Pharmaceutical analysis of synthetic lipid A-based vaccine adjuvants in poly (D,L-lactic-co-glycolic acid) nanoparticle formulations.
The present study had two main objectives. First, was to compare the immune stimulatory effect of two synthetic lipid A analogues (7-acyl lipid A and pentaerythritol-based lipid A (PET lipid A)) on maturation/stimulation of bone marrow derived dendritic cells (DCs). Our second objective was to develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of lipid A-based vaccine adjuvants. Treatment of immature DCs with 7-acyl lipid A and PET lipid A up regulated the surface expression of CD86 and CD40 molecules, and also induced similar profile of pro-inflammatory cytokine secretion. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, coupled to a Waters 2795 separations module with an autosampler. Calibration curves with R(2)>0.999 were constructed over the concentration range of 1.25-20 microg/ml for the solution of 7-acyl lipid A and PET lipid A. The method was tested in a 3 day validation protocol. The accuracy of the assay at different concentrations tested ranged from 89 to 108% and from 92 to 107% for 7-acyl lipid A and PET lipid A, respectively. The limit of quantification for both 7-acyl lipid A and PET lipid A was 1.25 microg/ml (signal/noise (S/N)) ratio >15:1. The sensitivity of the method (the limit of detection) was 0.35 and 0.15 ng for 7-acyl lipid A and PET lipid A, respectively (S/N ratio between 4:1 or 3:1). As a preliminary application, this method has been successfully applied to the determination of 7-acyl lipid A and PET lipid A content in poly (D,L-lactic-co-glycolic acid) nanoparticles (PLGA-NP).
Hamdy
Samar
S
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada. shamdy@pharmacy.ualberta.ca
Haddadi
Azita
A
Somayaji
Vishwa
V
Ruan
David
D
Samuel
John
J
D000276:Adjuvants, Immunologic / Q000032:analysis*; D000818:Animals; D051940:B7-2 Antigen / Q000096:biosynthesis; D001854:Bone Marrow Cells / Q000502:physiology; D019013:CD40 Antigens / Q000096:biosynthesis; D002626:Chemistry, Pharmaceutical; D002853:Chromatography, Liquid; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004339:Drug Compounding; D005434:Flow Cytometry; D019344:Lactic Acid / Q000032:analysis*; D008050:Lipid A / Q000031:analogs & derivatives / Q000032:analysis*; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D053758:Nanoparticles / Q000032:analysis*; D010641:Phenotype; D011100:Polyglycolic Acid / Q000032:analysis*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers / Q000032:analysis*; D011409:Propylene Glycols / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D015854:Up-Regulation / Q000187:drug effects
D000276:Adjuvants, Immunologic; D051940:B7-2 Antigen; D019013:CD40 Antigens; D016207:Cytokines; D008050:Lipid A; D011108:Polymers; D011409:Propylene Glycols; D012996:Solutions; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; C008783:pentaerythritol