Anti-vegf protein compositions and methods for producing the same
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsAndrew Tustian, Ankit Vartak, Thomas Daly, Erica Pyles, Nisha Palackal, Shunhai Wang, Ning Li
Life of this patent
Estimating the term…
Abstract
The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.
Claims30
A method for preparing a prefilled syringe comprising the step of loading a syringe with aflibercept at a concentration of about 8 mg/0.07 mL, wherein a process of producing aflibercept at a concentration of about 8 mg/0.07 mL comprises the steps of:
- (a) binding aflibercept to a first capture chromatography, wherein aflibercept is produced in a cell culture media including a chemically defined media (CDM), wherein said aflibercept includes oxidized species of aflibercept that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof, (b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate has a first color;
- (c) subjecting said affinity eluate to an ion exchange chromatography column;
- and (d) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color, and wherein said first color is a more intense yellow-brown color than said second color when said affinity eluate and said flowthrough fraction protein concentrations are normalized to 5.0 g/L.
A method of preparing a prefilled syringe comprising aflibercept at a concentration of about 8 mg/0.07 mL, wherein said aflibercept is produced in a cell culture media including a chemically defined media (CDM) by:
- (a) binding aflibercept produced in a cell culture media including a chemically defined media (CDM) to a first capture chromatography, wherein said aflibercept includes oxidized species of aflibercept that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof, (b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate has a first color;
- (c) subjecting said affinity eluate to an ion exchange chromatography column;
- and (d) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color, and wherein said first color is a more intense yellow-brown color than said second color when said affinity eluate and said flowthrough fraction protein concentrations are normalized to 5.0 g/L.
A method of preparing a prefilled syringe comprising aflibercept at a concentration of about 8 mg/0.07 mL, (a) producing said aflibercept in a cell culture media including a chemically defined media (CDM);
- and (b) purifying said aflibercept by a process that includes the following chromatography steps to obtain aflibercept having a BY value of at least 5 when the concentration of aflibercept is normalized to 5 g/L:
- (i) affinity chromatography;
- and (ii) ion exchange chromatography, wherein said aflibercept includes one of an oxidized histidine selected from the group consisting of His86, His110, His145, His209, His95, His19, His203, and a combination thereof, and an oxidized tryptophan selected from the group consisting of Trp58, Trp138, and combinations thereof.
A method for preparing a prefilled syringe comprising the step of loading a syringe with aflibercept at a concentration of about 110 mg/mL to about 120 mg/mL, wherein a process of producing aflibercept at a concentration of about 110 mg/mL to about 120 mg/mL comprises the steps of:
- (a) binding aflibercept produced in a cell culture media including a chemically defined media (CDM) to a first capture chromatography, wherein said aflibercept includes oxidized species of aflibercept that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof,
- (b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate has a first color;
- (c) subjecting said affinity eluate to an ion exchange chromatography column;
- and
- (d) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color, and wherein said first color is a more intense yellow-brown color than said second color when said affinity eluate and said flowthrough fraction protein concentrations are normalized to 5.0 g/L.
Dependencies are inferred from the claim text.