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Title: | Biophysical Characterization and Bioprocessing of Proteins |
Authors: | Sadavarte, Rahul |
Advisor: | Ghosh, Raja Filipe, Carlos. D. M. |
Department: | Chemical Engineering |
Publication Date: | 2022 |
Abstract: | Advances in biotechnology have opened up numerous new avenues for researchers in their study of proteins. These new technologies have not only resulted in a more robust understanding of protein characteristics which had hitherto only been achievable through the use of a handful of techniques - but they have also made this knowledge accessible to a wider audience. Although traditional approaches to studying proteins have performance limitations, they have remained the preferred options due to the absence of better alternatives that can accommodate similar scales of operation. Ideally such scenarios are commonly encountered during bioprocessing, which involves the extraction and purification of a pure protein from a host cell organism via the separation and removal of impurities. Here, the protein of interest undergoes through treatment to remove unwanted impure substances while at the same time preserving the targeted qualities. This thesis introduces approaches to protein characterization and bioprocessing and discusses their limitations. In addition, newer alternatives are also introduced, particularly those that provide improved efficiency and quality of information compared to the traditional approaches. Specifically, this thesis highlights various aspects of bioprocessing and the biophysical characterization of proteins, with a focus on areas such as the processing of aggregates, impurity detection in protein samples, and the structural stability of proteins. Overall, the results presented in this thesis were obtained with respect to the specific protein(s) under consideration. However, the nature of the presented processing techniques enables their use in the study of other similar proteins. As such, the results presented herein are not necessarily limited to a specific protein type rather, they can be generalized for application to a wide range of proteins, thus highlighting the true potential of these techniques. Finally, recommendations are made with regards to further exploring the wide scale application of these new approaches. In general, the new approaches presented in this thesis do not involve complex mechanisms; on the contrary, they are relatively easy to understand and perform. This thesis covers the following points and presents preliminary data supporting the hypotheses: (i) The problem of protein aggregation is very common in the bioprocessing industry and solution(s) to remedy this problem typically involves tedious and energy intensive approaches. In this thesis, a simple and easier to perform thermal-cycling method capable of disaggregating protein clusters and refolding the resultant isolated protein chains into functional protein molecules is presented and explained. (ii) Another aspect of bioprocessing examined in this thesis is the impact of prolonged interaction time between a protein and substrate post adsorption. During the study it was observed that prolonged contact between a protein and adsorptive surface causes structural changes in proteins. This change was identified based on chromatographic peak broadening within an interval of 10-15 minutes. (iii) Heterogeneity in proteins is introduced during processing and storage, and this can adversely impact the quality of the target product. Using a rapid analytical membrane chromatographic technique, it was possible to identify monoclonal antibody charge variants. While the obtained resolution was comparable to that of column chromatography, membrane chromatography provided faster processing overall. The work documented in this thesis provides a “proof-of-concept” methodology as an alternative to currently used protein analysis techniques. The new methods presented in this thesis lay out a platform for the further investigation of these ideas. There is immense scope for thorough and detailed studies to be performed to further bolster the principles discussed herein. Since the presented data is only a preliminary finding with a single test protein, more elaborate testing is required to validate the proposed hypotheses of this work. |
URI: | http://hdl.handle.net/11375/28053 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Sadavarte_Rahul_H_2022August_PhD.pdf | 1.83 MB | Adobe PDF | View/Open |
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