title: High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.
abstract: The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.
authors: Adimab Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
authors: Gavin C
authors: GC
authors: Barnard
authors: Angela R
authors: AR
authors: Kull
authors: Nathan S
authors: NS
authors: Sharkey
authors: Seemab S
authors: SS
authors: Shaikh
authors: Alissa M
authors: AM
authors: Rittenhour
authors: Irina
authors: I
authors: Burnina
authors: Youwei
authors: Y
authors: Jiang
authors: Fang
authors: F
authors: Li
authors: Heather
authors: H
authors: Lynaugh
authors: Teresa
authors: T
authors: Mitchell
authors: Juergen H
authors: JH
authors: Nett
authors: Adam
authors: A
authors: Nylen
authors: Thomas I
authors: TI
authors: Potgieter
authors: Bianka
authors: B
authors: Prinz
authors: Sandra E
authors: SE
authors: Rios
authors: Dongxing
authors: D
authors: Zha
authors: Natarajan
authors: N
authors: Sethuraman
authors: Terrance A
authors: TA
authors: Stadheim
authors: Piotr
authors: P
authors: Bobrowicz
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D019149:Bioreactors; D018929:Cell Culture Techniques; D002460:Cell Line; D004271:DNA, Fungal / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005818:Genetic Engineering; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008828:Microbiological Techniques; D010843:Pichia / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D004271:DNA, Fungal; D006023:Glycoproteins; D011994:Recombinant Proteins