Anti-VEGF protein compositions and methods for producing the same
Inventors
Wang, Shunhai • Li, Ning • Chen, Hunter • Bhupender Bhalla, Amardeep Singh • LAWRENCE, Shawn M. • JOHNSON, Amy S. • Casey, Meghan • Grapel, Jaimie
Assignees
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Publication Number
US-12338274-B2
Publication Date
2025-06-24
Expiration Date
Abstract
The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.
Core Innovation
The invention relates to aflibercept that includes oxidized variants having at least one oxidized amino acid residue selected from tryptophan, histidine, or a combination thereof, where the aflibercept is made in cell culture media comprising chemically defined media (CDM). The document further addresses formulations of such aflibercept with one or more pharmaceutically acceptable excipients selected from buffering agents, preservatives, detergents, and combinations thereof, and a pH between 5.0 and 8.0.
A key aspect described in the document is the control of oxidized, acidic, and PTM variant profiles associated with aflibercept and VEGF MiniTrap protein variants using ion-exchange chromatography strategies. The document describes anion exchange chromatography (AEX) and cation exchange chromatography (CEX) approaches, including bind-elute mode and flowthrough mode concepts to selectively enrich or deplete oxidized, acidic species and specific PTM variants.
The described chromatography approaches extend to mixed-mode chromatography (MM) and include downstream processing and analytical monitoring that link changes in oxidized residues and yellow-brown coloration to photostability concerns. The document also contextualizes subsequent pharmaceutical formulations and administration, including intravitreal use of aflibercept, and describes yellow-brown coloration measured relative to European Pharmacopeia BY standards and by CIELAB b* values.
Claims Coverage
The independent claims identified are clm-00001, clm-00020, and clm-00026, which collectively cover a formulation of aflibercept with oxidized variants produced using CDM, intravitreal administration of such aflibercept, and treating specified retinal diseases using such aflibercept.
Aflibercept formulation with oxidized tryptophan and/or histidine variants produced using CDM
A formulation comprising 40 to 200 mg/ml of aflibercept produced in cell culture media comprising chemically defined media (CDM), where the aflibercept includes oxidized variants with at least one oxidized amino acid residue selected from tryptophan, histidine, or a combination thereof, together with one or more pharmaceutically acceptable excipients selected from buffering agents, preservatives, detergents, and combinations thereof, wherein the formulation has a pH between 5.0 and 8.0.
Intravitreal administration of CDM-produced aflibercept with oxidized tryptophan and/or histidine variants
A method of administering a pharmaceutically effective amount of aflibercept by intravitreal injection wherein the aflibercept is made in cell culture media comprising chemically defined media (CDM) added to the cell culture media, and wherein the aflibercept includes oxidized variants with at least one oxidized amino acid residue selected from tryptophan, histidine, or a combination thereof and one or more pharmaceutically acceptable excipients.
Treating retinal diseases with CDM-produced aflibercept containing oxidized tryptophan and/or histidine variants
A method of treating Neovascular (Wet) Age-Related Macular Degeneration (AMD), Macular Edema Following Retinal Vein Occlusion (RVO), Diabetic Macular Edema (DME), or Diabetic Retinopathy with aflibercept wherein the aflibercept is made in cell culture media comprising chemically defined media (CDM) added to the cell culture media, and wherein the aflibercept includes oxidized variants that have at least one oxidized amino acid residue selected from tryptophan, histidine, or a combination thereof.
Across independent claims, the document’s claimed subject matter centers on aflibercept produced in cell culture media with CDM, where the aflibercept includes oxidized variants defined by oxidized residues of tryptophan and/or histidine, and on using that aflibercept in a specified pH formulation with excipients, intravitreal injection administration, and treatment of specified retinal diseases.
Stated Advantages
Enables detailed profiling of impurities and PTMs in VEGF MiniTrap constructs, including deamidation, succinimide/aspartate, methionine oxidation, and glycine loss.
Provides characterization of disulfide/trisulfide profiles, including free thiols and hinge disulfide isomerization.
Supports site-specific characterization of N-glycosylation occupancy and glycan distributions and includes O-glycosylation information for MT6.
Enables correlation of upstream media/feed process optimization effects with product coloration (b* values) and levels of oxidized residues (2-oxo-histidines and oxo-tryptophans).
Documented Applications
Formulations comprising CDM-produced aflibercept with oxidized variants for pH- and excipient-defined pharmaceutical preparation.
Intravitreal administration of CDM-produced aflibercept containing oxidized variants using pharmaceutically acceptable excipients.
Treatment of Neovascular (Wet) Age-Related Macular Degeneration (AMD), Macular Edema Following Retinal Vein Occlusion (RVO), Diabetic Macular Edema (DME), or Diabetic Retinopathy with CDM-produced aflibercept containing oxidized variants.
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