Mobile Phase Solvents and Reagents for LC-MS Biopharmaceutical Applications

Biopharmaceutical drugs are a unique class of therapeutic proteins that have far more complex structures than small molecule drugs. Liquid chromatography (LC) coupled with mass spectrometry (MS), LC-MS, has become increasingly popular for characterizing biopharmaceutical drugs. LC-MS is widely used for characterizing the primary structure of protein drugs such as amino acid sequencing for peptides/proteins, profiling of process-related impurities such as host cell proteins (HCPs), disulfide bond mapping, N-glycan profiling of monoclonal antibodies (mAbs), and nucleotide sequencing for oligonucleotides.

At the primary structure level, the protein post-translational modifications (PTMs) that arise at different stages of manufacturing and storage can impact drug efficacy and safety; thus, PTMs are classified as critical quality attributes (CQA) that should be monitored and controlled. Combining the power of LC separation with the sensitivity and specificity of MS detection, LC-MS is the tool of choice for peptide mapping to detect multiple PTMs in a single analytical run, i.e., the multi-attribute method (MAM) workflow. A critical component of the MAM workflow is to separate peptides of various lengths from the digested therapeutic protein. This is typically achieved by employing a long LC gradient of water and acetonitrile with formic acid added as a modifier, as shown in Figure. Mobile phase solvents and reagents must be of high purity with low organic impurities to ensure no interference with detection by electrospray (ESI) MS.

 Figure. Typical LC gradient in a MAM workflow for the separation of NIST mAb peptides with a binary mobile phase: A: 0.1% formic acid in water (Cat. No. LS118); B: 0.1% formic acid in acetonitrile (Cat. No. LS120), from Reference [1].