Research

Theoretical framework One of the first specific defense mechanisms against invading pathogens and self-antigens is the complement system, activated by immunoglobulins (Igs). Igs bind specifically to the antigen on the pathogen and thereby enable the docking of the C1q-complement initiation complex. Two factors influencing the complement activation were so far not investigated in detail. First, the format of the antigen, described by the chemical nature, the molecular size and the mode of presentation (as soluble substance or embedded in vesicles for mimicking the cell surface). Second, the huge difference in complement activation resulting from the degree of oligomerization of the IgMs. Objectives The overall goal of the pent/hexIgM project is to elucidate the activation sites of C1q and the IgM-Fc after binding of pentameric and hexameric IgMs to different antigen formats. Approach/methods We will produce recombinant IgMs and the C1q protein in mammalian cells and generate mutants thereof by yeast surface display. Next, we will confirm the biological activities of generated proteins in vitro by immunochemical and biophysical analyses as well as functionality tests. Most important, we will elucidate in influence of the antigen format and the degree of oligomerization of the IgMs (pentameric versus hexameric IgMs) on the activation of the complement system. Level of originality Although IgMs in combination with complement proteins have an important function in the human body, these proteins are not yet widely used in therapy and diagnosis. The results of the pent/hexIgM project will contribute to understanding the complex mechanisms underlying the activation of the complement cascade and will provide data of particular importance for developing new diagnostics and efficient treatment methods for various infectious, inflammatory, chronical and cancerous diseases.

HUMAN PLACENTA Collagen-I from THT Biomaterials GmbH is a novel biomaterial that due to his human source alleviates the downstream limitations associated with the use of animal-derived materials in research. Although the intrinsic fibrillogenesis capacity of THT HUMAN PLACENTA Collagen-I has shown to be sufficient for 2D coating applications, his polymerization ability is limited for the formation of stable 3D hydrogel structures that are indispensable for physiologically relevant cell culture strategies. In this regard, Prof. Cornelia Kasper´s research lab from the Universität für Bodenkultur Wien BOKU has the necessary expertise to support THT in adjusting the mechanical properties of HUMAN PLACENTA Collagen-I to obtain stable and functional hydrogels. Prof. Cornelia Kasper´s research lab suggests to functionalize the HUMAN PLACENTA Collagen-I with methacrylate groups, a common strategy used to modify of different proteins or sugar-based biopolymers. The presence of methacrylate-groups will enable the introduction of covalent bonds upon exposure to UV in the presence of photoinitiators, thus forming hydrogels that can be used subsequent used for different 3D applications. The newly functionalized product (HUMAN PLACENTA Collagen-I methacrylate) will expand THT portfolio allowing his straightforward the use for customers working in different 3D biological applications such as 3D cell culture (e.g. organoids culture), lab-on-a-chip, bioprinting and thus broaden the current applicability of HUMAN PLACENTA Collagen-I. Significantly, the envisioned biomaterial can also be used as ready-to-use bioink for trendy technologies such as light-based 3D bioprinting. Apart from possible publications and co-authorships abstracts, THT & Universität für Bodenkultur Wien BOKU can potentially obtained IP on HUMAN PLACENTA Collagen-I methacrylate generating value for both project partners. If for any reason the innovation check should be cancelled, BOKU reserves the right to charge for services provided in the meantime.

Energy and water consumption for buffer preparation can be substantial, especially if WFI is used. Whereas chromatographic process development can target minimal buffer use, the indirect ecologic impact of buffers in bioprocessing is completely unknown. This is because sustainability analysis and life cycle inventories are rudimentary and not fit-for-purpose to be used as development goal during process development. Traditionally, the reduction of buffer volumes is already part of the current implemented process development goals, as lower buffer volumes also correspond to lower costs and faster processes. Besides manufacturing of novel chromatographic formats (like filters, monoliths, etc.) this leaves very little room for further improvements in direct reduction of consumed buffers, but the sustainability influence of the buffer species and buffer strength is completely open for investigation, not addressed yet, and easy to implement in any scale as design and development goal. Buffer species, buffer preparation and buffer concentrations are currently selected by tradition, or by development goals completely unrelated to sustainability and we can tap into that underdeveloped area to select buffers and concentrations that are equally performant from a bioprocess viewpoint, but with a significantly lower ecological footprint. To tackle and quantify the influence of buffers and buffer preparation on downstream processing, we propose: (1) The generation of a comprehensive life cycle inventory of common buffer species used in biotechnological production (2) an assessment of the potential maximum savings through changes in buffer species and buffer concentrations that can be achieved by this approach (3) a fast to use tool for process development to assess the life cycle impact of buffers to implement a sustainability related key-performance-indicator that can be followed during process development (4) and finally the generation of physical demonstration use cases as a proof of concept.