Advanced materials for the extraction, purification and filtration of adenovirus type 5

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Dissertation_Dietl.pdf (23.51 MB)

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2024-12-18

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Dietl, Sandra

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Dissertation

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https://doi.org/10.18725/OPARU-54790

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Abstract

The binding of viruses to materials is of importance for a wide variety of applications. These include for example the separation of viral contaminants from biotechnological products such as therapeutic proteins and antibodies for biological drugs. For this purpose, suitable separation strategies are required to obtain high-purity end products. The requirements for the applied materials are substantial, as they should be sufficiently robust in order to perform at harsh conditions such as high pressures. In addition, they need to bind the target virus with high capacity. On the other hand, such scavenger materials should be easy and inexpensive to produce in order to save time and cost during synthesis. Since the use of natural receptors is usually expensive and complex, this thesis focuses on the development and optimization of synthetic polymer materials that provide the demanded properties for scavenging adenovirus. The developed materials are based on silica core particles providing chemical and mechanical stability next to simple synthesis routes. The particles were then coated with an organosilane polymer layer yielding robust core-shell particles with tailorable functionalities that may be readily incubated with biotechnologically relevant solutions followed by efficient removal from the suspensions via centrifugation. Human adenovirus type 5 is a human pathogen capable of causing infections in the human body and was used as the exemplary target species during this study. Infections include respiratory diseases, gastrointestinal tract infections, and ocular diseases. Hence, efficient removal of adenovirus from biological samples or waters is therefore an important protection against diseases. Conversely, adenovirus is among the most promising candidates serving as viral vectors in gene therapy, e.g., against cancer. For this purpose, the purification of such viruses after biotechnological production ensuring efficient separation from other cell lysate components is essential. Therefore, the devised polymer materials ideally should cater to both application scenarios and allow for scalable production. To maximize the specificity for binding a selected virus, the first part of this thesis deals with the synthesis of molecularly imprinted polymers for adenovirus type 5. Hereby, adenovirus served as a template species that is spatially fixed via functional monomers and crosslinkers polymerized into a binding matrix. After removal of the template, binding sites are created that correspond to the template in size, shape, and functionalities and ideally specifically rebind the target species. However, large templates such as viruses pose several challenges for imprinting strategies given the plethora of potential unspecific interactions resulting from the vast number of functional groups provided by the capsid proteins. This is evident when comparing virus binding to the imprinted polymers with the binding efficiency to non-imprinted control polymers. To overcome this challenge, a so-called ‘epitope imprinting strategy’ was applied. Here, a peptide fragment is selected that is characteristic for a prevalent surface protein and then used as a template during the imprinting process. Subsequently, the entire viruses may be rebound via molecular recognition of the surface-protein-specific peptides with the correspondingly created binding moieties. Using these polymer particles, the second part of the thesis focused on developing a method to purify adenovirus from cell lysate supernatant via an incubation-elution approach. As in such scenarios only one virus species is present, selectivity is not the main criterium but binding capacity for efficiently harvesting the produced virus and efficient clean-up. Hence, in essence non-imprinted polymer particles were synthesized that are in composition similar to the approach discussed above, yet, only relying on the pronounced interactions between the virus surface and the functional groups at the particles resulting in substantial virus scavenging properties. This approach allowed for an efficient harvesting strategy with almost the entire amount of the virus present in an incubation solution being bound to the particle surface while the majority of the matrix proteins were separated after elution. In the last part of this thesis, virus binding and removal using innovative acrylate-based materials was tested and successfully applied. The polymer materials were fabricated via 3D printing of porous polymer structures and then used as syringe inserts facilitating a flow-through centrifugation strategy for virus retention within the material. In summary, the developed methods have the common goal of simplified binding and removal of adenovirus without using bioreceptors and are entirely based on synthetic scavenger materials, which are in essence universally applicable, i.e., tailorable to other kinds of viral species. For the example of adenovirus, it could be shown that they may either be almost entirely scavenged from virus-containing suspensions, or – for the case of a purification method – that they may be eluted after trapping by the developed particles with efficient removal of the other matrix components.

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Faculties

Fakultät für Naturwissenschaften

Institutions

Institut für Analytische und Bioanalytische Chemie
ZE Elektronenmikroskopie

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DFG Project uulm

EU Project THU

Other projects THU

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Lizenz B (ohne Print-on-Demand)
https://oparu.uni-ulm.de/handle/licenseB_opod_v1

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S. Dietl, P. Walther, H. Sobek, B. Mizaikoff, Materials 2021, 14, 7692. https://doi.org/10.3390/ma14247692
 S. Dietl, H. Sobek, B. Mizaikoff, Trends in Analytical Chemistry 2021, 143, 116414. https://doi.org/10.1016/j.trac.2021.116414
 S. Dietl, F. Kiefer, S. Binder, P. Walther, H. Sobek, B. Mizaikoff, Journal of Biotechnology 2023, 361, 49-56. https://doi.org/10.1016/j.jbiotec.2022.11.015
 B. Keitel, S. Dietl, T. Philipp, G. Neusser, C. Kranz, H. Sobek, B. Mizaikoff, M. Dinc, Advanced Materials Technologies 2024, 2401178. https://doi.org/10.1002/admt.202401178

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DFG Project THU

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Keywords

Polymer particles, Adenovirus type 5, Epitope imprinted polymers, Polymere, Molecular imprinting, Imprinted polymers, DDC 540 / Chemistry & allied sciences, DDC 570 / Life sciences