title: Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies.
abstract: A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab'2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab'2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method.
authors.affiliation: Department of Analytical Sciences, Amgen Inc., Seattle WA, USA.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Eakin
authors.affiliation: Department of Analytical Sciences, Amgen Inc., Seattle WA, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Miller
authors.affiliation: Department of Analytical Sciences, Amgen Inc., Seattle WA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kerr
authors.affiliation: Department of Functional Biocharacterization, Amgen Inc., Thousand Oaks CA, USA.
authors.forename: James
authors.initials: J
authors.lastname: Kung
authors.affiliation: Department of Analytical Sciences, Amgen Inc., Seattle WA, USA.
authors.forename: Alison
authors.initials: A
authors.lastname: Wallace
keywords: IdeS; fabricator; hydrophobic interaction chromatography; isoaspartic acid; peptide map