Purpose: Insulin, in typical use, undergoes multiple changes in temperature; from refrigerator, to room temperature, to body temperature. Although long-term storage temperature has been well-studied, the short term changes to insulin are yet to be determined. Insulin detemir (IDet) is a clinically available, slow-acting, synthetic analogue characterised by the conjugation of a C14 fatty acid. The function of this modification is to cause the insulin to form multi-hexameric species, thus retarding the pharmacokinetic rate of action. In this investigation, the temperature dependence properties of this synthetic analogue is probed, as well as expiration. Methods: Dynamic light scattering (DLS) and viscometry were employed to assess the effect of temperature upon IDet. Mass spectrometry was also used to probe the impact of shelf-life and the presence of certain excipients. Results: IDet was compared with eight other insulins, including human recombinant, three fast-acting analogues and two other slow-acting analogues. Of all nine insulins, IDet was the only analogue to show temperature dependent behaviour, between 20°C and 37°C, when probed with non-invasive backscatter dynamic light scattering. Upon further investigation, IDet observed significant changes in size related to temperature, direction of temperature (heated/cooled) and expiration with cross-correlation observed amongst all 4 parameters. Conclusions: These findings are critical to our understanding of the behaviour of this particular clinically relevant drug, as it will allow the development of future generations of peptide-based therapies with greater clinical efficacy.

Protein polysaccharide complexes have been widely studied for multiple industrial applications and are popular due to their biocompatibility. Insulin degludec, an analogue of human insulin, exists as di-hexamer in pharmaceutical formulations and has the potential to form long multi-hexamers in physiological environment, which dissociate into monomers to bind with receptors on the cell membrane. This study involved complexation of two negatively charged bio-polymers xanthan and alginate with clinically-relevant insulin degludec (PIC). The polymeric complexations and interactions were investigated using biophysical methods. Intrinsic viscosity [η] and particle size distribution (PSD) of PIC increased significantly with an increase in temperature, contrary to the individual components indicating possible interactions. [η] trend was X > XA > PIC > A > IDeg. PSD trend was X > A > IDeg > XA > PIC. Zeta (ζ)-potential (with general trend of IDeg < A < XA < X ≈ PIC) revealed stable interaction at lower temperature which gradually changed with an increase in temperature. Likewise, sedimentation velocity indicated stable complexation at lower temperature. With an increase in time and temperature, changes in the number of peaks and area under curve were observed for PIC. Conclusively, stable complexation occurred among the three polymers at 4 • C and 18 • C and the complex dissociated at 37 • C. Therefore, the complex has the potential to be used as a drug delivery vehicle.Polymers 2020, 12, 390 2 of 15 Alginates (A) are linear polymers of 1,4-linked β-D-mannuronic acid residues and 1,4-linked α-L-guluronic residues, containing homo-polymeric sequences [7]. Xanthan (X) is composed of D-glucosyl, D-mannosyl, and D-glucuronyl acid residues in a 2:2:1 molar ratio and variable proportions of O-acetyl and pyruvyl residues. Its main chain consists of β-D-glucose units linked at the 1 and 4 positions. Side-chains consist of a tri-saccharide composed of mannose (β-1,4) glucuronic acid and (β-1,2) mannose, attached to alternate glucose residues in the backbone by α-1,3 linkages [8,9].Xanthan gum and alginate complexes (XA) have been studied previously [10] and used for functional foods [11], tissue engineering [3,12,13] and drug delivery. As negatively charged polymers, xanthan [14][15][16][17] and alginate [18][19][20] have been used separately, and in combination with positively charged polymers such as chitosan, as potential vehicles for insulin delivery.Interactions among different types of protein-polysaccharide complexes (PPCs) have been previously investigated using a variety of methods [21,22]. The formation and dissociation of PPCs and their solubility depend on many factors such as surface charge, pH, temperature and ionic strength of the solvent [23]. These factors greatly influence non-covalent interactions such as electrostatic, H-bonding, hydrophobic, and steric interactions, as well as covalent interactions [24]. Conjugation of polysaccharides with therapeutic proteins is a well-established phenomenon [25]. In particul...