BACKGROUND:Fast, efficient and reproducible digestion is critical for LC-MS/MS quantitative bioanalysis of therapeutic proteins. Traditional digestion methods require a pretreatment, such as sequential denaturation, reduction and alkylation, which are very time-consuming.RESULTS:Pellet digestion, which does not require the serial pretreatments of denaturation, reduction and alkylation, was evaluated using a test monoclonal antibody (mAb) with 16 disulfide bonds, and compared with traditional digestion methods. For the test mAb, pellet digestion provided much better digestion efficiency compared with direct digestion, and provided similar or better digestion efficiency compared with digestion-with-pretreatment. ... More
BACKGROUND:Fast, efficient and reproducible digestion is critical for LC-MS/MS quantitative bioanalysis of therapeutic proteins. Traditional digestion methods require a pretreatment, such as sequential denaturation, reduction and alkylation, which are very time-consuming.RESULTS:Pellet digestion, which does not require the serial pretreatments of denaturation, reduction and alkylation, was evaluated using a test monoclonal antibody (mAb) with 16 disulfide bonds, and compared with traditional digestion methods. For the test mAb, pellet digestion provided much better digestion efficiency compared with direct digestion, and provided similar or better digestion efficiency compared with digestion-with-pretreatment. In particular, for two peptides with very low digestion efficiency under direct digestion, pellet digestion improved the digestion yield by approximately 30-fold, which was similar to or better than what digestion-with-pretreatment offered. This method was then successfully applied to an LC-MS/MS assay of the test mAb in monkey serum.CONCLUSION:Pellet digestion will be a very useful technique for high-throughput and reliable LC-MS/MS bioanalysis of mAbs and other large proteins, including ones with multiple disulfide bonds.
