Correlation between Dynamic Light Scattering and Size Exclusion High Performance Liquid Chromatography for Monitoring the Effect of pH on Stability of Biopharmaceuticals.

Aggregate formation is a major problem affecting both safety and efficacy of biopharmaceuticals and is associated with protein immunogenicity. Size exclusion high performance liquid chromatography (SE-HPLC) has always been the gold standard technique for detection and determination of protein aggregates. However, large protein aggregates may be filtered off and build up on top of the column leading to deterioration in column performance. Moreover, low-affinity protein aggregates may dissociate during analysis and thus not detected. On the other hand, dynamic light scattering (DLS) is a simple and non-destructive technique that can detect high molecular weight physical and chemical aggregates in their native environment. Here, three model biopharmaceutical proteins of different physicochemical properties were selected; quadrivalent human papillomavirus virus like particles vaccine (HPV VLP, physically assembled subunit vaccine, 55kDa), pegylated Interferon (PegIFN, pegylated non-glycosylated protein, 31.3kDa) and Pegylated Erythropoietin (PegEPO, pegylated and glycosylated protein, 60kDa). Samples were subjected to forced degradation conditions previously shown to lead to aggregate formation (pH 4.0, 8.0 and 10.0, at 37°C for 24h) and samples were analyzed using DLS and SE-HPLC. Generally, good agreement between the results of DLS and SE-HPLC was noted, regardless of the differences in physicochemical properties of the studied biopharmaceuticals. Results showed that aggregate formation was not detected in some cases by SE-HPLC and the decrease in the concentration of the monomeric forms indicated that such aggregates might have been filtered off the column. Although no single techniques can reveal all aspects of protein stability, DLS can serve as a screening tool to detect aggregate formation and cross-validate SE-HPLC results during batch release testing. Owing to its simplicity and low-sample volume requirements, DLS can be used even by hospital pharmacists to confirm absence of protein aggregates immediately before drug administration.

Optimization and validation of ELISA immunoassay for the evaluation of in-vitro relative potency of a four-component human papillomavirus vaccine products.

A sandwich-type ELISA was optimized and validated to determine the in-vitro relative potency of the four-component prophylactic Human papillomavirus (HPV) vaccine. The vaccine contains the non-infectious virus like particles (VLP) corresponding to HPV Types 6, 11, 16 and 18. A modification of the desorption step required to release the VLPs from the aluminum adjuvant was carried out. Samples were incubated with citrate buffer for two hours at 37 °C instead of overnight incubation at room temperature. Assay validation was then carried out according to ICH guidelines. The assay was linear over a concentration range of 0.30-2000.00 ng/mL for the four HPV types. The assay was accurate and precise with a LOD of 0.092, 0.081, 0.086 and 0.068 ng/mL for type 6, 11, 16 and 18 respectively. Results were also statistically compared to those obtained using the reported ELISA assay and no significant difference was noted. In contrary to the reported ELISA protocol, this optimized immunoassay was superior with respect to analysis time, without affecting the accuracy and precision (RSD < 5%). This assay has proven to be useful for evaluating the efficacy of the quadrivalent HPV vaccine and is applicable for quality control and batch release purposes.

Immunoaffinity extraction using conformation-dependent antibodies coupled to SE-HPLC for the development of stability and potency-indicating assay for quadrivalent human papillomavirus vaccine.

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.