Identification and characterization of type-16 HPV-VLP capsomeres and monomers by two-dimensional liquid...

Analysis of Human Papillomavirus Virus-Like Particles (HPV-VLP) at various process points is complicated by multiple factors. First is the complexity of the moiety itself including its large size and the lack of available commercial solutions designed for analysis of VLP as they are typically large (50-60 nanometers) molecular assembly and, prone to clustering under various conditions. Second is the need to separate the VLP from cellular components if the sample is the fermentation broth, making it difficult to use traditional chromatographic columns. This study proposes the use of a two-dimensional liquid chromatography-hyphenated mass spectrometry (2DLC-MS) approach for the characterization of VLP. The proposed approach has been successfully demonstrated for quantification of in-house HPV-VLP L1 protein at different phases of product development with VLP titer observed to increase with respect to monomer species (39.07 %, 44.25 %, and 78.08 % of total protein) in crude, semi-purified, and purified samples, respectively. In addition to quantitative monomeric VLP identification using the stand-alone SEC-MS approach, a qualitative analysis was also conducted at the capsomere level. Peptide mapping as a confirmatory analysis was performed with all three in- process samples (crude-processed, semi-purified, and purified) of type-16 in-house HPV-VLP and with the commercially available Gardasil HPV-VLP. The results demonstrate that the proposed SEC-RPC-MS platform offers efficient and accurate quantification of HPV-VLP.