(385h) Experimental and Computational Insights into the Homogeneous and Heterogeneous Crystallisation Behaviour of Glycine Homopeptides

Peptide therapeutics have seen an upswing in popularity in recent years, due to their success in the treatment of a range of chronic diseases, such as diabetes, cancer, and osteoporosis. Insulin remains the most well-known member of the peptide family, but shorter chain peptides (<15 amino acids) have seen the most development in recent years. As a result, there have been significant advances in the maturity of both synthetic and recombinant peptide synthesis techniques. However, separation and purification technologies have not seen the same rate of improvement as the preceding synthesis steps. As such, the bulk of the purification workflow relies on costly chromatographic steps which suffer from high resin costs and significant solvent volume requirements.

Crystallisation therefore presents a potentially preferable alternative to industrially standard chromatography steps, as it does not suffer from the issues that trouble chromatography. As well as this, the crystalline form inherently allows for high purity solids, of a controlled size and shape, to be obtained, especially when using seeding protocols to inhibit nucleation. However, due to the vast number of peptide sequences possible, very little is known about the solubility, nucleation, and growth of peptide crystals; information which is necessary in order to design peptide crystallisation processes.

The addition of insoluble species, termed heterogeneous nucleants or “heteronucleants” into the crystallisation liquor has also been shown to increase the rate of crystallisation in many pharmaceutical systems. The mechanism of action of these heteronucleants is an area of active research, and both topography (including both the porosity of the heteronucleant and favourable epitaxial relations between heteronucleant and crystallising species) and surface chemistry (functional group matching and hydrogen bonding interactions) have been shown to be responsible for the increase in the nucleation rate. However, despite the promising utility of heteronucleants for promoting and controlling crystallisation, there has been little investigation into the applicability and mechanism of action of heteronucleants for peptide crystallisation.

The proposed presentation presents research into the crystallisation of the simplest homopeptides, composed solely of glycine residues. Initial work focused on the effect of chain length on the induction time and nucleation rate of glycine homopeptides. Supersaturated solutions of (poly)glycine were generated via cooling crystallisation of aqueous solutions containing these species to crystallisation temperatures of 5°C and 10°C, and the effect of relative supersaturation (S=C/C*, where C is the concentration in solution and C* is the solubility at the crystallisation temperature) on the nucleation rate and induction time was analysed. The results of this are presented in Figure 1.

Predictably, while the nucleation rate increases with increasing supersaturation, it was found that the induction time increases exponentially with peptide chain length, with the longest peptide studied, pentaglycine, having an induction time almost three orders of magnitude higher than that of glycine. Via usage of the induction time alongside the Classical Nucleation Theory (CNT), it was also possible to calculate and compare the interfacial energy of each species, as well as the activation Gibbs energy of crystallisation. Experimental observations also reveal the presence of unique phase behaviour during the crystallisation of triglycine and raise questions as to the applicability of CNT to the crystallisation of peptides. Specifically, triglycine is shown to form a dihydrate at lower temperatures, and solutions of triglycine exhibit initial gelation of the solution as a precursor to crystallisation, rather than the formation of the crystalline phase directly from the saturated solution.

From these initial studies on the crystallisation of (poly)glycine from otherwise pure water, subsequent investigations were performed into the applicability of heteronucleants for the enhancement of the crystallisation rate of glycine, diglycine, and triglycine. The heteronucleant selected was simple glass beads. As the addition of heteronucleants predictably creates a turbid solution, it was not possible to observe the induction time visually, and as such the concentration of the system was analysed by either ex-situ UV-Vis spectrophotometry or in-situ FTIR probes. Each species was crystallised in both the absence and presence of glass beads at three different temperatures, corresponding to three different supersaturations, and the induction time was calculated using the concentration curves obtained. The induction time was then used to calculate the nucleation rate, and the results of this analysis are presented in Table 1.

From this, it is evident that the presence of glass beads decrease the induction time and increase the nucleation rate for all three glycine species, regardless of supersaturation. It is observed that the rate of enhancement of the nucleation rate (given as the nucleation rate ratio, J_het/J_hom) increases with increasing chain length, i.e. triglycine nucleation is enhanced more than diglycine, which is enhanced more than glycine. It was postulated that in all cases, the mechanism responsible for the enhancement of the nucleation rate is hydrogen bonding between the surface silanol groups on the glass bead and the oxygen- and nitrogen-based groups in glycine, diglycine, and triglycine. These hydrogen bonds act to reduce the molecular mobility of the bonded solute molecule, thereby increasing the likelihood of collision between solute molecules and therefore the rate of nucleation. This hypothesis was investigated via molecular dynamics (MD) simulations, which looked at the distribution of interaction lifetimes between the carbonyl and carboxyl groups on (poly)glycine and silanol groups the silica surface. Through this analysis, it was shown that the expected residence time of a molecule of (poly)glycine increases with increasing chain length, and that this is due to increase hydrogen bonding propensity with increasing chain length, as shown in Figure 2.

Finally, through use of the Classical Nucleation Theory, the impact of the heteronucleant on nucleation parameters was studied. Classical heterogeneous nucleation theories propose that the presence of a heterogeneous surface enhances nucleation via reduction of the interfacial energy of the system. However, via induction time analysis, results indicate that the heteronucleant primarily acts via enhancement of the pre-exponential factor for nucleation (which increased by 5% for glycine, 85% for diglycine, and 75% for triglycine), rather than reduction of the effective interfacial energy (which reduced by 5% for glycine, 15% for diglycine, and 13% for triglycine), which is in line with the postulated hydrogen bonding mechanism of action. This result has also been observed by other researchers, and therefore the results raise interesting questions on the applicability of classical theories on heterogeneous nucleation.