SATIN: Separation and Analysis of Therapeutic and Industrial proteiNs

In the decades ahead of us industrial enzymes and therapeutic proteins will become undoubtedly key molecular entities used in the food industry and human/animal healthcare. Their role is rapidly increasing in this era of advanced biotechnology, with a large part of the newly approved drugs being already protein based. However, due to their large size, biology driven production, and complicated structural features therapeutic proteins and industrial enzymes form some of the most challenging molecular entities to be functionally and structurally characterized. Such a characterization ideally involves the charting of all functionally important proteoforms (i.e. all protein variants originating from one gene). Due to the plethora of modifications often occurring on a given protein backbone, such as disulphide bridges, widespread and extensive glycosylation, phosphorylation, proteolytic truncation, amidation, oxidation, glycation and conformeric variants, tens to thousands of proteoforms may co-occur for a single natural enzyme or engineered antibody, each with its own bioactivity and clearance spectrum. Moreover, another emerging trend in biotechnology is the chemical modification and/or bioengineering of industrial enzymes and therapeutic proteins to enhance their efficacy, with antibody-drug conjugates and “bio-betters” representing some of the well-known emerging classes of such future medicines. Evidently, the chemical moieties attached may even further increase the structural diversity of these proteins, requiring even more in-depth structural characterization. The main goal in this proposal is to develop much-needed new analytical strategies enabling the separation and characterization of all complex proteoforms of industrial enzymes and biotherapeutics in their intact native state. Distinctively, our novel analytical workflows are aimed at the genuine intact proteins and will allow us to simultaneously establish structure-function relationships of proteoforms and aggregation states of proteins. This information is typically lost in current conventional approaches. We aim through this proposal at developing new mass spectrometric methods, and analytical workflows, optimized to analyze complex and large glycoproteins, led by the group of Heck (UU). Our aim is to detect with higher sensitivity and selectivity intact native industrial and therapeutic glycoproteins and their relevant protein assemblies (e.g. aggregates). Hereby we will develop new approaches to obtain analytical breakthroughs allowing these native proteins to reach with more efficiency the detector. From a mass spectrometric angel, gains in mass resolving power, sensitivity, robustness, selectivity and sequencing efficiency will be some of the main deliverables. Development of intact native protein separation techniques forms a second pillar of this proposal, led by the group of Wuhrer (LUMC). We aim to achieve the separation of proteoforms prior to (native) MS analysis and functional characterization to reduce heterogeneity and allow structure-function relationship determination. Workflows for the separation of the heterogeneous glycoproteins require the full spectrum of separation techniques based on different separation principles. Making use of orthogonality in methods, smart hyphenation via new interfacing, inlet and ion transmission solutions, we aim at resolving, identifying and quantifying the plethora of functionally relevant protein modifications. In separating native proteoforms we foresee a major role for protein affinity chromatography exploring interactions that are relevant for the mode-of-action of biopharmaceuticals. We will work on preparative native separation to generate sufficient materials for relevant functional tests. The more established complementary bottom-up approaches, relying on proteolytic processing for protein backbone and glycosylation analysis, are well-established in the laboratories of the academic and industrial applicants and will be used to complement intact-level analysis. A major aim in this proposal is also to develop algorithms and software allowing us for the first time to integrate the information gathered by different levels of product characterization. Firstly, the group at the UU will extend their data-integration software suite to be able to integrate bottom-up, middle-down, top-down and native MS analysis data on structurally highly complex industrial enzymes and biotherapeutics, as this may provide the only means to get a complete qualitative and quantitative picture of the proteoform profiles of the investigated glycoproteins. An important next aim is to integrate also data obtained with the different applied separation technologies into such analyses as well as the qualitative and quantitative glycan profiles of the same products generated at the LUMC using dedicated glyco-profiling software. The most successful outcomes of the optimization process in mass spectrometry and chromatography will be hyphenated with each other through exchange of technologies and researchers (e.g. PhDs, Master and ASPT students) from UU and LUMC, and made available to the industrial partners within the consortium (DSM, FrieslandCampina and Roche) via secondments and training sessions, enabling the detailed characterization of structural and functional features of industrial and biopharmaceutical proteins for establishing structure-function relationships. These insights will speed up development of new or improved protein products of the different industrial partners. Our research is expected to have significant utilization impact in a wide variety of application fields, three of which are specifically addressed in this proposal: the pharmaceutical industry, represented by Roche, industrial enzymes, represented by DSM and milk proteins, represented by FrieslandCampina.