Early twentieth century, Paul Ehrlich reported on “magic bullets” to cure a wide range of diseases thereby visionarily refering to antibodies [1,2]. The development of the hybridoma technology in 1975 by Köhler and Milstein, enabling the production of monoclonal antibodies (mAbs), and the introduction of the first therapeutic murine mAb in 1986, bridged the gap between concept and clinical reality [3]. Four decades of further maturation resulted in the European and United States approval of well-beyond 100 therapeutic mAbs, spanning multiple formats, such as canonical mAbs, antibody-drug conjugates (ADCs), bispecifics and fragments [4], [5], [6], [7], [8]. With a sales value exceeding $ 200 billion in 2022, mAbs account for 80% of protein biopharmaceutical sales and are the fastest growing class of therapeutics [8]. Together with an enormous therapeutic and market potential comes an immense structural complexity. Unraveling the characteristics of these highly heterogeneous molecular giants, demands for a wide range of complementary analytical tools and methodologies with chromatography and mass spectrometry at the forefront [9], [10], [11].

In recent years, there has been a tendency to merge several of these methods in one analytical platform. This has been facilitated by the introduction of commercial and robust online multidimensional liquid chromatography (mD-LC) instrumentation. Various reports describe the combination of cation exchange chromatography (CEX), anion exchange chromatography (AEX), size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and affinity chromatography in online two-dimensional (2D) LC-MS set-ups for the automated characterization of mAbs [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. Recently, a true 3D-LC-MS configuration was presented with Protein A affinity chromatography in the first dimension (1D), a multi-method option in the second dimension (2D) (choice between SEC, CEX and HIC) and desalting SEC-MS in the third dimension (3D) [35]. 1D and 2D peaks are stored in loops installed on (multiple) heart-cutting valves prior to transfer to the next dimension. This multi-attribute analyzer allows simultaneous and sequential assessment of mAb titer, size/charge/hydrophobic variants, molecular weight (MW) and post-translational modifications (PTMs) directly from cell culture supernatants.

To unambigously identify mAb species, various groups explored the incorporation of chemical and enzymatic reactors in online mD-LC-MS set-ups allowing middle-up and/or bottom-up analysis [14,[36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]]. Indeed, while intact mAb measurement is indicative for identity and highlights dominant modifications having mass differences beyond the mass accuracy of the MS instrument, acquisition at subunit and, particularly, peptide level provides essential information related to amino acid sequence, modification sites and modifications resulting in zero/minor mass differences (aspartate isomerization, asparagine deamidation). This triggered Gstöttner et al. to pioneer a mD-LC-MS method incorporating 1D CEX, peak collection, 2D RPLC desalting, denaturation and reduction, 3D on-column trypsin digestion and 4D RPLC-MS based peptide mapping for the in-depth and automated characterization of mAb charge variants [36]. In comparison to the classical offline approach, this innovation substantially reduces turnaround time, sample manipulation, loss and artefacts. Subsequent manuscripts described various applications and several adaptations/extensions on this mD-LC-MS workflow [31,[37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]]. Goyon et al. and Camperi et al. replaced 1D CEX by, respectively, 1D SEC and 1D Protein A affinity chromatography [40,42]. The latter study reported on both bottom-up and middle-up analysis directly from cell culture harvest by diverting the 2D flow, carrying reduced mAbs, either to a 3D trypsin column or 3D HILIC-MS [42]. In two subsequent manuscripts, the same group reported on the use of mD-LC-MS bottom-up analysis using 1D Protein A chromatography to assess multiple quality attributes throughout the cell culture production process of a mAb and heavily glycosylated Fc-fusion protein including real-time monitoring in the bioreactor [45,46]. A variety of PTMs including oxidation, deamidation, succinimide and glycosylation (sialylation, fucosylation) were characterized. These studies also introduced on-column LysC digestion as complementary protease to trypsin to increase sequence coverage. Oezipek et al., described a parallel 3D trypsin and LysC on-column digestion set-up followed by combined peptide mapping to prevent loss of small and polar peptides [47]. A post-digestion 4D RPLC trapping pre-column was furthermore included thereby decoupling the 3D digestion step from 5D peptide mapping enabling the use of long sub 2 µm RPLC columns and high system pressures. Beyond trypsin and LysC, immobilized IdeS has been employed in mD-LC-MS set-ups for middle-up analysis [41] or for subunit bottom-up characterization [43]. The above described configurations are dependent on the availability of robust immobilized protease columns. To offer more flexibility Mayr et al. recently introduced a mD-LC-MS workflow with in-loop enzymatic digestion [50]. The set-up facilitates the addition of any enzyme to multiple heart-cuts for multilevel analysis, e.g. peptide mapping, fragment generation, deglycosylation, etc. and might be subject to further exploration in the coming years.

The current study adds to the above-reported flavours of mD-LC-MS and describes a protein analyzer with 1D multi-method option and parallel middle-up and bottom-up MS acquisition for the automated characterization of charge, size and hydrophobic variants. It differentiates from earlier studies in the option to select several 1D chromatographic modes via a column selector valve (CEX, SEC and HIC) and in the simultaneous LC-MS acquisition at subunit and peptide level allowing unprecedented structural characterization. The workflow is challenged by the analysis of a recombinant human immunoglobulin 1 (IgG1)-derived Fc fragment (residues D221-K447 according to EU numbering scheme) with enhanced affinity for the neonatal Fc receptor (FcRn) through the substitution of five amino acid residues (M252Y, S254T, T256E, H433K and N434F) [51]. Binding to the FcRn receptor extends serum half-life of IgGs, including disease causing autoantibodies, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases [51], [52], [53], [54].