Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries

Jostock, Thomas; Vanhove, Marc; Brepoels, Eddy; Gool, Rein van; Daukandt, Marc; Wehnert, A.; Hegelsom, Rob van; Dransfield, Dan; Sexton, Dan; Devlin, Mary; Ley, Art; Mullberg, Jürgen

doi:10.1016/j.jim.2004.03.014

Cited by 80 publications

(44 citation statements)

References 42 publications

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“…Purified Fab product was exchanged into a buffer containing 50 m m citrate/phosphate, 100 m m NaCl, pH 6.0, through an ultrafiltration/diafiltration process using a Millipore Amicon Ultra concentration unit (10,000-kDa molecular mass cutoff). Recombinant Fab fragments were reformatted into full-length human IgG1 antibodies (f-allotype) (39), expressed as stable transfections in CHO cells using a fed-batch fermentation strategy, and purified as previously described (40) into formulation buffer (100 m m citrate/phosphate buffer, pH 6.0, 50 m m NaCl, 2% (w/v) trehalose, and 0.01% Tween 80).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Kenniston¹,

Faucette²,

Martik³

et al. 2014

Journal of Biological Chemistry

103

110

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Background: Unregulated plasma kallikrein proteolytic activity can result from C1-inhibitor deficiency, causing excessive and potentially fatal edema.Results: The antibody DX-2930 potently and specifically inhibits plasma kallikrein and exhibits a long plasma half-life.Conclusion: An antibody protease inhibitor can lead to potent and specific bioactivity.Significance: DX-2930 could be an effective therapeutic for the prophylactic inhibition of plasma kallikrein-mediated diseases.

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Section: Methodsmentioning

confidence: 99%

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Kenniston¹,

Faucette²,

Martik³

et al. 2014

Journal of Biological Chemistry

103

110

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“…Moreover, transient mammalian antibody production can be scaled up by employing batch or fed-batch bioreactor processes to more than 150 liter production volumes [29]. Therefore, transient antibody production is suitable for small scale production for antibody screening [30], but also capable to generate even grams of antibodies [31-33]. …”

Section: Introductionmentioning

confidence: 99%

High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

et al. 2013

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BackgroundThe demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.ResultsIn this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.ConclusionTransient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

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“…Vectors with compatible restriction cassettes allow one step cloning of scFv fragments from phage display libraries into the scFv-Fc format. Small scale production in HEK293T cells was also used for the screening of functional IgG clones after two-step subcloning of Fab gene fragments immediately after phage display selection (146). Transient productions in tissue culture plates using adherent HEK293T cells yielded 1 and 20 mg/L of IgG and scFv-Fc, respectively (137 and our own unpublished results).…”

Section: 4mammalian Cellsmentioning

confidence: 99%

Production systems for recombinant antibodies

Schirrmann¹

2008

Front Biosci

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Recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. The increasing demand for antibodies with regards to amount and quality resulted in the development of a variety of recombinant production systems employing gram-negative and gram-positive bacteria, yeast and filamentous fungi, insect cell lines as well as mammalian cell lines. More recently, antibodies were also successfully produced in transgenic plants and animals. Currently, the production of recombinant antibodies for therapy is performed in mammalian cell lines to reduce the risk of immunogenicity caused by non-human post-translational modifications, in particular glycosylation. However, novel strategies already allow human-like glycosylation patterns in yeast, insect cell lines and transgenic plants. Furthermore, therapeutic strategies not requiring glycosylation of the Fc portion have been conceived, most prominently using bispecific antibodies or scFv fusion proteins, which can be produced in bacteria. Here, we review all current antibody production systems considering their advantages and limitations with respect to intended applications.

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Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries

Cited by 80 publications

References 42 publications

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

Production systems for recombinant antibodies

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