title: Production of monoclonal antibodies by glycoengineered Pichia pastoris.
abstract: The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.
authors.affiliation: GlycoFi Inc. (A wholly-owned subsidiary of Merck & Co Inc.), 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA. Thomas Potgieter@Merck.com
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Potgieter
authors.forename: Michael
authors.initials: M
authors.lastname: Cukan
authors.forename: James E
authors.initials: JE
authors.lastname: Drummond
authors.forename: Nga Rewa
authors.initials: NR
authors.lastname: Houston-Cummings
authors.forename: Youwei
authors.initials: Y
authors.lastname: Jiang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Heather
authors.initials: H
authors.lastname: Lynaugh
authors.forename: Muralidhar
authors.initials: M
authors.lastname: Mallem
authors.forename: Troy W
authors.initials: TW
authors.lastname: McKelvey
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Adam
authors.initials: A
authors.lastname: Nylen
authors.forename: Alissa
authors.initials: A
authors.lastname: Rittenhour
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zha
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D019149:Bioreactors; D002478:Cells, Cultured; D005818:Genetic Engineering; D042822:Genomic Instability; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000432:Methanol / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010100:Oxygen; D000432:Methanol