BsAb was generated by transient co-transfection of four expression plasmids encoding anti-mBCMA heavy/light chain and anti-mCD3 heavy/light chain into Expi293 cells (Thermo Fisher Scientific). Heavy chains (HC) and light chains (LC) were transfected with a DNA ratio (by weight) of 1:1:1:1 for HC_BCMA:HC_CD3:LC_BCMA:LC_CD3. Six days after transfection supernatant was harvested and purified on an AKTA Avant (GE Healthcare). The supernatant was loaded on MabSelect Sure LX (GE Healthcare) column, washed with Phosphate Buffered Saline (PBS), eluted in 100 mM pH 3.6 sodium citrate buffer, neutralized, and dialyzed into PBS. The bispecific quality was examined by intact LC-MS after PNGase F (Prozyme or New Englab Biolabs) deglycosylation. Control KLH/CD3 bsAb was generated following the same protocol as described above.
Mabselect sure lx
Manufactured by GE Healthcare
Sourced in Sweden
MabSelect SuRe LX is a chromatography resin designed for the purification of monoclonal antibodies (mAbs) and other Fc-containing proteins. It features a high-performance agarose-based matrix and a recombinant protein A ligand that provides reliable and consistent performance.
Automatically generated - may contain errors
Lab products found in correlation
Mabselect, by Cytiva (6 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Akta avant, by GE Healthcare (1 mentions)
Vivaspin 20 concentrator, by Sartorius (1 mentions)
Hiscreen, by Cytiva (1 mentions)
Freedom evo 200, by Tecan (1 mentions)
10k mwco slide a lyzer dialysis cassettes, by Thermo Fisher Scientific (1 mentions)
Centrifugal filter unit, by Merck Group (1 mentions)
Expicho system, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 f, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Freestyle f17 expression medium, by Thermo Fisher Scientific (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Complete his tag purification resin, by Roche (1 mentions)
Capto s, by GE Healthcare (1 mentions)
Capto q, by GE Healthcare (1 mentions)
Capto, by Cytiva (1 mentions)
8 protocols using mabselect sure lx
1
Bispecific Antibody Production in Expi293 Cells
2
PODXL-v2 Extracellular Domain Purification
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Example 7
Expression and Purification of the Extracellular Domains of PODXL-v2 and PODXL-v2-Del with Fc Fusion
The DNA fragment encoding the extracellular domain of PODXL-v2 or PODXL-v2-Del was fused in-frame to the human IgG1-Fc DNA fragment in the pFUSE-hIgG1-Fc1 expression vector (InvivoGen, San Diego, Calif.) and transfected into CHO-K1 cells to select stable cell lines expressing the fusion protein. Stable cell lines were subcloned and cultured in home-made serum-free medium. The rPODXL-v2-Fc and rPODXL-v2-Del-Fc proteins were purified by Protein-A chromatography using MabSelect™ SuRe™ LX following the manufacturer's protocol (GE Healthcare Bio-Sciences AB, Uppsala Sweden). Both recombinant PODXL-Fc proteins were highly glycosylated with an apparent molecular weight of 150 kDa in reduced SDS-PAGE gel, and they could interact with MAb1738 mAb, but not any other irrelevant mAb (isotype controls), in a dose-dependent manner in both direct and indirect ELISA assays (data not shown).
3
Antibody Purification for Clinical Use
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Antibodies used in clinical studies must be highly purified to maximise the therapeutic potency and to avoid possible side effects. To reduce impurities and aggregates, culture supernatant containing c4G12 was obtained as described above and purified in a series of steps. First, c4G12 was purified from the supernatant by affinity chromatography using a HiScale 26/20 column packed with MabSelect SuRe LX (GE Healthcare). Additional purification by hydroxyapatite chromatography was performed using a BioScale CHT20-I prepacked column (Bio-Rad), and fractions containing aggregates were further purified by anion exchange chromatography using a HiScreen Q-Sepharose HP prepacked column (GE Healthcare). Throughout the purification steps, an ÄKTA avant 150 chromatography system (GE Healthcare) was used. The buffer was exchanged with PBS using a Vivaspin20 concentrator with 50 kDa molecular weight cut of membrane (Sartorius, Gӧttingen, Germany) and stored at 4 °C until use.
4
Antibody Purification Using MabSelect SuRe LX
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The clarified cell culture supernatants were loaded onto a Tecan Freedom EVO 200 (Tecan Life Sciences, Männedorf, Switzerland) for antibody purification utilizing miniature columns manufactured by Repligen (Waltham, MA, USA) and packed with MabSelect™ SuRe™ LX (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The antibodies were eluted with 20 mM sodium acetate at pH 3.5 and immediately neutralized with 0.33 M Tris, 1 M sodium acetate pH 8.0 and buffer exchanged into 20 mM sodium acetate pH 5.5 using 10K MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA).
5
SARS-CoV-2 RBD Binding Antibody Screening
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Candidate antibody P17 was screened from phage-display fully human naive antibody library (simplified as ST-ST-HuNAL, constructed by Sanyoubio with the capacity of 2.2 × 1011, Fab format) by solid-phase immunotube screening method using recombinant RBD of SARS-CoV-2 as a target. The P17 antibody was prepared by ExpiCHO system (Thermo Fisher Scientific). Briefly, the plasmids harboring the heavy-chain and light-chain of the P17 antibody were mixed in ExpiFectamineTM CHO, and then were added to the prepared cell suspension and mixed with gently shaking. The cell culture was then transferred and incubated at 37 °C in the shaker with 7% CO2. About 18–22 h after transfection, ExpiCHOTM Enhancer and ExpiCHOTM Feed were added and the cell culture was transferred to another shaker for incubation at 32 °C supplied with 5% CO2. Seven to fifteen days after transfection, the culture supernatant containing P17 antibody was collected and centrifuged at 12,000 × g for 10 min and the resulting supernatant was subjected to affinity purification with MabSelect SuRe LX (GE). P17 antibody was eluted with 100 mM Glycine-HCl (pH 3.0) and then neutralized with 1 M Tris-HCl. Finally, the purified antibody was exchanged into PBS buffer through the centrifugal filter unit (Millipore). Aliquots were made and stored in −80 °C.
6
Recombinant Human IgG1 mAb Production
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DNA fragments encoding the antibody variable regions of hybridoma cells were cloned and sequenced with 5 ‘RACE method by a commercial sequencing service (Genewiz, Suzhou, China). Recombinant human IgG1 mAbs were synthesized using DNA fragments encoding heavy-chain and light-chain genes and their signal sequence by Genewiz, Inc. (Suzhou, China) and cloned into modified pcDNA3.4 vectors that contain human constant regions of IgG1 or light chains. HEK293F cells were transiently cotransfected with pairs of the heavy-chain and light-chain expression vectors by using EZ cell transfection reagent (Life-iLab, Shanghai, China) according to the instructions’ manual. After 7 days of culture, the cell supernatants were harvested and purified using MabSelect SuRe™ LX (GE Healthcare, Sweden).
7
Large-Scale Antibody Expression and Purification
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Example 15
The target antibody was expressed using Freestyle™ 293-F (Invitrogen) suspension cells. One day before transfection, cells were seeded at a density of 6×105 cells/mL in a 1 L shake flask containing 300 mL of F17 complete medium (Freestyle™ F17 expression medium, Gibco), grew overnight by shaken at 37° C., 5% CO2, 120 rpm at cell incubator. The next day, transfection of the antibody expression plasmid was carried out with PEI, wherein the ratio of plasmid:PEI was 2:1. One day after the transfection, the TN1 feed medium was added at 2.5% (v/v), and the culture was continued for 4 days, and the supernatant was collected by centrifugation.
The collected cell expression supernatant was eluted by a Protein An affinity chromatography column (Mabselect Sure LX, GE) eluting with 0.1 M citric acid (pH 3.0), and the captured antibody was treated with 1 M Tris-HCl (pH 9.0) and adjusted to pH 7.0 at 1/10 (v/v). Remove impurities such as multimers and endotoxin by gel filtration column SEC (Superdex 200, GE), and replace the antibody buffer with PBS (pH 7.4) at the same time, a sample of the target peak of UV280 nm was collected and concentrated to 2 mg/ml through an ultrafiltration centrifuge tube (30 KD, Pall Corporation). The target antibody monomer (PO %) obtained by this method was greater than 90% and was stored for subsequent experiments.
8
Purification of H11, H11-HLE, Ipi-WT, and Ipi-LALAPG
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The culture supernatants which contained H11 and H11-HLE were loaded onto cOmplete™ His-Tag Purification Resin (Roche), followed by Capto S (GE Healthcare), Capto Q (GE Healthcare), and Superdex 75 (GE Healthcare). The final concentrated proteins were stored in PBS. The culture supernatants which contained Ipi-WT and Ipi-LALAPG were loaded onto MabSelect SuRe LX (GE Healthcare). The eluted proteins were neutralized with 1 M trisodium citrate. The final concentrated proteins were stored in 25 mM sodium citrate, 125 mM NaCl pH 5.5. All purified proteins were > 95% pure, confirmed by SDS-PAGE. All endotoxin levels were below 0.02 E.U./mg.
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