title: Immunoaffinity extraction using conformation-dependent antibodies coupled to SE-HPLC for the development of stability and potency-indicating assay for quadrivalent human papillomavirus vaccine.
abstract: Quadrivalent human papillomavirus (HPV) vaccine is formulated of four types of non-infectious recombinant virus like particles (VLPs) that are structurally and immunologically similar to the corresponding infectious HPV virus types 6, 11, 16 and 18. With almost identical physical, chemical and structural properties of the four types of VLPs, ELISA remains the only approved in vitro potency testing assay. In this study, an alternative industry-friendly, stability- and potency-indicating assay protocol was developed and validated for the determination of HPV vaccine. Vacuum-driven immunoaffinity extraction (IAE) was employed using type-specific, conformation-dependent antibodies against each type of HPV VLPs. ELISA assay was employed to evaluate the ability of IAE columns to specifically separate each of the four types of VLPs from their quadrivalent mixture. Mean percentage recoveries of 76.76±2.69, 69.12±5.79, 84.86±5.25 and 71.14±4.50% were obtained for VLPs types 6, 11, 16 and 18, respectively with no significant interference in each case. Antigen content was then determined using SE-HPLC over a concentration range of 5.00-20.00μg/mL (r>0.998) for VLPs type 6, 11, 16 and 18, respectively. The SE-HPLC assay was found accurate and precise (RSD<10.00%) with LOD ranging from 1.23-3.85μg/mL. The assay protocol was found superior to conventional ELISA assay with respect to simplicity, total analysis time and cost. Good correlation between the results of analysis obtained using IAE-SE-HPLC and ELISA demonstrated the suitability of the suggested assay protocol for stability and potency assessment with a good potential for implementation for batch release. This approach should be applicable for quality assessment of other vaccine preparations based on VLPs.
authors.affiliation: National Organization for Research and Control of Biologicals, Egypt.
authors.forename: Moushira M
authors.initials: MM
authors.lastname: Mostafa
authors.affiliation: Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt; Bioanalysis Research Group, Faculty of Pharmacy, Cairo University, Egypt. Electronic address: medhat.alghobashy@cu.edu.eg.
authors.forename: Medhat A
authors.initials: MA
authors.lastname: Al-Ghobashy
authors.affiliation: National Organization for Research and Control of Biologicals, Egypt.
authors.forename: Faten A
authors.initials: FA
authors.lastname: Fathalla
authors.affiliation: Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt.
authors.forename: Maissa Y
authors.initials: MY
authors.lastname: Salem
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D053918:Papillomavirus Vaccines / Q000737:chemistry / Q000302:isolation & purification*; D058425:Vaccines, Virus-Like Particle / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D055910:Antibodies, Immobilized; D053918:Papillomavirus Vaccines; D058425:Vaccines, Virus-Like Particle
keywords: Human papillomavirus vaccine; Immunoaffinity extraction; Potency-indicating assay; Size exclusion chromatography; Virus like particles