TY  - JOUR
A1  - Gätjen, Dominic
A1  - Wieczorek, Marek
A1  - Listek, Martin
A1  - Tomszak, Florian
A1  - Nölle, Volker
A1  - Hanack, Katja
A1  - Droste, Miriam Susanna
T1  - A switchable secrete-and-capture system enables efficient selection of
Pichia pastoris clones producing high yields of Fab fragments
JF  - Journal of immunological methods
N2  - Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened. 
In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and -capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS).Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones. 
Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha -agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and -capture system offers a switchable scFv display. 
Efficient plasmid clearance upon removal of selective pres-sure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (fac-tor in the range of-21-49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone. 
Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of-2-3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.
KW  - Fab fragment production
KW  - Pichia pastoris
KW  - FACS
KW  - yeast surface display high throughput screening
Y1  - 2022
U6  - https://doi.org/10.1016/j.jim.2022.113383
SN  - 0022-1759
SN  - 1872-7905
VL  - 511
PB  - Elsevier Science
CY  - Amsterdam [u.a.]
ER  -