During the past decades, protein-based drugs, especially monoclonal antibodies, are one of highlights of drug discovery, because they generally offer high target specificity with low side effects. Unlike small molecule drugs, therapeutical products are more susceptible to various modifications during the procedures of manufacturing, shipping, and storage. Despite the maturation and robustness of biosynthesis platforms, specific product-related modifications may be present in the final drug products, at varying levels, which potentially affect safety and efficacy after dosing. Deamidation of asparagine (Asn) or glutamine (Gln) and isomerization of aspartic acid (Asp) represent routine degradation pathways for monoclonal antibodies. The spontaneous non-enzymatic deamidation, or isomerization may induce the alteration of protein properties through the introduction of negative charges, and/or the insertion of a methylene group in the peptide backbone, leading to reduced biological activity & product stability, and increase of immunogenicity. It is an intrinsic part of determining the critical quality attributes and appropriate control strategy for therapeutic monoclonal antibodies.
The rate of the deamidation reaction depends on the solution pH, temperature, solvent ionic strength, protein primary sequence and higher order structure. Researchers often rely on mass spectrometric based proteomic techniques to identify the deamidation/ isomerization. Because hydrophobicities of deamidated peptides and their corresponding native peptides are very similar, leading to similar in retention time and coelution or partial coelution. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping 13C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. Creative Proteomics is equipped with a nanoLC-ESI-quadrupole orbitrap (Q Exactive) mass spectrometer with high mass accuracy and resolving power, for proper assignment. Our experienced analysts and technicians can hep you to not only detect and identify deamidation sites, but also estimate the percentage of deamidation in specific sites.
Previous researches have proven that the deamidation would be increased significantly at high pH during sample preparation. To overcome the problems outlined above, Creative Proteomics developed a rapid peptide mapping method using a near neutral pH reversed phase separation and high resolution/accuracy MS, which can generate robust and reproducible analytical results.