Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Kirchhoff, Janina; Raven, Nicole; Boes, Alexander; Roberts, Jean L.; Russell, Sean; Treffenfeldt, Wiltrud; Fischer, Rainer; Schinkel, Helga; Schiermeyer, Andreas; Schillberg, Stefan

doi:10.1111/j.1467-7652.2012.00722.x

Cited by 69 publications

(42 citation statements)

References 55 publications

(101 reference statements)

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“…We have previously reported 10‐fold increase in productivity with a stable model protein GFP–HFBI in BY‐2 suspension cells (Reuter et al ., ). Several means for improving the productivity of BY‐2 suspension cells have been published recently, including improved culture media (Holland et al ., ), FACS‐based clone screening (Kirchhoff et al ., ), protease knockout lines (Mandal et al ., ) and development of culture systems (Raven et al ., ). However, improving the yield in the BY‐2 suspension cells was not the aim of this study.…”

Section: Discussionmentioning

confidence: 99%

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

Kurppa

Reuter

Ritala

et al. 2017

Plant Biotechnology Journal

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SummaryPurification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low‐cost and scalable antibody capturing in solutions. Immunoglobulin‐binding Protein A is widely used in affinity‐based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two‐phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35‐fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB‐induced protein body formation. We also demonstrated production of HFBI–Protein A fusion protein in tobacco BY‐2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody‐binding capacity of the Protein A block was similar to the nonfused Protein A. The best‐performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two‐phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.

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Section: Discussionmentioning

confidence: 99%

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

Kurppa

Reuter

Ritala

et al. 2017

Plant Biotechnology Journal

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“…Besides the transfection or transformation of whole plants or at least organs, monoclonal BY-2 tobacco cell lines that grow in suspension have been developed (219). Flow cytometric analysis has been used to enrich cells expressing a fluorescent marker which was located on the same T-DNA with the antibody gene.…”

Section: Transgenic Organismsmentioning

confidence: 99%

Expression of Recombinant Antibodies

Frenzel¹,

Hust²,

Schirrmann³

2013

Front. Immunol.

274

220

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Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

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“…1,2 Applications range from understanding complex heterogeneous biological samples, such as tumor biopsies, 3 to rare cell isolation, 4 or clone selection for biotechnological processes. 5 Conventionally, cells are analyzed by bulk measurements on cell populations consisting of several thousands to ten thousand individual cells. Inherently, bulk measurements yield only averaged results.…”

mentioning

confidence: 99%

Single-cell printing based on impedance detection

Schoendube

Wright²,

Zengerle

et al. 2015

Biomicrofluidics

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Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl-800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 lm Â 55 lm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%-17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% 6 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.

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Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Cited by 69 publications

References 55 publications

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

Expression of Recombinant Antibodies

Single-cell printing based on impedance detection

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