An overall conclusion of the results of the A4B project is that the topic of proteoforms is even much more challenging than the consortium assumed at the beginning of the project.
With bottom-up proteomics features like e.g. deamidated peptides from therapeutic recombinant monoclonal antibodies can be detected, which can have different proteoforms in common, as was demonstrated very well by ESR 9, ESR 10, ESR 14 and ESR 15. However, the bottom-up approach does NOT give access to the individual specific proteoforms because the proteins are digested. Thus, the analysis of intact proteoforms is only possible with transferring intact proteoforms into mass spectrometers (top-down mass spectrometry (TD-MS)), which is still in its infancy compared to mass spectrometry-based bottom-up analysis of proteins. Nevertheless, within the A4B project we improved the application of TD-MS significantly with respect to the quantification of proteoforms (ESR 1, ESR 3 and ESR 7). Even much more challenging than quantification of proteoforms with TD-MS is the identification of proteoforms. ESR 7 observed that the optimization of fragmentation is necessary for each individual TP for yielding optimal results with respect to the sequence coverage. The separation of proteoforms with liquid chromatography (LC) is even less well developed compared to mass spectrometry. ESR 2 demonstrated that the displacement chromatography mode is the most efficient elution mode compared to isocratic and gradient elution for separating proteoforms. Further, ESR6 has successfully worked on native-mode protein separation methods, with a focus on (1) capillary electrophoresis (CE-MS) online coupled to electrospray ionization MS and (2) isoelectric chromato-focusing on an anion exchange column with online electrospray MS coupling. In addition, Fc-gamma- receptor affinity chromatography, another native-mode separation technique was established by ESR5. For the investigation of pharmacokinetics and metabolism of TPs, enrichment methods prior to mass spectrometric analysis are needed due to the high complexity of biological samples. The combination of affimers and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. Regarding this ESR9 and ESR10 successfully established methods for the enrichment, separation and monitoring of TPs to investigate the in vivo biotransformation of TPs. For the optimization of the TPs production, a fully continuous operation process was developed by ESR12 and ESR13 by integrating the perfusion system with the capture unit, which means that the upstream and downstream unit were combined. In collaboration with ESR6, ESR12 and ESR13 tested the optimized process, confirming that a continuous reconstitution of the medium results in the same quality of the product compared to a batch process. A patent application for the Continuous Reconstitution of Process Materials From Solids is pending.
In summary, despite the many successful outcomes within the A4B project there is an urgent need for further improvement in the techniques for the separation of proteoforms. Improvements in TD-MS are required ensuring a much faster optimization of parameters for yielding high sequence coverages of proteoforms. All these necessary future improvements in LC and MS as well as LC-MS are not possible without advanced bioinformatic solutions and therefore must be accompanied with improvements in bioinformatics focusing on proteoform analysis.
As part of the A4B programme, the ESRs attended several conferences and presented their achieved results. A total of 33 publications could be published so far, which still shows the need for research on proteoforms.
