The variable light-chain and heavy-chain sequences were recovered from the 1F10 hybridoma and cloned into expression vectors in frame with constant regions to obtain a mouse IgG1/κ antibody harboring the N297Q mutation to abrogate binding to Fcγ receptors. The linearized vectors for the light chain and for the heavy chain were co-transfected into a CHO cell line using the Neon device (Invitrogen). The antibody was purified from the supernatant using the MabSelect PrismA resin (Cytiva). The antibody was eluted with citrate 0.1 M pH4.5 buffer, dialyzed overnight against PBS and 0.22 µM filtered. Size exclusion chromatography, SDS-PAGE and endotoxin levels quality controls were performed.
Mabselect prisma resin
Manufactured by Cytiva
Sourced in Sweden
MabSelect PrismA Resin is a chromatography resin designed for the purification of monoclonal antibodies. It is based on a rigid agarose matrix and features a recombinant Protein A ligand. The resin is optimized for high dynamic binding capacity and efficient antibody capture.
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4 protocols using mabselect prisma resin
1
Antibody Production in CHO Cells
2
Monoclonal Antibody Production and Characterization
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Monoclonal antibodies were expressed in ExpiCHO-S cells (Thermo Fisher) cultured and transfected according to the manufacturer’s specifications. Antibodies were purified from culture supernatant using GE AKTA Pure FPLC and MabSelect PrismA Resin (Cytiva) using 20mM Acetate, 30mM Glycine pH 3.75 elution buffer. Purified antibodies were buffer exchanged with dialysis cassettes into PBS pH 7.4 buffer and evaluated by SEC-HPLC at 220 nm on a Tosoh TSKgel G3000SWXL column using 0.2 M sodium phosphate pH 6.7 as the mobile phase to determine monomeric purity. Endotoxin levels were measured using a kinetic chromogenic LAL assay with the Charles River Endosafe PTS100. The VSTB174 antibodies were derived from the Janssen Pharmaceuticals VSTB174 sequence (WO2016207717) and were expressed as full human IgG1 antibodies.
3
Rapid Chromatography Membrane Device
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The ProtA chromatography membrane device used in this study was the Sartobind® Rapid A convective-diffusion membrane (Sartorius, Göttingen, Germany). A 1 mL membrane volume (MV) device (3 mL hold-up volume) was used for initial development work, with scaleup assessment conducted on a 75 mL device (200 mL hold-up volume). A fixed bed height of 4 mm applied to both membranes.
ProtA resin MabSelect™ PrismA resin (Cytiva, Uppsala, Sweden) was purchased in a HiScreen™ chromatography column format (10 cm bed height and 4.7 mL bed volume). This format is a good model for process-scale columns based on our experience. The resin was used as a benchmark reference for membrane chromatography.
ProtA resin MabSelect™ PrismA resin (Cytiva, Uppsala, Sweden) was purchased in a HiScreen™ chromatography column format (10 cm bed height and 4.7 mL bed volume). This format is a good model for process-scale columns based on our experience. The resin was used as a benchmark reference for membrane chromatography.
4
Expression and Purification of hVISTA-ECD-Fc
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DNA encoding human VISTA-ECD (33-194, Uniprot) was cloned into an Fc (human IgG1) construct (pcDNA3.3 vector) that contained a Factor Xa cleavage site at the N-terminus of the hinge region. The hVISTA-ECD-Fc mutants were generated using site-directed mutagenesis using a standard two-stage QuikChange PCR protocol. Human VISTA-ECD-Fc proteins were expressed in human Expi293F (Thermo Fisher) cultivated and transfected according to the manufacturer’s specifications. The hVISTA-ECD-Fc proteins were purified from culture supernatant using GE AKTA Pure FPLC and MabSelect PrismA Resin (Cytiva).
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