Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry

Okumura, Takeshi; Masuda, Kenji; Watanabe, Kazuhiko; Miyadai, Kenji; Nonaka, Kazuhiro; Yabuta, Masayuki; Ômasa, Takeshi

doi:10.1016/j.jbiosc.2015.01.007

Cited by 18 publications

(15 citation statements)

References 30 publications

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“…FITC-Anti-CD14 antibody (M5E2 clone; BD, Bioscience, USA) was used to recognize the lineage of mononuclear cells. PBMCs collected from 28 brucellosis patients and 55 non-Brucella-infected blood donors were stained for intracellular Brucellae by FACSCalibur flow cytometer with FITC-Anti-CD14 antibody (M5E2) and PE-6C12 specific to FCM Omp25 (Delaporte et al, 2008;Okumura et al, 2015). Briefly, PBMCs were treated with 5 µl FITC-M5E2 for 30 min at room temperature and cells were washed twice with sample buffer [PBS containing 1% bovine serum albumin (BSA;…”

Section: Flow Cytometry Assay (Fcm)mentioning

confidence: 99%

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Yang

et al. 2020

Front. Cell. Infect. Microbiol.

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Brucellosis is a serious zoonosis occurring mainly in developing countries, and its diagnosis is largely dependent on serologic detection and bacterial culture. In this study, we developed the murine monoclonal antibodies (mAbs) against a conserved and major outer membrane protein 25 (Omp25) of Brucella species (B. spp.) for use in clinical diagnosis. The mAbs to Omp25 were produced by hybridoma technique, which were utilized for developing various immunoassays for detection of Brucellae, including Western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunochemical staining (ICS), immunofluorescence staining (IFS), and flow cytometry assay (FCM). A number of five mAbs (2B10, 4A12, 4F10, 6C12, and 8F3) specific to Omp25 were selected, including 2 IgG1, 2 IgG2a, and 1 IgG2b. Among them, mAbs 6C12, 8F3, and 4A12 reacted highly with B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2). No cross-reactivity with Yersinia enterocolitica O:9, Salmonella spp., and Escherichia coli was found. By mapping Omp25 epitopes, mAb 6C12 was found as reacting with a semi-conformational epitope, and mAbs 4A12 and 8F3 as recognizing a different linear epitope, respectively. The paired mAbs were tested for detecting Brucella species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Brucella Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of Brucellae in clinical samples with various immunoassays. mAb 6C12-based FCM could be potentially used for the monitoring of therapeutic efficacy for brucellosis in clinical practice.

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Section: Flow Cytometry Assay (Fcm)mentioning

confidence: 99%

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Yang

et al. 2020

Front. Cell. Infect. Microbiol.

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“…FACS is commonly used for cloning antibody-producing cells (DeMaria et al, 2007), but several studies have reported that multiple rounds of screening and cloning are necessary to ensure the isolation of high producers (Okumura et al, 2015). FACS is commonly used for cloning antibody-producing cells (DeMaria et al, 2007), but several studies have reported that multiple rounds of screening and cloning are necessary to ensure the isolation of high producers (Okumura et al, 2015).…”

Section: Clonal Enrichment Of High-producer Cells Based On Surface mentioning

confidence: 99%

“…Generation of clonal manufacturing cell lines is a crucial step towards ensuring reproducible product quality for biopharmaceuticals. FACS is commonly used for cloning antibody-producing cells (DeMaria et al, 2007), but several studies have reported that multiple rounds of screening and cloning are necessary to ensure the isolation of high producers (Okumura et al, 2015). We sought to determine whether one round of clonal cell sorting based on amber suppression-dependent surface display is adequate to enrich for the highest antibody-producing cells.…”

Section: Clonal Enrichment Of High-producer Cells Based On Surface mentioning

confidence: 99%

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity

Chakrabarti¹,

Li²,

Roy³

et al. 2019

Biotech & Bioengineering

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Cell line development (CLD) for biotherapeutics is a time‐ and resource‐intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high‐yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence‐activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane‐associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is “switchable” in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high‐producer cells. The strategy provides a means for selecting high‐producing cells with potential applications to multiple biotherapeutic protein formats.

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“…Over the years, there have been various highthroughput methods developed to reduce man power and process timelines, as well as increase the overall efficiency of screening. Common methods include: flow cytometry based cloning methods [2,[5][6][7][8][9], automated future science group Review Keil colony picking systems [7,[10][11][12] that increase the recovery of high-producing transfected cells and the use of fully automated robotic selection systems that increase throughput and efficiency across multiple steps in the clone selection process [4,13].…”

Section: Automated High-throughput Clone Screening and Imagingmentioning

confidence: 99%

“…To further increase the specificity for selecting high producing cells during FACS cloning, detection antibodies against the protein of interest (in most cases this would be an IgG) are used to label the high-producing cells. The detection antibody is conjugated to a fluorochrome that can be detected when performing flow cytometry [2,[5][6][7][8][9], and after incubation with the antibody, single cells with the highest fluorescent intensity can be preferentially sorted into microtiter plates. This method increases the likelihood of isolating the highest producers from the transfection pool.…”

Section: Automated High-throughput Clone Screening and Imagingmentioning

confidence: 99%

High-throughput screening and automation approaches for the development of recombinant therapeutic proteins

Keil

2015

Pharmaceutical Bioprocessing

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Generation of recombinant therapeutic proteins involves the use of Chinese hamster ovary cells as one of the workhorses for complex protein production. This process requires screening large numbers of transfected cells to identify single clones that have high protein production and drug-specific quality attributes. Traditionally, this process was limited by manual operation; however, high-throughput screening methods and automation have made this process more efficient. Implementation of high-throughput screening and automation within bioprocess development have led to increased screening capacity, higher product yields, reductions in manual operation, reduction in human error and shorter development time lines. This review outlines the high-throughput methodologies and technologies currently used for clone screening, selection and evaluation in bioprocess development.

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Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry

Cited by 18 publications

References 30 publications

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity

High-throughput screening and automation approaches for the development of recombinant therapeutic proteins

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