Our goal is to develop binding proteins in E. coli in a process of directed evolution that can be utilized in diagnostic techniques and target-mediated drug delivery. The concept of our system subdivides into the following aspects: At first, we create a library of random binding protein sequences in bacteria forming the starting point of our project. After that we use a special plasmid which generates a higher mutation rate in the needed regions. Finally, we select proteins that show the best affinity to the target protein. Mediated by the binding of our protein and the target, the cell in concern is granted a selective advantage directly increasing its evolutionary fitness. The impact of antibodies in modern medicine is on a permanent rise but the cost and time factor as well as the immunization of animals, which die during the harvesting process, are still problematic. We want to establish an alternative using antibody-like binding proteins that are generated in vivo in E. coli. Profiting of the short generation cycle and the exponential growth of bacteria, we want to generate binding proteins in a much shorter period of time while also being more cost efficient. Furthermore, no animals have to suffer in the process. The binding proteins can subsequently be utilized in both, diagnostics and therapy against pathogens. Due to the ability of our system to quickly adapt to a certain target protein under evolutionary pressure it is especially useful in concern of quickly evolving and newly arising viral pathogens. The potential our project has is a quick response to emerging viruses. Whether the pathogens are new, mutated from an animal only pathogen to a human pathogen or already known viruses that changed or adapted in a way that the present treatment is no longer effective we provide a quick response to these threats. Our goal is to reduce the time for the generation of an antibody and also make the immunization of animals obsolete. This not only benefits humans in reducing the casualties but also stops the killing of animals. Due to its time efficiency the spread of the pathogen is inhibited earlier further reducing the number of people getting sick and preventing any damage caused by the pathogen. Our target audience is patients with a viral infection with doctors acting as the intermediates. We also plan to sell stocks to governments in order to fight a sudden outbreak in the fastest and most efficient way with a minimum of infections. Health is and will always be one of the greatest treasures in life and we want to preserve it. In the beginning the market was rather small due to its focus mostly on small research teams. But due to the recent of new viruses like Denge, Ebola and most recently Zika the demand has significantly grown. The more frequent appearances of several epidemic viruses are alarming and more and more researchers choose to further tackle the new challenges. So the demand is on the rise. The market itself is always changing as soon as a new method is introduced. Several groups are testing a new procedure and system and when it proves to be sufficient it is fast adapted by everybody in the field. The competitions are producers and license holder of current antibody production methods. These still involve immunization and extraction of animals like rabbits or mice. This can only be done by specially equipped labs or ordered by producers of antibodies. These already available methods are time consuming and expensive. We offer a cheap and fast method that can be applied even in small labs. Also the client can modify our system to his likes while current producers are limited in their product range. Because our product is aimed mostly at research groups there are very few barriers to enter the market. For research use only products have very few limitations for example the declaration of dangerous chemicals or GMOs which handling procedures have to be added. We intend to provide researchers with the sufficient armamentarium to start their research and quickly produce results that can be medically applied. The system was designed to give the widest range of targets for it can be applied in various fields of research and in all kinds of context. Our vision is an easy accessible system that produces quick results and can be applied even in small labs. Therefore even small research groups with a smaller budget can use it. Our short time goal is to quickly introduce our system to the market and collect as much feedback as possible to further improve our Evobodys. The longtime goal is to establish our system as the starting point for all target/ binder related research. What makes our system unique is that we do not need a specific binder sequence for our target. Due t other randomization and mutation we gradually generate the perfect binder regardless of the target. So anyone that is looking for a binder to its target can generate one himself in a simple and quick manner. Our system would be the next step in target related research that even spreads to the medical field because of its application in target drug delivery, the tagging of certain cells like cancer cells and the inactivation or killing of viruses and pathogens. We aim to provide the starting point and make fast responses to new threats possible. The process from a newly designed or found antibody to a real life application is quite time consuming due to the requirement of going through the three phases of approval. For example the FDA requires tests on animals, tests on volunteers (Phase 1) to determine its safety and side effects, phase 2 that tests its effectiveness by comparing it to different types of treatments and phase 3 that further examines its effectiveness in a large scale study that also explores the necessary dosage and combination with other drugs.1 Our product would be our E. colistrain with the necessary plasmids already transformed and a stock of plasmids for any further transformation. It would also include a detailed procedure with the basic steps, tips, tricks and possible modifications. The price will be affordable due to the main expenses being labor cost and the materials for purification. The contents of our product itself are cheap and produced by ourselves. We will distribute our product by mail order which provides a fast and efficient way. Advertising will also mainly take place on the internet on sites with scientific content in the field of microbiology and molecular biology as well as professional literature like magazines that publish scientific papers and articles. Because of this a rather small budget is needed and our product is presented specifically to subject related audience. This provides us a customer oriented and cost efficient form of marketing. The management structure of our company will contain all necessary assignments while keeping it simple to avoid unnecessary costs.

We will mostly employ molecular biologists, biotechnologists and biotechnical assistants. The most advantageous location would be an enterprise zone because of good accessibility to highways and thus faster transportation. For our laboratory we would need a safety level 1 laboratory which involves registration with the government, regularly controls and special equipment to ensure the safety of personnel, nature and humans. Aside from that no additional permit is necessary. We will call our company “Evobody Incorporated” which also indicates our legal form 1 http://www.fda.gov/drugs/resourcesforyou/consumers/ucm143534.htm The developmentof a flowchart of our process and procedures supports the inital overview on our project. Furthermore, this overview is a major requirement to successfully upscale our idea and bring it into actual use for researchers and diagnosticians all over the world. Therefore, we talked to several experts in large scale production within biotechnology. Results from these discussions were transferred to our procedures to reach a bigger scale. One major issue was identified easily: the whole process would be too complex. Therefore, the two central processes of generating of the Evobody encoding sequence and the final production of the Evobody itself are presented in separate flowcharts to present our unique system of in vivo mutagenesis and selection. Our starting point is the main procedure that gives an overview makes up the skeleton of our project. It describes the steps that are necessary to get to the generation of an Evobody sequence and how to continue after the production of the Evobody itself. It involves the procedures that are applied when an order for a new target is issued up to the finished product that is then send out (Fig. 1).

Second is the generation of an Evobody sequence that shows the greatest binding properties to our target. This is achieved with our system. It involves the generation of the library, the system for mutating the sequence of the Evobody and the constant selection for the best binder.

Last but not least is the actual process of producing our Evobody, the extraction and cleanup of the Evobody before it can be shipped. It starts with the amplification of the sequence for the best binder that is the result of the second process and ends with the purified and stabilized binding protein. Our Evobody.

In order to transfer our project to an industrial level we had to compare the differences between the scales of processes in production and a research group, respectively. For the process of upscaling we consulted different experts from different fields: Prof. Dr. Karl Friehs (fermentation), Prof Dr. Thomas Noll (cell culture), Dr. Joe Max Risse (fermentation) and Dr. Martin Schleef (Plasmid Factory ). Our main concerns were the approval by the government institutions and the efficient production of our Evobody protein. This takes the method of cultivation as well as the materials used into consideration. The most in important thing is that the process of creating of the best binder, our Evobody, is still rather small and can be done on the level of a single lab. But when it comes to producing the actual binder a large scale fermentation process is needed for production in large quantities. We discussed two approaches: a repeated batch and a continuous culture, respectively. The repeated batch is easier and we have no dilution, for example antibiotics, over time. In the continuous batch we face a lower my which means constant pressure on the cells, it is reproducible and we have constant conditions during the production . Suggested cultivation time was between 10 to 12 generations. Also adding another carbon source like glucose or glycerin ( that does not need autoclavation) is improving the yield and reducing the cost. Using a defined medium would just elongate the process in terms of analysis, control and preparation measures. Considering the generation of the sequence of the perfect binder the culture should be harvested in the stationary phase, because there is the peak in plasmid DNA.

Therefore, when using a repeated batch letting only the last batch grow into the stationary phase would speed up the process as well as the yield. The type of E. coli used in production is also crucial. A fast growing and well characterized strain has to be used in order to speed up production as well as approval. Also strain characteristics with our plasmids transformed have to be determined to further optimize the system. Another hurdle we had to consider was the approval by the government institutions. They differentiate between generics and biosimilars. Generics are a complete synthetic copy and thus the approval is shortened for its effect and side effects are already known. However, biosimialars root from a different amino acid sequence but the same target as the original. Because we are changing the sequence in search for the best binder, we fall into the second category. That means we have to go through all phases of testing. Aside from the pharmaceutical field our Evobodies can be freely used in research. In research our advantage is that it is in silico which means fast, flexible and a guaranteed outcome. Therefore research labs can instantly react to new or mutated pathogens. Additionally they can focus on how they plan to kill the pathogen instead of spending valuable time on finding ways to bind the target. Their possibilities are large because of the high stability of our Evobody various enzymes and proteins can be fused to it.