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

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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).

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