A study on the temperature dependency and time course of the cold capture antibody secretion assay

Pichler, Johannes; Hesse, Friedemann; Wieser, Matthias; Kunert, Renate; Galosy, Sybille; Mott, John E.; Borth, Nicole

doi:10.1016/j.jbiotec.2009.03.001

Cited by 35 publications

(26 citation statements)

References 10 publications

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“…The top FACS‐isolated clones can show a 5‐ to 7‐fold increase in yield. Further work showed that the same process can be performed at room temperature (21 °C) and that use of phycoerythrin (PE) as a fluorophore for detecting the secreted protein was superior to fluorescein isothiocyanate (FITC) in terms of stability of the signal [98].…”

Section: Advances In Selection Methodologymentioning

confidence: 99%

Recent advances in mammalian protein production

2013

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Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones.

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Section: Advances In Selection Methodologymentioning

confidence: 99%

Recent advances in mammalian protein production

2013

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“…After three rounds of reiterative sorting, a 100-fold enrichment of MTX-amplified CHO cells was observed with specific mAb productivity reaching 42 pg/cell/day [195]. The cells can be chilled to below room temperature to slow down protein release from the membrane and endocytosis of the product-label complex to maximize the signal intensity [196]. Another way to use surface labeling for sorting is to co-express a surface protein not found in CHO cells, such as CD20 [197].…”

Section: Clone Selectionmentioning

confidence: 99%

Generation of monoclonal antibody-producing mammalian cell lines

Ho¹,

Yang²

2013

Pharmaceutical Bioprocessing

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“…In order to enrich for stable cells with the exchanged promoter and the EpoFc cassette, selection pressure was applied by adding 10 μg mL −1 blasticidin (InvivoGen) and, after viability drop, the recovered culture was sorted for EpoFc‐positive, CD4‐negative cells. For sorting, 2 × 10 7 cells were stained with an anti‐CD4 Viobright‐FITC antibody (Miltenyi Biotec) and an anti‐EpoFc‐phycoerythrin (PE) antibody (Sigma‐Aldrich) as described by Pichler et al Cell sorting was done on an Astrios Cell Sorter (Beckman Coulter): FITC was detected with the 488 nm laser and a 526/52 bandpass filter and PE was detected with the 561 nm laser and a 579/16 bandpass filter. CD4‐negative and EpoFc‐positive cells were sorted directly into 24‐well plates for bulk sorting (40 000 cells per well) or as single cells into 96‐well plates.…”

Section: Methodsmentioning

confidence: 99%

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

Nguyen

Baumann

Dhiman

et al. 2019

Biotechnology Journal

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For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

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A study on the temperature dependency and time course of the cold capture antibody secretion assay

Cited by 35 publications

References 10 publications

Recent advances in mammalian protein production

Recent advances in mammalian protein production

Generation of monoclonal antibody-producing mammalian cell lines

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

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