The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pK values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

No abstract