Efficient On-Column Removal of Endotoxin from Immunoglobulins Such as AK23

Unattended endotoxin (ETX) contamination in biological samples constitute a major challenge for in vitro and in vivo applications. Besides being potentially life-threatening, ETX contamination is especially relevant in global transcriptome analyses, where competing ETX stimulation can significantly skew the final gene expression profile. Our studies in mice and cultured skin epithelial cells (epidermal keratinocytes) aiming to characterize the effect of antibodies such as AK23 immunoglobulins (IgG directed against the cell-cell adhesion molecule desmoglein [DSG] 3) in the autoimmune disease pemphigus vulgaris (PV) revealed that laboratory-produced and even commercial control antibodies can exhibit non-negligible ETX contaminations. Moreover, these contaminants are extremely difficult to remove. To overcome these challenges, we have devised a simple yet nontoxic and scalable two-step protocol to efficiently reduce ETX levels during or after the IgG purification process. It consists firstly of 0.5 M NaOH pre-treatment of all devices, including the protein A resin, used during IgG sanitization and purification, in parallel with meticulous in-process monitoring of ETX levels. Secondly, before IgG elution from protein A, ETX is stripped from IgG by ion-exchange with the common amino acid arginine. This two-step approach successfully reduces ETX by >95% from hybridoma-derived, laboratory-produced AK23 IgG, as well as patient PV and control IgG, resulting in an 85% IgG recovery rate and ETX levels compatible with U.S. Pharmacopeia guidelines. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sanitization of devices and protein A resin with 0.5 M NaOH Basic Protocol 2: On-column stripping of ETX from AK23 IgG Basic Protocol 3: Quality control of sanitized AK23 IgG.