Periodic Reporting for period 2 - A4B (Analytics for Biologics)
Okres sprawozdawczy: 2019-10-01 do 2022-03-31
The market of therapeutic proteins (TPs) is a growing field in the pharmaceutical industry. Production of TPs originates in a mixture of very similar biomolecules, called protein species respectively proteoforms (synonym). Some of these differences can have an impact on stability, half-life, safety, and efficacy of the TP, sometimes with harmful consequences for the patient.
Thus, methods for qualitative and quantitative the analysis and purification of TPs are urgently needed as well as specialists in the field of TP manufacturing. In an Europe-wide joint research and training network the graduate school A4B has offered a specific research and training program yielding improved and new methods for analysis and purificaton of TPs as well as the required specialists.
Within the joint A4B-project, students focused on the improvement of manufacturing TPs as well as the development of analytical tools for identification and quantification of TP-species (TP-S) and their separation. For controlling effectiveness and design of experiments, especially fast and reliable quantitative methods were developed. The improved and new tools comprise the analysis of many different types of posttranslational modifications (PTMs) including glycans, with which the TPs can be modifed. For this aim, advanced electrophoretic, liquid chromatographic, and mass spectrometric approaches in combination with bioinformatics were applied. The methods developed in the A4B project were used to optimise purification and characterisation of TP-S as well as to study the metabolism of TPs in vitro and in vivo.
In summary, the Analytics for Biologics (A4B) ITN was successfully training 15 early-stage researchers (ESRs) to become specialists production, purification and analysis of TP-S. All A4B students received a basic education in manufacturing and marketing of TPS. Through their research projects the students developed in depth expertise in one of the areas covered by the consortium.This project thus generated urgently needed new methods for an improved analysis and purification of TP-S, while at the same time ensuring that well qualified young researchers are available for this rapidly growing area of the pharmaceutical industry.
Thus, methods for qualitative and quantitative the analysis and purification of TPs are urgently needed as well as specialists in the field of TP manufacturing. In an Europe-wide joint research and training network the graduate school A4B has offered a specific research and training program yielding improved and new methods for analysis and purificaton of TPs as well as the required specialists.
Within the joint A4B-project, students focused on the improvement of manufacturing TPs as well as the development of analytical tools for identification and quantification of TP-species (TP-S) and their separation. For controlling effectiveness and design of experiments, especially fast and reliable quantitative methods were developed. The improved and new tools comprise the analysis of many different types of posttranslational modifications (PTMs) including glycans, with which the TPs can be modifed. For this aim, advanced electrophoretic, liquid chromatographic, and mass spectrometric approaches in combination with bioinformatics were applied. The methods developed in the A4B project were used to optimise purification and characterisation of TP-S as well as to study the metabolism of TPs in vitro and in vivo.
In summary, the Analytics for Biologics (A4B) ITN was successfully training 15 early-stage researchers (ESRs) to become specialists production, purification and analysis of TP-S. All A4B students received a basic education in manufacturing and marketing of TPS. Through their research projects the students developed in depth expertise in one of the areas covered by the consortium.This project thus generated urgently needed new methods for an improved analysis and purification of TP-S, while at the same time ensuring that well qualified young researchers are available for this rapidly growing area of the pharmaceutical industry.
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.
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.
In February 2022, the symposium “Bridging Genotypes with Phenotypes via Proteoforms” took place in Hamburg, organized by the Hartmut Schlüter (UKE) and his team as final meeting of the A4B project summarizing the work which was done by each beneficiary and for discussing the next steps after the A4B project ends. Several ESRs and PIs presented their work within the A4B project. It was concluded that an international network-building action (COST-action) is needed to speed up the progress in proteoform analysis. Seven members of the A4B consortium consequently decided to apply for a COST action grant in 2022 focusing on proteoforms for building a network, which will include the results of the A4B project as fruitful basis for further improving proteoform research on an interdisciplinary scale.