Quantification of Lipid and Peptide Content in Antigenic Peptide-loaded Liposome Formulations by Reversed-phase UPLC using UV Absorbance and Evaporative Light Scattering Detection

Jeroen Heuts,Celine Van Haaren,Stefan Romeijn,Ferry Ossendorp,Wim Jiskoot,Koen Van Der Maaden

doi:10.1016/j.xphs.2022.01.021

Abstract

Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantification was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 °C and 40 °C.

Highlights

Cationic liposomes have shown to be an efficient delivery vehicle for peptide-based cancer vaccines [1,2,3,4,5,6,7]
Reversed phase ultra-performance liquid chromatography (RP-UPLC) methods have been extensively used to separate peptides and lipids based on their hydrophobicity . 1,5,11,12,16 Upon separation, peptides are detectable by ultraviolet (UV) detection of the peptide bond at a wavelength of 214 nm, where the π → π* transition leads to light absorption [17,18]
The lipid calibration curves for both DOTAP and DOPC were prepared by automated injections of increasing volumes from the respective lipid stock solution, or by manual dilution followed by injection of a constant volume

Summary

Introduction

Cationic liposomes have shown to be an efficient delivery vehicle for peptide-based cancer vaccines [1,2,3,4,5,6,7]. We have shown that positively charged liposomes, composed of 1,2-dioleoyl-3trimethylammoniumpropane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), can accommodate a wide range of physicochemically diverse antigenic peptides 1 This enables the use of these liposomes in personalized cancer vaccines, where each patient will receive a personalized vaccine containing a unique set of tumor antigens [1,8,9,10]. A quantification method for liposomal peptide-based vaccines should not require extraction and should be suitable for any kind of peptide, whereas at the same time enables the quantification of the lipids. This implies that the presence of lipids and peptides should not mutually influence each other’s quantification. The ELSD responses are fitted by non-linear regression [20,21,22]

Methods

Results

Discussion

Conclusion