PEDS Advance Access originally published online on November 20, 2008
Protein Engineering Design and Selection 2009 22(3):169-174; doi:10.1093/protein/gzn068
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This article appears in the following Protein Engineering issue: Antibody Special Issue [View the issue table of contents]
A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells
1 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland 2Philogen SpA, Via Montarioso 11, 53035 Monteriggioni SI, Italy 3Philochem AG, c/o ETH Zürich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Strasse 10, HCI E520, CH-8093 Zürich, Switzerland
5 To whom correspondence should be addressed. E-mail: dario.neri{at}pharma.ethz.ch
The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering research.
Keywords: CHO/fluorescence activated cell sorting/recombinant protein production/selection
Received October 14, 2008; revised October 14, 2008; accepted October 18, 2008.
4 These authors contributed equally.