WO2016027859A1 - 蛋白質溶液の粘度測定方法 - Google Patents
蛋白質溶液の粘度測定方法 Download PDFInfo
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- WO2016027859A1 WO2016027859A1 PCT/JP2015/073392 JP2015073392W WO2016027859A1 WO 2016027859 A1 WO2016027859 A1 WO 2016027859A1 JP 2015073392 W JP2015073392 W JP 2015073392W WO 2016027859 A1 WO2016027859 A1 WO 2016027859A1
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- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
- G01N2011/006—Determining flow properties indirectly by measuring other parameters of the system
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- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
- G01N2011/006—Determining flow properties indirectly by measuring other parameters of the system
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- G01N2223/054—Investigating materials by wave or particle radiation by diffraction, scatter or reflection small angle scatter
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- G01N2223/60—Specific applications or type of materials
- G01N2223/612—Specific applications or type of materials biological material
Definitions
- the present invention relates to a method for measuring the viscosity of a protein solution, and more particularly to a method for measuring the viscosity of a protein solution that can be measured with a small amount of sample.
- SAXS Small angle X-ray scattering
- the object of the present invention is to measure the viscosity of a protein solution that can be carried out particularly with a small amount of sample, to predict the viscosity of a protein, to select a protein with controlled viscosity, and to produce a modified protein with controlled viscosity
- Another object of the present invention is to provide a method for producing a modified protein with reduced viscosity and a method for producing a protein having low viscosity.
- the viscosity of a protein solution such as an antibody is measured by a rheometer that calculates the viscosity from the proportional coefficient between the shear rate and the shear stress, or the EMS viscosity that calculates the viscosity from the rotation speed of the metal sphere in the sample solution.
- the mainstream is to use a meter, or a viscosity measuring device using a micro-channel that calculates the viscosity from the passage speed of the sample and the pressure loss at the time of passage.
- viscosity measurement using a rheometer or EMS viscometer requires a large amount of sample, and the sample preparation work is complicated. Therefore, it was difficult to measure many samples at the same time. Further, in the viscosity measurement using the microchannel, it is difficult to accurately measure the viscosity of the protein solution having high adsorptivity with high reproducibility. Accordingly, it has been impossible to screen modified proteins using viscosity as an index, such as measuring many kinds of small samples.
- the present inventors have made use of the technique of small-angle X-ray scattering (SAXS) that has been used for molecular shape analysis in protein solutions.
- SAXS small-angle X-ray scattering
- the measured values of the viscosity at a high concentration were obtained by changing the kind of the antibody and the solvent at room temperature (25 ° C.).
- the apparent particle size was evaluated at a sub-low concentration (60 mg / mL) under the same conditions by SAXS measurement, they were found to correlate very well.
- SAXS measurement at a low temperature we considered that the difference in associability between samples could be evaluated at a lower concentration, and when evaluating the apparent particle size and apparent molecular weight at 5 ° C / 15 mg / mL, It was found to correlate well with viscosity at 25 ° C / 160 mg / mL.
- [1] Method for measuring viscosity of protein solution including the following steps: 1) Apparent particle size (apparent particle) using X-ray small angle scattering method (SAXS) or X-ray solution scattering method by irradiating X-rays to a sample whose protein concentration is 1-100mg / ml Measuring the maximum length (D max app ) or apparent particle radius of inertia (R g app )) or apparent molecular weight; 2) A step of calculating the viscosity of the protein from the measured value based on a calibration curve obtained in advance.
- SAXS X-ray small angle scattering method
- R g app apparent particle radius of inertia
- a method for predicting the viscosity of a protein which comprises irradiating a sample having a protein concentration of 1 to 100 mg / ml with X-rays, X-ray small angle scattering (SAXS) or X-ray solution scattering Measuring the apparent particle size (apparent particle maximum length (D max app ) or apparent particle inertia radius (R g app )) or apparent molecular weight.
- SAXS X-ray small angle scattering
- X-ray solution scattering Measuring the apparent particle size (apparent particle maximum length (D max app ) or apparent particle inertia radius (R g app )) or apparent molecular weight.
- the method of selection comprising measuring an apparent particle size (apparent particle maximum length (D max app ) or apparent particle inertia radius (R g app )) or apparent molecular weight using a method.
- a method for producing a modified protein with controlled viscosity comprising the following steps: 1) A step of obtaining a modified protein by modifying a part of the amino acid of the original protein; 2) A step of predicting the viscosity of the protein using the prediction method of [11].
- a method for producing a low-viscosity protein comprising the following steps: 1) A step of selecting a low-viscosity protein using the prediction method of [11].
- a method for producing a modified protein with controlled viscosity comprising the following steps: 1) A step of obtaining a modified protein by modifying a part of the amino acid of the original protein; 2) A step of selecting a protein whose viscosity is controlled from the modified protein using the selection method of [12].
- a method for producing a modified protein with reduced viscosity comprising the following steps: 1) A step of obtaining a modified protein by modifying a part of the amino acid of the original protein; 2) A step of selecting a modified protein having a viscosity lower than that of the original protein from the modified protein using the selection method of [12].
- 3 is a graph showing the results of plotting the viscosity of each antibody solution as a function of R g app at 60 mg / mL, 15 to 35 ° C. obtained by SAXS measurement.
- 3 is a graph showing the results of plotting the viscosity of each antibody solution as a function of D max app at 60 mg / mL, 15 to 35 ° C. obtained by SAXS measurement.
- 3 is a graph showing the results of plotting the viscosity of each antibody solution as a function of I (0) at 15 mg / mL, 5 ° C. obtained by SAXS measurement.
- FIG 3 is a graph showing the results of plotting the viscosity of each antibody solution as a function of R g app at 15 mg / mL, 5 ° C. obtained by SAXS measurement.
- the viscosity of each antibody solution is a graph showing the results plotted as a function of D max app in 15 mg / mL, 5 °C obtained by SAXS measurement.
- It is a figure which shows the crystal structure of Fab of Mab5-A.
- the figure on the left shows the Fab-Fab interaction of Mab5-A.
- the right figure is an enlarged view of the part surrounded by the ellipse in the left figure, and shows that His at position 97 in one Fab main chain is at a distance that can form a hydrogen bond with the other Fab main chain. ing.
- the present invention produces a method for measuring the viscosity of a protein solution (estimation method), a method for predicting the viscosity of a protein, a method for selecting a protein with controlled viscosity, and a modified protein with controlled viscosity.
- the present invention relates to a method, a method for producing a modified protein with reduced viscosity, and a method for producing a protein with low viscosity.
- the term “protein with controlled viscosity” or “protein with controlled viscosity” means a protein that exists in a desired viscosity range in a solution, and preferably has a viscosity increased with increasing concentration.
- a protein within a viscosity range suitable as an active ingredient in a protein solution formulation (preferably a solution formulation for subcutaneous administration) to be used as a medicament can be mentioned.
- the viscosity range is preferably a viscosity range with high manufacturability and usability of the protein solution preparation, and can be appropriately set by those skilled in the art.
- the viscosity range is 2000 mPa ⁇ s from room temperature to body temperature (15 to 40 ° C.).
- it is preferably 1500 mPa ⁇ s or less, more preferably 0.1 to 1000 mPa ⁇ s, and particularly preferably 1 to 700 mPa ⁇ s.
- the modified protein in the method for producing a modified protein of the present invention is a modified protein in which a part of amino acid residues is substituted with a histidine (His) residue.
- the present invention also relates to a modified protein produced by such a production method.
- the protein is preferably a mutated antibody, more preferably a mutated antibody in which a part of the amino acid residues of the original antibody is placed in the histidine (His) residue, and the 97th amino acid in the Kabat numbering system It is a mutant antibody whose residue is not a His residue, wherein the amino acid residue at position 97 is preferably the amino acid residue of the original antibody. More preferably, the antibody is less viscous than an antibody in which the amino acid residue at position 97 is substituted with His.
- “original protein” and “original antibody” are “protein before artificially modifying part of an amino acid” or “protein before artificially modifying part of an amino acid”. is there.
- a low-viscosity protein or “a modified protein with reduced viscosity” or “a modified protein with reduced viscosity” means a protein within the above viscosity range.
- the amount of the sample used in the method of the present invention is not particularly limited as long as the apparent particle size or the apparent molecular weight can be measured. For example, it is 3 to 100 ⁇ L, preferably 3 to 50 ⁇ L, particularly preferably. 5-30 ⁇ L.
- the protein concentration in the sample used in the method of the present invention is not particularly limited as long as the apparent particle size or the apparent molecular weight can be measured, but is preferably 1 to 100 mg / ml, more preferably 10 to 100 mg. / ml, more preferably 15 to 30 mg / ml.
- a protein solution having a lower concentration can be measured.
- the modified protein with controlled viscosity, reduced viscosity, or low viscosity is used. Can be efficiently selected (screened), and such a modified protein can be efficiently produced.
- the viscosity of the protein solution correlates well with the above particle size parameter (apparent particle maximum length (D max app ) or apparent particle inertia radius (R g app )) or apparent molecular weight.
- the viscosity of the protein solution is estimated from the measured value of the apparent particle size or the apparent molecular weight.
- the correlation is a positive correlation in which an increase in the viscosity of the protein solution correlates with an increase in apparent particle size or apparent molecular weight, and is preferably an exponential correlation or a linear correlation.
- the temperature condition for the measurement of the particle size parameter is not limited as long as the temperature range allows the measurement, but is, for example, 40 ° C. or less, preferably room temperature or less, more preferably 0 to 25 ° C., particularly preferably 3 ⁇ 10 ° C.
- D max app the maximum length of the apparent particle
- R g app apparent radius of inertia
- the maximum particle length (D max app ) and the apparent particle inertia radius (R g app ), which are the particle size parameters of the protein solution, can be calculated as described in the examples of the present application.
- the apparent molecular weight is given as a value proportional to the scattering intensity (I (0)) at a scattering angle of 0. Detection of the scattering pattern of the sample irradiated with X-rays used for calculation of these parameters can be performed by a method known to those skilled in the art by the X-ray small angle scattering method (SAXS) or the X-ray solution scattering method.
- SAXS X-ray small angle scattering method
- the calibration curve used in the step (2) can be obtained by analyzing the correlation between the viscosity measured for various protein solution and SAXS measurement sample pairs and the apparent particle size or apparent molecular weight. .
- the protein type and the solvent in the pair of the protein solution for viscosity measurement and the sample for SAXS measurement are the same, and the concentration and measurement temperature of the protein used for both measurements are not necessarily the same. Therefore, the viscosity to be analyzed for correlation is not limited to the viscosity under the same measurement conditions as in the step (1).
- the viscosity under the conditions (for example, the viscosity at room temperature when the concentration is increased) can be estimated. Since the calibration curve can be applied to solutions of different proteins, the protein in the solution whose correlation is analyzed in this step is the protein in the solution used in the steps (1) to (2). The correlation may be analyzed using measurement results of protein solutions composed of various proteins and solvents.
- the protein used in the present invention is not particularly limited as long as it is a protein capable of measuring the apparent particle size or the apparent molecular weight by the X-ray small angle scattering method (SAXS) or the X-ray solution scattering method. It is a protein for which development of a concentrated protein solution preparation is desired, and examples thereof include albumin such as BSA and antibodies.
- the antibody used in the present invention is not particularly limited as long as it binds to a desired antigen, and may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced. .
- the monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, monkeys, but also artificial antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Modified genetically modified antibodies are also included. In addition, antibody properties may be modified to modify antibody molecule physical properties (specifically, isoelectric point (pI) modification, Fc receptor affinity modification, etc.) for the purpose of improving blood retention and pharmacokinetics. Also included are genetically engineered antibodies in which the constant region and the like are artificially modified.
- the immunoglobulin class of the antibody used in the present invention is not particularly limited, and may be any class such as IgG1, IgG2, IgG3, IgG4, IgG, IgA, IgD, IgE, IgM, etc. IgM is preferred.
- the antibodies used in the present invention include not only antibodies having constant regions and variable regions (whole antibodies), but also antibody fragments such as Fv, Fab, F (ab) 2 and variable regions of antibodies as peptide linkers. These include low-molecular-weight antibodies such as single-body Fv (scFv, sc (Fv) 2 ) and dibodies such as scFv dimers linked by a linker such as preferable.
- a hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method.
- the hybridoma can be prepared, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the immunogenicity of the antigen is low, immunization may be performed by binding to an immunogenic macromolecule such as albumin.
- a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, introducing it into a host, and using a gene recombination technique can be used (for example, Carl, A). K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
- cDNA of the variable region (V region) of the antibody is synthesized from the hybridoma mRNA using reverse transcriptase.
- DNA encoding the V region of the target antibody When DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
- DNA encoding an antibody V region may be incorporated into an expression vector containing antibody C region DNA. It is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter.
- host cells can be transformed with this expression vector to express the antibody.
- a recombinant antibody that has been artificially modified for the purpose of reducing the heteroantigenicity to humans such as a chimeric antibody or a humanized antibody
- modified antibodies can be produced using known methods.
- a chimeric antibody is a mammal other than a human, for example, a mouse antibody heavy chain, light chain variable region and a human antibody heavy chain, light chain constant region, and a DNA encoding the murine antibody variable region.
- a humanized antibody also called a reshaped human antibody, is a non-human mammal, such as a mouse antibody complementarity-determining region (CDR) grafted to the complementarity-determining region of a human antibody.
- CDR complementarity-determining region
- an amino acid of an antibody in order to improve the activity, physical properties, pharmacokinetics, safety, etc. of the antibody, for example, the techniques described below are also known, and the antibody used in the present invention includes such a technique. Also included are antibodies with various amino acid substitutions (including deletions and additions).
- the technology of amino acid substitution in the variable region of IgG antibody is humanized (Tsurushita N, Hinton PR, Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax., Methods. 2005 May; 36 (1): 69 -83.), Affinity complementation region (CDR) amino acid substitution (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci U S A. 2005 Jun 14; 102 (24): 8466-71.
- CDR Affinity complementation region amino acid substitution
- a method for obtaining a human antibody is also known.
- human lymphocytes are sensitized with a desired antigen or cells expressing the desired antigen in vitro, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to have a desired human antibody having an antigen-binding activity.
- a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with an antigen (WO 93/12227, WO 92/03918, WO 94/02602, WO 94 / 25585, WO 96/34096, WO 96/33735).
- variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected.
- scFv single chain antibody
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, a suitable expression vector containing the sequence can be prepared and a human antibody can be obtained.
- an antibody gene When an antibody gene is once isolated and introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
- animal cells When eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used.
- Animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, (2) amphibian cells, such as Xenopus oocytes, or (3) insect cells.
- sf9, sf21, Tn5, etc. are known.
- plant cells cells derived from the genus Nicotiana (for example, Nicotiana tabacum) are known, and these may be cultured in callus.
- Known fungal cells include yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, and filamentous fungi such as the genus Aspergillus, such as Aspergillus niger.
- yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae
- filamentous fungi such as the genus Aspergillus, such as Aspergillus niger.
- prokaryotic cells there are production systems using bacterial cells.
- Known bacterial cells include E. coli and Bacillus subtilis.
- An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.
- the antibodies used in the present invention include antibody fragments, low molecular weight antibodies, and modified antibodies.
- Fab, F (ab ′) 2, Fv, or a mono-chain or bivalent or more single chain Fv (scFv, Fv of H chain and L chain linked by an appropriate linker) sc (Fv) 2 etc.) Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).
- the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then an appropriate host cell.
- an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then an appropriate host cell.
- Antibodies modified with various molecules such as polyethylene glycol (PEG) and cytotoxic drugs can also be used as modified antibodies (Farmaco. 1999 Aug 30; 54 (8): 497-516., Cancer J. 2008 May-Jun; 14 (3): 154-69.).
- the antibodies used in the present invention also include these modified antibodies.
- Such a modified antibody can be obtained by chemically modifying the antibody. These methods are already established in this field.
- the antibodies used in the present invention include anti-tissue factor antibody, anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb / IIIa antibody, anti-TNF Antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2 / neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, anti-HM1.24 antigen antibody
- Anti-parathyroid hormone related peptide antibody anti-PTHrP antibody
- anti-ganglioside GM3 antibody anti-TPO receptor agonist antibody
- coagulation factor VIII substitute antibody anti-IL31 receptor antibody, anti-HLA antibody, anti-AXL antibody, anti-CXCR4 antibody
- Examples include, but are not limited to, anti-NR10 antibodies, Bi-specific antibodies of Factor IX and Factor X, and the like.
- Preferred reconstituted humanized antibodies for use in the present invention include humanized anti-interleukin 6 (IL-6) receptor antibodies (tocilitumab, hPM-1 or MRA, see WO92 / 19759), humanized anti-HM1.24 antigen monoclonals.
- IL-6 interleukin 6
- hPM-1 or MRA humanized anti-HM1.24 antigen monoclonals.
- Antibody see WO98 / 14580
- humanized anti-parathyroid hormone related peptide antibody see WO98 / 13388
- humanized anti-tissue factor antibody see WO99 / 51743
- anti-glypican-3 humanized IgG1 ⁇ Antibody see codrituzumab, GC33, WO2006 / 006693
- anti-NR10 humanized antibody see WO2009 / 072604
- Bi-specific humanized antibody of factor IX and factor X see ACE910, WO2012 / 067176
- Particularly preferred as humanized antibodies for use in the present invention are humanized anti-IL-6 receptor antibodies, anti-NR10 humanized antibodies, and Bi-specific humanized antibodies of Factor IX and Factor X.
- the human IgM antibody is preferably an anti-ganglioside GM3 recombinant human IgM antibody (see WO05 / 05636).
- an anti-TPO receptor agonist Diabody see WO02 / 33072
- an anti-CD47 agonist Diabody see WO01 / 66737
- the like are preferable.
- the antibody used in the present invention includes a modified antibody that binds to an antigen in an ion concentration-dependent manner (for example, pH-dependent or calcium ion concentration-dependent).
- An antibody that binds to an antigen in a pH-dependent manner that binds strongly to the antigen under neutral pH conditions in plasma, and dissociates from the antigen under acidic pH conditions in the endosome.
- An antibody that binds to an antigen in a calcium ion concentration-dependent manner an antibody that binds to an antigen under a high calcium ion concentration condition and dissociates under a low calcium ion concentration condition) It is possible to dissociate from the antigen within the endosome.
- An antibody that binds to an antigen in a pH-dependent manner or an antigen that binds to an antigen in a calcium ion concentration-dependent manner can be bound to the antigen again after being dissociated and then recycled into plasma by FcRn.
- one molecule of antibody can repeatedly bind to a plurality of antigen molecules.
- the antigen bound to the antigen-binding molecule is dissociated from the antibody in the endosome and is not recycled into the plasma but is degraded in the lysosome.
- an antibody having a low isoelectric point refers to an antibody having a low isoelectric point, which is hardly present in nature.
- the isoelectric point of such an antibody include, but are not limited to, 3.0 to 8.0, preferably 5.0 to 7.5, more preferably 5.0 to 7.0, and particularly preferably 5.0 to 6.5.
- natural (or normal) antibodies are usually considered to have an isoelectric point in the range of 7.5 to 9.5.
- the antibody used in the present invention is preferably a pI-modified antibody in which the pI of the antibody is lowered by modifying amino acid residues exposed on the surface of the antibody.
- a pI-modified antibody refers to an antibody having a pI lowered by 1 or more, preferably 2 or more, more preferably 3 or more, than the pI of the antibody before modification.
- the isoelectric point of SA237 MAb2 in the present example
- the amino acid sequence of tocilitumab isoelectric point: 9.4 was modified to control the isoelectric point
- a fully humanized NS22 antibody produced by the method described in Example 12 of WO2009 / 072604 and having an isoelectric point controlled to 5.6 by modifying the amino acid sequence is also included.
- antibodies having an improved isoelectric point examples include SA237 (MAb2, H chain / SEQ ID NO: 1, L chain / SEQ ID NO: 2), an anti-IL-6 receptor antibody described in WO2009 / 041621, Examples include, but are not limited to, a humanized NR10 antibody and a fully humanized NS22 antibody produced by the method described in Example 12 of WO2009 / 072604.
- amino acid residues exposed on the surface of the antibody in the case of the heavy chain variable region, amino acid residues based on Kabat numbering are H1, 5H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H31, H39, H42, H43, H44, H46, H61, H62, H64, H65, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, Examples include, but are not limited to, amino acid residues selected from H108, 112H110, and H112.
- modification refers to substitution of an original amino acid residue with another amino acid residue, deletion of the original amino acid residue, addition of a new amino acid residue, etc. Preferably, it refers to substitution of the original amino acid residue with another amino acid residue.
- amino acids there are charged amino acids among amino acids.
- lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids).
- negatively charged amino acid negatively charged amino acid
- aspartic acid D
- glutamic acid E
- Other amino acids are known as non-charged amino acids.
- amino acid residue after modification in the present invention is preferably selected from amino acid residues included in any of the following groups (a) or (b), but is not particularly limited to these amino acids.
- A Glutamic acid (E), aspartic acid (D)
- B Lysine (K), Arginine (R), Histidine (H)
- the modification in the present invention includes (1) substitution from an amino acid having a charge to an amino acid having no charge, (2) substitution from an amino acid having a charge to an amino acid having an opposite charge to the amino acid, ( 3) Substitution from an amino acid having no charge to an amino acid having a charge.
- the value of the isoelectric point can be measured by isoelectric focusing known to those skilled in the art.
- the value of theoretical isoelectric point can be calculated using gene and amino acid sequence analysis software (Genetyx etc.).
- An antibody having a modified amino acid residue charge can be obtained by modifying a nucleic acid encoding the antibody, culturing the nucleic acid in a host cell, and purifying the antibody from the host cell culture.
- “modifying a nucleic acid” refers to modifying a nucleic acid sequence to be a codon corresponding to an amino acid residue introduced by the modification. More specifically, it means that the base sequence of the nucleic acid is modified so that the codon of the amino acid residue before modification becomes the codon of the amino acid residue introduced by modification. That is, the codon encoding the amino acid residue to be modified is replaced by the codon encoding the amino acid residue introduced by the modification.
- Such nucleic acid modification can be appropriately performed by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis.
- the pharmaceutical composition of the present invention can be a solution preparation (antibody-containing solution preparation) or a lyophilization agent.
- the solution preparation of the present invention includes a solution before lyophilization treatment or a solution after re-dissolution in the production process of the lyophilized preparation.
- the solution preparation of the present invention is preferably a solution preparation produced without including a freeze-drying step in the production process.
- the lyophilizing agent of the present invention can be obtained by lyophilizing the solution preparation of the present invention by a method known to those skilled in the art.
- the preparation of the present invention includes, as necessary, a cryoprotectant, a suspending agent, a solubilizing agent, an isotonic agent, a preservative, an adsorption inhibitor, a diluent, an excipient, a pH adjuster, a soothing agent.
- a cryoprotectant such as an agent, a sulfur-containing reducing agent, and an antioxidant can be included.
- cryoprotective agents include, but are not limited to, sugars such as trehalose, sucrose, and sorbitol.
- solution adjuvant examples include, but are not limited to, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, tuna gol, castor oil fatty acid ethyl ester.
- isotonic agents include, but are not limited to, sodium chloride, potassium chloride, calcium chloride and the like.
- preservatives include, but are not limited to, methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, and the like.
- adsorption inhibitor examples include, but are not limited to, human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol and the like.
- sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, carbon atoms
- sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, carbon atoms
- sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thio
- Antioxidants such as erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite
- the chelating agent include, but are not limited to, sodium, triamyl gallate, propyl gallate, disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.
- the preparation of the present invention can be administered either orally or parenterally, but is usually administered by a parenteral route. Specifically, it is administered by injection, transdermal, transmucosal, nasal, transpulmonary, or the like. As an example of an injection form, it can be administered systemically or locally by, for example, subcutaneous injection, intravenous injection, intramuscular injection, or the like. In the case of subcutaneous injection, the amount of injection solution is limited, but the dose of antibody per administration can be large (about 100 to 200 mg-). Therefore, the preparation of the present invention is particularly suitable for subcutaneous administration (injection). In addition, all prior art documents cited in the present specification are incorporated herein by reference.
- ACE910 has an activity to substitute for the function of coagulation factor VIII as described in the above-mentioned patent document.
- MAb2 An anti-IL-6 receptor antibody described in WO 2009/041621, which is an antibody (SA237) having a pI value of 5.8 by modifying the amino acid of tocilizumab.
- the amino acid sequence of MAb2 antibody is represented by H chain / SEQ ID NO: 1, L chain / SEQ ID NO: 2.
- MAb3 anti-glypican 3 humanized antibody (GC33, generic name: codrituzumab, antibody humanized by the method described in Example 24 of WO2006 / 006693 and modified in the L chain by the method of Example 25), antibody class is IgG1 .
- FIG. 1 and FIG. 2 show the results of plotting as a function of R g app or D max app .
- a good correlation was confirmed between the viscosity for any particle size parameter.
- the correlation was well fitted by an exponential function, but a linear correlation was confirmed in the low viscosity region.
- Example 2 Correlation between particle size parameter and molecular weight parameter and viscosity in MAb1, MAb3, MAb4 MAb1, MAb3 (all the same as those described in Example 1), MAb4 (humanized anti-IL-6 receptor antibody, (Generic name: tocilizumab), a sample having an antibody concentration of 15 mg / mL was prepared in the formulation solution of Table 2. About 10 ⁇ L of the sample is filled in a microcapillary cell, and the apparent radius of inertia R g app (nm) and the maximum length of the apparent particle D max app (particle size parameters) are measured with SAXSess mc 2 (Anton Paar).
- the viscosity at the time of increasing the concentration can be predicted by using the particle size parameter obtained from the SAXS measurement as an index. Even when various modified proteins in which a part of the amino acid sequence of a protein such as an antibody is substituted with a different amino acid are prepared and the modified proteins with controlled viscosity are selected, the parameters and viscosity obtained by the method of the present invention are selected. Since the good correlation can be obtained in the same manner, the method of the present invention is also useful as a method for selecting a modified protein with a desired viscosity controlled.
- Example 3 Correlation between particle size parameter and viscosity in MAb2 and MAb4 Using MAb2 and MAb4 (same as those described in Examples 1 and 2, respectively), the antibody (Mab4) before the amino acid sequence was modified It was investigated whether the difference in viscosity from an antibody (Mab2) in which a part of the amino acid sequence was substituted with another amino acid could be evaluated using the measurement method of the present invention. Using each antibody, a sample having an antibody concentration of 15 mg / mL was prepared in the formulation solution shown in Table 3.
- Example 4 Correlation between particle size parameters and viscosity in MAb1, MAb2, MAb3, MAb4, and BSA MAb1, MAb2, MAb3, MAb4 (all the same as those described in Examples 1-2), and BSA A sample having a protein concentration of 15 mg / mL was prepared in the formulation solution shown in Table 4. About 10 ⁇ L of the sample is packed in a microcapillary cell, and the SAXSess mc 2 (Anton Paar) is used to measure the scattering intensity I (0) at the scattering angle 0, which is the particle size parameter, and the apparent radius of inertia R g app of the apparent particle. The maximum length D max app (nm) of the particles was calculated.
- the particle size parameters (I (0), R g app , and D max app ) obtained at 15 mg / mL and 5 ° C obtained from SAXS measurement are certainly correlated with the viscosity at 160 mg / mL and 25 ° C. It has been confirmed that the viscosity evaluation method of the present invention can be applied to proteins other than antibodies.
- Example 5 Correlation between particle size parameter and viscosity in MAb5 and its modified antibody MAb5-A (anti-IgE antibody) and MAb5-A, a modified antibody in which four amino acid residues at specific positions of MAb5 are substituted with histidine The viscosity was measured. Residues before and after modification are listed in Table 5. The modified positions in Table 5 are indicated based on Kabat numbering. Samples with protein concentrations of 151 and 155 mg / mL were prepared in the formulation solutions shown in Table 6. Using 90 ⁇ L of the sample, the viscosity ⁇ (mPa ⁇ s) of the sample was measured with an EMS viscometer (Kyoto Electronics) (J Artif Organs. 16: 359-367 (2013)). The experimental temperature was 25 ° C. The measurement results are shown in Table 6. Substantial increases in viscosity were observed with 4-residue histidine substitution.
- MAb5-A In order to identify the alteration that causes this increase in viscosity and to design a low-viscosity modified antibody, MAb5, MAb5-A, and MAb5-A are substituted with histidine in 4 residues, one MAb5 residue at a time.
- a sample having a protein concentration of 30 mg / mL was prepared in the formulation solution shown in Table 6. Residues before and after modification are listed in Table 5.
- D max app (nm) of the apparent particle was calculated with SAXSess mc 2 (Anton Paar). This value is considered to represent the particle size of the protein in consideration of the association state.
- measurement was performed at a low temperature of 5 ° C. The measurement results are shown in Table 6.
- D max app for MAb5-A, MAb5-B, MAb5-D, MAb5-E compared to clearly greater than the MAb5 of D max app before modification, is D max app for MAb5-C of 21 nm The value was close to the D max app of MAb5 before modification.
- FIG. 6 shows that Mab5-A molecules interact with each other through the Fab domain
- the right figure shows that the side chain of His at position 97 in the Fab domain of the Mab5-A molecule is different. It is shown that it is at a distance that can form a hydrogen bond with the main chain of Trp in the Fab domain of Mab5-A molecule.
- such a Fab-Fab interaction is not observed in Mab5. Therefore, the increase in viscosity due to substitution of Pro at position 97 of Mab5 with His may be attributed to the formation of hydrogen bonds between different Fabs.
- the results of crystal structure analysis also suggested the validity of this screening method.
- a method for easily measuring the viscosity of a protein solution with a small amount of sample is provided.
- the present invention facilitates the development of a low-viscosity protein solution formulation, further enables selection (screening) of proteins with low viscosity in the solution, and provides a protein solution formulation such as an antibody having superior physical properties. It became possible.
- the method of the present invention is particularly useful in the production of low viscosity biopharmaceuticals.
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Abstract
Description
一般的に、抗体等の蛋白質溶液の粘度の測定には、せん断速度とせん断応力との比例係数から粘度を算出するレオメーターや、試料溶液中の金属球の回転速度から粘度を算出するEMS粘度計、あるいは試料の通過速度や通過の際の圧力損失から粘度を算出するマイクロ流路を利用した粘度測定器を用いるのが主流である。
〔1〕以下の工程を含む、蛋白質溶液の粘度の測定方法:
1)該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定する工程;
2)予め求めた検量線に基づき、上記測定値から該蛋白質の粘度を算出する工程。
〔2〕試料中の蛋白質濃度が10~100mg/mlである、〔1〕の測定方法。
〔3〕試料中の蛋白質濃度が10~30mg/mlである、〔1〕または〔2〕の測定方法。
〔4〕試料中の蛋白質濃度が15~30mg/mlである、〔1〕~〔3〕のいずれかの測定方法。
〔5〕測定温度の条件が0~40℃である、〔1〕~〔4〕のいずれかの測定方法。
〔6〕測定温度の条件が0~25℃である、〔1〕~〔5〕のいずれかの測定方法。
〔7〕測定温度の条件が3~10℃である、〔1〕~〔6〕のいずれかの測定方法。
〔8〕測定する試料の量が1~100μLである、〔1〕~〔7〕のいずれかの測定方法。
〔9〕測定する試料の量が5~30μLである、〔1〕~〔8〕のいずれかの測定方法。
〔10〕前記蛋白質が抗体である、〔1〕~〔9〕のいずれかの測定方法。
〔11〕蛋白質の粘性を予測する方法であって、該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定することを含む、前記方法。
〔12〕粘性を制御した蛋白質の選抜方法であって、該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定することを含む、前記選抜方法。
〔13〕以下の工程を含む、粘性の制御された改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)〔11〕の予測方法を用いて、当該蛋白質の粘性を予測する工程。
〔14〕以下の工程を含む、粘性の低い蛋白質を製造する方法:
1)〔11〕の予測方法を用いて、粘性の低い蛋白質を選抜する工程。
〔15〕以下の工程を含む、粘性の制御された改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)〔12〕の選抜方法を用いて、当該改変蛋白質から粘性の制御された蛋白質を選抜する工程。
〔16〕以下の工程を含む、粘性を低下させた改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)〔12〕の選抜方法を用いて、当該改変蛋白質から元の蛋白質よりも粘性が低下した改変蛋白質を選抜する工程。
〔17〕元の蛋白質が、アミノ酸残基の一部がヒスチジン(His)残基に置換された改変蛋白質である、〔13〕、〔15〕または〔16〕の方法:
〔18〕蛋白質が抗体である、〔11〕~〔16〕のいずれかの方法。
〔19〕元の抗体のアミノ酸残基の一部がヒスチジン(His)残基に置換された改変抗体であって、Kabat numbering systemにおける97位のアミノ酸残基がHis残基でない改変抗体。
〔20〕97位のアミノ酸残基が元の抗体のアミノ酸残基である、〔19〕の改変抗体。
〔21〕元の抗体のアミノ酸残基の一部が別のアミノ酸に置換されている、抗原に対してイオン濃度依存的に結合する改変抗体であって、Kabat numbering systemにおける97位のアミノ酸残基がHis残基でない改変抗体。
〔22〕97位のアミノ酸残基が元の抗体のアミノ酸残基である、〔21〕に記載の改変抗体。
本発明は、少量の試料で実施可能な蛋白質溶液の粘度の測定方法(推定方法)、蛋白質の粘性を予測する方法、粘性を制御した蛋白質の選抜方法、粘性の制御された改変蛋白質を製造する方法、粘性を低下させた改変蛋白質を製造する方法、および粘性の低い蛋白質を製造する方法に関する。
具体的には、(1)蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定し、(2)予め求めた検量線に基づき、上記測定値から該蛋白質の粘度を算出することにより、該蛋白質を含む溶液を高濃度化した際の粘度を推定することができる。
なお、ここで「元の蛋白質」や「元の抗体」とは、「人為的にアミノ酸の一部を改変する前の蛋白質」若しくは「人為的にアミノ酸の一部を改変する前の蛋白質」である。
(1)前記蛋白質の濃度が1~100mg/mlである試料にX線を照射し、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、前記溶液中の蛋白質の見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定する工程;および
(2)予め求めた検量線に基づき、上記測定値から該蛋白質の粘度を算出する工程。
低分子化抗体としては、抗TPO受容体アゴニストDiabody(WO02/33072参照)、抗CD47アゴニストDiabody(WO01/66737参照)などが好ましい。
(a)グルタミン酸(E)、アスパラギン酸(D)
(b)リジン(K)、アルギニン(R)、ヒスチジン(H)
すなわち、本発明における改変としては、(1)電荷を有するアミノ酸から電荷を有さないアミノ酸への置換、(2)電荷を有するアミノ酸から当該アミノ酸とは反対の電荷を有するアミノ酸への置換、(3)電荷を有さないアミノ酸から電荷を有するアミノ酸への置換が挙げられる。
なお、本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
MAb1:血液凝固第IX因子および/または活性化血液凝固第IX因子ならびに血液凝固第X因子および/または活性化血液凝固第X因子を認識する二重特異性抗体である。非特許文献(PLoS One. 2013;8(2):e57479)及び特許文献(WO 2012/067176)に記載の二重特異性抗体であるACE910 (Q499-z121/J327-z119/L404-k)(配列番号:3に記載のアミノ酸配列からなるH鎖と配列番号:5に記載のL鎖が会合し、配列番号:4に記載のアミノ酸配列からなるH鎖と配列番号:5に記載のL鎖が会合している二重特異性抗体)を、前記非特許文献又は特許文献の記載に従って作製した。尚、ACE910は前記特許文献に記載されているように凝固第VIII因子の機能を代替する活性を有している。
MAb2:WO 2009/041621に記載された抗IL-6レセプター抗体で、トシリツマブのアミノ酸を改変してpI値5.8とした抗体(SA237)である。MAb2抗体のアミノ酸配列はH鎖/配列番号:1、L鎖/配列番号:2で表される。
MAb3:抗グリピカン3ヒト化抗体(GC33、一般名:codrituzumab、WO2006/006693の実施例24に記載の方法でヒト化し、実施例25の方法でL鎖が改変された抗体)、抗体クラスはIgG1。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対してギニエプロットを行い、q*Rg app<1.3を満たす条件下においてRg appを得る。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対して間接フーリエ変換法(J Appl Cryst. 13 :577-584 (1980))を適用し、散乱を与えている粒子の二体距離分布関数(p(r))を得る。p(r)のx軸切片からDmax appを得る。
MAb1, MAb3(いずれも実施例1に記載のものと同じ), MAb4(ヒト化抗IL-6レセプター抗体、一般名:トシリツマブ)を用い、表2の処方溶液において、抗体濃度が15 mg/mLの試料を調製した。当該試料約10 μLをマイクロキャピラリーセルに充てんし、SAXSess mc2(アントンパール)にて粒子サイズパラメータであるみかけの粒子の慣性半径Rg app (nm)とみかけの粒子の最大長Dmax app (nm)に加え,みかけの分子量に比例する値として散乱角0における散乱強度I(0) (a.u.)を算出した。これらの値が大きいほど抗体の会合性が高いことを表すと考えられる。試料間での会合性の違いをより顕著に検出するために、低温である5 ℃にて測定を実施した。測定結果を表2に記す。I(0)、Rg app、及びDmax appの算出方法を以下に記す。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対してギニエプロットを行い、q*Rg app<1.3を満たす条件下においてRg appを得る。同時にy切片からI(0)を得る。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対して間接フーリエ変換法(J Appl Cryst. 13 :577-584 (1980))を適用し、散乱を与えている粒子の二体距離分布関数(p(r))を得る。p(r)のx軸切片からDmax appを得る。
また、抗体等の蛋白質の一部アミノ酸配列を異なるアミノ酸に置換した種々の改変蛋白質を作製して、粘度が制御された改変蛋白質を選抜する場合においても、本発明の方法によって得られるパラメータと粘度の実測値について、同様に良好な相関がえられるので、所望とする粘度が制御された改変蛋白質の選抜方法としても本発明の方法は有用である。
MAb2、MAb4(それぞれ実施例1、2に記載のものと同じ)を用いて、アミノ酸配列を改変する前の抗体(Mab4)と一部のアミノ酸配列を別のアミノ酸に置換した抗体(Mab2)との粘度の違いを本発明の測定方法を用いて評価できるかどうか検討を行った。それぞれの抗体を用い、表3の処方溶液において,抗体濃度が15 mg/mLの試料を調製した。当該試料約10 μLをマイクロキャピラリーセルに充てんし、SAXSess mc2(アントンパール)にて,粒子サイズパラメータである散乱角0における散乱強度I(0)、みかけの粒子の慣性半径Rg app、みかけの粒子の最大長Dmax app (nm)を算出した。これらの値が大きいほど抗体の会合性が高いことを表すと考えられる。試料間での会合性の違いをより顕著に検出するために、低温である5 ℃にて測定を実施した。測定結果を表3に記す。I(0)、Rg app、及びDmax appの算出方法を以下に記す。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対してギニエプロットを行い、q*Rg app<1.3を満たす条件下においてRg appを得る。同時にy切片からI(0)を得る。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対して間接フーリエ変換法(J Appl Cryst. 13 :577-584 (1980))を適用し、散乱を与えている粒子の二体距離分布関数(p(r))を得る。p(r)のx軸切片からDmax appを得る。
SAXS測定より得られた15 mg/mL、5 ℃における粒子サイズパラメータ(I(0)、Rg app、及びDmax app)と、160 mg/mL、25 ℃における粘度は確かに相関することが確認された。よって、本発明の方法を用いた、粘度を指標とする蛋白質のアミノ酸置換体のスクリーニング法の妥当性が実証された。
MAb1、MAb2、MAb3、MAb4(いずれも実施例1~2に記載のものと同じ)、及びBSAを用い、表4の処方溶液において、蛋白質濃度が15 mg/mLの試料を調製した。当該試料約10 μLをマイクロキャピラリーセルに充てんし、SAXSess mc2(アントンパール)にて、粒子サイズパラメータである散乱角0における散乱強度I(0)、みかけの粒子の慣性半径Rg app、みかけの粒子の最大長Dmax app (nm)を算出した。これらの値は会合状態を考慮した蛋白質の粒子サイズを表すと考えられる。試料間での会合性の違いをより顕著に検出するために、低温である5 ℃にて測定を実施した。測定結果を表3に記す。I(0)、Rg app、及びDmax appの算出方法を以下に記す。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対してギニエプロットを行い、q*Rg app<1.3を満たす条件下においてRg appを得る。同時にy切片からI(0)を得る。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対して間接フーリエ変換法(J Appl Cryst. 13 :577-584 (1980))を適用し、散乱を与えている粒子の二体距離分布関数(p(r))を得る。p(r)のx軸切片からDmax appを得る。
MAb5(抗IgE抗体)及びMAb5の特定の位置の4つのアミノ酸残基をヒスチジンに置換した改変抗体であるMAb5-Aの粘度を測定した。改変前後の残基を表5に記載した。なお、表5における改変位置は、Kabatナンバリングに基づき表記した。表6の処方溶液において、蛋白質濃度が151、155 mg/mLの試料を調製した。当該試料90 μLを用いてEMS粘度計(京都電子)(J Artif Organs. 16:359-367 (2013))にて試料の粘度η (mPa・s)を測定した。実験温度は25 ℃とした。測定結果を表6に記す。4残基のヒスチジン置換により粘度の大幅な上昇が認められた。
1) 試料及び溶媒(ブランク)の散乱パターンを2次元のイメージングプレートを用いて検出する。
2) 2次元の散乱パターンをSAXSQuantソフトウェア(アントンパール)によって散乱ベクトルqを変数とした1次元の散乱強度に変換し、散乱曲線を得る。
3) ビームストッパーを透過したq=0における散乱強度で散乱曲線を規格化する。
4) 試料の散乱曲線からブランクの散乱曲線を差し引く(ブランク補正)。
5) デスメア補正(光学系補正)を行う。
6) 吸収補正、ブランク補正及びデスメア補正後の散乱曲線に対して間接フーリエ変換法(J Appl Cryst. 13 :577-584 (1980))を適用し、散乱を与えている粒子の二体距離分布関数(p(r))を得る。p(r)のx軸切片からDmax appを得る。
Claims (22)
- 以下の工程を含む、蛋白質溶液の粘度の測定方法:
1)該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定する工程;
2)予め求めた検量線に基づき、上記測定値から該蛋白質の粘度を算出する工程。 - 試料中の蛋白質濃度が10~100mg/mlである、請求項1に記載の測定方法。
- 試料中の蛋白質濃度が10~30mg/mlである、請求項1または2に記載の測定方法。
- 試料中の蛋白質濃度が15~30mg/mlである、請求項1~3のいずれか一項に記載の測定方法。
- 測定温度の条件が0~40℃である、請求項1~4のいずれか一項に記載の測定方法。
- 測定温度の条件が0~25℃である、請求項1~5のいずれか一項に記載の測定方法。
- 測定温度の条件が3~10℃である、請求項1~6のいずれか一項に記載の測定方法。
- 測定する試料の量が1~100μLである、請求項1~7のいずれか一項に記載の測定方法。
- 測定する試料の量が5~30μLである、請求項1~8のいずれか一項に記載の測定方法。
- 前記蛋白質が抗体である、請求項1~9のいずれか一項に記載の測定方法。
- 蛋白質の粘性を予測する方法であって、該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定することを含む、前記方法。
- 粘性を制御した蛋白質の選抜方法であって、該蛋白質の濃度が1~100mg/mlである試料にX線を照射することによる、X線小角散乱法(SAXS)またはX線溶液散乱法を用いて、見かけの粒子サイズ(見かけの粒子の最大長(Dmax app)もしくは見かけの粒子の慣性半径(Rg app))または見かけの分子量を測定することを含む、前記選抜方法。
- 以下の工程を含む、粘性の制御された改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)請求項11に記載の予測方法を用いて、当該蛋白質の粘性を予測する工程。 - 以下の工程を含む、粘性の低い蛋白質を製造する方法:
1)請求項11に記載の予測方法を用いて、粘性の低い蛋白質を選抜する工程。 - 以下の工程を含む、粘性の制御された改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)請求項12に記載の選抜方法を用いて、当該改変蛋白質から粘性の制御された蛋白質を選抜する工程。 - 以下の工程を含む、粘性を低下させた改変蛋白質を製造する方法:
1)元の蛋白質のアミノ酸の一部を改変して、改変蛋白質を得る工程;
2)請求項12に記載の選抜方法を用いて、当該改変蛋白質から元の蛋白質よりも粘性が低下した改変蛋白質を選抜する工程。 - 元の蛋白質が、アミノ酸残基の一部がヒスチジン(His)残基に置換された改変蛋白質である、請求項13、15または16に記載の方法:
- 蛋白質が抗体である、請求項11~16のいずれか一項に記載の方法。
- 元の抗体のアミノ酸残基の一部がヒスチジン(His)残基に置換された改変抗体であって、Kabat numbering systemにおける97位のアミノ酸残基がHis残基でない改変抗体。
- 97位のアミノ酸残基が元の抗体のアミノ酸残基である、請求項19に記載の改変抗体。
- 元の抗体のアミノ酸残基の一部が別のアミノ酸に置換されている、抗原に対してイオン濃度依存的に結合する改変抗体であって、Kabat numbering systemにおける97位のアミノ酸残基がHis残基でない改変抗体。
- 97位のアミノ酸残基が元の抗体のアミノ酸残基である、請求項21に記載の改変抗体。
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US11851486B2 (en) | 2017-05-02 | 2023-12-26 | National Center Of Neurology And Psychiatry | Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils |
US11692037B2 (en) | 2017-10-20 | 2023-07-04 | Hyogo College Of Medicine | Anti-IL-6 receptor antibody-containing medicinal composition for preventing post-surgical adhesion |
JP2022511150A (ja) * | 2019-01-02 | 2022-01-31 | エム アンド ジェイ サイエンティフィック エルエルシー | 光散乱検出器及び光散乱検出器のための方法 |
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WO2020204055A1 (ja) | 2019-04-02 | 2020-10-08 | 中外製薬株式会社 | 標的特異的な外来遺伝子の導入方法 |
Also Published As
Publication number | Publication date |
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EP3185004A4 (en) | 2018-05-30 |
JP6858559B2 (ja) | 2021-04-14 |
JPWO2016027859A1 (ja) | 2017-06-01 |
US20170362304A1 (en) | 2017-12-21 |
EP3185004A1 (en) | 2017-06-28 |
EP4056993A1 (en) | 2022-09-14 |
JP2020101555A (ja) | 2020-07-02 |
US11059881B2 (en) | 2021-07-13 |
JP6936883B2 (ja) | 2021-09-22 |
US20200131250A1 (en) | 2020-04-30 |
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