Side Effect-Free Biotherapeutic Protein Sample Preparation for Peptide Mapping

Part # PS322

Abstract

Sergei Saveliev, Lyndsey Jager, Chris Hosfield, Mike Rosenblatt and Marjeta Urh
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711

Non-enzymatic modifications such as deamidation, disulfide bond scrambling and oxidation can affect both the stability and efficacy of biotherapeutic proteins. Peptide mapping is the primary analytical method used to monitor these modifications. Unfortunately, steps involved in peptide mapping sample preparation are also a source of these modifications. We address this problem by performing the entire sample preparation at mildly acidic conditions which suppress deamidation and disulfide bond scrambling. Artificial oxidation is suppressed with a reactive oxygen scavenger. Procedural steps of the method are optimized to match the efficiency of the steps in conventional procedure.

The procedure has been adapted for use in common peptide mapping sample preparation procedures based on protein denaturation with GuHCl or Urea and size exclusion clean-up. It is readily amenable to automation.

Printed in USA.