Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions
Published: April 30, 2023
Abstract
Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.
Protocol
1. Produce and Characterize the Antibody Express the antibody Thaw a vial of HEK suspension cells in a 250 mL flask containing 50 mL of expression medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B. Keep the cells at 37 °C with 8% CO2 in humidified incubators equipped with a shaker at 125 rpm. Split cells to 0.3-0.5 x 106 cells/mL (every 2-3 days) at least 2 times before transfecting. When…
Disclosures
The authors have nothing to disclose.
Materials
| Expi293F | ThermoFisher Scientific | A14527 | HEK suspension cells |
| Expi293 Expression Medium | ThermoFisher Scientific | A1435101 | Expression medium |
| Antibiotic-antimycotic | ThermoFisher Scientific | 15240062 | Penicillin-streptomycin-amphotericin B |
| 125mL Polycarbonate Erlenmeyer Flask with Vent Cap | Corning | 431143 | Shake flasks |
| Brunswick S41i incubator | Eppendorf | S41I230011 | CO2 incubator with a shaker |
| Sodium hydroxide 4 mol/l (4 N) in aqueous solution | VWR | 191373M | |
| Cyclopropene lysine | Sichem | SC-8017 | In this study it was synthesized as described by Elliot et al. 2014 |
| Steriflip-GP, 0.22 µm, polyethersulfone, gamma irradiated | Merck Millipore | SCGP00525 | |
| Opti-MEM, Reduced Serum Medium | ThermoFisher Scientific | 31985070 | Reduced serum medium |
| ExpiFectamine 293 Transfection Kit | ThermoFisher Scientific | A14525 | Transfection reagent |
| 5810 R centrifuge | Eppendorf | 5811000460 | |
| Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, gamma sterilized | Merck Millipore | SLGP033RS | |
| Protein A resin | Sino Biological | 10600-P07E-RN-25 | |
| Poly-Prep Chromatography Columns, Pkg of 50 | Bio-Rad | 7311550 | Polypropylene chromatography column |
| Econo-Column Funnel | Bio-Rad | 7310003 | |
| Sodium citrate | Fluka | 71635 | |
| ÄKTA explorer FPLC | GE Healthcare | ||
| HiTrap HIC Selection Kit | GE Healthcare | 28-4110-07 | Includes HiTrap 1 mL Butyl HP |
| Ammonium sulfate | VWR | 2133.296 | |
| Isopropanol | Honywell | 34863-2.5L | |
| Dymethyl sulfoxide | Sigma-Aldrich | D8418-50ML | |
| Tetrazine-vcMMAE | ChemPartner | – | Costum synthesized |
| Tetrazine-5-TAMRA | Jena Bioscience | CLK-017-05 | |
| NuPAGE 4-12% Bis-Tris Gel 1.0mm x 10 well | ThermoFisherScientific | NP0321BOX | |
| Xcell SureLock Mini-Cell | ThermoFisherScientific | EI0001 | |
| UltiMate 3000 HPLC | ThermoFisherScientific | ||
| Thermo Scientific MAbPac, HIC-20, 4.6 x 100 mm, 5 µm | ThermoFisherScientific | 88553 | |
| NanoAcquity | Waters | ||
| Human serum | Sigma-Aldrich | H4522-20mL | |
| CO2 incubator | Panasonic | ||
| Monomethyl Auristatin E | Cayman Chemical | 16267 | |
| NanoDrop 2000 | ThermoFisherScientific | Microvolume spectrophotometer | |
| IntantBlue | Expedeon | ISB1L | Coomassie-based stain |
| Tube 50 mL, 114x28mm, PP | Sarstedt | 62.547.254 | Conical tube |
| Amicon Ultra-15 Centrifugal Filter Units 50,000 NMWL | Merck | UFC905024 | Centrifugal filtration concentrator (after protein A pull down) |
| Amicon Ultra-4 Centrifugal Filter Units 50,000 NMWL | Merck | UFC805024 | Centrifugal filtration concentrators (after FPLC or HPLC purification) |
| Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL | ThermoFisherScientific | 89882 | Size exclusion spin columns |
| Tube PCR 0.2ml Flat Cap | Thistle Scientific Ltd | AX-PCR-02-C-CS | PCR tubes |
Tags
Cite This Article
Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions. J. Vis. Exp. (Pending Publication), e21099, doi: (2023).