Protein a affinity chromatography

Manufactured by Cytiva

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Protein A affinity chromatography is a purification technique used to separate and purify specific proteins, such as antibodies, from complex mixtures. It utilizes the high-affinity interaction between Protein A, a bacterial protein, and the Fc region of antibodies. This method allows for efficient and selective capture of the target protein, enabling its isolation and concentration for further analysis or applications.

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Lab products found in correlation

Hiload 16 600 superdex 200 pg column, by Cytiva (2 mentions) Superose 6 increase 10 300 gl column, by Cytiva (1 mentions) Endosafe pts, by Charles River Laboratories (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Capto l, by Cytiva (1 mentions) 0.22 μm filter, by Merck Group (1 mentions) Capto, by Cytiva (1 mentions) Opti cho, by Thermo Fisher Scientific (1 mentions) Puromycin, by InvivoGen (1 mentions) Papain, by Merck Group (1 mentions)

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Protein A chromatography (2 citations)

7 protocols using protein a affinity chromatography

Monoclonal Antibody Development Against Glycoprotein B

Cited 1 time

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Eight-week-old female BALB/c mice were immunized with gB (100 μg/mouse) emulsified with Freund’s adjuvant subcutaneously 3 times at 2-week intervals. Two weeks after the final immunization, mice were boosted again. Three days later, spleen cells of immunized mice were collected and fused with mouse myeloma Sp2/0 cells. The hybridomas secreting gB-specific MAbs were sequentially screened by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. The hybridomas were cloned three times via limiting dilution, and purified from mouse ascites using protein A affinity chromatography (Cytiva).
293F cells were transfected with plasmids encoding heavy and light chains of AMMO5 (48 (link)). AMMO5 was further purified by protein A affinity chromatography (Cytiva).

Hong J., Wei D., Zhong L., Wu Q., Chen K., Zhang W., Yang Y., Chen J., Xia N., Zhang X, & Chen Y. (2022). Glycoprotein B Antibodies Completely Neutralize EBV Infection of B Cells. Frontiers in Immunology, 13, 920467.

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HIV Env gp140 Trimer Production and Antibody Purification

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The native-like, soluble HIV Env gp140 trimer BG505 SOSIP.664 construct with ‘SOS’ mutations (A501C_gp120, T605C_gp41), the ‘IP’ mutation (I559P_gp41), N-linked glycosylation site mutation (T332N_gp120), an enhanced furin protease cleavage site (REKR to RRRRRR), and truncation after the C-terminus of gp41 residue 664^{9 (link)} was cloned into pTT5 vector (National Research Council of Canada) and expressed in transiently-transfected Expi293F cells. For DEER experiments involving nitroxide spin labeling, BG505 SOSIP.664 was modified to include a free cysteine at residue Ser174_gp120 (S174C) by site-directed mutagenesis as described^{38 (link)}. BG505 and BG505 S174C mutant Env trimers were purified from transfected cell supernatants as described^{48 (link)} by 2G12 immunoaffinity and SEC with a HiLoad 16/600 Superdex 200 pg column followed by Superose 6 Increase 10/300 GL column (Cytiva).
The heavy and light chains of 6x-His tagged Ab1303 and Ab1573 Fabs were expressed in transiently-transfected Expi293F cells and purified by Ni-NTA chromatography followed by SEC as described^{19 (link)}. IgG proteins were expressed in transiently-transfected Expi293F cells and purified by protein A affinity chromatography (Cytiva) followed by SEC as described^{21 (link),48 (link)}.

Yang Z., Dam K.M., Bridges M.D., Hoffmann M.A., DeLaitsch A.T., Gristick H.B., Escolano A., Gautam R., Martin M.A., Nussenzweig M.C., Hubbell W.L, & Bjorkman P.J. (2022). Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer. Nature Communications, 13, 732.

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Recombinant Monoclonal Antibody Production

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Mammalian expression vectors encoding the respective heavy chain (HC) and light chain (LC) genes of tralokinumab (CAS: 1044515–88-9) and cendakimab (CAS: 2151032–62-9) were generated and transiently transfected into Chinese Hamster Ovary cells (Lonza). The mAbs were purified from the harvest media using Protein-A affinity chromatography (Cytiva) followed by cation exchange chromatography (Thermo Fisher). Purified mAbs were buffer exchanged into phosphate-buffered saline (PBS), pH7.2, and evaluated for purity and identity using SDS-PAGE, aSEC and LC/MS analysis. Endotoxin was measured at < 1 EU/mg using Endosafe ® PTS (Charles River Laboratories). At the time of this study marketed tralokinumab and cendakimab were not available for purchase. Therefore, these mAbs were expressed and purified at Eli Lilly and Company. Lebrikizumab and IgG control antibody were expressed using Chinese Hamster Ovary cells and were also purified at Eli Lilly and Company.

Okragly A.J., Ryuzoji A., Wulur I., Daniels M., Van Horn R.D., Patel C.N, & Benschop R.J. (2023). Binding, Neutralization and Internalization of the Interleukin-13 Antibody, Lebrikizumab. Dermatology and Therapy, 13(7), 1535-1547.

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Bispecific Antibody Manufacturing Protocol

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Relevant expression vectors containing the heavy and light chains of parental antibodies were transfected into CHO host cells to prepare recombinant CHO cell lines, respectively. Each production process included independent cell line seed training and fed-batch cell culture in a 200L GE Xcellerex XDR-200 single-use bioreactor (as workflow described in Figure 7). The chemical-defined basal medium of BalanCD CHO Growth A and feed mediums of Cell boost 7a, 7b (Cytiva, Cat. No. SH31026 and SH31027) were used as production media, respectively. The harvested cell fluid was then clarified and filtered, followed by Protein A affinity chromatography (Cytiva, Cat. No. 175474) and intermediate depth filtration. The target bsAb was then assembled in vitro from parental antibodies via 2-MEA (2-mercaptoethylamine) cFAE reaction. The target bsAb was subjected to a platform antibody manufacturing process, including ultrafiltration/diafiltration (UF/DF), oxidation, low pH virus inactivation, chromatograph polishing, and nano virus filtration. UF/DF was used to concentrate the protein before the final formulation and excipients were added to formulate the final drug substance.

Yao X., Xie M., Ben Y., Zhu Y., Yang G., Kwong S.C., Zhang Z, & Chiu M.L. (2024). Large scale controlled Fab exchange GMP process to prepare bispecific antibodies. Frontiers in Bioengineering and Biotechnology, 11, 1298890.

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BsAb Production via Split Intein Splicing

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The CD3 and EpCAM fragments were purified by Capto L chromatography (Cytiva, Uppsala, Sweden) through the Akta150 system (Cytiva, Uppsala, Sweden). The supernatant was centrifuged and flirted through a 0.45 μm filter membrane. The column was loaded with binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), supernatants, wash buffer (100 mM sodium citrate, pH 5.0), and eluted with elution buffer (100 mM sodium citrate, pH 2.8). The elution was neutralized to pH 7.0 with Tris-HCl buffer (1 M, pH 8.0). Fragment A and B samples were dialyzed into phosphate buffer (PBS, pH 7.4) followed by splicing reaction catalyzed mediated by split intein.
After dialyzing all of the fragments in PBS buffer and adding 2 mM dithiothreitol (DTT, Sigma Aldrich, Shanghai, China), 100 mM fragment A was combined with 375 mM fragment B and incubated at 37°C for 2 h. The mixture was dialyzed to PBS to remove DTT, sterilized using a 0.22 μm filter (Millipore, Shanghai, China), and the oxidation reaction was carried out at room temperature for 2–3 days. The CD3×EpCAM BsAb was isolated through Protein A affinity chromatography (Cytiva, Uppsala, Sweden). The eluates were adjusted to pH 7.0 using Tris-HCl buffer, dialyzed to PBS, and sterilized through a 0.22 μm filter (Millipore, Shanghai, China).

Wang L., Qiao Y., Zong H., Han L., Ke Y., Pan Z., Chen J., Lu J., Li J., Ying T., Zhang B, & Zhu J. (2022). IgG-like Bispecific Antibody CD3×EpCAM Generated by Split Intein Against Colorectal Cancer. Frontiers in Pharmacology, 13, 803059.

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Production and Purification of Recombinant ACE2-Fc

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A DNA fragment for the amino acids 1–740 of the human ACE2 (NG_012575.3) was cloned into the pDAD2 vector containing the Fc-domain of human IgG1 (P01857). The vector was transfected into CHO DG44 cells via nucleofector. Transfected cells were transferred to selection medium (OptiCHO (Gibco) with 5 µg/mL puromycin (Invivogen)). Single cell studies were carried out from the pooled cells that grew in the selection medium. Selected clones were tested in fed batch. All supernatants were collected and purified via Protein A affinity chromatography (Cytivia). Affinity-purified ACE2-Fc was subjected to size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with PBS (pH 7.4) for polishing. Recombinant protein separated by SDS-PAGE was detected by Coomassie Blue staining and western blot with an anti-human IgG (H + L)-horseradish peroxidase antibody (Life technology). Recombinant protein aliquots were stored in PBS at − 80 °C.

Kalyoncu S., Yilmaz S., Kuyucu A.Z., Sayili D., Mert O., Soyturk H., Gullu S., Akinturk H., Citak E., Arslan M., Taskinarda M.G., Tarman I.O., Altun G.Y., Ozer C., Orkut R., Demirtas A., Tilmensagir I., Keles U., Ulker C., Aralan G., Mercan Y., Ozkan M., Caglar H.O., Arik G., Ucar M.C., Yildirim M., Yildirim T.C., Karadag D., Bal E., Erdogan A., Senturk S., Uzar S., Enul H., Adiay C., Sarac F., Ekiz A.T., Abaci I., Aksoy O., Polat H.U., Tekin S., Dimitrov S., Ozkul A., Wingender G., Gursel I., Ozturk M, & Inan M. (2023). Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris. Scientific Reports, 13, 5224.

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QSOX1trx-hαQSOX1.4 Fab Complex Crystallization

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Purified QSOX1trx was concentrated to 10 mg/ml. The purified CUMAb antibody hαQSOX1.4 was concentrated to 1.5 mg/mL in PBS and digested at 37 °C with papain (Sigma) at a 1:20 papain:hαQSOX1.4 molar ratio. Prior to use, papain was activated by dissolving in PBS supplemented with 20 mM EDTA and 20 mM cysteine. Digestion was stopped after 4 h using leupeptin, and the digested antibody was dialyzed against PBS, pH 8. The Fab fragment of hαQSOX1.4 was purified by protein A affinity chromatography (Cytiva). Purified hαQSOX1.4 Fab was incubated for 1 h at 4 °C with a 2-to 3-fold excess of QSOX1trx, and the complex was isolated by size-exclusion chromatography in 10 mM Tris, pH 7.5, and 100 mM NaCl. The complex was concentrated using a centrifugal concentrator to 10 mg/mL. Crystals were grown by hanging-drop vapor diffusion at 293 K by mixing 1 μl of protein complex solution with 1 μl of well solution and suspending over well solution. Initial branched, feather-like crystals were observed when well conditions were 14% w/v polyethylene glycol 4 kDa and 0.1 M ammonium phosphate dibasic. These crystals were seeded into drops in similar conditions (11-14% w/v polyethylene glycol 4 kDa and 0.05-0.15 M ammonium phosphate dibasic) to produce individual crystal rods. These crystals were soaked in a similar solution with an addition of 20% glycerol and flash frozen in liquid nitrogen.

Tennenhouse A., Khmelnitsky L., Khalaila R., Yeshaya N., Noronha A., Lindzen M., Makowski E.K., Zaretsky I., Sirkis Y.F., Galon-Wolfenson Y., Tessier P.M., Abramson J., Yarden Y., Fass D, & Fleishman S.J. (2024). Computational optimization of antibody humanness and stability by systematic energy-based ranking. Nature biomedical engineering, 8(1).

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