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 characterisation of the mAb would help in optimising the production steps. Efficiency in mAb characterisation can be obtained by automating analysis and reducing hands-on time. Although mass spectrometry (MS) is an essential technique for detailed characterisation 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 characterisation 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 characterisation of mAb subunits was made possible by digestion of the mAb online by an immobilised IdeS enzyme. The addition of a disulfide bridge reduction step online led to analysing smaller molecules to access fine characterisation.
Two approaches were developed to characterise mAbs with mass spectrometry using innovative two-dimensional liquid chromatography workflows. For both approaches, detailed information on monoclonal antibodies can be obtained at the intact or subunit levels without any sample preparation, using a fully automated and GMP-compliant 2D-LC–MS system.
With these techniques, optimisation of mAb production process is made easier and faster by analysing samples without manual purification. The second approach shows the potential for automated QC multiple quality attributes monitoring of mAbs without any sample preparation.
These methodologies open the way to fully compliant at-line monitoring of mAb quality attributes.
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