TABLE-US-00002 TABLE 2 Composition of the test solution Component Concentration (mg/mL) I2S-anti-hTfR antibody 5 NaCl 0.8 NaH.sub.2PO.sub.4—2H.sub.2O 2.136 Na.sub.2HPO.sub.4 12 H.sub.2O 2.256 sucrose 75 poloxamer 188 0.325

[0191] 2.5 mL of each of the test solution was filled in a vial made of borosilicate glass, with a rubber stopper (made of chlorinated butyl) half-capped, and lyophilized. In the lyophilization process, the gas phase in the vials was replaced with nitrogen, and then the whole rubber stopper was capped to seal the vials. The lyophilized products formed a white mass in vials.

[Example 7] Evaluation of Stability of Lyophilized Formulation Containing I2S-Anti-hTfR Antibody

[0192] The lyophilized products prepared in Example 6 were allowed to stand in a dark environment at 5° C. or 25° C. for 6 months. The lyophilized products in the vials at the start of standing, after 1 month, after 3 months, and after 6 months, were dissolved in 2.5 mL of pure water and the solutions were used as sample solutions. The pH of each sample solution was measured and the presence or absence of foreign matter was visually examined at a brightness of 2000 to 3750 lx under a white light source. The number of fine particles, whose particle sizes were 1 to 100 μm, was measured by the method shown in Example 8. The ratio of I2S-anti-hTfR antibody polymers in each sample solution was determined by the method shown in Example 9. In addition, the affinity of the I2S-anti-hTfR antibody contained in each sample solution with human TfR was determined by the method shown in Example 10. Furthermore, the enzymatic activity of the I2S-anti-hTfR antibody contained in each sample solution was determined by the method shown in Example 11.

[0193] Table 3 shows the results of the stability measurements of lyophilized formulations containing I2S-anti-hTfR antibody after left for 6 months in the dark at 5° C. As shown in Table 3, the pH of the sample solutions was kept at approximately pH 6.5 during the 6 month standing period of the lyophilized product. There was no foreign insoluble matter in the sample solutions during the same time period, which could be clearly observed optically. The ratio of polymers to whole I2S-anti-hTfR antibody in the sample solutions during the same time period was almost constant. Further, the number of fine particles, whose particle sizes were 1 to 100 μm, did not increase significantly during the same time period. An increase in the number of particles was observed in the sample solution left for 6 months standing, but it was within an acceptable range when the storage period of the lyophilized formulation was set at 2 years in a cold place. The enzyme activity of I2S-anti-hTfR antibody in the sample solutions remained almost constant during the same time period. Further, the affinity of I2S-anti-hTfR antibody contained in the sample solutions for human TfR did not decrease significantly during the same time period (Data not shown).

TABLE-US-00003 TABLE 3 Results of stability evaluation of lyophilized formulation stored in the dark at 5° C. At the 1 2 3 6 Test item start month Months Months Months pH 6.5 6.5 6.5 6.5 6.5 Foreign insoluble none none none none none matter (visual observation) Ratio of polymers 1.13 1.09 1.08 1.02 1.19 (%) Number of fine 1110 1500 1550 730 3240 particles (/vial) Enzyme activity 419 436 425 422 515 (mU/mg) Affinity for hTfR — — — — — (K.sub.D)

[0194] Table 4 shows the results of stability measurements of lyophilized formulations containing I2S-anti-hTfR antibodies after 6 months of standing in the dark at 25° C. As shown in Table 4, the pH of the sample solution was kept at approximately pH 6.5 during the 6 month standing period of the lyophilized product. Any foreign insoluble matter was not observed in the sample solutions during the same period, which could be clearly observed optically. The ratio of polymer was almost constant during the same period. The number of fine particles, whose particle sizes were 1 to 100 μm, did not increase significantly during the same time period. An increase in the number of fine particles was observed after 6 months, which was within an acceptable range when the storage period of the lyophilized formulation was set at 2 years in a cold place. The enzyme activity of I2S-anti-hTfR antibody in the sample solutions remained almost constant during the same period. Further, the affinity of I2S-anti-hTfR antibody contained in the sample solutions for human TfR did not decrease significantly during the same period (Data not shown).

TABLE-US-00004 TABLE 4 Results of stability evaluation for lyophilized formulation stored in the dark at 25° C. At the 1 2 3 6 Test item start month Months Months Months pH 6.5 6.5 6.5 6.5 6.5 Foreign insoluble none none none none none matter (visual observation) Ratio of polymers 1.13 1.09 1.09 1.02 1.19 (%) Number of fine 1110 2140 3130 1060 2620 particles (/vial) Enzyme activity 419 445 430 430 506 (mU/mg) Affinity for hTfR — — — — — (K.sub.D)

[0195] These results above indicate that a drug containing I2S-anti-hTfR antibody as an active ingredient can be provided to the market in a pharmacologically stable state by using the lyophilized formulation with the composition shown in Table 2, provided that the drug is placed in a cold place in the distribution process and in the storage.

[Example 8] Measurement of the Number of Particles Contained in the Sample Solution (Particle Size: 1 to 100 μm)

[0196] The measurement of the number of particles contained in the sample solutions was conducted by using a flow imaging particle analyzer FlowCAM™ (Fluid Imaging Technologies Inc.). The flow imaging particle analyzer enables to measure the number of particles contained in the sample solution by drawing the sample solution into a flow cell disposed perpendicularly to the optical system by a syringe pump, and to photograph the particles passing through the flow cell in real time. The measurement was carried out by setting the particle size to be detected to 1 to 100 μm.

[Example 9] Measurement of the Amount of Polymers of I2S-Anti-hTfR Antibodies Contained in a Sample Solution

[0197] TSKgel UltraSW Aggregate 3 μm column, a size exclusion column chromatography, (7.8 mm in diameter×30 cm in length, TOSOH Inc.) was set on Shimazu HPLC system LC-20A (Shimadzu Corporation). A spectrophotometer was installed downstream of the column to measure the absorbance (Measurement wavelength: 215 nm) of the effluent from the column continuously. After equilibrating the column by loading 0.2 M aqueous sodium phosphate buffer solution at a flow rate of 0.5 mL/min, a sample solution containing 3 to 20 μg of I2S-anti-hTfR antibody was loaded onto the column, and a further 0.2 M aqueous sodium phosphate buffer solution was loading at the same flow rate. During this time, the elution profile was obtained by measuring the absorbance (Measurement wavelength: 215 nm) of the effluent from the column. From the obtained elution profiles, the peak area of the monomer of the I2S-anti-hTfR antibody (monomer peak area) and the peak area of the polymer of the I2S-anti-hTfR antibody appearing before the monomer peak (polymer peak area) were determined. The amount (%) of the polymer was determined by the following equation.

Amount of polymer (%)={Polymer peak area/(monomer peak area+polymer peak area)}×100

[Example 10] Measurement of Affinity of I2S-Anti-hTfR Antibody in Sample Solution to Human TfR

[0198] The affinity of the I2S-anti-hTfR antibody to human TfR was determined by using OctetRED 96 (ForteBio Inc., a division of Pall Corporation), to which bio-layer interferometry (BLI) is applied. The basic principles of bio-layer interferometry are briefly explained below. When a layer of a biomolecule immobilized on the surface of a sensor tip is irradiated with light of a certain wavelength, the light is reflected from two of the surfaces, the one of the biomolecule and the other of inner, reference layer, producing interfering light waves. A molecule in the sample being measured binds to the biomolecule on the surface of the sensor tip and thus increases the thickness of the layers on the sensor tip, which results in a shift between the interfering waves. By measuring the variations of this shift between the interfering waves, determination of the number of the molecules bound to the layer of the biomolecules immobilized to the sensor tip surface and kinetic analysis of it can be performed in real time. The measurement was performed according generally to the operating manual attached to Octet RED96. As a human TfR, a recombinant human TfR (rhTfR: Sino Biological Inc.) was used, which had the amino acid sequence of the hTfR extracellular region, i.e., the cysteine residue at the position 89th from the N-terminal side to the phenylalanine at the C-terminus, of the amino acid sequence set forth as SEQ ID NO:1, with a histidine tag attached to the N-terminus.

[0199] The Sample solutions prepared in Example 6 were subjected to 2-fold dilution steps with HBS-P+ (10 mM HEPES containing 150 mM NaCl, 50 μM EDTA, and 0.05% surfactant P20) to prepare 7 step diluted solutions, whose concentrations were 0.78125 to 50 nM (0.117 to 7.5 μg/mL). The rhTfR-ECD (Histag) solution was prepared by diluting hTfR with HBS-P+ to adjust the concentration to 25 μg/mL.

[0200] Each of the sample solutions prepared above by 2-fold dilution steps was added, 200 μL/well, to a 96-well plate, black (Greiner Bio-One Inc.). Each of the rhuman TER-ECD (Histag) solution prepared above was added, 200 μL/well, to predetermined wells. To respective wells for baseline, dissociation and washing were added HBS-P+, 200 μL/well. To wells for regeneration were added 10 mM Glycine-HCl, pH 1.7, 200 μL/well. To wells for activation was added 0.5 mM NiCl.sub.2 solution, 200 μL/well. The plate and biosensor (Biosensor/Ni-NTA: ForteBio Inc., a division of Pall Corporation) were set in the prescribed positions of Octet RED96.

[0201] Octet RED96 was run under the conditions shown in Table 5 below to collect data, on which then, using the analyzing software attached to Octet RED96, and fitting the binding reaction curve to 1:1 binding model or 2:1 binding model, the association rate constant (k.sub.on) and dissociation rate constant (k.sub.off) of anti-hTfR antibody to r human TfR were measured and the dissociation constant (K.sub.D) was calculated. The measurement was performed at 25 to 30° C.

TABLE-US-00005 TABLE 5 Operating conditions of OctetRED 96 Contact time Speed Step (sec) (rpm) Threshold 1 Baseline 1 60 1000 — 2 Load 600 1000 1.5~2.0 3 Baseline 2 60 1000 — 4 Associate 180 1000 — 5 Dissociate 540 1000 — 6 Regenerate 5 1000 — 7 Wash 5 1000 — Steps 6~7 repeated 6~7 times. 8 activate 60 1000 — Steps 1~8 repeated until all the samples measured.

[Example 11] Measurement of Enzymatic Activity of I2S-Anti-hTfR Antibodies in Sample Solution

[0202] The sample solutions were desalted by membrane filtration using a vertical polyethersulfone membrane (VIVASPIN2 5,000 MWCO PES, Saltrius Inc.) as an ultrafiltration membrane, and then desalted samples were diluted to approximately 100 ng/mL with Reaction Buffer (5 mM sodium acetate, 0.5 mg/L BSA, 0.1% Triton X-100, pH 4.45). To each well of a 96-well microtiter plate (FluoroNunc Plate, Nunc Inc.), 10 μL of each rhI2S sample was added and pre-incubated for 15 minutes at 37° C. Substrate solutions were prepared by dissolving 4 methylumbelliferyl sulfate (SIGMA) in substrate buffer (5 mM sodium acetate, pH 4.45, containing 0.5 mg/mL BSA) to a final concentration of 1.5 mg/mL. 100 μL of Substrate solutions was added to each well containing rhI2S sample solution, and the plates were allowed to stand in the dark at 37° C. for 1 hour. After the incubation, 190 μL of stop buffer (0.33 M glycine, 0.21 M sodium carbonate buffer solution, pH 10.7) was added to each well containing the sample. 150 μL of 0.4 μmol/L 4-methylumbelliferone (4-MUF, Sigma) solution and 150 μL of the stop buffer were added to a well as the standard, then the plate was read on a 96-well plate reader with excitation light at the wavelength of 330 nm and fluorescent light at the wavelength of 440 nm.

[0203] A standard curve was produced by measuring fluorescence intensity at various concentrations of 4-MUF in solution. The fluorescence intensity of each sample was extrapolated to the standard curve. Results were calculated as activity in Units/mL where one Unit of activity was equal to 1 μmol of 4-MUF produced per minute at 37° C. A published US patent application (publication No. 2004-0229250) was referred to for conducting this measurement.

INDUSTRIAL APPLICABILITY

[0204] According to the present invention, a lyophilized formulation containing a protein in which an antibody and a lysosomal enzyme are combined as an active ingredient can be provided to the market in a pharmacologically stable state.

SEQUENCE LISTING FREE TEXT

[0205] SEQ ID NO:2: Amino acid sequence of the light chain of humanized anti-hTfR

[0206] SEQ ID NO:3: Nucleotide sequence encoding amino acid sequence of the light-chain of humanized anti-hTfR antibody No. 1, synthetic sequence

[0207] SEQ ID NO:4: Amino acid sequence of the light-chain of humanized anti-hTfR antibody No. 2

[0208] SEQ ID NO:5: Nucleotide sequence encoding amino acid sequence of the light chain of humanized anti-hTfR antibody No. 2, synthetic sequence

[0209] SEQ ID NO:6: amino acid sequence of the light chain of humanized anti-hTfR antibody No. 3

[0210] SEQ ID NO:7: Nucleotide sequence encoding amino acid sequence of the light chain of humanized anti-hTfR antibody No. 3, synthetic sequence

[0211] SEQ ID NO:8: Amino acid sequence of the heavy chain of humanized anti-hTfR antibody No. 1

[0212] SEQ ID NO:9: Amino acid sequence of the heavy chain of humanized anti-hTfR antibody No. 2

[0213] SEQ ID NO:10: Amino acid sequence of the heavy chain of humanized anti-hTfR antibody No. 3

[0214] SEQ ID NO:11: Primer Hyg-Sfi5′, synthetic sequence

[0215] SEQ ID NO:12: Primer Hyg-BstX3′, synthetic sequence

[0216] SEQ ID NO:13: Amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 1 and hI2S

[0217] SEQ ID NO:14: Nucleotide sequence encoding amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 1 and hI2S, synthetic sequence

[0218] SEQ ID NO:15: Amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 2 and hI2S

[0219] SEQ ID NO:16: Nucleotide sequence encoding amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 2 and hI2S, synthetic sequence

[0220] SEQ ID NO:17: Amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 3 and hI2S

[0221] SEQ ID NO:18: Nucleotide sequence encoding amino acid sequence of fused protein of the heavy chain of humanized anti-hTfR antibody No. 3 and hI2S, synthetic sequence

[0222] SEQ ID NO:19: Amino acid sequence of an exemplified linker 1

[0223] SEQ ID NO:20: Amino acid sequence of an exemplified linker 2

[0224] SEQ ID NO:21: Amino acid sequence of an exemplified linker 3

[0225] SEQ ID NO:23: Amino acid sequence of the light chain variable region of humanized anti-hTfR antibody No. 1

[0226] SEQ ID NO:24: Amino acid sequence of the heavy chain variable region of humanized anti-hTfR antibody No. 1

[0227] SEQ ID NO:25: Amino acid sequence of the light chain variable region of humanized anti-hTfR antibody No. 2

[0228] SEQ ID NO:26: Amino acid sequence of the heavy chain variable region of humanized anti-hTfR antibody No. 2

[0229] SEQ ID NO:27: Amino acid sequence of the light chain variable region of humanized anti-hTfR antibody No. 3

[0230] SEQ ID NO:28: Amino acid sequence of the heavy chain variable region of humanized anti-hTfR antibody No. 3

[0231] SEQ ID NO:29: Amino acid sequence 1 of CDR1 in the light chain of anti-hTfR antibody No. 1

[0232] SEQ ID NO:30: Amino acid sequence 2 of CDR1 in the light chain of anti-hTfR antibody No. 1

[0233] SEQ ID NO:31: Amino acid sequence 1 of CDR2 in the light chain of anti-hTfR antibody No. 1

[0234] SEQ ID NO:32: Amino acid sequence 2 of CDR2 in the light chain of anti-hTfR antibody No. 1

[0235] SEQ ID NO:33: Amino acid sequence of CDR3 in the light chain of anti-hTfR antibody No. 1

[0236] SEQ ID NO:34: Amino acid sequence 1 of CDR1 in the heavy chain of anti-hTfR antibody No. 1

[0237] SEQ ID NO:35: Amino acid sequence 2 of CDR1 in the heavy chain of anti-hTfR antibody No. 1

[0238] SEQ ID NO:36: Amino acid sequence 1 of CDR2 in the heavy chain of anti-hTfR antibody No. 1

[0239] SEQ ID NO:37: Amino acid sequence 2 of CDR2 in the heavy chain of anti-hTfR antibody No. 1

[0240] SEQ ID NO:38: Amino acid sequence 1 of CDR3 in the heavy chain of anti-hTfR antibody No. 1

[0241] SEQ ID NO:39: Amino acid sequence 2 of CDR3 in the heavy chain of anti-hTfR antibody No. 1

[0242] SEQ ID NO:40: Amino acid sequence 1 of CDR1 in the light chain of anti-hTfR antibody No. 2

[0243] SEQ ID NO:41: Amino acid sequence 2 of CDR1 in the light chain of anti-hTfR antibody No. 2

[0244] SEQ ID NO:42: Amino acid sequence 1 of CDR2 in the light chain of anti-hTfR antibody No. 2

[0245] SEQ ID NO:43: Amino acid sequence 2 of CDR2 in the light chain of anti-hTfR antibody No. 2

[0246] SEQ ID NO:44: Amino acid sequence of CDR3 in the light chain of anti-hTfR antibody No. 2

[0247] SEQ ID NO:45: Amino acid sequence 1 of CDR1 in the heavy chain of anti-hTfR antibody No. 2

[0248] SEQ ID NO:46: Amino acid sequence 2 of CDR1 in the heavy chain of anti-hTfR antibody No. 2

[0249] SEQ ID NO:47: Amino acid sequence 1 of CDR2 in the heavy chain of anti-hTfR antibody No. 2

[0250] SEQ ID NO:48: Amino acid sequence 2 of CDR2 in the heavy chain of anti-hTfR antibody No. 2

[0251] SEQ ID NO:49: Amino acid sequence 1 of CDR3 in the heavy chain of anti-hTfR antibody No. 2

[0252] SEQ ID NO:50: Amino acid sequence 2 of CDR3 in the heavy chain of anti-hTfR antibody No. 2

[0253] SEQ ID NO:51: Amino acid sequence 1 of CDR1 in the light chain of anti-hTfR antibody No. 3

[0254] SEQ ID NO:52: Amino acid sequence 2 of CDR1 in the light chain of anti-hTfR antibody No. 3

[0255] SEQ ID NO:53: Amino acid sequence 1 of CDR2 in the light chain of anti-hTfR antibody No. 3

[0256] SEQ ID NO:54: Amino acid sequence 2 of CDR2 in the light chain of anti-hTfR antibody No. 3

[0257] SEQ ID NO:55: Amino acid sequence of CDR3 in the light chain of anti-hTfR antibody No. 3

[0258] SEQ ID NO:56: Amino acid sequence 1 of CDR1 in the heavy chain of anti-hTfR antibody No. 3

[0259] SEQ ID NO:57: Amino acid sequence 2 of CDR1 in the heavy chain of anti-hTfR antibody No. 3

[0260] SEQ ID NO:58: Amino acid sequence 1 of CDR2 in the heavy chain of anti-hTfR antibody No. 3

[0261] SEQ ID NO:59: Amino acid sequence 2 of CDR2 in the heavy chain of anti-hTfR antibody No. 3

[0262] SEQ ID NO:60: Amino acid sequence 1 of CDR3 in the heavy chain of anti-hTfR antibody No. 3

[0263] SEQ ID NO:61: Amino acid sequence 2 of CDR3 in the heavy chain of anti-hTfR antibody No. 3