Although small molecules represent the majority of compounds in development as drug candidates, there is an increasing tendency to more complex molecular structures to address unmet molecular needs. This has been reflected in the types of molecules that the LC-MS/MS Discovery team at KCAS has increasingly been working with. This means that the typical small molecule research grade assay is not necessarily applicable to discovery work because the methods for molecules themselves require much more focused development work than for a typical rapid turnaround LC-MS/MS assay.
Peptide based drugs have been shown to have potential therapeutic applications but their chemical properties can work against them. The need to enhance the delivery as well as the targeting and binding of peptide-based drugs has prompted addition of other chemical moieties to alter their physicochemical properties. An example change is the addition of lipid-based chains to the peptide of interest.
Glucagon-like peptide 1 agonists (GLP-1 agonists) have been approved for patients with Type 2 diabetes to help control blood sugar levels. The first of these medications to be approved, exenatide, is a synthetic version of Exendin-4 which is found in Gila Monster saliva. Other GLP-1 agonists include liraglutide and semaglutide amongst others. Moving from exenatide to liraglutide and then semaglutide involves going from a peptide molecule to others with increasingly large lipid additions. All three work with the same mode of action but have markedly different chemical properties.
From a bioanalytical perspective it would seem that adapting an existing assay for exenatide to liraglutide or semaglutide should be relatively straightforward but the increasing non-polar portions of the overall molecule introduce a number of different issues. The detection and ionization efficiency is very different from the “native peptide” so achieving comparable sensitivity is not possible without additional work. Similarly, the chromatographic conditions for exenatide do not translate directly to semaglutide so further optimization is a necessary part of the development process.
In common with GLP-1 agonists like liraglutide and semagluitde, the biggest challenges that we’ve encountered with other lipid-substituted peptides result from adsorption throughout the bioanalytical process. Lo-bind tubes are our default approach during sample preparation. Post extraction, carryover is the primary concern during chromatographic runs and it can occur anywhere in the sample flow path from injector to valves to the analytical column. This can drastically reduce the calibration range of the assay or prompt complex washing procedures that in turn lead to long run times.
These types of molecules are analytically challenging but having good background information can really help when looking to develop an LC-MS/MS assay. It’s really helpful to have information on solubility, particularly solvents that are applicable and relevant pH range. The solubility information that is provided for dosing in tox. studies can also help inform the bioanalytical team for preparation of standards. It is also useful to know if there are solvent systems that mitigate adsorption so that can help with a strategy to address carryover.