(600c) Optimising the Nucleation of Polypeptides Using Nanotemplates: The Case Study of Insulin

Frederik J. Link*^a, Jerry Y.Y. Heng^a

^aDepartment of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK

*f.link18@imperial.ac.uk

Keywords: insulin, nanotemplates, crystallisation

Bio-active polypeptides have attracted interest in research because of their significant use in medical, biological and pharmaceutical industries. Thereby, the crystalline form offers significant pharmacokinetic advantages with regards to achieving higher bioavailability and better release control [1]. In the case of insulin, which is widely used for treating diabetes, the most common route of administration is by subcutaneous injections of either pure microcrystals or a mixture of microcrystals and amorphous insulin. Due to the slow dissolution of the crystals, the crystalline administration leads to a slow release of insulin into the blood stream [2]. However, one of the main limitation is the lack of controllability of the nucleation mechanism. However, nanotemplates are remarkable in controlling nucleation. Recent studies showed that by using small inorganic particle with a highly porous surface the induction time of nucleation of polypeptides can be significantly accelerated even if the supersaturation is low [3]. Furthermore, it is believed that nucleation is enhanced if the pore diameter has the same order of magnitude as the radius of gyration of the polypeptide [4]. This stems from the suggestion that nucleation in porous surfaces gets enhanced due to local increases in supersaturation which is controlled by the diffusibility of the protein into the pores. However, little is known about this mechanism and the impact of the chemical properties of the surface of the nanotemplates on controlling nucleation. It is believed that porous surfaces trigger nucleation due to the local increase in supersaturation. Therefore, investigating the non-covalent interaction between the surface patches of the nanotemplates and insulin will open up new routes for controlling the crystallisation of insulin.

Insulin has a molecular weight of 5.8 kDa and consist of 2 polypeptide-chains, A and B, with combined 51 amino acids. Furthermore, the two chains are inter-linked by two disulfide bonds and, additionally, chain A is intra-linked with an additional disulfide bond. Citrate buffer with a pH near the isoelectric point of insulin was used with zinc-sulphate as precipitant. Different concentrations were tested to identify the most promising ones. Since insulin is stored in a rhombohedral crystal configuration in combination with zinc-sulphate in the pancreas, zinc-sulphate was selected as the most appropriate precipitate. The impact of nanotemplates on crystallisability is investigated by mean of measuring induction time and crystal yield. Therefore, batch crystallisation experiments will be carried out in which the crystal yield and induction time will be tracked via measuring the concentration of insulin within the solution over time. To achieve this, the concentration of insulin is determined via UV-vis absorption in combination with Bradford’s method [5]. Different types of nanotemplates will be investigated which differ in their surface chemistry and porosity, amount and size.

By using nanotemplates, the induction time of crystallising a complex molecule such like insulin can be reduced significantly even at low supersaturation. Although the nanotemplates do not have a notable influence on the crystal quality, multiple configurations can be observed which is due to the local availability of dissolved zinc-sulphate. When in the vicinity of zinc-sulphate insulin crystallises in its rhombohedral form, whereas otherwise it crystallises in a cubic shape.

Further work will be done to characterise the impact of the nanotemplates with regards to understanding the impact of the chemistry of the surface patches and whether the crystallisation can be enhanced solely due to local supersaturation or also because of specific non-covalent interaction between the surface patches and insulin. Additionally, the impact of the seeds on the solubility of insulin has to be investigated to evaluate the impact of the nanotemplates on the overall yield. All these findings should ultimately result in the ability of engineering appropriate nanotemplates to enhance the crystallisability of insulin. Further work will also be done in investigating the potential of scaling up from 1 ml to 10 and 100 ml scale.

References

1. Pechenov, S., et al., Injectable controlled release formulations incorporating protein crystals. J Control Release, 2004. 96(1): p. 149-58.
2. Norrman, M. and G. Schluckebier, Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH. BMC Struct Biol, 2007. 7: p. 83.
3. Shah, U.V., et al., Crystallization of Proteins at Ultralow Supersaturations Using Novel Three-Dimensional Nanotemplates. Crystal Growth & Design, 2012. 12(4): p. 1772-1777.
4. Shah, U.V., D.R. Williams, and J.Y.Y. Heng, Selective Crystallization of Proteins Using Engineered Nanonucleants. Crystal Growth & Design, 2012. 12(3): p. 1362-1369.
5. M., B.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Prinicple of Protein-Dye Binding. Analytical Biochemistry, 1976. 72: p. 248-254.