(369d) Developing a Scalable Templating-Platform for Enhancing Crystallisation of Bio-Active Proteins

Over the last decade, bio-active peptides have emerged as the next generation of bio therapeutics with a rapidly growing demand [1]. A lack of cost-effective and reliable processes for downstream purification is a main industrial challenge for meeting the growing demand of bio-active peptides [2]. Crystallisation of peptides is a novel strategy to debottleneck downstream purification, offering a more economical and efficient technique than expensive traditional techniques [3].

Due to their low solubility and large molecular size, one of the main challenges in peptide crystallisation is the lack of controllability of the nucleation. However, very little has been done in using templates for controlling nucleation to achieve a consistent and reproducible crystallisation process. Chen et al. recently reported that highly mesoporous inorganic surfaces, referred to as “hard templates”, can remarkably induce significantly faster and more controlled nucleation, even at low supersaturation [4]. In our recent work, we have successfully demonstrated a “soft templating” strategy by using soft, organic molecules, such as amino acids, to enhance nucleation. In buffer solution, amino acids form various interactions with peptides, whereby electrostatic interactions have been identified as most important. Therefore, amino acids that exhibit strong electrically charged residual groups for the relevant buffer conditions were investigated. In this presentation, the impact of types, amount and surface charge of the amino acids on the crystallisation performance of insulin at various supersaturation will be reported. With the aim of providing a strategy suitable for high-throughput peptide crystallisation, the possibility of scaling this templating strategy up, from the microlitres to the tens of millilitres, will be evaluated.

Insulin was used as model peptide and comprises of two chains, A and B, with a combined 51 amino acids and an overall molecular weight of 5.8 kDa. The impact of amino acids on crystallisation is evaluated by means of measuring induction time, crystal yield, and size distribution as a function of time. Citric buffer with a pH between 5 and 7 was used with zinc sulphate as the precipitant. The impact of amino acids on crystallisation is evaluated by monitoring the peptide concentration with UV-vis spectroscopy every 20-30 minutes. Using this, the induction time and crystal yield are obtained. Furthermore, the pH is measured at the start and the end of the crystallisation. Images obtained with a microscope are analysed to evaluate the crystal sizes and shapes. The experiments were carried out in batch crystallisers with volumes up to 15 millilitres to demonstrate the scalability.

Our recent results show that amino acids reduce the induction time of insulin crystallisation by up to 40% when compared to the induction time with no amino acids. The faster decrease in insulin concentration when amino acids are added also implies an increase in the crystal growth rate. As amino acids accelerate the crystallisation process, a yield of 90% can be achieved in a shorter period of time in crystallisers with volumes of up to 15 millilitres. Preliminary investigations show that the type and amount of amino acid added have only a minor impact on solubility, leading to a slight increase. Instead of impacting the solubility significantly, the charged residual-groups of the added amino acids increase the pH significantly. This increase in pH leads to an increase in stability of the tertiary structure of insulin by altering its physio-chemical surface properties which result in a faster nucleation. As a result of the more controlled crystallisation with amino acids, a narrower crystal size distribution can be obtained while achieving the same mean crystal size (~20 µm) when compared to the mean crystal size obtained with no amino acids. The insulin crystals obtained with amino acids show a more uniform crystal size distribution with a smaller span for crystalliser volumes reaching from the scale of microliters to the tens of millilitres. The soft templating strategy could be successfully demonstrated for crystalliser volumes of up to 15 millilitres while identifying mixing as one of the main limiting factors of scaling-up.

It has been demonstrated that amino acids as organic templates have the potential of enhancing the controllability of insulin crystallisation even at low supersaturations and to the scales of tens of millilitres. These includes a significant reduction of residence time needed in the crystalliser to obtain a desired crystal yield by maintaining a high level of consistency. Although different polar amino acids were investigated, the magnitude of advancing crystallisation differs dramatically between them as well as the ratio of amino acids concentration to insulin supersaturation.

Bibliography

1. Lau, J.L. and M.K. Dunn, Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg Med Chem, 2018. 26(10): p. 2700-2707.
2. Yang, H., et al., Development and Workflow of a Continuous Protein Crystallization Process: A Case of Lysozyme. Crystal Growth & Design, 2019. 19(2): p. 983-991.
3. Roque, A.C.A., et al., Anything but Conventional Chromatography Approaches in Bioseparation. Biotechnol J, 2020.
4. Chen, W., et al., High Protein-Loading Silica Template for Heterogeneous Protein Crystallization. Crystal Growth & Design, 2019. 20(2): p. 866-873.