title: Analytical Characterization of an Oil-in-Water Adjuvant Emulsion.
abstract: Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.
authors.affiliation: Analytical Biotechnology, MedImmune Inc., 20878, Gaithersburg, Maryland, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Sun
authors.affiliation: Analytical Biotechnology, MedImmune Inc., 20878, Gaithersburg, Maryland, USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Remmele
authors.affiliation: Analytical Biotechnology, MedImmune Inc., 20878, Gaithersburg, Maryland, USA. sanyalg@medimmune.com.
authors.forename: Gautam
authors.initials: G
authors.lastname: Sanyal
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000494:pharmacology; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology; D002853:Chromatography, Liquid / Q000379:methods; D004339:Drug Compounding / Q000379:methods*; D004655:Emulsions / Q000737:chemistry; D005079:Excipients / Q000737:chemistry / Q000494:pharmacology; D005960:Glucosides / Q000737:chemistry / Q000494:pharmacology; D008050:Lipid A / Q000737:chemistry / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D064166:Vaccine Potency; D014612:Vaccines / Q000737:chemistry / Q000493:pharmacokinetics; D024502:alpha-Tocopherol / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000276:Adjuvants, Immunologic; D000975:Antioxidants; D004655:Emulsions; D005079:Excipients; D005960:Glucosides; D008050:Lipid A; D014612:Vaccines; C000608161:glucopyranosyl lipid-A; D024502:alpha-Tocopherol
keywords: adjuvant; evaporative light scattering detection (ELSD); excipient; glucopyranosyl lipid A; liquid chromatography with mass spectrometry (LC-MS); vaccine