Methods of controlling the formation of disulfide bonds in protein solutions
AssigneeGenzyme Corporation
InventorsKevin P. Brower, Chris Hwang, Rao Koduri, Konstantin B. Konstantinov, Veena Warikoo, Marcella Yu, Jin Yin
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Abstract
Disclosed herein are methods that have been developed to control the formation of disulfide bonds between polypeptides of a multimeric protein produced by a bioprocess. Also disclosed are protein solution parameters that allow for controlling the formation of disulfide bonds. In one example, the methods disclosed herein can be used to control the proportion of half antibody molecules in an antibody solution.
Claims31
A method of controlling the number of disulfide bonds between polypeptides of a multimeric protein produced by a bioprocess, the method comprising:
- (a) contacting the polypeptides with a conditioned solution at a specific time point during the bioprocess, wherein the conditioned solution comprises a redox reagent, and
- (b) incubating the conditioned solution comprising the polypeptides for a predetermined time at a predetermined temperature;
- wherein the incubation of the polypeptides with the conditioned solution controls the formation of disulfide bonds between the polypeptides of the protein;
- wherein the number of disulfide bonds between the polypeptides of the protein is increased;
- wherein the redox reagent is 2-mercaptoethylamine (2-MEA);
- wherein the conditioned solution has a pH between 3.4 to 6;
- and
- wherein the predetermined temperature of the incubation is between 2° C. and 8° C.
A method of controlling the proportion of half antibody molecules in a solution comprising a population of antibody molecules, the method comprising:
- (a) contacting the solution comprising the population of antibody molecules with a conditioned solution, wherein the conditioned solution comprises a redox reagent;
- and
- (b) incubating the conditioned solution comprising the antibody molecules for a predetermined time at a predetermined temperature;
- wherein the incubation of the antibody molecules with the conditioned solution regulates the proportion of half antibody molecules in the conditioned antibody solution;
- wherein the regulation of the proportion of half antibody molecules consists of decreasing the proportion of half antibody molecules;
- wherein the redox reagent is 2-mercaptoethylamine (2-MEA);
- wherein the conditioned solution has a pH between 3.4 to 6;
- and
- wherein the predetermined temperature of the incubation is between 2° C. and 8° C.
Dependencies are inferred from the claim text.