The challenge of formulating high concentration therapeutic peptides
July 14-17, 2019
Therapeutic peptides offer significant opportunities to treat serious and debilitating diseases, with several purported advantages over proteins, such as a simpler characterization process and a reduced regulatory burden. However, many peptides are known to be susceptible to aggregation, and surprisingly little work has been published on designing formulation principles for these therapeutic agents.
In this paper the focus will be on the challenges encountered in rational development of a stabilization strategy for two model peptides formulated at high concentration (>50 mg/ml). The first model peptide studied was carbetocin, a 9-amino acid long analogue of oxytocin with improved chemical and enzymatic stability. At high concentrations (70 mg/mL, ca 70 mM) carbetocin is sensitive to interface-induced stress, causing the formation of large amorphous aggregates. The addition of uncharged or charged surfactants surprisingly did not stabilize against this interface-induced stress; instead, positively and negatively charged surfactants steered the aggregation pathway towards the formation of amyloid-like fibrils . The addition of very low concentrations of the uncharged surfactant Span 60 appeared to result in the formation of aggregates with a refractive index close to that of water, creating a false impression of stabilization. For carbetocin, only the removal of the air-water interface by fully filling the vials resulted in a significant stabilization against aggregation .
The second model peptide was a 33-amino acid negatively charged peptide which formed amyloid-like fibrils when exposed to heat stress (40 degrees C) at 50 mg/mL concentration. The limited impact of shaking indicated this fibrillation was primarily induced through peptide-peptide interactions in the bulk solution. Peptide-peptide interactions were therefore determined using Taylor Dispersion Analysis. The latter method suggested significant repulsive interactions for this peptide formulation in aqueous solution. Addition of salt (NaCl) or the amino acids Arg or Glu at 150 mM shielded these repulsive interactions. In contrast, addition of 250 mM of the amino acids Val or Ile had a very limited shielding effect. However, none of the added excipients appeared to stabilize the peptide against aggregation, although infrared spectroscopic analysis indicated that the aggregates in the NaCl, Arg and Glu containing solutions consisted of native-like peptides. The aggregates formed in the presence of Val and Ile, or in the absence of any excipients, contained a significant amount of intermolecular beta-sheet .
Our results illustrate the challenges in protecting high concentration peptides against aggregation and indicates a strong dependence of the aggregation pathway on the excipients added and their potential interaction with the peptide. A novel formulation toolbox may therefore be required for peptides. The high structural flexibility introduces a significant complicating factor in understanding their interactions with their local environment.
 Høgstedt UB, Østergaard J, Weiss T, Sjögren H, van de Weert M. Manipulating aggregation behavior of the uncharged peptide carbetocin. Journal of Pharmaceutical Sciences. 2018 Mar;107(3):838-847.
 Høgstedt UB. Formulation of concentrated peptide solutions – physical stability challenges and the impact of peptide-peptide interactions. PhD thesis University of Copenhagen, 2018.
Marco van de Weert, "The challenge of formulating high concentration therapeutic peptides" in "Biological and Pharmaceutical Complex Fluids III: Protein Self-Assembly, Rheology and Interfacial Properties", Samiul Amin, Manhattan College, USA Miguel Rodrigues, University of Lisbon, Portugal Paolo Arosio, ETHZ, Switzerland Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/bpcf_iii/33