Analysis of Tryptic Digests of a mAb and an ADC with the Agilent 1290 Infinity II LC

by | Mar 23, 2017

Application note describing analysis of tryptic digests of a mAb and an ADC with the Agilent 1290 Infinity II LC.

Agilent Technologies has produced an application note describing the analysis of tryptic digests of the monoclonal antibody (mAb) trastuzumab, or trade name Herceptin, and the antibody-drug conjugate (ADC) ado-trastuzumab emtansine, or trade name Kadcyla using the Agilent 1290 Infinity II LC.

Monoclonal antibodies (mAbs) have emerged as important therapeutics for the treatment of cancer and autoimmune diseases, among others. The successes of mAbs have triggered the development of various next-generation formats including antibody-drug conjugates (ADCs), which enable highly toxic drugs to be selectively delivered to tumor cells, thereby substantially lowering side effects typically experienced with classical chemotherapy.

This application note describes how an Agilent 1290 Infinity II LC in combination with an Agilent AdvanceBio Peptide Map column is successfully applied to tackle challenging separations.

Sample Preparation
A 100 µg amount of protein, diluted in 0.05 % Rapigest/100 mM TrisHCl, pH 8, was reduced at 60 °C for 30 minutes by the addition of 5 mM dithiothreitol, and alkylated at 37 °C for 1 hour by adding 10 mM iodoacetamide. Trypsin was subsequently added at an enzyme-to-substrate ratio of 1:25 (w:w). Digestion proceeded for 16 hours at 37 °C. The final concentration was 0.5 µg/µL.

High peak capacities can be achieved for the analysis of tryptic digests of mAbs and ADCs with an Agilent 1290 Infinity II LC in combination with an Agilent AdvanceBio Peptide Map column. The Agilent V380 Jet Weaver mobile phase mixer is effective in reducing the noise caused by the TFA modifier. This opens opportunities to quantify low abundant species in complex mixtures. The excellent precision obtained with the current setup suggests that this is a valuable tool for detailed analysis of protein biopharmaceuticals such as mAbs and ADCs.


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