Protein quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an increasingly popular field for pharmacokinetics study in the drug discovery phase. Traditional method development using a ligand binding assay usually requires 2-3 months. By using LC-MS/MS techniques, development times can be reduced to one week. In this white paper, we discuss testing the robustness of membrane affinity versus magnetic beads by using both methods to analyze antibody samples.
The magnetic bead approach and membrane approach produced comparable validation parameters including linearity, intra-, inter-day accuracy and precision, carryover, dilution integrity, matrix effect, specificity, and selectivity.
The two validated strategies for protein immunoaffinity purification were compared and both methods offered the same LLOQ of 0.05 μg/mL. The main differences was with the sample processing time. Specifically, the sample immunoaffinity purification (IP) processing time for a 96-well plate with the magnetic bead method was 3-4 hours in comparison to the 10-20 minutes by the membrane approach. The magnetic bead method was also more prone to operational pipetting errors.
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