New 2D-LC–MS Approaches for the Analysis of In-Process Samples and for the Characterization of mAbs in a Regulated Environment

Biologics, and in particular monoclonal antibodies (mAbs), are an important class of therapeutics, and their market share keeps growing. The production of antibodies is a complex and lengthy process. In-process characterization of the mAb would help in optimizing the production steps. Efficiency in mAb characterization can be obtained by automating analysis and reducing hands-on time. Although mass spectrometry (MS) is an essential technique for detailed characterization of biomolecules, its use is limited to purified samples. However, the hyphenation of an MS system to two-dimensional liquid chromatography (2D-LC) allows for the analysis of more complex samples. The first dimension of a 2D-LC system can be used to purify the sample from its matrix or separate compounds using mobile phases that are not MS-compatible, whereas the second dimension coupled to MS can be used to desalt or separate the different variants or species obtained on the first dimension. A 2D-LC–MS system installed in a full good manufacturing practice (GMP)-compliant environment using validated software was used for the characterization of mAbs in complex mixtures at the intact and subunit levels using a Protein A affinity column with no sample preparation steps. In the second application, MS characterization of mAb subunits was made possible by digestion of the mAb online by an immobilized IdeS enzyme. The addition of a disulfide bridge reduction step online led to analyzing smaller molecules to access fine characterization.