Selection of Pichia pastoris strains expressing recombinant immunoglobulin G by cell surface labeling

Song, Ligang; Shen, Zheng; Zha, Dongxing; Sharkey, Nathan; Prinz, Bianka; Hamilton, Stephen R.; Pavoor, Tej V.; Bobrowicz, Beata; Shaikh, Seemab S.; Rittenhour, Alissa; Potgieter, Thomas I.; Bobrowicz, Piotr; Stadheim, Terrance A.

doi:10.1016/j.jim.2010.03.007

Cited by 6 publications

(4 citation statements)

References 18 publications

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“…The cold capture technique offers a direct selection procedure via secreted recombinant protein that is associated with the cell glycocalyx at low temperature. Cold capture has been successfully demonstrated in CHO, NSO, and Pichia cells for isolating high expressing clones 27, 28, 29…”

Section: Resultsmentioning

confidence: 99%

“…A cold‐capture technique using fluorescence activated cell sorting (FACS) has been demonstrated for the isolation of high‐expressing clones for a secreted, recombinant protein 27, 28, 29. This protocol takes advantage of a decreased rate of protein secretion from the cell at lower temperatures, rendering secreted proteins to be temporarily ‘captured’ on the cell surface glycocalyx.…”

Section: Introductionmentioning

confidence: 99%

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Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high‐throughput FACS screening

Song

Raphaelli

Jones

et al. 2011

J of Chemical Tech & Biotech

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BACKGROUND: Human embryonic kidney-293 (HEK-293) cells are commonly used as a transient expression host but their application in stable therapeutic protein production is limited. This is presumably due to the absence of a suitable amplifiable expression system and hence limited protein output compared with other mammalian cells such as Chinese hamster ovary cells. This paper describes a rapid clonal selection method for isolating HEK293 cell lines with high specific productivity, for a non-amplifiable expression system, to achieve high-level, scalable expression of recombinant antibodies.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high‐throughput FACS screening

Song

Raphaelli

Jones

et al. 2011

J of Chemical Tech & Biotech

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“…However, antibodies produced in E. coli are not glycosylated and this severely limits its use as a manufacturing platform. Antibodies with specific human N-glycan structures have been expressed in glycoengineered lines of the yeast Pichia pastoris and its utility as a general platform for producing recombinant antibodies with human N-glycosylation is being developed (Li et al, 2006a;Lin et al, 2010). Antibody has been expressed in engineered chicken eggs and in plants (Zhu et al, 2005;Cox et al, 2006).…”

Section: Manufacturingmentioning

confidence: 99%

Monoclonal antibodies — a proven and rapidly expanding therapeutic modality for human diseases

2010

Protein Cell

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The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and intermittent until recently. The first antibody therapy, murine-derived murononab OKT3 for acute organ rejection, was approved by the US Food and Drug Administration (FDA) in 1986, more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975. As a result of the scientific, technological, and clinical breakthroughs in the 1980s and 1990s, the pace of therapeutic antibody discovery and development accelerated. Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development. Despite the progress, need for improvement exists at every level. Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.

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“…For antibodies, a linkage between secretion capacity and residual surface‐bound antibodies on yeast or mammalian cells has been used to isolate high‐producing strains, but the system so far lacks applicability for other proteins. [ 25–27 ] Additionally, several systems which enable partial covalent or high‐affinity capturing of secreted proteins on cell surfaces have been proposed but those require substantial modifications to the host cell. [ 28–31 ] Intracellular protein production is, in principle, compatible with single‐cell analysis, but since the majority of fluorescent labels are cell‐membrane‐impermeable, substantial modifications of the protein of interest such as fusions to a reporter protein are required to obtain fluorescent readouts.…”

Section: Introductionmentioning

confidence: 99%

High‐Throughput Optimization of Recombinant Protein Production in Microfluidic Gel Beads

Napiorkowska

Pestalozzi

Panke

et al. 2020

Small

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Efficient production hosts are a key requirement for bringing biopharmaceutical and biotechnological innovations to the market. In this work, a truly universal high‐throughput platform for optimization of microbial protein production is described. Using droplet microfluidics, large genetic libraries of strains are encapsulated into biocompatible gel beads that are engineered to selectively retain any protein of interest. Bead‐retained products are then fluorescently labeled and strains with superior production titers are isolated using flow cytometry. The broad applicability of the platform is demonstrated by successfully culturing several industrially relevant bacterial and yeast strains and detecting peptides or proteins of interest that are secreted or released from the cell via autolysis. Lastly, the platform is applied to optimize cutinase secretion in Komagataella phaffii (Pichia pastoris) and a strain with 5.7‐fold improvement is isolated. The platform permits the analysis of >106 genotypes per day and is readily applicable to any protein that can be equipped with a His6‐tag. It is envisioned that the platform will be useful for large screening campaigns that aim to identify improved hosts for large‐scale production of biotechnologically relevant proteins, thereby accelerating the costly and time‐consuming process of strain engineering.

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Selection of Pichia pastoris strains expressing recombinant immunoglobulin G by cell surface labeling

Cited by 6 publications

References 18 publications

Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high‐throughput FACS screening

Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high‐throughput FACS screening

Monoclonal antibodies — a proven and rapidly expanding therapeutic modality for human diseases

High‐Throughput Optimization of Recombinant Protein Production in Microfluidic Gel Beads

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