The project concerns the establishment the first platform in Sapienza with the capability to meet modern-day challenges in therapeutic protein production, for both the Research and Development (R&D) cycle and Quality Control (QC) process. With the ambitious goal to create a multidisciplinary network for biotechnological applications, the project is centered on the acquisition of equipment combining precise mass measurements of proteins, high-throughput of analyses, user-friendly sample preparation, and researchers self-using. MALDI-ToF/ToF is the preferred solution, being the indispensable technique for a large number of purposes in biomolecules analyses.
Among several applications, the most challenging commitment for PREMISE is the physical chemical characterization of biosimilars from plants. The development pathway of a biosimilar is somewhat different from a novel biotherapeutic with a greatly increased requirement for physicochemical analytics compared to a novel biological molecule. The recently revised EMA quality guideline provides some additional clarification about analytical strategies. State-of-the-art analytical methods must be used to assess composition, physical properties, primary and higher order structure, purity, product-related substances and impurities to be compared between the biosimilar and the originator. Advances in MALDI instrumentation and proteomics/glycomics strategies enable rapid identification of structural data such as monosaccharide composition, glycan populations, antennary profiles, antennae linkages and glycosylation sites.
Within PREMISE these issues will addressed settled on competence from three Sapienza Departments. Interaction with internal and external academic groups and with non-academic components will be implemented to offer a cutting-edge tool for a precise and fast structural screening of molecules in different areas of basic and translational research.
Currently, several developing expression systems based on heterologous cultures are used for production of recombinant proteins. A high proportion is produced in bacteria, where secretion out of the cytoplasm is a preferred strategy. However, prokaryotic platforms have severe limitations and there is an overwhelming need for new production systems (yeast, insects) that can deliver these products in greater yields, with higher quality and at lower costs.
Sapienza has a key position in the R&D (Research and Development) of "molecular factories". Several Departments at the Life Science Area are operating at an intersection employing advanced knowledge of the mechanisms of functioning of living organisms with the most advanced technologies available in biology, chemistry, physics and other disciplines to meet challenging issues in production of pharmacological proteins. Collaborative projects, public/private financed, proved the attractiveness of Sapienza in this area in these last years. Moreover, successes in development and refinement of expression technologies are reflected in a large body of literature and patents over the last decade.
The extensive use of recombinant host systems for the protein production demonstrated that the unnatural bioreactor environment might promote mistranslation events. On the other hands, the requirement of stable protein products demands for structural engineering by chemical modifications (e.g, PEGylation, glycosylation, etc). Therefore, high variability in terms of biomass accumulation and structural integrity/fidelity of protein products are highly compelling issues toward the development of QC (quality control) platforms reliable to implement an accurate and high-throughput evaluation of the protein expression.
"PREMISE" aims to provide Sapienza with a cutting-edge platform, which could meet for the first time these challenges through the provision of informatics packages and instrumentation with unique capabilities. At the core of this platform is the MALDI-ToF/ToF technology, appropriate to i) sustain a high number of analyses on crude samples, ii) carry out structural characterization of target molecules with high accuracy, iii) combine specificity, sensitivity, speed, automation and convenience in QC processes.
Comparing this proposal with cutting-edge ESI-based mass spectrometry platforms already within Sapienza, the exclusive features of MALDI source (desorption/ionization on dried samples) and of the Time of Flight, ToF, analyser (the largest dynamic range in mass measurement by MS) will be the requisite for flexible analyses. PREMISE will be covered all the analytical requirements in the pipeline of a biopharmaceutical production, from bioreactor monitoring through metabolite profiles to the screening of drug interactions. Moreover, MALDI-ToF is a user-friendly technology: this remarkable trait will allow a "free-usage" access to all groups supporting this proposal (see Regulations).
At present, another MALDI-ToF/ToF-based platform is present in Sapienza (Dept. of Cellular Biotechnology and Hematology), although using discontinued equipment and mainly focused in translational medicine. The innovation of our proposal is to create a specialized facility, based on a MALDI-ToF/ToF of new-generation, for the QC in the production of therapeutic proteins. Competences in protein chemistry, protein stability and in protein mass spectrometry at the Dept. of Biochemical Sciences will be the cornerstone of this project. Building upon these there will be the expertise in techniques and methodologies required by the QC processes from the Dept. of Chemistry and Technology of Drugs. The last few years have seen pharmaceutical companies invest significantly in plant-based technologies ("molecular farming"). Indispensable linchpins in development and optimization of protocols for screening integrity/fidelity of mammalian protein products expressed in plant systems will arise by competences from the Dept. of Biology and Biotechnologies.
In conclusion, PREMISE intends to represent the novelty of being a multidisciplinary tool for each Protein Factory/Farming in Sapienza, fully integrated with the R&D and QC areas, and suitable to meet modern-day challenges in therapeutic protein production. Nevertheless, PREMISE, performing high-throughput and flexible mass measurements, will suitable to support also life science research in Sapienza (listed above).
The proposed platform is structured for extensive collaboration (see Regulations), and then could be for Sapienza the "PREMISE" for the additional opportunity to be an attractive funding point. The cross-service cooperation through PREMISE will be the basis for being awarded grants provided by the nationwide funding programs. Moreover, PREMISE stands for biotechnology-oriented regional alliance, and for cooperation in R&D projects undertaken by several companies or between companies and Sapienza.