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.
Highlights
There is an increasing number of monoclonal antibody (IgG) based protein therapeutics undergoing clinical trials, or under development making them one of the fastest growing classes of protein therapeutics (Beck et al, 2008)
A correlation between the levels of isoaspartic acid (isoAsp) detected in the peptide maps and the digested-hydrophobic interaction chromatography (HIC) method suggest that the digested-HIC method could be routinely used to monitor Asp isomerization as a higher throughput alternative to peptide mapping, for monitoring trends in Asp isomerization
Peptide mapping coupled with mass spectrometry is the single most powerful analytical technique for detection and quantitation of isoAsp species
Summary
There is an increasing number of monoclonal antibody (IgG) based protein therapeutics undergoing clinical trials, or under development making them one of the fastest growing classes of protein therapeutics (Beck et al, 2008). Formation of isoAsp inserts an extra methylene group into the protein backbone with a corresponding shortening of the Asp side chain by one methylene group (Geiger and Clarke, 1987; Johnson et al, 1989; Oliyai and Borchardt, 1994; Reissner and Aswad, 2003). This results in deformation in the protein structure which can impact protein function. Conversion of Asp to isoAsp in the complementary-determining regions (CDRs) of therapeutic antibodies has been shown to decrease receptor binding and efficacy (Cacia et al, 1996; Harris et al, 2001)
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