Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

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

1. Express the antibody
   1. 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.
   2. When a density of 2.5 x 10⁶ cells/mL is reached (2-3 days after splitting), prepare a fresh solution of 100 mM CypK. For this purpose, wei....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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