Anti-vegf protein compositions and methods for producing the same

A method of producing aflibercept, comprising:

• a. binding aflibercept from a clarified harvest cultured in a chemically defined medium (CDM) to a Protein A resin, 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, phenylalanine, tyrosine and a combination thereof;
• b. eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate comprises oxidized species of aflibercept;
• c. subjecting said eluted aflibercept to anion exchange (AEX) chromatography column;
• and
• d. collecting a flowthrough fraction, wherein the percent of oxidized species of aflibercept in said affinity eluate is greater than the percent of oxidized species of aflibercept in said flowthrough fraction when the concentrations of protein in said eluate and flowthrough fraction are normalized, and
• wherein said oxidized species of aflibercept is measured by subjecting said affinity eluate and said flowthrough fractions to digestion, followed by their analysis using reverse-phase ultra-performance chromatography (UPLC), detection at wavelengths of 280 nm, 320 nm and 350 nm and mass spectrometry analysis using a first mobile phase of 0.1% v/v formic acid in water and a second mobile phase of 0.1% v/v formic acid in acetonitrile