Capito, Florian (2014)
Development and monitoring of a novel monoclonal antibody purification strategy.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung
Kurzbeschreibung (Abstract)
The studies presented in the cumulative part of this thesis illustrate the different steps to develop a polymer-driven antibody purification process. These peer-reviewed reports show in detail fundamental research, additional method development useful in the development of such a purification process as well as implementation of the final process. A strategy for analyzing copolymers, synthesized by a lab in house, was implemented with particular emphasis on copolymer composition analysis. This was especially important in the context of understanding how copolymer composition affects precipitation yield and selectivity. Compared to 1H-NMR composition analysis, the use of ATR infrared spectro-scopy enabled a cost-effective and fast analysis of copolymers and according adjustment of synthesis parameters.Besides the benefits for this project, the similar analytical power of IR compared to NMR was shown, also allowing small-scale companies to use such a technique. Following synthesis and synthesis optimization, basic research experiments were conducted, elucidating how ionic strength, polymer chain length, polymer chain flexibility, pH, pyhsico-chemical properties of copolymer and protein as well as copolymer composition affect precipitation behavior. Similar to relevant work in the literature, cited in the introduction section, increasing ionic strength led to reduced precipitation yields. Additionally to these known aspects, low ionic strength also resulted in reduced precipitation yield.We concluded polyelectrolyte chain conformation being rather stiff at low ionic strength to be the reason for these findings. At low ionic strength, charges at polyelectrolyte sub-units are not sufficiently shielded anymore, leading to a more expanded conformation of the polyelectrolyte. This would then impede interaction with the proteins. Comparing precipitation behavior of different anti-bodies, the required polyelectrolyte flexibility to allow for high precipitation yields depended on the charge density of the protein.[56] These new insights could also help in the design of polyelectrolytes with defined flexibility to control precipitation selectivity. Another important factor is polymer chain length, which does not only influence yield and selectivity. It also affects precipitation efficiency, meaning the number of polymer chains required to obtain precipitation of an antibody molecule.The use of polymer standards with defined molecular weight distribution revealed that the polymer chain length required per precipitated protein molecule is up to 25-times larger than the actual diameter of the specific protein. Moreover, comparing different types of polymers within this context, the defined length of polymer chain length differed among these copolymers. Under precipitation conditions strongly charged polymers allowed precipitation even with short defined lengths, meaning they enabled efficient precipitation. Moreover, a further adjustment of selectivity and yield can be achieved altering the copolymer composition. Compared to results by other working groups, cited in the introduction section, the use of these copolymers allowed higher salt tolerance and higher yields without prior dilution of the cell culture fluid. Simultaneously, infrared spectroscopy was used as a process-assessment tool, determining the amount of antibody, host cell proteins as well as aggregated antibody before and after precipitation to analyze selectivity and yield.This was particularly important, as e.g. the use of ELISA-assays for HCP quantification is quite costly, especially during process development which requires a large number of these assays to be used. Regarding aggregation analysis and quantification, IR has already been used in the past, as shown by literature cited in the introduction section and was further advanced not only to provide information about the presence of aggregation but also to quantify the amount of aggregates in a sample. The results of these peer-reviewed reports were suitable in the context of this thesis and precipitation process development. Additionally as the here developed quantification procedures for antibody titer, aggregate level and host cell protein amount may also find suitable application as a general fast and cost-effective process-monitoring technique, they are currently under consideration for a patent application. Some further experiments were conducted showing that infrared spectroscopy can be used to distinguish antibodies from other proteins based on differences in secondary structure. These findings were, together with additional denaturation monitoring experiments, submitted for a book chapter. Moreover, they allowed comparison of the secondary structure of monoclonal antibodies before and after precipitation, to elucidate any changes to the protein secondary structure and thus harmful effects of this process.Integration of all these findings helped to implement a protein purification process based on precipitation. This process was then compared to protein A chromatography with respect to costs and effectiveness.The developed process can be used within the purification cascade and may replace at least in part existing initial chromatography-based purification processes. Compared to protein A affinity chromatography, costs are lower for future high titer cell culture systems used for antibody production.Yet, additional applications beyond antibody production are also feasible.
Typ des Eintrags: | Dissertation | ||||
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Erschienen: | 2014 | ||||
Autor(en): | Capito, Florian | ||||
Art des Eintrags: | Erstveröffentlichung | ||||
Titel: | Development and monitoring of a novel monoclonal antibody purification strategy | ||||
Sprache: | Englisch | ||||
Referenten: | Kolmar, Dr. Harald ; Schmitz, Dr. Katja | ||||
Publikationsjahr: | 22 Januar 2014 | ||||
Datum der mündlichen Prüfung: | 20 Januar 2014 | ||||
URL / URN: | http://tuprints.ulb.tu-darmstadt.de/3769 | ||||
Kurzbeschreibung (Abstract): | The studies presented in the cumulative part of this thesis illustrate the different steps to develop a polymer-driven antibody purification process. These peer-reviewed reports show in detail fundamental research, additional method development useful in the development of such a purification process as well as implementation of the final process. A strategy for analyzing copolymers, synthesized by a lab in house, was implemented with particular emphasis on copolymer composition analysis. This was especially important in the context of understanding how copolymer composition affects precipitation yield and selectivity. Compared to 1H-NMR composition analysis, the use of ATR infrared spectro-scopy enabled a cost-effective and fast analysis of copolymers and according adjustment of synthesis parameters.Besides the benefits for this project, the similar analytical power of IR compared to NMR was shown, also allowing small-scale companies to use such a technique. Following synthesis and synthesis optimization, basic research experiments were conducted, elucidating how ionic strength, polymer chain length, polymer chain flexibility, pH, pyhsico-chemical properties of copolymer and protein as well as copolymer composition affect precipitation behavior. Similar to relevant work in the literature, cited in the introduction section, increasing ionic strength led to reduced precipitation yields. Additionally to these known aspects, low ionic strength also resulted in reduced precipitation yield.We concluded polyelectrolyte chain conformation being rather stiff at low ionic strength to be the reason for these findings. At low ionic strength, charges at polyelectrolyte sub-units are not sufficiently shielded anymore, leading to a more expanded conformation of the polyelectrolyte. This would then impede interaction with the proteins. Comparing precipitation behavior of different anti-bodies, the required polyelectrolyte flexibility to allow for high precipitation yields depended on the charge density of the protein.[56] These new insights could also help in the design of polyelectrolytes with defined flexibility to control precipitation selectivity. Another important factor is polymer chain length, which does not only influence yield and selectivity. It also affects precipitation efficiency, meaning the number of polymer chains required to obtain precipitation of an antibody molecule.The use of polymer standards with defined molecular weight distribution revealed that the polymer chain length required per precipitated protein molecule is up to 25-times larger than the actual diameter of the specific protein. Moreover, comparing different types of polymers within this context, the defined length of polymer chain length differed among these copolymers. Under precipitation conditions strongly charged polymers allowed precipitation even with short defined lengths, meaning they enabled efficient precipitation. Moreover, a further adjustment of selectivity and yield can be achieved altering the copolymer composition. Compared to results by other working groups, cited in the introduction section, the use of these copolymers allowed higher salt tolerance and higher yields without prior dilution of the cell culture fluid. Simultaneously, infrared spectroscopy was used as a process-assessment tool, determining the amount of antibody, host cell proteins as well as aggregated antibody before and after precipitation to analyze selectivity and yield.This was particularly important, as e.g. the use of ELISA-assays for HCP quantification is quite costly, especially during process development which requires a large number of these assays to be used. Regarding aggregation analysis and quantification, IR has already been used in the past, as shown by literature cited in the introduction section and was further advanced not only to provide information about the presence of aggregation but also to quantify the amount of aggregates in a sample. The results of these peer-reviewed reports were suitable in the context of this thesis and precipitation process development. Additionally as the here developed quantification procedures for antibody titer, aggregate level and host cell protein amount may also find suitable application as a general fast and cost-effective process-monitoring technique, they are currently under consideration for a patent application. Some further experiments were conducted showing that infrared spectroscopy can be used to distinguish antibodies from other proteins based on differences in secondary structure. These findings were, together with additional denaturation monitoring experiments, submitted for a book chapter. Moreover, they allowed comparison of the secondary structure of monoclonal antibodies before and after precipitation, to elucidate any changes to the protein secondary structure and thus harmful effects of this process.Integration of all these findings helped to implement a protein purification process based on precipitation. This process was then compared to protein A chromatography with respect to costs and effectiveness.The developed process can be used within the purification cascade and may replace at least in part existing initial chromatography-based purification processes. Compared to protein A affinity chromatography, costs are lower for future high titer cell culture systems used for antibody production.Yet, additional applications beyond antibody production are also feasible. |
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Alternatives oder übersetztes Abstract: |
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URN: | urn:nbn:de:tuda-tuprints-37691 | ||||
Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 540 Chemie | ||||
Fachbereich(e)/-gebiet(e): | 07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie 07 Fachbereich Chemie |
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Hinterlegungsdatum: | 16 Feb 2014 20:55 | ||||
Letzte Änderung: | 16 Feb 2014 20:55 | ||||
PPN: | |||||
Referenten: | Kolmar, Dr. Harald ; Schmitz, Dr. Katja | ||||
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 20 Januar 2014 | ||||
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