Sequences encoding light chain (Vκ-Cκ) and heavy chain fragment (VH-CH1) of h38C2 Fab containing a Lys99Cys (K99C) and Lys99Tyr (K99Y) mutation in VH and an N-terminal human CD5 signal peptide (MPMGSLQPLATLYLLGMLVASVLA) were cloned into mammalian expression vector pCEP4 (Thermo Fisher) via NheI/XhoI (New England Biolabs). Following maxipreparation (Qiagen), plasmids encoding Vκ-Cκ and VH-CH1 were co-transfected into Expi293F cells (grown to a density of 3 ⅹ 106 cells/mL in 300 mL) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher). After continued culturing at 37°C, 5% CO2 for 5 days, the culture supernatant was collected and subjected to purification by affinity chromatography using a 1-mL HiTrap KappaSelect column (Cytiva) on an ÄKTA FPLC instrument (Cytiva). The Pierce BCA Protein Assay Kit (Thermo Fisher) was used to determine the yield of Fab (~15 mg/L). Further purification of the Fab was done by size-exclusion chromatography (SEC) using a Superdex 200 10/300 GL column (Cytiva) on an ÄKTA FPLC instrument. Following SEC, Fab peak fractions were concentrated using an Amicon Ultra 0.5-mL Centrifugal Filter (MilliporeSigma) and brought into 0.1 M sodium acetate (pH 5.5).
Hitrap kappaselect column
Manufactured by Cytiva
The HiTrap KappaSelect column is a chromatography column designed for the purification of kappa light chain-containing antibodies. The column contains a pre-packed agarose-based medium with recombinant protein L ligands that bind to the kappa light chain of antibodies. This column can be used as part of a protein purification workflow to isolate and concentrate kappa light chain-containing antibodies from complex samples.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pcep4, by Thermo Fisher Scientific (1 mentions)
Maxi preparation, by Qiagen (1 mentions)
Nhei xhoi, by New England Biolabs (1 mentions)
Kta fplc instrument, by Cytiva (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl column, by Cytiva (1 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)
Hitrap protein a column, by Cytiva (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
2 protocols using hitrap kappaselect column
1
Recombinant Fab Production and Purification
2
Engineered Antibody Production in CHO-K1 Cells
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IgG and IgA3.0 (hereafter called IgA) antibodies targeting GD2 (ch14.18, dinutuximab) were produced by WuXi Biologics in CHO-K1 cells. To block CD47, we either used an engineered human SIRPα D1 domain with high affinity for both mouse and human CD47 fused to IgG1 L234A/L235A/P329G (LALAPG) as described in Chernyavska et al33 (link) or an IgG1 LALAPG anti-CD47 (Clone 2.3D11) produced and purified in-house. Antibodies were produced and purified as described previously.20 34 (link) In short, antibodies were produced through transient transfection of Expi-CHO-S cells (Thermo Fisher Scientific). Subsequently, supernatant containing the antibody was affinity purified using a HiTrap Protein A column (Cytiva) for IgG antibodies or a HiTrap KappaSelect column (Cytiva) for IgA antibodies, coupled to a ÄKTA liquid chromatography system (Cytiva). The captured IgG was eluted and subsequently dialyzed against PBS, while IgA was additionally purified by size exclusion chromatography. The eluate was filtered using a 0.22 µm filter and the antibody concentration was measured by UV absorbance at 280 nm using the corresponding extinction coefficient (ε280).
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