High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.
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.
Barnard
Gavin C
GC
Adimab Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
Kull
Angela R
AR
Sharkey
Nathan S
NS
Shaikh
Seemab S
SS
Rittenhour
Alissa M
AM
Burnina
Irina
I
Jiang
Youwei
Y
Li
Fang
F
Lynaugh
Heather
H
Mitchell
Teresa
T
Nett
Juergen H
JH
Nylen
Adam
A
Potgieter
Thomas I
TI
Prinz
Bianka
B
Rios
Sandra E
SE
Zha
Dongxing
D
Sethuraman
Natarajan
N
Stadheim
Terrance A
TA
Bobrowicz
Piotr
P
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
D000911:Antibodies, Monoclonal; D004271:DNA, Fungal; D006023:Glycoproteins; D011994:Recombinant Proteins