Humanized Antibody Expression and Characterization

The stable expression of the humanized antibody was obtained as previously described [47 (link)]. Briefly, YB2/0 were stably transfected with the linearized expression vectors. The humanized IgGs were produced in YB2/0 over 5 to 7 days using EMS (Invitrogen, Carlsbad, USA), 5% Ultra low IgG FCS (PAA) and 0.5 g/L G418. The antibodies were purified from culture supernatant by affinity chromatography onto protein A sepharose (GE-Healthcare, Chalfont St Giles, UK). The level of aggregates and endotoxins were determined by gel filtration on Superdex HR/200 (GE-Healthcare, Chalfont St Giles, UK) and by LAL (limulous amoebocyte lysate) testing [48 (link)], respectively. Antibody quality and purity was also monitored 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) [49 (link),50 (link)] confirming the EMABling characteristic.

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Miethe S., Mazuet C., Liu Y., Tierney R., Rasetti-Escargueil C., Avril A., Frenzel A., Thullier P., Pelat T., Urbain R., Fontayne A., Sesardic D., Hust M, & Popoff M.R. (2016). Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B. PLoS ONE, 11(8), e0161446.

Publication 2016

Ultra low IgG FCS protein A sepharose Superdex HR/200

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

Corresponding Organization : LFB (France)

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Variable analysis

independent variables

Stable transfection of YB2/0 cells with linearized expression vectors

dependent variables

Antibody production levels in YB2/0 cells over 5-7 days
Antibody aggregation levels
Antibody endotoxin levels
Antibody quality and purity
Antibody glycosylation patterns and core fucose percentage

control variables

Use of EMS (Invitrogen, Carlsbad, USA)
Use of 5% Ultra low IgG FCS (PAA)
Use of 0.5 g/L G418

controls

Positive control: Affinity chromatography onto protein A sepharose (GE-Healthcare, Chalfont St Giles, UK) for antibody purification
Negative control: Not explicitly mentioned

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