#	Pagina
attuale pagina	/open-h2020/projects/211895/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - A4B (Analytics for Biologics)

Teaser

Summary

The Analytics for Biologics (A4B) ITN aims the training of 15 ESRs to become specialists in purification and analysis of therapeutic proprotein species (TP-S).
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. 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.
Using state of the art chromatographic methods, mass spectrometry (MS) and capillary electrophoresis, the A4B-network aims to develop faster and more economic bioanalytical tools for the effective separation and analysis TP-S.
The project aims the development of, fast, automated, reproducible, sensitive, selective and cost-effective tools for qualitative and quantitative analysis of TP-S, including determination of their chemical composition. As well as the development of software tools needed for the processing of the complex MS data, efficient and cost-effective methods for purification of target TP-S and removal of undesired TP-S. The developed methods will be applied to optimize the purification and characterization processes of TP-S upon protein metabolism in vitro and in vivo.

Work performed

At the TP production facilities at BOKU, two ESRs are specializing in the optimization of TP production and downstream processing. Newly acquired bioreactors were designed and optimized by ESR13 according to the needs of the in-house cell line and different operational modes (batch and continuous) were tested. New prototypes were designed by 3D-printing and evaluated based on their potential to extend or even substitute conventional process units. First results of a prototype were promising and patent processing is pending. Preliminary evaluation of a second prototype shows promising results. A soft sensor in periodic counter current chromatography (PCCC) which functions an on-line monitoring tool for continuous bioreactors, allows the prediction of the concentration of antibody in the perfusion fermentation was implemented. PCCC generates an information feedback loop rendering titer determination in the fermentation obsolete.
Different approaches in the analysis of TP-S are being developed and tested. ESR1 is working on intact analysis of model proteins, investigating the most appropriate parameters for the fast quantification of the TP-S and has established a flow injection analysis (FIA) method that is applied as a read out for the experiments of ESR2. ESR2 is screening chromatography parameters to separate TP-S by sample displacement batch chromatography (SDBC).
Focusing on the analysis of intact TP-S, ESR6 at SDU applied advanced MS technologies to fragment intact proteins and large protein fragments in the MS, to obtain detailed information about the sequence and coexisting post translational modifications (PTMs) on different TP-S.
To determine the masses of intact TP-S, an ultrafast and high-quality novel algorithm (â€œFLASHDeconvâ€) was developed by ESR3 at EKUT and published online.
Using bottom-up MS near to full sequence coverage of the TP filgrastim using chymotrypsin has been achieved by ESR1. Furthermore, unreported PTMs on cysteine residues have been observed. A novel reagent with unique cleavage specificity to the amino acids serine and threonine was established and revealed >90% sequence coverage of filgrastim.
ESR14, has developed a multi-attribute monitoring (MAM) method based on peptide mapping of reduced protein samples for the characterization of monoclonal antibodies attributes like, glycosylation, deamidation and oxidation. A MAM method involving characterization and quantification of protein disulfide bridges is in development.
The TP-S are not only characterized by their primary amino acid sequence but also by their PTMs. ESR 15 has established a parallel reaction monitoring (PRM) method to analyze PTMs (e.g. deamidation and oxidation) occurring in Pertuzumab from serum samples. Due to their variety, glycosylations are of the most challenging PTMs to study. ESR8 has been developing novel tags with simultaneous fluorescence and MS detection capabilities (VTAGS) that allow rapid glycan characterisation of TPs. The tags allow site specific analysis of TP glycosylation from released glycans and glycopeptides. Therefore, ESR8 has synthesized the ITAG-NHS tag and used it to label glycans after release from the TP. In addition, the compound was used to label monoclonal IgG glycopeptides. Based on the gained knowledge, a third phase of VTAG synthesis will begin.
ESR10 is developing novel chromatographic methods to separate TP-S of Trastuzumab and Pertuzumab. The separated TP-S will be analysed by peptide mapping for modifications as well as by a Her-2 receptor binding assay. ESR9 focusses on the enrichment of Trastuzumab and Pertuzumab TP-S from blood plasma using recombinant Her-2 anti-idiotypic antibodies for Trastuzumab and custom-made affimers for Pertuzumab (Avacta Life Sciences). The affimers are being evaluated for their ability to enrich different species of the targeted TPs.

Final results

By the end of this project we expect to obtain fast and robust methods to separate, quantify and characterize TP-S by MS. Therefore, the separation techniques like SDBC will help to enrich TP-S for in-depth analysis and for removing species from the product.Fast quantification of intact species with MS will be applied for optimisation and controlling of the manufacturing of TPs including up- and down-stream processing and ensure a high quality of the TP products in the biopharmaceutical industry. Hence, decreasing the time and the amount of protein needed to fully characterize the protein species.
Newly developed powerful algorithms and a complete data analysis workflow will enable efficient yet accurate quantitative TP-S analytics from complex top-down MS data.
In depth characterization of the TPs will be achieved by very high or full sequence coverage including quantification of TP-S using stable isotopic labeling, as well as the application of MAM methods for comprehensive characterization and quantification of challenging TP-S, like disulfide variants, and the synthesis of the next generation of VTAGs that can be used for simultaneous fluorescence and MS analysis of gycans and glycopeptides. Unlike the current commercial offerings these VTAGs critically maintain the ability to be digested with core fucosidases. This approach, fluorescent labelling of glycopeptides for site analysis with MS has never been published and represents a truly novel approach to biotherapeutic glycan characterization.
To understand the processing of TP-S in the treated patient, an in vitro system able to simulate in vivo conditions for testing and predicting modifications of TPs will be developed and validated, that might enable the prediction of the degree of modifications in serum with possible impact on pharmacokinetics, safety and efficacy.
The first detailed study of the biotransformation of monoclonal antibodies in patients treated with TPs will be conducted. We expect to discover modifications that abrogate activity and establish a link between such modifications and response to therapy. The final goal is to use MS as a novel approach to monitor such forms in patient plasma in view of establishing the proper dosage, and patient response in the context of personalised medicine.