Codon-optimized genes corresponding to the 10E8 heavy and light chain intermediates were synthesized with a 5’ secretion leader sequence and sub-cloned into the mammalian expression vector pVRC-8400. Transient expression of the antibodies and variants was undertaken in 293F cells or 293 Expi cells as per manufacturer’s instructions (Life Technologies) by co-transfection of equal amounts of the respective heavy and light chain plasmids. Antibody IgG’s were purified from expression supernatants by capture with Protein A sepharose (Pierce) followed by elution with low pH elution buffer (Pierce) and adjustment of eluate pH with use of Tris-Cl (pH 8.0).
293 expi cells
Manufactured by Thermo Fisher Scientific
The 293 Expi cells are a human embryonic kidney (HEK) cell line derived from the 293 cell line. They are suspension-adapted and designed for high-density transfection and recombinant protein production. The 293 Expi cells provide a robust and scalable platform for transient gene expression in biopharmaceutical research and development.
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Lab products found in correlation
293f cells, by Thermo Fisher Scientific (2 mentions)
Endoproteinase lys c, by Roche (2 mentions)
Protease inhibitor tablet, by Roche (2 mentions)
Endoproteinase lysc, by New England Biolabs (1 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Superdex 75 increase, by GE Healthcare (1 mentions)
Freestyle max 293 expression system, by Thermo Fisher Scientific (1 mentions)
Sec uplc, by Waters Corporation (1 mentions)
Human vegfr2, by R&D Systems (1 mentions)
T200 evaluation software, by Cytiva (1 mentions)
6 protocols using 293 expi cells
1
Mammalian Expression of Antibody Variants
2
Expression and Purification of Nanoparticles and Monoclonal Antibodies
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All H3ssF and H7ssF nanoparticles and FI6v3, CT149, CR8020, CR6261, MEDI8852, 315-53-1F12, 54-1G07, 04-1D02, and 09-1B12 MAbs were expressed in 293 Expi cells (Life Technologies) and purified by affinity chromatography (Galanthus nivalis lectin for the nanoparticles and protein A for MAbs), using previously described methods (13 (link), 17 (link)). Assembly of nanoparticles was assessed by gel filtration with a Superose 6 increase 10/300 GL column (GE Healthcare). To make MEDI8852 and CR8020 Fabs, IgG was digested with endoproteinase Lys-C (New England Biolabs) overnight at room temperature (RT). To make CT149 Fab, CT149 IgG containing an HRV-3C cleavage site engineered in the heavy-chain hinge region was digested using HRV-3C enzyme overnight at RT. The reactions were assessed by SDS-PAGE and, upon completion, were quenched by the addition of protease inhibitor cocktail (MilliporeSigma), and Fc and Fab antibodies were separated by protein A affinity chromatography.
3
Production and Purification of 10E8 Antibody
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Transient expression of the 10E8 antibody was undertaken in 293F cells or 293 Expi cells per the manufacturer’s instructions (Life Technologies) by co-transfection of equal amounts of the 10E8 heavy and light chain plasmids, as previously described66 (link). 10E8 IgG was purified by capture with Protein A sepharose (Pierce) followed by elution in low pH (Pierce) with adjustment of eluate pH with Tris-Cl pH 8.0. The 10E8 Fab was prepared using endoproteinase Lys-C (Roche) digestion, as described66 (link). The LysC protease was added at concentrations of 1:1,000 to 1:10,000 and the digestion undertaken at 37°C for 4–6 h. Digestion reactions were stopped with protease inhibitor tablets (Roche) and cleaved products were run over a Protein A column to segregate the Fc fragment. 10E8 Fab was then subjected to cation exchange (Mono S) and size-exclusion (S200) chromatography, followed by dialysis of peak fractions into 150 mM NaCl, 2.5 mM Tris, pH 7.5, 0.02% NaN3.
4
Production and Purification of 10E8 Antibody
5
Isolation and Purification of VEGFR2-Binding Fab
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KD035 was identified by panning of Dyax FAB‐310 phagemid library on immobilized human VEGFR2 (R&D systems) followed by affinity maturation using a light chain shuffling method. The constructs carrying KD035 antigen‐binding fragment (Fab) domain were cloned into the mammalian expression vector pBh1 (Dyax) and transiently expressed in human 293 Expi cells (Invitrogen). Fabs were purified from cell culture supernatant by passing it several times through a protein A Sepharose HP column (GE Healthcare) using a peristaltic pump for a period of 8 h and eluting with 1 M Glycine pH 2.0. The eluate was further purified by size exclusion chromatography (SEC) using a Superdex 75 increase (GE Healthcare) column.
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).
6
SPR-based Antibody Concentration Analysis
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To determine the concentration of 1G2 and its point mutants in cell culture supernatant, an SPR-based concentration analysis method was used. Briefly, a calibration curve was set-up using purified 1G2 at concentrations ranging from 10 μg ml−1 to 3.2 ng ml−1 in fivefold dilutions by capturing the antibody on a human IgG binder chip described above. A 1:100 dilution of cell culture supernatant from 293 Expi cells (Invitrogen Inc.) transfected with WT or point mutants of 1G2 was then flowed over the chip and the resulting capture level recorded. The final concentration of the antibodies was determined by the concentration analysis using calibration program within the Biacore T200 evaluation software.
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