Method for measuring viscosity of protein solution

Abstract

The inventors discovered that viscosity of a protein solution can be estimated by measuring the apparent particle size or apparent molecular weight by a small angle X-ray scattering (SAXS) method or X-ray solution scattering method, which enables measurement of small amounts of samples, and then correlating those measurement results with viscosity of the protein solution.

1 .- 22 . (canceled) 
     
     
         23 . A method of determining viscosity, at a given temperature, of a protein solution of interest, the method comprising:
 (a) irradiating, at the given temperature, a sample of the protein solution of interest with X-ray to obtain a scattering pattern;   (b) determining, from the scattering pattern, a value for a parameter selected from: apparent maximum particle diameter (D max   app ) of the protein in the sample, apparent particle radius of gyration (R g   app ) of the protein in the sample, and apparent molecular weight of the protein in the sample; and   (c) using the value for the selected parameter to determine, from a predetermined calibration curve, viscosity at the given temperature of the protein solution of interest, wherein the predetermined calibration curve correlates:
 (i) viscosity, measured at the given temperature, of a sample of each of a plurality of different test solutions, each comprising a test protein, to 
 (ii) a value for the selected parameter of each of the test solutions, measured at the given temperature; 
 wherein the test proteins in the test solutions can be the same or different, and 
 wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are the same, and are within the range of 1 mg/mL to 100 mg/mL. 
   
     
     
         24 . The method of  claim 23 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 10 mg/mL to 100 mg/mL. 
     
     
         25 . The method of  claim 23 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 10 mg/mL to 30 mg/mL. 
     
     
         26 . The method of  claim 23 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 15 mg/mL to 30 mg/mL. 
     
     
         27 . The method of  claim 23 , wherein the given temperature is in the range of 0° C. to 40° C. 
     
     
         28 . The method of  claim 23 , wherein the given temperature is in the range of 0° C. to 25° C. 
     
     
         29 . The method of  claim 23 , wherein the given temperature is in the range of 3° C. to 10° C. 
     
     
         30 . The method of  claim 23 , wherein the amount of the sample of the protein solution of interest is in the range of 1 μL to 100 μL. 
     
     
         31 . The method of  claim 23 , wherein the amount of the sample of the protein solution of interest is in the range of 5 μL to 30 μL. 
     
     
         32 . The method of  claim 23 , wherein the protein in the protein solution of interest is an antibody. 
     
     
         33 . A method of determining viscosity, at a first temperature, of a protein solution of interest, the method comprising:
 (a) irradiating, at a second temperature, a sample of the protein solution of interest with X-ray to obtain a scattering pattern;   (b) determining, from the scattering pattern, a value for a parameter selected from: apparent maximum particle diameter (D max   app ) of the protein in the sample, apparent particle radius of gyration (R g   app ) of the protein in the sample, and apparent molecular weight of the protein in the sample; and   (c) using the value for the selected parameter to determine, from a predetermined calibration curve, viscosity at the first temperature of the protein solution of interest, wherein the predetermined calibration curve correlates:
 (i) viscosity, measured at the first temperature, of a sample of each of a plurality of different test solutions, each comprising a test protein, to 
 (ii) a value for the selected parameter of each of the test solutions, measured at the second temperature; 
 wherein the test proteins in the test solutions can be the same or different, and 
 wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are the same, and are within the range of 1 mg/mL to 100 mg/mL. 
   
     
     
         34 . The method of  claim 33 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 10 mg/mL to 100 mg/mL. 
     
     
         35 . The method of  claim 33 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 10 mg/mL to 30 mg/mL. 
     
     
         36 . The method of  claim 33 , wherein the protein concentration of each of the test solutions and the protein concentration of the protein solution of interest are within the range of 15 mg/mL to 30 mg/mL. 
     
     
         37 . The method of  claim 33 , wherein the second temperature is in the range of 0° C. to 40° C. 
     
     
         38 . The method of  claim 33 , wherein the second temperature is in the range of 0° C. to 25° C. 
     
     
         39 . The method of  claim 33 , wherein the second temperature is in the range of 3° C. to 10° C. 
     
     
         40 . The method of  claim 33 , wherein the amount of the sample of the protein solution of interest is in the range of 1 μL to 100 μL. 
     
     
         41 . The method of  claim 33 , wherein the amount of the sample of the protein solution of interest is in the range of 5 μL to 30 μL. 
     
     
         42 . The method of  claim 33 , wherein the protein in the protein solution of interest is an antibody. 
     
     
         43 . A method of predicting viscosity, at a given temperature, of a protein solution of interest having a first concentration of a protein, the method comprising:
 (a) irradiating, at the given temperature, a sample solution having a second concentration of the protein with X-ray to obtain a scattering pattern, wherein the sample solution and the protein solution of interest are identical except for protein concentration;   (b) determining, from the scattering pattern, a value for a parameter selected from: apparent maximum particle diameter (D max   app ) of the protein in the sample solution, apparent particle radius of gyration (R g   app ) of the protein in the sample solution, and apparent molecular weight of the protein in the sample solution; and   (c) using the value for the selected parameter to determine, from a predetermined calibration curve, viscosity at the given temperature of the protein solution of interest,
 wherein the predetermined calibration curve correlates, for each of a plurality of test solution/corresponding solution pairs: 
 (i) a value for the selected parameter, measured at the given temperature, for the test solution, to 
 (ii) viscosity, measured at the given temperature, of the corresponding solution, 
 wherein each test solution comprises a test protein that can be the same or vary among the plurality of test solutions, 
 wherein each test solution and its paired corresponding solution comprise the same test protein and are identical except for concentration of the test protein, 
 wherein the concentration of test protein in all of the test solutions is the second concentration and the concentration of test protein in all of the corresponding solutions is the first concentration, and 
 wherein the second concentration is a concentration in the range of 1 mg/mL to 100 mg/mL. 
   
     
     
         44 . The method of  claim 43 , wherein the second concentration is within the range of 10 mg/mL to 100 mg/mL. 
     
     
         45 . The method of  claim 43 , wherein the second concentration is within the range of 10 mg/mL to 30 mg/mL. 
     
     
         46 . The method of  claim 43 , wherein the second concentration is within the range of 15 mg/mL to 30 mg/mL. 
     
     
         47 . The method of  claim 43 , wherein the given temperature is in the range of 0° C. to 40° C. 
     
     
         48 . The method of  claim 43 , wherein the given temperature is in the range of 0° C. to 25° C. 
     
     
         49 . The method of  claim 43 , wherein the given temperature is in the range of 3° C. to 10° C. 
     
     
         50 . The method of  claim 43 , wherein the amount of the sample solution is in the range of 1 μL to 100 μL. 
     
     
         51 . The method of  claim 43 , wherein the amount of the sample solution is in the range of 5 μL to 30 μL. 
     
     
         52 . The method of  claim 43 , wherein the protein in the protein solution of interest is an antibody. 
     
     
         53 . A method of predicting viscosity, at a first temperature, of a protein solution of interest having a first concentration of a protein, the method comprising:
 (a) irradiating, at a second temperature, a sample solution having a second concentration of the protein with X-ray to obtain a scattering pattern, wherein the sample solution and the protein solution of interest are identical except for protein concentration and temperature;   (b) determining, from the scattering pattern, a value for a parameter selected from: apparent maximum particle diameter (D max   app ) of the protein in the sample solution, apparent particle radius of gyration (R g   app ) of the protein in the sample solution, and apparent molecular weight of the protein in the sample solution; and   (c) using the value for the selected parameter to determine, from a predetermined calibration curve, viscosity of the protein solution of interest at the first temperature, wherein the predetermined calibration curve correlates, for each of a plurality of test solution/corresponding solution pairs:
 (i) a value for the selected parameter, measured at the second temperature, for the test solution, to 
 (ii) viscosity, measured at the first temperature, of the corresponding solution, 
 wherein each test solution comprises a test protein that can be the same or vary among the plurality of test solutions, 
 wherein each test solution and its paired corresponding solution comprise the same test protein and are identical except for temperature and concentration of the test protein, 
 wherein the concentration of test protein in all of the test solutions is the second concentration and the concentration of test protein in all of the corresponding solutions is the first concentration, and 
 wherein the second concentration is a concentration in the range of 1 mg/mL to 100 mg/mL. 
   
     
     
         54 . The method of  claim 53 , wherein the second concentration is within the range of 10 mg/mL to 100 mg/mL. 
     
     
         55 . The method of  claim 53 , wherein the second concentration is within the range of 10 mg/mL to 30 mg/mL. 
     
     
         56 . The method of  claim 53 , wherein the second concentration is within the range of 15 mg/mL to 30 mg/mL. 
     
     
         57 . The method of  claim 53 , wherein the second temperature is in the range of 0° C. to 40° C. 
     
     
         58 . The method of  claim 53 , wherein the second temperature is in the range of 0° C. to 25° C. 
     
     
         59 . The method of  claim 53 , wherein the second temperature is in the range of 3° C. to 10° C. 
     
     
         60 . The method of  claim 53 , wherein the amount of the sample solution is in the range of 1 μL to 100 μL. 
     
     
         61 . The method of  claim 53 , wherein the amount of the sample solution is in the range of 5 μL to 30 μL. 
     
     
         62 . The method of  claim 53 , wherein the protein in the protein solution of interest is an antibody. 
     
     
         63 . A method of selecting a protein that possesses desired viscosity characteristics, the method comprising:
 (a) providing a solution comprising a given protein;   (b) carrying out the method of  claim 23 , wherein the solution of (a) is the protein solution of interest, thereby determining the viscosity of the protein solution of interest at the given temperature;   (c) determining that the viscosity of the protein solution of interest at the given temperature is a desired viscosity; and   (d) selecting the given protein as possessing the desired viscosity characteristics.   
     
     
         64 . A method of selecting a protein that possesses desired viscosity characteristics, the method comprising:
 (a) providing a solution comprising a given protein;   (b) carrying out the method of  claim 33 , wherein the solution of (a) is the protein solution of interest, thereby determining the viscosity of the protein solution of interest at the first temperature;   (c) determining that the viscosity of the protein solution of interest at the first temperature is a desired viscosity; and   (d) selecting the given protein as possessing the desired viscosity characteristics.   
     
     
         65 . A method of selecting a protein that possesses desired viscosity characteristics, the method comprising:
 (a) providing a solution comprising a given protein;   (b) carrying out the method of  claim 43 , wherein the solution of (a) is the protein solution of interest, thereby predicting the viscosity of the protein solution of interest at the given temperature;   (c) determining that the predicted viscosity of the protein solution of interest at the given temperature is a desired viscosity; and   (d) selecting the given protein as possessing the desired viscosity characteristics.   
     
     
         66 . A method of selecting a protein that possesses desired viscosity characteristics, the method comprising:
 (a) providing a solution comprising a given protein;   (b) carrying out the method of  claim 53 , wherein the solution of (a) is the protein solution of interest, thereby predicting the viscosity of the protein solution of interest at the first temperature;   (c) determining that the predicted viscosity of the protein solution of interest at the first temperature is a desired viscosity; and   (d) selecting the given protein as possessing the desired viscosity characteristics.   
     
     
         67 . A method of producing a protein that possesses desired viscosity characteristics, the method comprising:
 (a) selecting an original protein;   (b) producing a second protein that is a mutated version of the original protein;   (c) preparing a solution comprising the second protein;   (d) carrying out the method of  claim 23 , using the solution of (c) as the protein solution of interest, thereby determining the viscosity of the protein solution of interest at the given temperature;   (e) determining that the viscosity of the protein solution of interest at the given temperature is a desired viscosity, thereby determining that the second protein as a protein that possesses the desired viscosity characteristics.   
     
     
         68 . The method of  claim 67 , wherein the desired viscosity is 2000 mPa·s or less. 
     
     
         69 . The method of  claim 67 , wherein the desired viscosity is in the range of 0.1 mPa·s to 1000 mPa·s. 
     
     
         70 . The method of  claim 67 , wherein the desired viscosity is in the range of 0.1 mPa·s to 700 mPa·s. 
     
     
         71 . The method of  claim 67 , wherein the viscosity of the protein solution of interest is lower than the viscosity of a reference protein solution, wherein the reference protein solution is identical to the protein solution of interest except that the reference protein solution comprises the original protein instead of the second protein. 
     
     
         72 . The method of  claim 67 , wherein the second protein and the original protein are identical except that the amino acid sequence of the second protein comprises histidine (His) at one or more positions that are not His in the amino acid sequence of the original protein. 
     
     
         73 . The method of  claim 67 , wherein the original protein is an antibody. 
     
     
         74 . The method of  claim 73 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein is not a His residue. 
     
     
         75 . The method of  claim 73 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein and the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the original protein are the same. 
     
     
         76 . The method of  claim 73 , wherein the antibody is an ion concentration-dependent antigen binding antibody. 
     
     
         77 . A method of producing a protein that possesses desired viscosity characteristics, the method comprising:
 (a) selecting an original protein;   (b) producing a second protein that is a mutated version of the original protein;   (c) preparing a solution comprising the second protein;   (d) carrying out the method of  claim 33 , using the solution of (c) as the protein solution of interest, thereby determining the viscosity of the protein solution of interest at the first temperature;   (e) determining that the viscosity of the protein solution of interest at the first temperature is a desired viscosity, thereby determining that the second protein as a protein that possesses the desired viscosity characteristics.   
     
     
         78 . The method of  claim 77 , wherein the desired viscosity is 2000 mPa·s or less. 
     
     
         79 . The method of  claim 77 , wherein the desired viscosity is in the range of 0.1 mPa·s to 1000 mPa·s. 
     
     
         80 . The method of  claim 77 , wherein the desired viscosity is in the range of 0.1 mPa·s to 700 mPa·s. 
     
     
         81 . The method of  claim 77 , wherein the viscosity of the protein solution of interest is lower than the viscosity of a reference protein solution, wherein the reference protein solution is identical to the protein solution of interest except that the reference protein solution comprises the original protein instead of the second protein. 
     
     
         82 . The method of  claim 77 , wherein the second protein and the original protein are identical except that the amino acid sequence of the second protein comprises histidine (His) at one or more positions that are not His in the amino acid sequence of the original protein. 
     
     
         83 . The method of  claim 77 , wherein the original protein is an antibody. 
     
     
         84 . The method of  claim 83 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein is not a His residue. 
     
     
         85 . The method of  claim 83 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein and the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the original protein are the same. 
     
     
         86 . The method of  claim 83 , wherein the antibody is an ion concentration-dependent antigen binding antibody. 
     
     
         87 . A method of producing a protein that possesses desired viscosity characteristics, the method comprising:
 (a) selecting an original protein;   (b) producing a second protein that is a mutated version of the original protein;   (c) preparing a solution comprising the second protein;   (d) carrying out the method of  claim 43 , using the solution of (c) as the protein solution of interest, thereby predicting the viscosity of the protein solution of interest at the given temperature;   (e) determining that the predicted viscosity of the protein solution of interest at the given temperature is a desired viscosity, thereby determining that the second protein as a protein that possesses the desired viscosity characteristics.   
     
     
         88 . The method of  claim 87 , wherein the desired viscosity is 2000 mPa·s or less. 
     
     
         89 . The method of  claim 87 , wherein the desired viscosity is in the range of 0.1 mPa·s to 1000 mPa·s. 
     
     
         90 . The method of  claim 87 , wherein the desired viscosity is in the range of 0.1 mPa·s to 700 mPa·s. 
     
     
         91 . The method of  claim 87 , wherein the viscosity of the protein solution of interest is lower than the viscosity of a reference protein solution, wherein the reference protein solution is identical to the protein solution of interest except that the reference protein solution comprises the original protein instead of the second protein. 
     
     
         92 . The method of  claim 87 , wherein the second protein and the original protein are identical except that the amino acid sequence of the second protein comprises histidine (His) at one or more positions that are not His in the amino acid sequence of the original protein. 
     
     
         93 . The method of  claim 87 , wherein the original protein is an antibody. 
     
     
         94 . The method of  claim 93 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein is not a His residue. 
     
     
         95 . The method of  claim 93 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein and the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the original protein are the same. 
     
     
         96 . The method of  claim 93 , wherein the antibody is an ion concentration-dependent antigen binding antibody. 
     
     
         97 . A method of producing a protein that possesses desired viscosity characteristics, the method comprising:
 (a) selecting an original protein;   (b) producing a second protein that is a mutated version of the original protein;   (c) preparing a solution comprising the second protein;   (d) carrying out the method of  claim 53 , using the solution of (c) as the protein solution of interest, thereby predicting the viscosity of the protein solution of interest at the first temperature;   (e) determining that the predicted viscosity of the protein solution of interest at the first temperature is a desired viscosity, thereby determining that the second protein as a protein that possesses the desired viscosity characteristics.   
     
     
         98 . The method of  claim 97 , wherein the desired viscosity is 2000 mPa·s or less. 
     
     
         99 . The method of  claim 97 , wherein the desired viscosity is in the range of 0.1 mPa·s to 1000 mPa·s. 
     
     
         100 . The method of  claim 97 , wherein the desired viscosity is in the range of 0.1 mPa·s to 700 mPa·s. 
     
     
         101 . The method of  claim 97 , wherein the viscosity of the protein solution of interest is lower than the viscosity of a reference protein solution, wherein the reference protein solution is identical to the protein solution of interest except that the reference protein solution comprises the original protein instead of the second protein. 
     
     
         102 . The method of  claim 97 , wherein the second protein and the original protein are identical except that the amino acid sequence of the second protein comprises histidine (His) at one or more positions that are not His in the amino acid sequence of the original protein. 
     
     
         103 . The method of  claim 97 , wherein the original protein is an antibody. 
     
     
         104 . The method of  claim 103 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein is not a His residue. 
     
     
         105 . The method of  claim 103 , wherein the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the second protein and the amino acid residue at position 97 (Kabat numbering) of the heavy chain of the original protein are the same. 
     
     
         106 . The method of  claim 103 , wherein the antibody is an ion concentration-dependent antigen binding antibody. 
     
     
         107 . A modified antibody produced by substituting a portion of the amino acid residues of an original antibody with histidine (His) residues, whose amino acid residue at position 97 of the heavy chain according to the Kabat numbering system is not a His residue. 
     
     
         108 . The modified antibody of  claim 107 , wherein the amino acid residue at position 97 of the heavy chain is the amino acid residue of the original antibody. 
     
     
         109 . A modified antibody showing ion concentration-dependent antigen binding, which is produced by substituting a portion of the amino acid residues of an original antibody with other amino acids, wherein the amino acid residue at position 97 of the heavy chain according to the Kabat numbering system is not a His residue. 
     
     
         110 . The modified antibody of  claim 109 , wherein the amino acid residue at position 97 of the heavy chain is the amino acid residue of the original antibody.