Method of purifying glycosylated protein from host cell galectins and other contaminants

1. A method for purifying a glycosylated recombinant protein of interest from a contaminant, comprising:

(a) loading onto a Protein A matrix, at about neutral pH, a host cell culture supernatant or filtrate comprising the glycosylated recombinant protein of interest (POI) and a galectin host cell protein (HCP) contaminant, comprising galectin-3, wherein the POI comprises the C H 2 and C H 3 domains of an immunoglobulin Fc domain and one or more terminal beta-galactosyl residues to which the galectin-3 is reversibly bound;

(b) washing the Protein A matrix that has the POI bound to it, with a buffer at about pH 6.0-6.5, comprising 1-3 M calcium chloride;

(c) eluting the POI from the Protein A matrix with a buffer comprising citric acid or a citrate salt below about pH 4 into an eluant pool, and:

(i) if the eluant pool is more basic than the pH range of pH 3.3-3.7, titrating the eluant pool to pH 3.3-3.7 with citric acid, and

(ii) optionally, keeping the eluant pool at pH 3.3-3.7 for a period sufficient for viral inactivation;

(d) binding the POI in the eluant pool from (c) to a cation exchange matrix (CEX) in a low conductivity buffer of about 2-15 mS, at pH 5.0-5.5;

(e) eluting the POI from the cation exchange matrix with an electrolyte concentration gradient of increasingly higher conductivity, up to about 40-100 mS, into a CEX eluant pool; and

(f) loading the CEX eluant pool onto a hydrophobic interaction chromatography (HIC) matrix in a high conductivity buffer, about 40-100 mS in conductivity, or greater, at pH 5.0-6.0 and washing the HIC matrix, whereby the POI is separated from the galectin-3.

2. The method of claim 1 , wherein in (b) washing the Protein A matrix that has the POI bound to it, is with a buffer comprising 2 to 2.7 M calcium chloride.

3. The method of claim 1 , wherein the POI is selected from aflibercept, alefacept, etanercept, abatacept, belatacept, rFVIIIFc, rFIXFc, and rilonacept.

4. The method of claim 3 , wherein the POI is aflibercept.

5. The method of claim 1 , wherein the host cell culture supernatant or filtrate further comprises a metallic cation contaminant, and wherein the POI is also separated from the metallic cation contaminant.

6. The method of claim 5 , wherein the metallic cation contaminant is selected from aluminum, barium, calcium, cobalt, copper, zinc, iron, magnesium, manganese, mercury, nickel, and strontium cations.

7. The method of claim 6 , wherein the metallic cation contaminant is an iron cation.

8. The method of claim 1 , further comprising: before binding the POI in the eluant pool from (c) to a cation exchange matrix, subjecting the eluant pool to depth filtration comprising filtration through diatomaceous earth.

9. The method of claim 8 , wherein the diatomaceous earth was pre-conditioned with a citrate buffer.