Effect of Lipidation on the Structure, Oligomerization, and Aggregation of Glucagon-like Peptide 1

Lipidated analogues of glucagon-like peptide 1 (GLP-1) have gained enormous attention as long-acting peptide therapeutics for type 2 diabetes and also antiobesity treatment. Commercially available therapeutic lipidated GLP-1 analogues, semaglutide and liraglutide, have the great advantage of prolonged half-lives in vivo of hours and days instead of minutes as is the case for native GLP-1. A crucial factor in the development of novel lipidated therapeutic peptides is their physical stability, which greatly influences manufacturing and drug product development. This work provides a systematic study of the solubility, structure, oligomerization, and long-term stability of five different lipidated analogues of GLP-1, varying in the position of the lipidation site and the nature of lipid attachment. The lipidation was found to negatively impact the peptide solubility, in all cases, limiting it to a specific pH range. An increase in the α-helical secondary structure was observed upon lipidation, and the lipidated analogues were found to form larger and more stable oligomeric species compared to nonlipidated GLP-1. Importantly, the distributions and populations of oligomeric species formed were regulated by both the position and the nature of the lipidation. During the 6 days of sample aging, several lipidated analogues formed aggregates with variable morphologies ranging from elongated mature fibrils to amorphous structures. The kinetics of aggregation often showed multiple steps and did not follow a standard nucleation-propagation mechanism. A wide range of behaviors was observed, and while our observations indicate that the formation of a single stable oligomer results in the greatest physical stability, positioning the lipid group toward the N-terminus of the peptide results in extremely rapid amyloid formation. We believe that our study provides important findings for the development of long-acting lipidated analogues of peptide therapeutics.