This article from issue 16 of the Analytix Reporter explores an HPLC-UV method for three different monoclonal antibodies in standards and simulated matrix, to enable fast and reproducible results while handling high matrix loads.
INTRODUCTION
Monoclonal antibod (mAbs) manufacturing is often done by fermentation processes where the amount and quality of the expressed product are essential and are monitored via affinity chromatography. This antibody titer determination can be carried out by using a Chromolith® WP 300 Protein A HPLC column.
In this work, bind and elute experiments are shown for three different monoclonal antibodies: cetuximab, trastuzumab, and the universal antibody standard, human, on a Chromolith® WP 300 Protein A column.
EXPERIMENTAL PROCEDURE
Three mAbs in standard solutions and three simulated matrix samples were prepared using SILu™ Lite SigmaMAb standards and the matrix standard, SigMatrix Serum diluent, a 6% recombinant HSA (Human Serum Albumin) in phosphate-buffered saline (PBS) solution, pH 7.4, that can be used as a blank or matrix/diluent for proteinaceous analytes in LC-MS/MS calibrators, controls, or samples. The standard solutions and matrix samples were analyzed on a 25 x 2 mm I.D. Chromolith® WP 300 Protein A column under the conditions shown in.
See the full article for reagent and sample preparation specifications.
RESULTS & DISCUSSION
Read the full article for comprehensive figures and tables detailing the results.
CONCLUSION
It could be shown that all three antibodies (universal antibody standard, human, cetuximab, trastuzumab) can be analyzed reproducibly using the Chromolith® WP 300 Protein A column.
A fast separation, within about 2 min, can be achieved, with high linearity values, for a broad range of injected sample amounts.
Furthermore, it was demonstrated that the column can handle a high matrix load for multiple injections. A standardized matrix was used for this study (SigMatrix Serum diluent).
*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.