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Abstract
The quantitation of L1 protein is an important metric during the pharmaceutical manufacturing of human papilloma virus (HPV) vaccines, as they are critical components of virus like proteins (VLPs) that form the core of the vaccine matrix. The L1 protein is present in multiple states, including monomer, multimer, capsomere, fully formed VLPs, as well as aggregate species, and their expression levels provides an important read-out of upstream productivity and downstream purification efficiency through the measurement of step yields. However, quantitation of total L1 is challenging not only due to its presence in multiple states, but also due to the matrix complexity and purification stage of the samples which spans complex lysate to cleaner post purification material. Current analytical methods typically implemented for L1 quantitation includes simple western, which has its advantages, but requires sensitive and robust antibodies and are highly dependent on binding to accessible and linear regions of the L1. Additionally, conventional one-dimensional separation of L1 is ineffective where L1 and host cell protein or cellular components coelute, particularly in high-complexity lysate samples and intermediates prior to chromatographic capture steps. Herein, we present a sample preparation strategy and analytical method that is capable of total L1 quantitation regardless of its multimeric state and is compatible with sample matrices ranging from crude lysate to purified samples, without the use of complicated and serotype-specific reagents. We employ reduction and heat-denaturation during sample preparation to simplify the multimeric states of L1 to its monomer form and utilize two-dimension liquid chromatography (2D-LC) with first dimension (1D) size exclusion and second dimension (2D) reversed phase separation modes and demonstrate a suitable method for total L1 quantitation in multiple process intermediate steps.
