The expression plasmids for FI-3A, FD-11A, FD-5D, EY-6A and FI-1C were created as previously described 14 (link). The VH and VL genes of C121 15 (link), REGN10933 10 (link),33 (link), REGN10987 10 (link),33 (link) and S309 16 (link) were synthesized as cDNA fragments and subcloned into the AbVec Heavy, AbVec Kappa or AbVec Lambda vector as previously described 14 (link). Control mAb Z2-B3 is an anti-influenza neuraminidase mAb that was described previously 20 (link). Monoclonal antibodies were produced in ExpiCHO cell line according to manufacturer's instructions and affinity purified using a MabSelect SuRe (Cytiva) pre-packed column. Purified mAbs were then desalted using Zeba Spin Desalting Column (ThermoFisher) or a PD-10 Desalting Column (GE Healthcare).
Mabselect sure
Manufactured by Cytiva
Sourced in New Zealand
MabSelect SuRe is a protein A chromatography resin designed for the purification of monoclonal antibodies (mAbs) and Fc-fusion proteins. It features a highly rigid, cross-linked agarose base matrix and a recombinant protein A ligand that provides enhanced alkaline stability and expanded life cycle.
Automatically generated - may contain errors
Lab products found in correlation
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Superdex 200, by Cytiva (2 mentions)
Kta pure, by Cytiva (2 mentions)
Histrap excel column, by Cytiva (2 mentions)
Histrap ff crude column, by Cytiva (2 mentions)
Kta go, by Cytiva (2 mentions)
Hitrap fibro prisma, by Cytiva (2 mentions)
Superdex 200 increase 10 300 gl, by Cytiva (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (2 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Ficin, by Merck Group (1 mentions)
Mono q 5 50, by GE Healthcare (1 mentions)
Freestyle 293 f system, by Thermo Fisher Scientific (1 mentions)
Hispur ni nta, by Thermo Fisher Scientific (1 mentions)
Captureselect kappaxl, by Thermo Fisher Scientific (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Hitrap sp hp column, by Cytiva (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Capto q, by Cytiva (1 mentions)
22 protocols using mabselect sure
1
Monoclonal Antibody Production and Purification
2
Purification of IgG mAbs and Fabs
3
Expression and Purification of Bispecific Antibody
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Full length human IgG1 and 3F8 BiTE were expressed in Expi293F cells [Thermo Scientific, Inc.] as per manufacturer guidelines. Briefly, cells were transfected at 3x106/mL with >95% viability at linear growth. Enhancers were added on the following day and culture was harvested on day 5. Media was cleared by centrifugation and filtering through a 0.22μm membrane. Cleared supernatants were processed for protein purification using either MabSelect SuRe [IgG] or His-trap excel resin (BiTE) (Cytiva, former GE healthcare).
4
Recombinant Receptor-Fc Fusion Proteins
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Human BMPR2 cDNA (Q13873) and synthetic genes for human ACVR2A (P27037) and human IgG1-Fc were used to generate the receptor GBD-Fc fusions by two-step PCR. NCBI-protein accession numbers are shown in parentheses. Fusion constructs included the extracellular domains of human ACVR2A (1–120) or BMPR2 (1–138), a 20 amino acid linker containing TEV and enterokinase cleave sites, and a C-terminal Fc. The TEV site immediately followed the receptor GBD and the enterokinase site immediately preceded the Fc moiety. PAH variants were generated from the parental BMPR2-Fc construct via two-step PCR. Fusion proteins were expressed in stably transfected CHO cells and purified from conditioned medium (CM) using Protein A capture (MabSelect SuRe, Cytiva) as described (28 (link)) followed by SEC in PBS, pH 7.5. SEC removed aggregates ensuring that a monodisperse population of expected apparent molecular weight was used in all downstream studies. Purified proteins were stored at −80 °C.
5
Expression and Purification of CD19-HSA Fusion
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Antibodies and Fab were expressed in CHO cells (44 (link)). Capture from cell culture media was accomplished by affinity chromatography using either MabSelect SuRE (Cytiva Life Sciences) for antibodies or CaptureSelect KappaXL (Thermo Fisher Scientific) for Fab. Subsequent purification was done by size exclusion chromatography using Superdex 200 for antibodies and Superdex 75 for Fab (Cytiva Life Sciences).
Human CD19 ECD (amino acids 21–289) was fused to the N-terminus of C-terminally His-tagged human serum albumin (HSA) and expressed in the FreeStyle 293-F system (Thermo Fisher Scientific). Protein was captured from cell culture media by HisPur Ni-NTA (Thermo Fisher Scientific) and further purified by Superdex 200 (Cytiva Life Sciences). Purified CD19-HSA-His was biotinylated with EZ-Link Sulfo-NHS-LC biotin (Thermo Fisher Scientific).
Human CD19 ECD (amino acids 21–289) was fused to the N-terminus of C-terminally His-tagged human serum albumin (HSA) and expressed in the FreeStyle 293-F system (Thermo Fisher Scientific). Protein was captured from cell culture media by HisPur Ni-NTA (Thermo Fisher Scientific) and further purified by Superdex 200 (Cytiva Life Sciences). Purified CD19-HSA-His was biotinylated with EZ-Link Sulfo-NHS-LC biotin (Thermo Fisher Scientific).
6
Purification of IFN-alpha15/A and IFN-beta
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Recombinant mouse IFN-alpha15/A (Cat# 12100-9) and mouse IFN-beta (Cat#12410) were purchased from PBL Biosciences. Anti-mouse XCR1 mAb (clone MARX10), anti-human AGP3 peptide mAb (clone 4D2), anti-mouse CD11c mAb (clone N418) and Ab-IFN-alpha15/A (Ab-IFN) fusions were made on a mIgG1-SEFL2 N297A backbone with a G3G4S linker. Molecules were cloned into pTT5 expression vector and produced similar to a process described previously (27 (link)). The fusion proteins were purified from conditioned medium using MabSelect SuRe (Cytiva) and Size Exclusion Chromatography/Superdex 200 Hiload 26/600 (GE Healthcare), with a final formulation of HBSS (pH 7.6).
7
Purification and Reoxidation of THIOMAB Antibodies
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The antibodies in THIOMAB format with S400C mutated cysteine were expressed in Expi293 or CHO cell lines, and purified by MabSelect SuRe (Cytiva) followed by size exclusion or SP cation exchange chromatography. The purified antibodies were adjusted to pH 8.0 using 1 M Tris pH 8.5, and supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA), and 50-fold molar excess of dithiothreitol (DTT) to reduce the blocked cysteine residues. After LC-MS confirmation of complete blocker removal, the antibodies were diluted 10 times with Buffer A (10 mM Succinate, pH 5.0), and loaded onto a 5 ml HiTrap SP HP column. The column was then washed with 10 column volumes of Buffer A, and eluted with 50 mM Tris, 150 mM NaCl, pH 8.0. The purified reduced antibodies were then re-oxidized with 15-fold molar excess of dehydroascorbic acid (DHAA) and 2 mM EDTA. The samples were then loaded onto a 5 ml HiTrap SP HP column (Cytiva) and washed with 10 column volumes of Buffer A. The antibodies were then gradient eluted with 30–100% Buffer B (10 mM Succinate, 300mM NaCl, pH 5.0) in 20 column volumes.
8
Purification of Properdin Antibodies
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Controls were isotype-matched non-targeting mouse Ig (IgG-control) and mouse anti-properdin (anti-P) (Supplementary Figure S1 ). Proteins were generated using pVEK vectors and Expi293 cells (Thermo Fisher Scientific, Waltham, MA), purified using protein A (MabSelect SuRe, Cytiva, Marlborough, MA).
9
Purification of 18/12/TxM from CHO Cells
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Purification of 18/12/TxM was from clarified CHO cell culture supernatant by the following steps. Supernatant was loaded onto a MabSelect SuRe (from Cytiva, Marlborough, MA, formerly GE Healthcare) equilibrated with 4 volumes of 50 mM Tris 1M NaCl, pH 7.0, followed by washing the column with 4 volumes of the same buffer. The column was further washed with 4 volumes of 0.1M sodium citrate, pH 5.0 to remove contaminating proteins. The 18/12/TxM protein was eluted off the column with 0.1M glycine-HCl, pH 3.5. The eluted protein was then adjusted to pH 3.6 with 1M citric acid and incubated at room temperature for 1 h to inactivate viruses. A 1M Tris base was then added to this sample to bring the pH to 4.2 and then loaded onto a Capto Q (from Cytiva) column equilibrated with 4 volumes of 50 mM sodium acetate, pH 4.2, followed by washing the column with the same buffer until A280 reached to baseline. Protein peak (containing 18/12/TxM) of the Capto Q column flow through during sample loading and a wash step was collected and then formulated in 0.1M glycine and 0.2M sorbitol, pH 4.0. Sodium dodecyl sulfate (SDS) PAGE and size-exclusion chromatography–high-performance liquid chromatography (SEC-HPLC) analysis indicated that 18/12/TxM is at least 90% pure, with approximately 70% of protein existing as a dimer and rest as oligomers.
10
Recombinant Antibody Expression and Purification
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The light and heavy chain sequences of antibodies and TV variants were cloned into a pRK5 expression vector, using anti-DNP47 (link) or anti-BACE153 (link) Fabs, as specified, and expressed in Expi293F™ cells (Gibco/Thermo Fisher A14527), Horizon CHO, or CHOK1 GS knock-out cells using standard methods. In some cases, as indicated, cloned sequences also encoded mutations for the desired effector attenuating function L234A, L235A62 (link) and P329G63 (link), (EU numbering) TV variant, or knob/hole64 (link) combinations. Recombinant ATV variants were subsequently affinity purified from clarified culture supernatants using protein A chromatography (Mab Select SuRe, Cytiva), followed by size-exclusion chromatography (Superdex 200, Cytiva), and stored in PBS at 4 °C or 10 mM sodium acetate with 6% sucrose, pH5.5 as previously described12 . The identity of purified, intact ATV molecules was confirmed by LC/MS, and a purity of >95% was confirmed by SDS-PAGE and analytical HPLC-SEC.
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