Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants

Abstract

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC–MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC–MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC–MS and mD-LC–MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

Sample transformation in online separations: how chemical conversion advances analytical technology

“…An example is the work of Mayr et al where an automated in-capillary digestion was used to perform various enzymatic reactions within a heart-cut multi-dimensional system. 50 Up to 6 fractions from an ion exchange chromatography (IEC) charge variant separation of monoclonal antibodies were collected and in-capillary digestion allowed for fragment generation (bottom or middle-up), or deglycosylation followed by RPLC-MS. The digestion times varied between minutes and hours depending on the reaction performed.…”

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…As an alternative to immobilized protease cartridges, a novel mD-LC-MS analytical set-up was reported, involving looped enzymatic digestion between the 1 st dimension column and transfer to the 2 nd dimension, followed by MS or MS/MS analysis [38]. One of the main benefits of this configuration was that it facilitated the spike-in of any enzyme to multiple heart-cuts for multi-level analysis.…”

“…(H) TIC view of the heart‐cut IEX main peak after LysC/trypsin digestion. Adapted [38], with permission.…”