DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions

Abstract

Source: Knödler, M. et al., Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study. J. Vis. Exp. (2019).

This video describes an affinity chromatography technique for purifying 2F5 monoclonal antibodies using the DsRed-2F5-Epitope or DFE ligands. The purified 2F5 monoclonal antibodies are essential drugs in immunotherapy.

Protocol

1. Testing the Purification of mAbs from Clarified Plants Extracts

1. Prepare 100 mL of clarified plant extract containing 2F55 or the supernatant from the preferred cell-based expression system, also containing 2F5.
2. Prepare equilibration buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4), low-pH elution buffer (0.05 M citrate, 0.05 M sodium chloride, pH 4.0–3.25), and high-ionic-strength elution buffer (1.0–4.0 M magnesium chloride, 0.1 M HEPES, pH 8.0)
3. Flush the chromatography system with the buffers. Mount a DFE affinity column (Refer to the main protocol text) on the chromatography system and e....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DsRed	Fraunhofe IME	n/a	Standart
HiTrap NHS-activated Sepharose HP, 1 mL	GE Helthcare	17-0716-01	Chromatography columns
HEPES	Carl Roth GmbH	9105.3	Buffer component
Magnesium chloride	Carl Roth GmbH	KK36.2	Buffer component
PH 3110	WTW	2AA110	pH meter
Sephadex G-25 fine, cross linked dextran	GE Helthcare	17003301	Chromatography resin
Sodium chloride	Carl Roth GmbH	P029.2	Buffer component
Sodium citrate	Carl Roth GmbH	HN13.2	Buffer component