Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody

Abstract

The thorough characterization of biopharmaceuticals is essential for ensuring their quality and safety since many potential variations can cause changes to the properties of a drug that may be detrimental to the patient such as decreased efficacy, shorter half-life or increased immunogenicity. Prior to approval and release, protein-based drugs are subject to a battery of analyses to assess the nature of those parameters that are considered critical quality attributes. In some cases the analytical method used may itself cause modifications that are impossible to distinguish from those induced by the intended test conditions (e.g. storage time/temperature, light exposure) which are used to assess drug stability. It is therefore important to develop and utilize analytical methods which impose as few artifactual modifications as possible. Asparagine deamidation is a common protein modification and it is known to be induced during tryptic digestion. Therefore we examined common tryptic digestion protocols and compared their propensities towards asparagine modification. Since microwave assisted hydrolysis techniques are often used to shorten digestion times and the effect on deamidation is unknown we sought to compare this method against alternate digestion protocols.

Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies

“…Artificial deamidation from tryptic digestion facilitated by high temperature and long digestion time at basic pH has been widely reported. [9][10][11]31,32 After decreasing the digestion time of anti-CRTh2 from 3 h at 37 °C to 1 h at room temperature, we observed a significant decrease in artificial deamidation. By searching for the overall intensities of the peptides as well as uncleaved proteins, we observed that digestion efficiency was high when sufficient denaturant (8 M urea) and tryptic enzyme (1:10 w/w ratio) were used (results not shown).…”

Identification of Microorganisms by High Resolution Tandem Mass Spectrometry with Accurate Statistical Significance

“…This indicates that tryptic digestion of proteins can be improved. Based on recent studies [ 74 , 75 ], we believe that increasing the digestion time from 15 to 60 min at 50°C can be our next immediate improvement. In addition to increasing the number of CIPs, better tryptic digestion reduces the occurrences of missed cleavages, allowing the analyses to be done with a smaller allowed MCS.…”