Separation method of antibody
AssigneeTosoh Corporation
InventorsYuta HAYAKAWA, Naomasa TANIGUCHI, Yosuke TERAO
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Abstract
An antibody separation method including a step of applying a solution containing an antibody to a column filled with an antibody adsorption material containing an insoluble porous carrier and an Fc-binding protein immobilized on the carrier and adsorbing the antibody onto the antibody adsorption material; and a step of applying a buffer solution to the column and eluting the antibody adsorbed onto the antibody adsorption material with a pH gradient, wherein the pH gradient is a step gradient in which a pH of the buffer solution is lowered in steps, and wherein the magnitude of pH decrease is 0.1 or more and less than 1.
Claims16
A method for separating an antibody, comprising:
- applying a solution containing an antibody to a column filled with an antibody adsorption material containing an insoluble porous carrier and an Fc-binding protein immobilized on the carrier and adsorbing the antibody onto the antibody adsorption material;
- and
- applying a buffer solution to the column and eluting the antibody adsorbed onto the antibody adsorption material with a pH gradient,
- wherein the pH gradient is a step gradient in which a pH of the buffer solution is lowered in steps, and
- wherein the magnitude of pH decrease is 0.1 or more and less than 1.
An antibody composition containing 15 mass % or more of an antibody which is eluted in the following citrate buffer solution with a pH of 4.0 or less when a monoclonal antibody solution is fractionated under the following measurement conditions, based on a total mass of the antibody composition:
- (Measurement Conditions)
- Column:
- Tricorn 5/50 (q5.0 mm×50 mm, commercially available from Cytiva)
- Adsorption material:
- FcR36i_Cys immobilized gel obtained by reacting 30 mg of Fc-binding protein FcR36i_Cys (SEQ ID NO:
- 2) with 1 g of a porous hydrophilic vinyl polymer for an adsorption material (TOYOPEARL (filler for liquid chromatography, commercially available from Tosoh Corporation), volume-based Mean particle Diameter:
- 45 μm)
- Flow rate:
- 0.20 mL/min
- Solvent:
- phosphate buffered saline (pH 7.4)
- Step gradient:
- add 9.82 mL each of a 50 mM citrate buffer solution (pH 6.0), a 50 mM citrate buffer solution (pH 5.5), a 50 mM citrate buffer solution (pH 5.0), and a 50 mM citrate buffer solution (pH 4.8) in that order, add 29.46 mL each of a 50 mM citrate buffer solution (pH 4.6), a 50 mM citrate buffer solution (pH 4.4), a 50 mM citrate buffer solution (pH 4.2), a 50 mM citrate buffer solution (pH 4.0), and a 50 mM citrate buffer solution (pH 3.8) in that order, and add 9.82 mL each of a 50 mM citrate buffer solution (pH 3.6) and a
- 50 mM citrate buffer solution (pH 3.4) in that order 50 mM citrate buffer solution:
- a mixed solution containing a 50 mM citrate aqueous solution and a 50 mM trisodium citrate aqueous solution
- Column temperature:
- 25° C.
- Detection:
- UV (280 nm)
- Injection amount:
- 1000-8000 μL (antibody amount:
- 5-40 mg).
An antibody composition containing 15 mass % or more of an antibody which is eluted in the following citrate buffer solution with a pH of 4.2 or more when a monoclonal antibody solution is fractionated under the following measurement conditions, based on a total mass of the antibody composition:
- (Measurement Conditions)
- Column:
- Tricorn 5/50 (q5.0 mm×50 mm, commercially available from Cytiva)
- Adsorption material:
- FcR36i_Cys immobilized gel obtained by reacting 30 mg of Fc-binding protein FcR36i_Cys (SEQ ID NO:
- 2) with 1 g of a porous hydrophilic vinyl polymer for an adsorption material (TOYOPEARL (filler for liquid chromatography, commercially available from Tosoh Corporation), volume-based Mean particle Diameter:
- 45 μm)
- Flow rate:
- 0.20 mL/min
- Solvent:
- phosphate buffered saline (pH 7.4)
- Step gradient:
- add 9.82 mL each of a 50 mM citrate buffer solution (pH 6.0), a 50 mM citrate buffer solution (pH 5.5), a 50 mM citrate buffer solution (pH 5.0), and a 50 mM citrate buffer solution (pH 4.8) in that order, add 29.46 mL each of a 50 mM citrate buffer solution (pH 4.6), a 50 mM citrate buffer solution (pH 4.4), a 50 mM citrate buffer solution (pH 4.2), a 50 mM citrate buffer solution (pH 4.0), and a 50 mM citrate buffer solution (pH 3.8) in that order, and add 9.82 mL each of a 50 mM citrate buffer solution (pH 3.6) and a
- 50 mM citrate buffer solution (pH 3.4) in that order 50 mM citrate buffer solution:
- a mixed solution containing a 50 mM citrate aqueous solution and a 50 mM trisodium citrate aqueous solution
- Column temperature:
- 25° C.
- Detection:
- UV (280 nm)
- Injection amount:
- 1000-8000 μL (antibody amount:
- 5-40 mg).
Dependencies are inferred from the claim text.