title: Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris.
abstract: A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.
authors.affiliation: GlycoFi Inc., A Wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
authors.forename: Youwei
authors.initials: Y
authors.lastname: Jiang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zha
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Potgieter
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Renée
authors.initials: R
authors.lastname: Moore
authors.forename: Michael
authors.initials: M
authors.lastname: Cukan
authors.forename: Nga Rewa
authors.initials: NR
authors.lastname: Houston-Cummings
authors.forename: Adam
authors.initials: A
authors.lastname: Nylen
authors.forename: James E
authors.initials: JE
authors.lastname: Drummond
authors.forename: Troy W
authors.initials: TW
authors.lastname: McKelvey
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Natarajan
authors.initials: N
authors.lastname: Sethuraman
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins