Characterisation of a peptide conjugate and impurities

Although much focus in the biologics market is on antibody-based therapies, smaller peptide therapies are another interesting species. Peptide therapeutics are generally well tolerated and metabolised by the body and are used to mimic functions such as hormones, growth factors, and neurotransmitters. These peptides can be conjugated with a variety of functional groups to increase half-life such as polyethylene glycol, sugars, or lipids. As these peptides are often produced using solid phase synthesis, as opposed to expression, there is the potential for process impurities as well as amino acid modifications.

Here we outline an analysis strategy to confirm the intact molecular weight and potential impurities for a GLP-1 receptor antagonist peptide with a palmitic acid conjugated to the lysine residue. All data was collected on a SCIEX X500B mass spectrometer with an EXION HPLC. The chromatography was performed with a bioZen XB-C18 Column (1.7 µm, 150 x 2.1 mm).

The peptide was measured on a 90-minute LC gradient that provided separation between the major peptide and truncations. -HA and -H were the major observed truncations and eluted after the major peak. Overall, the major species showed 95% purity by MS. Figure 3 shows the reconstructed peaks from 54-55 minutes and from 59-62 minutes. Underneath the major peak we observe Liraglutide with one oxidation and various adducts. In the later eluting species we can see the reconstructions of -H, -HA, -HAE (not labelled) and -HAEG.