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Protein-based drugs – stability evaluation by confocal raman imaging
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Metodologias rápidas e eficazes são essenciais para atender às necessidades da indústria farmacêutica. Desta forma, o presente trabalho aborda uma importante necessidade de existir uma metodologia analítica que permita a caracterização da estrutura conformacional de uma proteína. Raman Confocal é uma metodologia poderosa para análise farmacêutica de proteínas no estado sólido que pode ser utilizada para testes de identidade e estabilidade, sendo útil para determinar mudanças na estrutura secundária e terciária de proteínas em diferentes matrizes. A identificação de bandas específicas associadas à estrutura secundária foi realizada utilizando proteínas com elementos α-hélice e folha-β. A espectroscopia Raman tem vantagens potenciais significativas na indústria biofarmacêutica, como a especificidade molecular intrinsecamente alta, flexibilidade de configurações de amostragem e a necessidade de pré-tratamento mínimo (ou nenhum) de amostra, podendo ser explorada para fornecer soluções analíticas úteis e exclusivas. No entanto, os métodos baseados em Raman, quando aplicáveis a proteínas, podem ser limitados pela complexidade química das mesmas. No entanto, com conhecimento suficiente sobre a estrutura das proteínas e um bom projeto experimental, métodos analíticos robustos podem ser desenvolvidos.
O presente trabalho tem como objetivo definir uma abordagem sistemática para o desenvolvimento de métodos de espectroscopia Raman Confocal através da metodologia AQbD que poderia ser usada para uma ampla gama de moléculas baseadas em proteínas visando detetar mudanças discretas na estrutura conformacional. Nesta dissertação, é apresentado o trabalho realizado no desenvolvimento de um método de identificação e caracterização da estrutura secundária de proteínas, seguindo uma abordagem AQbD.
Os resultados demostraram que a metodologia AQbD aplicada foi bem-sucedida. Através desta metodologia foi possível criar condições experimentais robustas que permitiram identificar a estrutura secundária nas proteínas selecionadas para o estudo. No entanto, para algumas das proteínas selecionadas, os resultados não ficaram dentro dos critérios de aceitação estabelecidos no estudo, a fim de identificar as bandas de interesse. Por fim, também foi possível realizar um estudo de degradação forçada termicamente em que foi possível demonstrar ligeiras alterações na estrutura conformacional da proteína selecionada.
Quick and effective methodologies are essential to meet the needs of the pharmaceutical industry. In this way, the present work addresses an important need of having an analytical methodology that allows the characterization of the conformational structure of a protein. Confocal Raman imaging is a powerful methodology for solid-state protein pharmaceutical analysis that can be used for identity and stability testing, being useful to determine changes in the secondary and tertiary structure of proteins in different matrices. The identification of specific bands associated with the secondary structure was achieved using proteins with α-helix and β-sheet elements. Raman spectroscopy has significant potential advantages in Biopharmaceutical industry, as intrinsically high molecular specificity, flexibility of sampling configurations and the requirement for minimal (or no) sample pre-treatment and can be exploited to deliver unique and useful analytical solutions. However, Raman-based methods, when applicable to proteins, can be limited by the chemical complexity of those. Nevertheless, with enough knowledge on the proteins structure and good experimental design, robust analytical methods can be developed. The present work aims to define a systematic approach for method development of Confocal Raman spectroscopy by means of Analytical Quality by Design (AQbD) that could be used to a wide range of protein-based molecules aiming at detecting discrete changes in conformational structure. In this dissertation, the work conducted regarding the development of an identification and characterization method for the secondary structure of proteins, following an AQbD approach, is shown. The results demonstrate the successful applicability of the methodology. Through this methodology it was possible to create robust experimental conditions that allowed to identify the secondary structure in the proteins selected for the study. However, for some of the selected proteins, the results were not within the acceptance criteria established in the study, in order to identify the bands of interest. Finally, it was also possible to carry out a study of thermally forced degradation in which it was possible to demonstrate slight changes in the conformational structure of the selected protein.
Quick and effective methodologies are essential to meet the needs of the pharmaceutical industry. In this way, the present work addresses an important need of having an analytical methodology that allows the characterization of the conformational structure of a protein. Confocal Raman imaging is a powerful methodology for solid-state protein pharmaceutical analysis that can be used for identity and stability testing, being useful to determine changes in the secondary and tertiary structure of proteins in different matrices. The identification of specific bands associated with the secondary structure was achieved using proteins with α-helix and β-sheet elements. Raman spectroscopy has significant potential advantages in Biopharmaceutical industry, as intrinsically high molecular specificity, flexibility of sampling configurations and the requirement for minimal (or no) sample pre-treatment and can be exploited to deliver unique and useful analytical solutions. However, Raman-based methods, when applicable to proteins, can be limited by the chemical complexity of those. Nevertheless, with enough knowledge on the proteins structure and good experimental design, robust analytical methods can be developed. The present work aims to define a systematic approach for method development of Confocal Raman spectroscopy by means of Analytical Quality by Design (AQbD) that could be used to a wide range of protein-based molecules aiming at detecting discrete changes in conformational structure. In this dissertation, the work conducted regarding the development of an identification and characterization method for the secondary structure of proteins, following an AQbD approach, is shown. The results demonstrate the successful applicability of the methodology. Through this methodology it was possible to create robust experimental conditions that allowed to identify the secondary structure in the proteins selected for the study. However, for some of the selected proteins, the results were not within the acceptance criteria established in the study, in order to identify the bands of interest. Finally, it was also possible to carry out a study of thermally forced degradation in which it was possible to demonstrate slight changes in the conformational structure of the selected protein.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Biologic product Quality characterization Confocal raman spectroscopy Stability Teses de mestrado - 2021