Humanized Antibody Production and Characterization

The stable expression of the humanized antibody was obtained as described previously [57 (link)]. Briefly, YB2/0 cells (ATCC CRL1662) were stably transfected with the linearized expression vectors. The hu8ELC18 antibody was produced in YB2/0 over 5–7 days using EMS (Invitrogen, Villebon Sur Yvette, France), 5% ultra low IgG FCS (PAA) and 0.5 g/L G418. The monoclonal hu8ELC18 was purified from culture supernatant by affinity chromatography onto protein A sepharose (GE-Healthcare, Vélizy-Villacoublay, France, 28-9365-47). The level of aggregates and endotoxins were determined by gel filtration on Superdex HR/200 (GE-Healthcare, 17-5175-01) and by LAL (limulous amoebocyte lysate) testing [58 (link)], respectively, and were always below 2%. Antibody quality and purity was also controlled by SDS-PAGE and Coomassie staining. In addition, glycosylation patterns and the core fucose percentage were determined for each purified antibody by high performance capillary electrophoresis laser induced fluorescence (HPCE-Lif) [59 (link),60 (link)] confirming the characteristic with EMABling platform (LFB, Alès, France).

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Derman Y., Selby K., Miethe S., Frenzel A., Liu Y., Rasetti-Escargueil C., Avril A., Pelat T., Urbain R., Fontayne A., Thullier P., Sesardic D., Lindström M., Hust M, & Korkeala H. (2016). Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody. Toxins, 8(9), 257.

Publication 2016

protein A sepharose Superdex HR/200

Affinity chromatography Amoebocyte lysate Antibody Capillary electrophoresis Cells Endotoxins Fluorescence Fucose G418 Gel filtration Glycosylation Protein a sepharose Sds page Vectors

Corresponding Organization : Technische Universität Braunschweig

Other organizations : National Institute for Biological Standards and Control, LFB (France), Eurasanté

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Variable analysis

independent variables

Stable transfection of YB2/0 cells with linearized expression vectors

dependent variables

Production of the hu8ELC18 antibody in YB2/0 cells over 5-7 days
Purification of the monoclonal hu8ELC18 antibody from the culture supernatant by affinity chromatography
Determination of the level of aggregates and endotoxins in the purified antibody
Evaluation of antibody quality and purity by SDS-PAGE and Coomassie staining
Determination of glycosylation patterns and core fucose percentage for the purified antibody by HPCE-LIF

control variables

Use of YB2/0 cells (ATCC CRL1662) as the host cell line
Culture conditions: EMS (Invitrogen, Villebon Sur Yvette, France), 5% ultra low IgG FCS (PAA), and 0.5 g/L G418

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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