Periodic Reporting for period 2 - NERS (Novel Electro-Responsive Protein Separation Method with Magnetic Nanoparticles) Reporting period: 2022-01-01 to 2022-12-31 Summary of the context and overall objectives of the project Purification is a critical step in drug manufacturing, which helps minimise undesired materials that compromise drug efficacy. Protein tags – peptide sequences attached to proteins – are commonly used to detect and purify expressed proteins. The EU-funded NERS project will explore more efficient protein purification approaches to help retain properties of pharmaceutically relevant proteins such as antibodies. It will extend prior work on the design of protein tags that had high binding affinity and selectivity for iron oxide magnetic materials. The project will develop an elution process that will help separate proteins containing peptide tags from those interacting with electric fields.Pharmaceuticals for cancer therapies and other diseases are very since the production of therapeutic molecules such as antibodies is costly and every production process needs to be developed individually. Especially purification processes, which make up to 80-90% of the whole production, need to be improved or new ideas need to be developed. Short peptide sequences, so called “tags”, can be used to create new purification strategies based on the biomolecule recognition of these sequences. Magnetic iron oxide nanoparticles are an interesting counterpart for peptide tags as their properties facilitate an easy handling and manipulation. I developed such a magnetite-binding peptide tag which allows the purification of tagged model proteins from fermentation broths by changing the surrounding media. However, such pH and buffer switches might also alter the properties of pharmaceutically relevant proteins such as antibodies. The challenge of this project is to establish a novel elution process based on an electrical potential switch instead of a pH switch. The process contains the magnetic separation of proteins containing the peptide tags and the elution of proteins based on the change of tag-particle interactions with electric fields. The use of this system will help to understand the binding of proteins to iron oxide nanoparticles and the formation of an electrochemical double layer in external fields. The electrical double layer formation is not only interesting in biotechnological processes but for the understanding of electrochemical catalysis and energy storage. This idea might pave the way to completely new approaches in biomolecule recognition, protein detection and purification.Objectives:1. The development of a suitable electrode set-up to which magnetic nanoparticles can be magnetically transported and where a potential can be applied which influences the electrochemical double layer around the magnetic nanoparticles. This set-up represents the proof of principle for influencing the electrochemical double layer of nanomaterials magnetically deposited to an electrode and will be the basis for further improvements and applications.2. The evaluation and development of suitable nanomaterials for the binding of tagged proteins, the magnetic separation and the control of the electrochemical double layer with a potential switch at the electrode set-up. This objective aims to prove the possibility to bind and elute proteins by a potential switch in a similar manner as by a pH shift.3. The application and exploitation of this technique for new processes. Investigation of the protein separation and purification principles under “real” conditions in complex media such as crude cell lysates. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Different electrode set-ups and different conditions were investigated towards their effect on the surface potential of iron oxide nanoparticles. Two and three electrode set-ups (with reference electrode) will be tested towards size of electrode, how to transport particles to the electrode, distance between electrodes, electrode materials (carbon, iron, copper, silver) and applied potentials. Impedance spectroscopy was conducted in order to evaluate the capacity of the electrochemical double layer. The surface with deposited nanoparticles was investigated with impedance spectroscopy. Beforehands, nanoparticles have been characterized with TEM, zeta potential, DLS and IR spectroscopy.Verification of model protein binding to the nanoparticle surface in accordance with previous works was conducted with negatively charged amino acids and peptides (Glu8). Evaluation of similar transport properties between particles with and without bound proteins was investigated with Gul4Gly4Glu4)-tagged GFP proteins. The aggregation of iron oxide nanoparticles will be investigated with dynamic light scattering for different salt concentrations and physiological pH conditions. Magnetic nanoparticles are characterised with magnetometry and transmission electron microscopy. The agglomeration behaviour of iron oxide nanoparticles with bound proteins was studied with dynamic light scattering. The separation within the electrochemical set-up is tested and agglomeration as well as binding is compared to standard binding studies with UV/Vis spectroscopy of the supernatant. Different buffer environments (pH, ionic strength and composition) were tested for the protein elution with the electrode set-up. A focus of these studies was the different concentration of the ionic strength which affects the electrochemical double layer as well as the ohmic resistance of the suspension. Elution efficiency of experiments in triplicates was evaluated with UV/Vis spectroscopy. Particle leaching was monitored with ICP-OES. 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) I was able to develop new social and management skills by attending training courses on time, data and project management, on rhetoric and on entrepreneurial thinking I improved my language skills by communicating with scientists at conferences, group meetings, in the lab and by preparing and holding a lecture for the course LEAPS (Leadership and Professional Strategies and Skills) for PhD students and postdocs at MIT with the help of Prof. Angeliki Rigos and Prof. Anna Frebel. This improved my pedagogical and leadership skills as well. All the skills mentioned are vital for taking on full responsibility for a research group as an independent group-leader at a European research facility. My long-term goal is to establish my own research group and attain a permanent position as a full professor at a leading European university. Being awarded with a Marie Sklodowska Curie Fellowship lead to the fulfilment of my habilitation midterm goals and thus directly lead to an improved employability in academic positions in Europe.The separation process as well as further applications (e.g. detection of micro-organisms) based on this technology have been patented and a commercialization is planned. The project results have been disseminated at conferences such as the MSB conference (August 2021), ACS national meeting (Fall 2021), AIChE meeting (October 2021). These meetings provided excellent possibilities to meet chemists and chemical engineers from all fields who work in Northern America.