Alkaline-tolerant RNA aptamers useful to purify acid-sensitive antibodies in neutral conditions

Abstract

Recently, several oligonucleotides have been launched for clinical use and a number of therapeutic oligonucleotides are under clinical trials. Aptamer is one of the oligonucleotide therapeutics and has received a lot of attention as a new technology and an efficacious pharmaceutical compound comparable to antibody. Aptamer could be used for various purposes, not only therapeutics but also diagnostics, and applicable to affinity chromatography as a carrier molecule to purify proteins of interest. Here we demonstrate the usage and advantages of RNA aptamer to Fc region of human IgG (i.e., IgG aptamer) for purification of human antibodies. IgG aptamer requires divalent cations for binding to IgG and bound IgG dissociates easily upon treatment with chelating reagent, such as EDTA, under neutral conditions. This elution step is very mild and advantageous for maintaining active conformations of therapeutic antibodies compared to the widely used affinity purification with Protein A/G, which requires acidic elution that often damages the active conformation of antibodies. In fact, of several monoclonal antibodies tested, three antibodies were prone to aggregate on acidic elution from the Protein A/G resin, while remained fully active upon neutral elution from the IgG aptamer resin. The IgG aptamer was fully manipulated to alkaline resistant by ribose 2'-modifications, and thereby reusable numerous times with 1 N NaOH washing. The capacity of the aptamer resin to bind IgG was equivalent to that of the Protein A/G resin. Therefore, the IgG aptamer will provide us with a unique tool to uncover and purify human monoclonal antibodies, which hold therapeutic potential but lose the activity upon acidic elution from Protein A/G-based affinity resin.

Keywords: Aggregation; Antibody purification; IgG; Protein A/G; RNA aptamer.