Mabselect sure resin

Manufactured by GE Healthcare

Sourced in United States

MabSelect SuRe resin is a chromatography medium designed for the purification of monoclonal antibodies (mAbs) and antibody fragments. It is based on a cross-linked agarose matrix and features a recombinant Protein A ligand that provides high binding capacity and selectivity for mAbs. The resin is well-suited for use in large-scale purification processes and can be regenerated and reused multiple times.

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Lab products found in correlation

31 protocols using mabselect sure resin

Recombinant IgG and FAB Expression

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Constructs for mammalian expression of IgGs and FAbs for R1MAb1, R1MAb2, and IMC-H7 were generated in house by gene synthesis. Plasmids encoding for the light chain and heavy chain were cotransfected into HEK293 cells and purified using a HiTrap column (GE Healthcare) with MabSelect Sure resin (GE Healthcare) followed by size-exclusion chromatography (SEC) with a Superdex S200 10/300 Gl size exclusion column (GE Healthcare) (56 (link)). To generate protein for epitope binning, a construct encoding Asn¹⁴³-Ser³⁷¹ of FGFR1b followed by a C-terminal His tag was used. The plasmid was transfected into HEK293 cells and purified using Ni-NTA Superflow resin (Qiagen) followed by SEC with a Superdex S200 10/300 Gl size exclusion column (GE Healthcare). To generate the FGFR1c receptor as a ligand trap, a construct encoding Met¹-Lys³⁶³ of FGFR1c followed by the sequence of human IgG1 Fc (EU numbering, Asp²²¹-Lys⁴⁴⁷) was used. The plasmid was transfected into HEK293 cells and purified using a HiTrap column (GE Healthcare) with MabSelect Sure resin (GE Healthcare) followed by SEC with a Superdex S200 10/300 Gl size exclusion column (GE Healthcare).

Chan J., Chan J., Shao L., Stawicki S.S., Pham V.C., Akita R.W., Hafner M., Crocker L., Yu K., Koerber J.T., Schaefer G, & Comps-Agrar L. (2022). Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action. The Journal of Biological Chemistry, 299(1), 102729.

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Recombinant Human, Mouse, and Canine IGF2R Domains

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Gene fragments encoding the domains 11–13 of human IGF2R (aa 1511-1989, Uniprot P11717), murine IGF2R (aa 1504-1982, Uniprot Q07113) and canine IGF2R (aa 1515-1993, Uniprot B1H0W0) were generated using gene synthesis. These sequences (Figure S1) were cloned into pFUSE-hIgG1-Fc2 vector (Invivogen, San Diego, CA, USA) for generation of soluble Fc fusion proteins. Recombinant proteins were expressed in Expi293F cells (Invitrogen) and purified using MabSelectSure resin (GE Healthcare, Chicago, IL, USA), using manufacturer recommended protocols.

Broqueza J., Prabaharan C.B., Andrahennadi S., Allen K.J., Dickinson R., MacDonald-Dickinson V., Dadachova E, & Uppalapati M. (2021). Novel Human Antibodies to Insulin Growth Factor 2 Receptor (IGF2R) for Radioimmunoimaging and Therapy of Canine and Human Osteosarcoma. Cancers, 13(9), 2208.

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Monoclonal Antibody Production and Characterization

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Anti-β7 (FIB504) and anti-α4β7 (DATK32) antibodies were ordered from ATCC and their sequences were individually cloned from hybridomas. Antigen-binding domain sequences from the parent hybridomas were fused to mouse IgG1 Fc in a pRK expression vector and transfected into HEK293 cells. Transfected supernatants were purified on a HiTrap column (GE healthcare) with Mabselect Sure resin (GE healthcare) with a phosphate-buffered saline (PBS) loading buffer. Antibodies were eluted with 0.1 M citrate (pH 3.0) and neutralized with 3 M Tris, pH 8.0, to a final pH of ∼7.0 prior to dialysis against PBS, pH 7.2. Each antibody was run on a Superdex S200 10/300 GL size exclusion column (SEC) (GE Healthcare) using PBS, pH 7.2, load buffer at a flow rate of 1 mL/min (30 cm/h) to remove any aggregates. Pooled fractions were filtered using a 0.2 mm filter and the final antibody preparation was assessed by analytical SEC carried out with a TSK-GEL, Super SW3000, 4.6 mm × 30 cm, 4 mm (Tosoh Bioscience) column using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) to confirm > 95% homogeneity of monomeric antibody. Anti-αE blocking antibody (clone number M290) was from BioXcell and anti-E-cadherin antibody (clone number ECCD-2) was from Invitrogen.

Dai B., Hackney J.A., Ichikawa R., Nguyen A., Elstrott J., Orozco L.D., Sun K.H., Modrusan Z., Gogineni A., Scherl A., Gubatan J., Habtezion A., Deswal M., Somsouk M., Faubion W.A., Chai A., Sharafali Z., Hassanali A., Oh Y.S., Tole S., McBride J., Keir M.E, & Yi T. (2021). Dual targeting of lymphocyte homing and retention through α4β7 and αEβ7 inhibition in inflammatory bowel disease. Cell Reports Medicine, 2(8), 100381.

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Protein A Purification of Fc-FGF21 Mutants

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Fc-FGF21 mutants were purified from HEK293-EBNA1 cell culture medium by protein A affinity chromatography using MabSelect SuRe resin (GE Healthcare), eluted with 0.5% (v/v) acetic acid, pH 3.5, 150 mM NaCl and neutralized with 10% (v/v) of 1 M Tris-HCl, pH 8.0. Protein purity was confirmed by SDS-PAGE and immunoblotting.

Shi S.Y., Lu Y.W., Richardson J., Min X., Weiszmann J., Richards W.G., Wang Z., Zhang Z., Zhang J, & Li Y. (2018). A systematic dissection of sequence elements determining β-Klotho and FGF interaction and signaling. Scientific Reports, 8, 11045.

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Production and Purification of COVID-19 Antibodies

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SAD-S35 was purchased from ACROBiosystems (cat# SAD-S35-100ug). ACE2-Fc (ACE2) was made according to Liu et al. 2018^{40 (link)}. For CR3022, CR3015^{41 (link)}, CR3046, and S309^{34 (link)} the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody. The antibodies were made by transfecting the IgG1 expression construct using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher) in Expi293F (ThermoFisher) cells according to the manufacturer specifications. Antibodies were purified from serum-free culture supernatants using mAb Select SuRe resin (GE Healthcare) followed by rapid desalting using a HiPrep 26/10 Desalting column (GE Healthcare). The final formulation buffer was 20 mM NaAc, 75 mM NaCl, 5% Sucrose pH 5.5. Convalescent serum (SER-0743-00001) was obtained from Sanquin, the Netherlands.

Bos R., Rutten L., van der Lubbe J.E., Bakkers M.J., Hardenberg G., Wegmann F., Zuijdgeest D., de Wilde A.H., Koornneef A., Verwilligen A., van Manen D., Kwaks T., Vogels R., Dalebout T.J., Myeni S.K., Kikkert M., Snijder E.J., Li Z., Barouch D.H., Vellinga J., Langedijk J.P., Zahn R.C., Custers J, & Schuitemaker H. (2020). Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses. NPJ Vaccines, 5, 91.

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Perfusion-Based Monoclonal Antibody Production

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A TFF‐based perfusion cell culture was directly connected to the Cadence BioSMB (Pall). The perfusion was conducted with a Asahi Kasei Microza 0.2 µm UMP‐1147R membrane at a 1500 s⁻¹ crossflow shear rate and 1.3 L·m⁻²·hr⁻¹ permeate flux. The capture step utilized four OPUS prepacked chromatography columns with 1.2 cm i.d. × 10 cm length (Repligen, Waltham, MA) with MabSelect Sure resin (GE Healthcare, Marlborough, MA). The multicolumn capture chromatography step operated for 18 consecutive days, and it was initiated on 11th day of cell culture after a state of control had been established.

Pinto N.D, & Brower M. (2020). Wide‐surface pore microfiltration membrane drastically improves sieving decay in TFF‐based perfusion cell culture and streamline chromatography integration for continuous bioprocessing. Biotechnology and Bioengineering, 117(11), 3336-3344.

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Bispecific Antibody Production and Purification

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Antibody scFv-Fc, HC, and LC were cloned into single-gene plasmids each having an identical CMV promoter. Plasmids were transfected into ExpiCHO cells (ThermoFisher) at a ratio of 2:1:3 of scFv-Fc: HC:LC, according to the manufacturer’s protocol. Cells were pelleted by centrifugation at 4000 × g for 12 minutes and the culture supernatants were harvested. Supernatants were applied to either mAbSelect Sure resin (GE Healthcare) or CaptureSelect CH1-XL affinity matrix (ThermoFisher) and eluted according to the manufacturers’ protocols.
Eluates containing purified BsAbs were dialyzed into 1 × PBS, pH 7.2 prior to analysis. Purity of antibody solutions was assayed by analytical size-exclusion chromatography (SEC) using a BioAssist G3SW_XL column (Tosoh). Capillary electrophoresis was performed using a Caliper LapChip GX II instrument according to the manufacturer’s protocol. CH1-XL resin was regenerated by incubation with 40 mM NaOH for 30 min.

Nesspor T.C., Kinealy K., Mazzanti N., Diem M.D., Boye K., Hoffman H., Springer C., Sprenkle J., Powers G., Jiang H., La Porte S.L., Ganesan R., Singh S, & Zwolak A. (2020). High-Throughput Generation of Bipod (Fab × scFv) Bispecific Antibodies Exploits Differential Chain Expression and Affinity Capture. Scientific Reports, 10, 7557.

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Recombinant ACE2-Fc Fusion Protein

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To generate a recombinant ACE2 Fc-fusion protein, we cloned the ectodomain of human ACE2 (GenBank ID: BAB40370.1 residues 18-740) with a C-terminal Fc tag into the pVRC8400 vector containing the human IgG1 Fc. We transfected the construct (pVRC8400-hACE2) into Expi293F™ cells using an ExpiFectamine™ transfection kit (ThermoFisher Cat# A14525) according to the manufacturer’s protocol. The supernatant was harvested after 5 days and purified using a MabSelect SuRE Resin (GE Healthcare Cat# GE17-5438-01) followed by size exclusion purification on a Superose 6 Increase column (GE Healthcare). The supernatant was harvested 5 days after transfection and purified with a CaptrueSelect KappaXL Affinity Matrix (ThermoFisher Cat# 194321005) followed by size exclusion chromatography on a Superdex200 Increase column (GE Healthcare).

Wellner A., McMahon C., Gilman M.S., Clements J.R., Clark S., Nguyen K.M., Ho M.H., Hu V.J., Shin J.E., Feldman J., Hauser B.M., Caradonna T.M., Wingler L.M., Schmidt A.G., Marks D.S., Abraham J., Kruse A.C, & Liu C.C. (2021). Rapid generation of potent antibodies by autonomous hypermutation in yeast. Nature chemical biology, 17(10), 1057-1064.

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Recombinant Antibody Expression in CHO Cells

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The variable domain genes of CR3022 (ref. ^{23 (link)}) and REGN10933 (ref. ^{32 (link)}) heavy and light chains were inserted into a pTT5 (Thermo Fisher Scientific) vector containing the constant region of the human IgG. The recombinant antibodies were expressed in Chinese hamster ovary (CHO) cells through transient transfection and purified from culture media by affinity chromatography using MabSelect Sure resin (GE Healthcare).

Li T., Xue W., Zheng Q., Song S., Yang C., Xiong H., Zhang S., Hong M., Zhang Y., Yu H., Zhang Y., Sun H., Huang Y., Deng T., Chi X., Li J., Wang S., Zhou L., Chen T., Wang Y., Cheng T., Zhang T., Yuan Q., Zhao Q., Zhang J., McLellan J.S., Zhou Z.H., Zhang Z., Li S., Gu Y, & Xia N. (2021). Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants. Nature Communications, 12, 5652.

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High-Throughput Antibody Production Optimization

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The top twenty clones were selected based on antibody titer from a 96 well plate. The selected clonal cell lines were tested for productivity in an enhanced fed batch overgrowth study. For each clone, duplicate T75 shake flasks were seeded with 300,000 viable cells per mL in 60 mL working volume of HyClone™ HyCell™ media (GE Healthcare) and incubated in a humidified (70–80 %) shaking incubator at 120 rpm with 5 % CO₂ at an initial temperature of 37 °C. Cultures were fed on designated days. A temperature shift was also performed on Day 4. Cultures were terminated when viabilities were ≤ 50 %. Antibody expression was determined by Protein A HPLC using a generic IgG standard. After production, antibodies were purified from the conditioned medium using Protein A chromatography with MabSelect SuRe™ resin (GE Healthcare).

York D., Baker J., Holder P.G., Jones L.C., Drake P.M., Barfield R.M., Bleck G.T, & Rabuka D. (2016). Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II). BMC Biotechnology, 16, 23.

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