Hek293f cell

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, China

HEK293F cells are an established human embryonic kidney cell line commonly used for recombinant protein production and viral vector manufacturing. These cells are adherent and can be adapted to grow in suspension culture. HEK293F cells provide a well-characterized system for research and development purposes.

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HEK293F (78 citations) HEK293F suspension cells (21 citations) HEK293F cell line (16 citations) HEK293F suspension-cultured cells (5 citations) HEK293F suspension-grown cells (2 citations) HEK293F suspension adapted cells (2 citations)

231 protocols using hek293f cell

Production and Purification of IgG1 Antibodies

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The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector containing the hIgG₁ Fc regions (hFc) at the C-terminus as described previously [67 (link)]. The expression vectors were transfected into HEK293F cells (Invitrogen), and the fusion proteins were purified by Protein A affinity chromatography as described previously [67 (link)].
For the expression of IgG₁, genes encoding V_H and V_L were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgG₁ was purified by Protein A affinity chromatography as described previously [68 (link)]. Then the eluate containing IgG₁ was subjected to gel filtration chromatography. A total of 4 mg of IgG₁ was injected at a flow rate of 1 mL/min and purified by gel filtration using a XK16/100 column packed with Superdex 200 pg at pH 7.4 (ÄKTA pure, GE Healthcare). The chromatogram was recorded at a UV absorbance of 280 nm. The fractions containing IgG₁ were pooled by collection criteria and concentrated.

Kim S.I., Kim S., Kim J., Chang S.Y., Shim J.M., Jin J., Lim C., Baek S., Min J.Y., Park W.B., Oh M.D., Kim S, & Chung J. (2019). Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody. International Journal of Molecular Sciences, 20(20), 5073.

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Rhesus Macaque Antibody Production

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The sequences encoding rhesus macaque IgG1 heavy chain of RM19R and the rhesus macaque kappa chain of RM19R were codon optimized, synthesized, and separately cloned into the pcDNA3.4 vector using the GeneArt service from Invitrogen. The RM19R IgG was produced by transient transfection of Expi293 cells and purified using Protein A affinity chromatography by Invitrogen. Human VRC01 and PGT145 were produced in HEK293F cells (Thermo Fisher) and purified using Protein A affinity resin (Thermo Fisher) as previously described^{58 (link),59 (link)}. The RM19R Fab heavy-chain plasmid was made introducing two stop codons following residue D234 in the RM19R IgG1 heavy-chain vector using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Agilent). The RM19R FAb was expressed in HEK293F cells (Invitrogen) by co-transfecting with the FAb heavy and kappa chain plasmids (1:1 ratio) using PEImax. Transfection supernatant was harvested after 6 days and passed through a 0.45 µm filter. The RM19R FAb was purified using CaptureSelect™ CH1-XL (Thermo Fisher) affinity chromatography.

Martin J.T., Cottrell C.A., Antanasijevic A., Carnathan D.G., Cossette B.J., Enemuo C.A., Gebru E.H., Choe Y., Viviano F., Fischinger S., Tokatlian T., Cirelli K.M., Ueda G., Copps J., Schiffner T., Menis S., Alter G., Schief W.R., Crotty S., King N.P., Baker D., Silvestri G., Ward A.B, & Irvine D.J. (2020). Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations. NPJ Vaccines, 5, 72.

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Recombinant Protein Production and Antibody Purification

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Soluble MET741 protein^{28 (link)} was produced from CHO Lec3.2.8.1 cells and purified by affinity chromatography (NiNTA Superflow, Qiagen) followed by cation exchange (MonoS, GE Life Sciences). 107_A07 and D1.3 Fabs and IgG –formatted antibodies were produced by transfection of suspension HEK293F cells (Invitrogen) with Valproic Acid (Sigma) added to 4 mM following transfection. Fab proteins were purified using affinity resins KappaSelect and/or GammaBind Plus (GE Life Sciences). IgG formatted antibodies were purified by Protein A affinity chromatography. For cell cycle analysis, 107_A07 and D1.3 Fabs were further purified by gel filtration chromatography (Superdex 200 10/300 (GE Life Sciences). Unless stated otherwise, 7A2 scFv was produced in Pichia pastoris and purified by Ni-NTA chromatography followed by gel filtration. Anti-MET antibody 5D5 sequences were obtained from US Patent No. 7,476,724 B2. Heavy and light chains were synthesized (GeneArt, Thermo Fisher Scientific) with restriction sites that allowed cloning into Fab vectors pBIOCAM1-3F and pBIOCAM3-3F, expressed in HEK293F cells and the Fab purified by Ni-NTA chromatography.

DiCara D.M., Chirgadze D.Y., Pope A.R., Karatt-Vellatt A., Winter A., Slavny P., van den Heuvel J., Parthiban K., Holland J., Packman L.C., Mavria G., Hoffmann J., Birchmeier W., Gherardi E, & McCafferty J. (2017). Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain. Scientific Reports, 7, 9000.

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Monoclonal Antibody Production Protocol

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All ACS101 variants were made using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). MAbs were produced by co-transfection of plasmids encoding for the HC and LC (1:1 ratio) into HEK293F cells (Invitrogen, cat no. R79009) using 1 mg/ml PEImax (Polysciences Europe GmBH, Eppelheim, Germany). HEK293F cells (Invitrogen, cat no. R79009) were maintained at a density of ∼1 million cells/ml in FreeStyle Medium (Life Technologies) prior to transfection. The supernatant was harvested five days after transfection, centrifuged and filtered using 0.22 µm pore size Steritops (Millipore, Amsterdam, The Netherlands). The supernatant was then flowed (0.5–1.0 ml/min) over a protein G agarose (Pierce) affinity chromatography column, washed with PBS and eluted with 0.1 M glycine pH 2.5 into 1 M Tris pH 8. MAbs were buffer exchanged into PBS using Vivaspin20 centrifugal filters with 100 kDa MW cut-off (Sartorius, Gӧttingen, Germany).

van Schooten J., Farokhi E., Schorcht A., van den Kerkhof T.L., Gao H., van der Woude P., Burger J.A., Meesters T.G., Bijl T., Ghalaiyini R., Turner H.L., Dorning J., van Schaik B.D., van Kampen A.H., Labranche C.C., Stanfield R.L., Sok D., Montefiori D.C., Burton D.R., Seaman M.S., Ozorowski G., Wilson I.A., Sanders R.W., Ward A.B, & van Gils M.J. (2022). Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein. Nature Communications, 13, 4515.

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Recombinant Anti-TSH Antibody Production

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Heavy and light chains of TU1.20 (mouse monoclonal antibody against TSH) were cloned into standard mammalian expression vectors featuring a CMV promotor (46 ) and including different conjugation tags (LLQGA and GGGSYRYRQGGGS) at the heavy chain C terminus. Both plasmids encoding heavy and light chain were co-transfected into suspension-adapted human embryonic kidney HEK293-F cells (Life Technologies/Thermo Fischer Scientific). HEK293-F cells were cultured in shaker flasks at 37 °C in FreeStyle 293 expression medium (Thermo Fisher Scientific) under serum-free medium conditions. The cells were transfected at ∼2 × 10⁶ vital cells/ml with the expression plasmids (0.5 mg/liter of cell culture) complexed by PEIpro (Polyplus) transfection reagent (1.3 ml/liter of cell culture) in PBS buffer. The culture supernatant was collected at day 7 post-transfection by centrifugation. IgG was purified via one-step protein A affinity purification (HiTrap MabSelect SuRe, GE Healthcare) according to the supplier's instructions.

Steffen W., Ko F.C., Patel J., Lyamichev V., Albert T.J., Benz J., Rudolph M.G., Bergmann F., Streidl T., Kratzsch P., Boenitz-Dulat M., Oelschlaegel T, & Schraeml M. (2017). Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins. The Journal of Biological Chemistry, 292(38), 15622-15635.

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Purification of Recombinant Trimeric HA

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HA proteins were transiently expressed in HEK 293F cells (Thermo Fisher) at a density of 1.0 × 10⁶ cells/mL with a 1:3 ratio of DNA to PEIMax. HEK 293F cells were maintained in 293FreeStyle expression medium (Life Technologies) and cultured at 37°C, 8% CO₂, and shaken at 125 rpm. Six days after transfection, cells were harvested and spun down. HAs were purified by a HisTrap column (Cytiva). After elution, HA trimers were purified by size exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Healthcare). Fractions corresponding to trimeric HA were pooled, concentrated, and buffer exchanged to TBS using 50 kDa Amicon concentrators.

Yang Y.R., Han J., Perrett H.R., Richey S.T., Jackson A.M., Rodriguez A.J., Gillespie R.A., O’Connell S., Raab J.E., Cominsky L.Y., Chopde A., Kanekiyo M., Houser K.V., Chen G.L., McDermott A.B., Andrews S.F, & Ward A.B. (2023). Immune memory shapes human polyclonal antibody responses to H2N2 vaccination. bioRxiv.

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Purification of Mouse and Chimeric Antibodies

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To prepare mouse Ab proteins, mouse hybridoma cells were cultured in RPMI1640 media (ThermoFisher Scientific; cat# 11875-093) supplemented with 10% fetal bovine serum (FBS) at 37 °C in an atmosphere of 5% CO₂. Mouse 3D8 IgG2a/κ and 6C407 IgG2a/κ were purified from the supernatant of hybridoma cells, and 3D8 IgG and 6C407 IgG were purified by affinity chromatography using Protein L-agarose resin that binds to the Vκ region of Abs (GE Healthcare; cat# 17-5478-01). Chimeric Ab proteins were prepared from cultures of FreeStyle 293-F serum-free and suspension-adapted HEK293F cells (ThermoFisher Scientific; cat# R79007). HEK293F cells were cultured in serum-free FreeStyle 293 media (ThermoFisher Scientific; cat# 12338018) with 8% CO₂ and shaking at 130 rpm in the 37 °C incubator. FreeStyle 293-F cells (100 ml) at a density of 2 × 10⁶ cells/ml were transfected with 200 μg of KV10 plasmid encoding an Ab gene using 400 μg of polyethylenimine (PEI) reagent with a molecular weight of ~25 kDa (Polyscience; cat# 23966-2), a final PEI concentration of 4 μg/ml^{46 (link)}. After 7 days, the culture supernatants were harvested by centrifugation, and two chimeric MC-IgY and two chimeric MH-IgG proteins were purified by affinity chromatography using Protein L (GE Healthcare; cat# 17-5478-01) and Protein A column (GE Healthcare; cat# 17-1279-01), respectively.

Choi J., Kim M., Lee J., Seo Y., Ham Y., Lee J., Lee J., Kim J.K, & Kwon M.H. (2019). Antigen-binding affinity and thermostability of chimeric mouse-chicken IgY and mouse-human IgG antibodies with identical variable domains. Scientific Reports, 9, 19242.

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Recombinant Fab and PD-1 Protein Expression

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Plasmids pMax‐heavy_chain and pMax‐light_chain of tislelizumab Fab were transiently cotransfected into HEK293F cells (Thermo Fisher) for protein expression. The supernatant was collected, and the tislelizumab Fab was purified by His tag affinity resin, followed by further purification with Superdex 200 (GE Healthcare, Shanghai, China) in a buffer containing 20 mm Tris and 150 mm NaCl (pH 8.0).
Plasmid pMax‐PD‐1 was transiently transfected into HEK293F cells (Thermo Fisher) for protein expression. The supernatant was collected, and the PD‐1 was purified by protein A, followed by treatment with TEV Protease to remove the Fc tag. Then, the Fc‐removed PD‐1 was further purified with Superdex 200 (GE Healthcare) in a buffer containing 20 mm Tris and 150 mm NaCl (pH 8.0). The mutated PD‐1 proteins were expressed and purified at similar condition as described above. The Fc tags of all PD‐1 proteins used for SPR analysis were not removed.

Hong Y., Feng Y., Sun H., Zhang B., Wu H., Zhu Q., Li Y., Zhang T., Zhang Y., Cui X., Li Z., Song X., Li K., Liu M, & Liu Y. (2021). Tislelizumab uniquely binds to the CC′ loop of PD‐1 with slow‐dissociated rate and complete PD‐L1 blockage. FEBS Open Bio, 11(3), 782-792.

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Transient Expression of A2M Variants

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Example 9

A2M variants were expressed in HEK293F cells (Gibco) by transient transfection of each construct in suspension cells. Cells were grown to a density of 550,000 cells/mL in a Erlenmeyer cell culture flask containing 20 mL of FreeStyle F17 medium (Invitrogen) containing 1× GlutaMax (Gibco) on a rotator at a speed of 125 rpm inside a 37° C. incubator containing an 8% CO2/air mixture. Cells were transfected by mixing 20 μg of plasmid DNA of each construct (wild-type or variant) in a 1:2 (w/v) ratio with TransIT Pro plus 10 μL TransIT Boost (Mirus) 15 minutes before addition to media. Cells were maintained in the same conditions for three days after transfection before the media containing secreted recombinant protein was removed for protein purification (FIG. 3).

US10889631B2. Therapeutic variant alpha-2-macroglobulin compositions (2021-01-12). CYTONICS CORPORATION [US]. Inventors: Lewis Hanna [US], John David Laughlin [US], Shawn Robert Browning [US].

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Transient Transfection of A2M Variants in HEK293F Cells

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Example 9

US10940189B2. Systems, compositions, and methods for transplantation (2021-03-09). Cytonics Corporation [US]. Inventors: Lewis Hanna [US], John David Laughlin [US], Shawn Robert Browning [US].

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