Periodic Reporting for period 3 - iConsensus (Integrated control and sensing platform for biopharmaceutical cultivation process high-throughput development and production) Reporting period: 2020-05-01 to 2021-04-30 Summary of the context and overall objectives of the project 1. What is the problem/issue being addressed?Biopharmaceuticals are large molecules, most often proteins, used for therapy, e.g. antibodies. The biopharmaceuticals are produced using cells in well-regulated processes ('bioprocess') in bioreactor. Today health technology is highly profitable but spending the highest percentage of its revenues on R&D. The development of a new drug costs about 1 billion dollars and takes ≥ 10 years. Reducing cost and time for the development of the manufacturing process represent very large savings and is essential to be first to market. The worldwide community of bioprocessing is in quest for more rational tools to understand, predict and optimize industrial biopharmaceutical manufacturing. 2. Why is it important for society?iConsensus aims to bring a disruptive new technology platform for mammalian-cell based production of biopharmaceuticals. It is expected to reduce the time and costs for the process development and lead to a better manufacturing control. This improvement influences the time-to-market. Competitiveness advantage, higher consistency, efficacy and robustness will be achieved for the biopharmaceutical European industry. All these factors will generate as well reduced cost, failure and risk for product shortage. The project clearly supports European Innovation, competitiveness and industrial leadership while giving tools to improve the European well-being and societal challenges. 3. What are the overall objectives?Mammalian cell cultures, e.g. Chinese Hamster Ovary cells (CHO), used for the production of a biopharmaceutical are complex mixtures of components. Monitoring all the medium components (≥ 70) could provide a better knowledge of the process however it is a very large and tedious task. All these components can have an effect on the process performance, the purification process or on the product quality, which is the reason why monitoring all these compounds is advantageous. To address this, iConsensus provides innovative analytical, hardware, software and high-throughput (HTP) solutions for the development, monitoring and control of mammalian cell cultivation process producing biopharmaceuticals. iConsensus develops an analytical platform with on-line/at-line sensors and detection methods measuring multiple factors in the cell culture for an antibody taken as model. Under advanced data management, the analytical platform is integrated to new micro-bioreactors for HTP screening and to bioreactors. To power the process development and improve manufacturing, iConsensus creates new mathematical models for simulation, optimization or control. Possibility for implementation in GMP production is also a major goal. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far High-throughput microbioreactors (MBR) Novel microbioreactors for mammalian cell culture based on microtiter plates have been designed and tested by creating prototypes for validation. The gassing system allows for simultaneous gas regulation in individual wells. CE chip platformNew methods based on capillary electrophoresis (CE) to measure different components in the culture, e.g. amino acids, sugars, vitamins, are developed and transferred to chip format. The transfer of capillary electrophoresis methods to CE chip has been successful for three types of metabolites. Optical on-line sensorsSeveral sensors based on optical properties are created and proof-of-concept's have been demonstrated. New formats have also been created for pH, oxygen and CO2 sensors for different bioreactors and vessels, including autoclavable sensors. Spectrometry and chemometry supportA generic chemometric partial least square (PLS) regression prediction model was created to predict glucose, glutamate and lactic acid concentration with errors of less than 5 % by using Raman spectra from different cultures and different Raman spectrometers. A generic model for glucose quantification was created without requirement of process specific model. Holographic methodsIprasense has developed a new concept for the quantification of the cell density based on holographic image. The evaluation performed at GSK indicated very close correlation (<10% error) to the reference. The method detects also early viability drop and early apoptotic cells. The system is amenable to integration to HTP bioreactors such as Ambr system. Affinity-based microfluidics The development and validation of the microfluidic bead-based strategy immunoassays were achieved with Rituximab-producing CHO cells samples. Micronit’s devices in cyclic olefin co-polymer, a widely-used plastic, were used for the integration of colorimetric assay for HCP detection and fluorescent assay for IgG titer. DNA quantification was addressed using a bead-based strategy with sample preparation followed by detection with a DNA intercalator. Analysis of glycosylationPAIABio has continued the validation of its lectin based detection technology with samples from Bayer on different antibody glycoforms. Simplified glycan assay workflow has been created to facilitate the transfer into microfluidics. First results from samples of EFPIA partners show that this approach is feasible. Total integration and data managementAn integrated analytical system, 'At-Line Integrated Analytical System' or 'ALIAS', was created. The architecture and functionalities of the ALIAS were defined and put in place, together with the monitoring information database, that we call MID. The MID is today a working tool to gather the data generated by the on/at-line detection systems. In the future, it will be aimed to be publicly available. Modelling of cell culturesKTH developed a fed-batch process, for which the data of the main metabolites were used for modelling. Mechanistic kinetic model by column generation and statistical approach for the kinetics as well as data-driven models were generated. The first results show the capacity to predict the behaviour of the process, and to design software sensors predicting unmeasurable variables. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) iConsensus aims at developing a new platform for real time analysis of the components. Nowadays, the development of cultivation processes uses empirical methods supported by statistics, and high-throughput, where only very few parameters are easily quantified, leading to suboptimal outcome. When more efforts are put on the analytical technology, this requires enormous resources and time. To address this, iConsensus brings solutions by far pioneer, with no equivalent product today, with real-time solutions and advanced MBR. Besides the importance of the use of innovative tools in development it is also crucial to have reliable, fast and qualified analytical tools for commercial manufacturing processes. Data and knowledge gained during development should be usable in a straight line in the final manufacturing process. The possibility to use the same analytical tools throughout the development and life cycle management of a process/product will be highly beneficial for the control and robustness of the final manufacturing process (Process Analytical tools, Design Space). The data-driven systematic generation of models with minimal and managed complexity, applied to the high variable scale datasets will by far be an original achievement. This will namely be supported by the automatic rejection of insignificant parameters (in terms of information content) to reduce the model size. The impacts are given above in Section 2. Overview of iConsensus work packages and partners Development of microbioreactors with spectroscopic online-monitoring Multiplexed microfluidic cartridge for at-line protein monitoring in mammalian cell culture process
