Periodic Reporting for period 1 - ASPIRATION (Soft Template Mediated Peptide Crystallisation)
Reporting period: 2021-10-01 to 2023-09-30
The action “Soft Template Mediated Peptide Crystallisation” involves establishing a program for process intensification and alleviating downstream processing bottlenecks. This is achieved through the implementation of a soft template strategy for continuous crystallisation, specifically designed for peptide purification. The overarching goal of this project is to revolutionise the personalised medicine sector by reducing production costs and enhancing the efficacy of peptide-based drugs. This is achieved by tailoring the amount of required peptide drugs based on individual patient needs.
Recent breakthroughs in using peptides as agonists or antagonists for various hormones and antibodies have spurred a growing need for an advanced downstream processing system for peptide drugs. This development aims to replace the costly multistep chromatography-based platform. This ambition is motivated by the attention that in peptide manufacturing, separation and purification processes remain a challenge from the aspect of product purity and process yield and represent time and cost-intense downstream operations. The manufacturing strategies to improve processability, reduce hygroscopicity, increase stability, and lower purification cost provide the opportunity to expedite bringing peptide drugs to the market. However, pharmaceutical companies have been facing considerable challenges in overcoming the manufacturing hurdles related to the peptide crystallisation process.
The overall objectives of ASPIRATION have been to 1) control the peptide co-crystallisation in the presence of soft templates, 2) gain a more in-depth understanding of soft template-mediated peptide crystallisation by exploring different soft templates using experiments and molecular modelling, and 3) continuously produce peptide crystals with improved physicochemical properties such as physical and chemical stability, and solubility. A parallel goal of this action is to foster the development of the researcher i.e. myself, and help to achieve my future goals to be an independent researcher.
Recent breakthroughs in using peptides as agonists or antagonists for various hormones and antibodies have spurred a growing need for an advanced downstream processing system for peptide drugs. This development aims to replace the costly multistep chromatography-based platform. This ambition is motivated by the attention that in peptide manufacturing, separation and purification processes remain a challenge from the aspect of product purity and process yield and represent time and cost-intense downstream operations. The manufacturing strategies to improve processability, reduce hygroscopicity, increase stability, and lower purification cost provide the opportunity to expedite bringing peptide drugs to the market. However, pharmaceutical companies have been facing considerable challenges in overcoming the manufacturing hurdles related to the peptide crystallisation process.
The overall objectives of ASPIRATION have been to 1) control the peptide co-crystallisation in the presence of soft templates, 2) gain a more in-depth understanding of soft template-mediated peptide crystallisation by exploring different soft templates using experiments and molecular modelling, and 3) continuously produce peptide crystals with improved physicochemical properties such as physical and chemical stability, and solubility. A parallel goal of this action is to foster the development of the researcher i.e. myself, and help to achieve my future goals to be an independent researcher.
ASPIRATION comprised of five work packages (WP), with three focusing on technical aspects marked by significant milestones. The first milestone in WP1 involved the 'experimental validation of peptide crystallization control in the presence of soft templates.' This was achieved by investigating the nucleation of glycine and diglycine with and without glass beads as templates. The study provided insights into the solubility of both peptides in water and revealed nucleation rate variations in the presence and absence of templates. Notably, the presence of a template reduced the nucleation time. These findings were published in the 'Faraday Discussions' Journal and presented at the 'Understanding Crystallisation Faraday Discussion' meeting in 2022.WP2 achieved a milestone in comprehending peptide-template interactions by investigating glycine/diglycine interactions with glass beads from WP1. These interactions confirmed that larger peptide sizes lead to more interactions, accelerating nucleation on templates. Simulating triglycine with glass beads reinforced this, predicting its fastest crystallization. Experimental results mirrored this prediction. Published in the 'Journal of Chemical Engineering and Technology,' this research was presented at the '26th International Congress of Chemical and Process Engineering (2022)' and the 'Solvay Meeting (2022).'
The milestone of WP3 was the ‘continuous production of peptide crystals’, which was partially achieved by studying the crystallisation of diglycine in the presence of porous silica templates using a semi-continuous process using an automated reactor and an in-line IR probe for concentration measurement. The study confirmed that the pore size significantly influences diglycine crystallisation. Silica with a 6nm pore size notably enhanced nucleation compared to 50nm pore size silica. Despite varying crystallisation times, the tabletability, and permeability of diglycine crystals remained consistent. Published in the 'Pharmaceutics' Journal, the findings were presented at the 'International Symposium on Industrial Crystallization (2023)' and discussed at the 'Gamlen User Meeting (2023).' This concept extended to insulin crystallization, exploring the impact of precipitant and glass chemistry in a continuous setup, with results published in the 'Chemical Engineering Journal (2023).' Furthermore, ongoing investigations into the crystallisation of the anti-cancer peptide Leuprolide acetate, stemming from this MSCA action, have yielded promising results, with the findings slated for future publication.
The milestone of WP3 was the ‘continuous production of peptide crystals’, which was partially achieved by studying the crystallisation of diglycine in the presence of porous silica templates using a semi-continuous process using an automated reactor and an in-line IR probe for concentration measurement. The study confirmed that the pore size significantly influences diglycine crystallisation. Silica with a 6nm pore size notably enhanced nucleation compared to 50nm pore size silica. Despite varying crystallisation times, the tabletability, and permeability of diglycine crystals remained consistent. Published in the 'Pharmaceutics' Journal, the findings were presented at the 'International Symposium on Industrial Crystallization (2023)' and discussed at the 'Gamlen User Meeting (2023).' This concept extended to insulin crystallization, exploring the impact of precipitant and glass chemistry in a continuous setup, with results published in the 'Chemical Engineering Journal (2023).' Furthermore, ongoing investigations into the crystallisation of the anti-cancer peptide Leuprolide acetate, stemming from this MSCA action, have yielded promising results, with the findings slated for future publication.
This action has successfully reported that peptides can crystallise faster in the presence of templates even at a bigger scale of 40ml. Results highlighted the significance of the pre-exponential factor, which is contingent on the number of nucleation sites present within the metastable solution. Molecular dynamics simulation suggested that larger peptide, provides more number of interaction points resulting in stronger binding with the templates, thereby enhancing the rate of crystallisation. This is the first time someone has reported the tabletability, compressibility, and compactability of peptide crystals. Although this action only studied the simplest known peptide, more research must be done in the area to fully understand this field of research. This information will encourage the researchers to work on peptide crystallisation to improve their stability.
Additionally, templated crystallisation was extended to other longer chain and complex peptides, namely, Leuprolide acetate, and anti-cancer peptide. Leuprolide acetate was crystallised from HEPES buffer using sodium citrate tribasic dihydrate salt and 2-propanol as a soft template. This is the first time that this peptide has been crystallised and studying the variation in these conditions will enable the researchers to other homologous peptides from the same family. The crystallisation of these peptides at a large scale will enable the industry to consider crystallisation as an alternative to the costly and unsustainable purification of peptides, thereby taking a step closer to achieving the net-zero mission in biopharmaceutical manufacturing. This study is ongoing, and its report will be published soon.
Beyond its scientific implications, the fellow consistently engaged in outreach activities throughout this action. The fellow took the lead at events such as 'Imperial Late' and the 'Great Exhibition Road Festival,' where he not only presented their research to a broader audience but also inspired the younger generation to pursue careers in science and engineering. The fellow received support from their research group's Ph.D. candidates, facilitating the enhancement of their interpersonal skills.
The fellow's active involvement in the broader scientific community remained notable during this initiative. They contributed by reviewing several journal articles for peer-reviewed publications and are presently involved in editing a special issue on 'Pharmaceutical Solids: Advanced Manufacturing and Characterization' for the 'Pharmaceutics’ Journal. In summary, this action has left a substantial impact both within the scientific community and in broader public engagement efforts.
Additionally, templated crystallisation was extended to other longer chain and complex peptides, namely, Leuprolide acetate, and anti-cancer peptide. Leuprolide acetate was crystallised from HEPES buffer using sodium citrate tribasic dihydrate salt and 2-propanol as a soft template. This is the first time that this peptide has been crystallised and studying the variation in these conditions will enable the researchers to other homologous peptides from the same family. The crystallisation of these peptides at a large scale will enable the industry to consider crystallisation as an alternative to the costly and unsustainable purification of peptides, thereby taking a step closer to achieving the net-zero mission in biopharmaceutical manufacturing. This study is ongoing, and its report will be published soon.
Beyond its scientific implications, the fellow consistently engaged in outreach activities throughout this action. The fellow took the lead at events such as 'Imperial Late' and the 'Great Exhibition Road Festival,' where he not only presented their research to a broader audience but also inspired the younger generation to pursue careers in science and engineering. The fellow received support from their research group's Ph.D. candidates, facilitating the enhancement of their interpersonal skills.
The fellow's active involvement in the broader scientific community remained notable during this initiative. They contributed by reviewing several journal articles for peer-reviewed publications and are presently involved in editing a special issue on 'Pharmaceutical Solids: Advanced Manufacturing and Characterization' for the 'Pharmaceutics’ Journal. In summary, this action has left a substantial impact both within the scientific community and in broader public engagement efforts.