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Expicho cells

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ExpiCHO cells are a mammalian cell line designed for use in the transient production of recombinant proteins. They are derived from Chinese Hamster Ovary (CHO) cells and are optimized for high-yield protein expression.

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Lab products found in correlation

Expicho expression medium, by Thermo Fisher Scientific (15 mentions) Expi293f cells, by Thermo Fisher Scientific (14 mentions) Expifectamine cho transfection kit, by Thermo Fisher Scientific (8 mentions) Hek293f cell, by Thermo Fisher Scientific (6 mentions) Expi293f expression medium, by Thermo Fisher Scientific (6 mentions) Hek293t, by American Type Culture Collection (6 mentions) Vero e6, by Thermo Fisher Scientific (5 mentions) Hek293t, by Thermo Fisher Scientific (4 mentions) Pcr based mycoplasma detection kit, by American Type Culture Collection (4 mentions) Freestyle 293 f cells, by Thermo Fisher Scientific (4 mentions) Vero e6, by American Type Culture Collection (4 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions) Mabselect, by GE Healthcare (3 mentions) Hiload 16 60 superdex 200, by GE Healthcare (3 mentions) Superdex 200 increase 10 300 gl, by GE Healthcare (3 mentions) Freestyle 293 f, by Thermo Fisher Scientific (3 mentions) Expicho, by American Type Culture Collection (3 mentions) Hek293, by American Type Culture Collection (3 mentions) Ptwist mcis vector, by Twist Bioscience (2 mentions) Hitrap mabselect sure column, by Cytiva (2 mentions)

93 protocols using expicho cells

1

Production and Purification of SARS-CoV-2 RBD and S Proteins

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RBD and S proteins were produced by using mammalian expression plasmids [9 (link),10 (link)] that were transiently transfected into expiCHO™ cells (ThermoFisher, A29133) via manufacturer’s instructions. Briefly, expiCHO cells were transfected with plasmid DNA (1 μg/ml of cell volume) at 6x106 cells/ml in suspension culture using the Expifectamine reagent (ThermoFisher, A14525). Transfected expiCHO cells are then cultured per the manufacturers ‘max titer’ protocol at 32 degrees shaking at 125 rpm for 12 days. Cell culture supernatants were harvested and filtered through a 0.2 μM membrane and both S protein and RBD were purified using a 20 mL Ni2+-charged HiPrep IMAC FF 16/10 column (Cytiva) to bind the His-tagged region engineered into each protein [9 (link),10 (link)]. A 10 kDa MWCO centrifugal filter unit (Amicon, ACS501024) was used to concentrate fractions containing RBD. Protein purity was validated by SDS-PAGE and western blotting using a PENTA-His antibody (Qiagen, ID:34660). Purified RBD and S proteins were characterized by sedimentation velocity analytical ultracentrifugation using a Beckman Coulter XL-I. Data were analyzed using SEDFIT’s continuous c(s) distribution model [13 (link)], SEDANAL version 7.45 [14 (link)], or DCDT+ version 2.4.3 [15 (link)]. Purified protein was stored at -20°C in 50% glycerol with 5 mM sodium azide.
Davis G., York A.J., Bacon W.C., Lin S.C., McNeal M.M., Yarawsky A.E., Maciag J.J., Miller J.L., Locker K.C., Bailey M., Stone R., Hall M., Gonzalez J., Sproles A., Woodle E.S., Safier K., Justus K.A., Spearman P., Ware R.E., Cancelas J.A., Jordan M.B., Herr A.B., Hildeman D.A, & Molkentin J.D. (2021). Seroprevalence of SARS-CoV-2 infection in Cincinnati Ohio USA from August to December 2020. PLoS ONE, 16(7), e0254667.
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2

Production and Purification of B7-H3xCD3

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B7-H3xCD3 and its isotype control MOPCxCD3 were generated as described previously (13 (link)). In brief, the constructs were produced in ExpiCHO cells (Gibco, Carlsbad, CA, USA) and purified from culture supernatant by affinity chromatography on Mabselect affinity columns (GE Healthcare, Munich, Germany) followed by analytical and preparative size exclusion chromatography using Superdex S200 Increase 10/300GL and HiLoad 16/60 columns (GE Healthcare). Endotoxin levels were measured with EndoZyme II (BioMerieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions and < 0.5 EU/ml.
Lutz M.S., Zekri L., Weßling L., Berchtold S., Heitmann J.S., Lauer U.M., Jung G, & Salih H.R. (2023). IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer. Frontiers in Immunology, 14, 1163136.
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3

Engineered AZD8895 Antibody Variants

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Struturally-important residues in the AZD8895 heavy chain sequence were identified as D108, P99, and the disulfide bond in HCDR3. The D108 residue was mutated to alanine, asparagine, and glutamic acid. The P99 residue was mutanted to valine, asparagine, and glycine. The disulfide bond was removed by introducing C101A/C106A mutations. Additionally, the germline revertant (GRev) forms of AZD8895 were generated by aligning the sequence to identified germline sequences using IgBlast, and reverting back the residues that were not germline-encoded. DNA fragments corresponding to the AZD8895 mutant heavy chain variable domains with human IgG1 and light chain variable domain with human kappa chain constant domain were synthesized and cloned into the pTwist_mCis vector (Twist Bioscience) as previously described21 (link). Constructs were transformed into E. coli, and DNA was purified. Antibodies then were produced by transient tranfection of ExpiCHO cells following the manufacturer’s protocol (Gibco). Supernatants were filter-sterilized using 0.45 µm pore size filters and samples were applied to HiTrap MabSelect Sure columns (Cytiva).
Dong J., Zost S.J., Greaney A.J., Starr T.N., Dingens A.S., Chen E.C., Chen R.E., Case J.B., Sutton R.E., Gilchuk P., Rodriguez J., Armstrong E., Gainza C., Nargi R.S., Binshtein E., Xie X., Zhang X., Shi P.Y., Logue J., Weston S., McGrath M.E., Frieman M.B., Brady T., Tuffy K., Bright H., Loo Y.M., McTamney P.M., Esser M.T., Carnahan R.H., Diamond M.S., Bloom J.D, & Crowe JE J.r. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nature microbiology, 6(10), 1233-1244.
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4

Purification and Characterization of SDIE Antibodies

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293C3-SDIE and iso-SDIE were produced as described previously [19 (link),20 (link)]. In brief, plasmids for heavy and light chains (in the case of iso-SDIE derived from MOPC21) were used to express antibodies in ExpiCHO cells (Gibco, Carlsbad, CA, USA) according to the manufacturer’s recommendations and purified by affinity (Mabselect; GE Healthcare, Chicago, IL, USA). This was followed by subsequent preparative size exclusion chromatography (HiLoad 16/60 Superdex 200; GE Healthcare, Chicago, IL, USA). Prior to use in functional experiments, the mAbs were cleared from endotoxins using the Endotrap HD kit from Hyglos (Bernried, Germany). Ultimately, the antibodies were run on analytical size exclusion columns (Superdex 200 Increase 10/300 GL; GE Healthcare; Chicago, IL, USA) and 4–12% gradient SDS-PAGE gels (Invitrogen; Carlsbad, CA, USA) using the gel filtration and Precision Plus standard from Bio-Rad (Hercules, CA, USA), respectively.
Riegg F., Lutz M.S., Schmied B.J., Heitmann J.S., Queudeville M., Lang P., Jung G., Salih H.R, & Märklin M. (2021). An Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity against B Cell Acute Lymphoblastic Leukemia. Cancers, 13(7), 1632.
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5

Purification and Characterization of mAbs

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293C3-SDIE and Iso-SDIE were produced as described previously [14 (link)]. In brief, plasmids for HC and LC were generated using the EndoFree Plasmid Maxi kit from Qiagen (Hilden, Germany) according to the manufacturer’s protocol. Antibodies were expressed in ExpiCHO cells (Gibco, Carlsbad, CA, USA) according to the manufacturer’s recommendations and purified by affinity (Mabselect; GE Healthcare, Chicago, IL, USA) and subsequent preparative size exclusion chromatography (HiLoad 16/60 Superdex 200; GE Healthcare, Chicago, IL, USA). Prior to use in functional experiments, mAbs were cleared from endotoxins using the Endotrap HD kit from Hyglos (Bernried, Germany). Ultimately, antibodies were run on analytical size exclusion columns (Superdex 200 Increase 10/300 GL; GE Healthcare; Chicago, IL, USA) and 4–12% gradient SDS-PAGE gels (Invitrogen; Carlsbad, CA, USA) using the gel filtration and Precision Plus standard from Bio-Rad (Hercules, CA, USA), respectively.
Schmied B.J., Riegg F., Zekri L., Grosse-Hovest L., Bühring H.J., Jung G, & Salih H.R. (2019). An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity Against Colorectal Cancer. Cancers, 11(6), 789.
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6

Fc-optimized Anti-FLT3 Antibody Production

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4G8-SDIE and iso-SDIE were generated by chimerization (human immunoglobulin G1/Κ constant region) and Fc-optimization (S239D/I332E modification) of the anti-FLT3 mAb 4G8 and control mAb MOPC21, respectively [12 (link),18 (link)]. 4G8-SDIE slightly differs from 4G8-SDIEM (FLYSYN) by the fact that it does not contain an M-tag. In brief, plasmids for the respective heavy and light chains were obtained using the EndoFree Plasmid Maxi kit from Qiagen (Hilden, Germany) according to the manufacturer’s recommendations. Antibodies were produced in ExpiCHO cells (Gibco, Carlsbad, CA, USA) according to the manufacturer’s recommendations and purified by affinity (Mabselect; GE Healthcare, Chicago, IL, USA) as well as subsequent preparative size exclusion chromatography (HiLoad 16/60 Superdex 200; GE Healthcare). Before use in functional experiments, mAb were cleared of endotoxins with the Endotrap HD Kit from Hyglos (Bernried, Germany). For structural analyses, mAb were investigated by analytical size exclusion chromatography (Superdex 200 Increase 10/300 GL; GE Healthcare) and SDS-PAGE (4%–12% gradient gels; Invitrogen, Carlsbad, CA, USA) using the gel filtration and Precision Plus standards from Bio-Rad (Hercules, CA, USA), respectively. The chimeric version of 4G8 with wildtype Fc part (4G8-WT) was described in [12 (link)].
Schmied B.J., Lutz M.S., Riegg F., Zekri L., Heitmann J.S., Bühring H.J., Jung G, & Salih H.R. (2019). Induction of NK Cell Reactivity against B-Cell Acute Lymphoblastic Leukemia by an Fc-Optimized FLT3 Antibody. Cancers, 11(12), 1966.
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7

Adherent and Suspension CHO Cell Culture

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Adherent Flp-In CHO cell line (Invitrogen, RRID:CVCL_U424) was cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 2 mM GlutaMAX (Gibco) and 10% fetal bovine serum (Sigma-Aldrich) at +37°C, 5% CO2 according to the manufacturer’s recommendations. The serum-free suspension adapted landing pad cell line was cultured in FectoCHO CD Medium (Polyplus), supplemented with 2 mM GlutaMAX at +37°C, 5% CO2 at 120 rpm orbital shaker with 19 mm throw. ExpiCHO cells (Gibco, RRID:CVCL_5J31) were cultured in ExpiCHO Expression Medium (Gibco) at +37°C, 8% CO2.
Huhtinen O., Salbo R., Lamminmäki U, & Prince S. (2023). Selection of biophysically favorable antibody variants using a modified Flp-In CHO mammalian display platform. Frontiers in Bioengineering and Biotechnology, 11, 1170081.
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8

Recombinant Antibody Production in CHO Cells

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The polypeptide sequences of VH and VK were codon‐optimized for CHO cell expression and synthesized by Integrated DNA Technologies (Coralville, IA). The synthesized VH and VK fragments were cloned into the human IgG1 containing effector knockout mutations (L234A, L235A, and P329G) and human kappa chain expression vectors, respectively. ExpiCHO cells (Gibco, Carlsbad, CA) were transfected with the antibody expression plasmids. The culture medium containing the expressed recombinant antibody was harvested 5 days after transfection. The recombinant antibody was purified by MabSelect SuRe (GE Life Sciences, Pittsburgh, PA). The purity of 4D9 recombinant antibody was analyzed by SDS–PAGE (NuPAGE 4–12% Bis–Tris, Invitrogen, Carlsbad, CA) and size exclusion chromatography (Tosoh TSKgel, Tosoh Biosciences, Japan).
Schlepckow K., Monroe K.M., Kleinberger G., Cantuti‐Castelvetri L., Parhizkar S., Xia D., Willem M., Werner G., Pettkus N., Brunner B., Sülzen A., Nuscher B., Hampel H., Xiang X., Feederle R., Tahirovic S., Park J.I., Prorok R., Mahon C., Liang C., Shi J., Kim D.J., Sabelström H., Huang F., Di Paolo G., Simons M., Lewcock J.W, & Haass C. (2020). Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Molecular Medicine, 12(4), e11227.
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9

Antibody and Fab Production from ExpiCHO Cells

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Antibodies were produced by transient transfection of ExpiCHO cells (Gibco) based on the manufacturer’s high titer protocol. To purify the antibodies, ExpiCHO medium was centrifuged (12,000 × g, 30 min, 4°C), followed by passage through 0.45 micron and 0.22 micron filters. The clarified medium was then applied to protein G resin (GenScript), rinsed with 20 column volumes of phosphate buffered saline (PBS), and eluted with 10 column volumes of 100 mM glycine buffer, pH 3.0. Eluted antibodies were immediately neutralized with 1 M Tris, pH 9.0. Antibodies were further purified by size exclusion chromatography on an S200 26/60 (GE Healthcare) and stored in PBS at 4°C. For Fab purification, ExpiCHO medium was clarified and then purified using protein G resin as stated above. Monomeric Fabs were further purified by size exclusion chromatography using an S75 26/60 (GE Healthcare).
Kuo Y.C., Kuo C.F., Jenkins K., Hung A.F., Chang W.C., Park M., Aguilar B., Starr R., Hibbard J., Brown C, & Williams J.C. (2022). Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells. Journal for Immunotherapy of Cancer, 10(6), e003752.
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10

Engineered AZD8895 Antibody Variants

Cited 1 time
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Struturally-important residues in the AZD8895 heavy chain sequence were identified as D108, P99, and the disulfide bond in HCDR3. The D108 residue was mutated to alanine, asparagine, and glutamic acid. The P99 residue was mutanted to valine, asparagine, and glycine. The disulfide bond was removed by introducing C101A/C106A mutations. Additionally, the germline revertant (GRev) forms of AZD8895 were generated by aligning the sequence to identified germline sequences using IgBlast, and reverting back the residues that were not germline-encoded. DNA fragments corresponding to the AZD8895 mutant heavy chain variable domains with human IgG1 and light chain variable domain with human kappa chain constant domain were synthesized and cloned into the pTwist_mCis vector (Twist Bioscience) as previously described21 (link). Constructs were transformed into E. coli, and DNA was purified. Antibodies then were produced by transient tranfection of ExpiCHO cells following the manufacturer’s protocol (Gibco). Supernatants were filter-sterilized using 0.45 µm pore size filters and samples were applied to HiTrap MabSelect Sure columns (Cytiva).
Dong J., Zost S.J., Greaney A.J., Starr T.N., Dingens A.S., Chen E.C., Chen R.E., Case J.B., Sutton R.E., Gilchuk P., Rodriguez J., Armstrong E., Gainza C., Nargi R.S., Binshtein E., Xie X., Zhang X., Shi P.Y., Logue J., Weston S., McGrath M.E., Frieman M.B., Brady T., Tuffy K., Bright H., Loo Y.M., McTamney P.M., Esser M.T., Carnahan R.H., Diamond M.S., Bloom J.D, & Crowe JE J.r. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nature microbiology, 6(10), 1233-1244.
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