(1303-D) High Throughput Analytics: Automation of HPV Yeast Sample Preparation for Intracellular HPV Protein Flow Cytometry
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Joshua Barton Sr. Scientist Merck & Co. West Point, PA
Co-Authors Nicole Smiddy 1, Amy Bowman 1, Emilie Yen 1, Rachel Thompson 1, Sijia Yi 2, Malini Mukherjee 3, Melissa Whiteman 2, and John Loughney 1 Merck & Co., Inc., Analytical Research and Development, 1. West Point, PA – High-Throughput Analytics; 2. Cell-Based Sciences; 3. Separation-Based Analytics
The Gardasil Human Papilloma Virus (HPV) vaccine is comprised of HPV protein virus-like particles produced during fermentation in genotype specific recombinant yeast cells. Previously, protein content had only been measured in cell lysates after harvest by Simple Western and knowledge of protein kinetics in whole cells during fermentation was never studied due to lack of appropriate analytical tools. Thus, a novel assay involving yeast cell intracellular protein staining, and flow cytometry was developed enabling protein measurement during fermentation for the first time. Despite the assays’ novelty, manual sample preparation prior to flow cytometry is labor intensive and time-consuming. The assay requires an initial measurement of cell optical density (OD) used to calculate concentration and dilute to a target density. Then, the cell wall is removed by enzymatic digestion, and the cells are fixed, permeabilized and immunostained with protein specific antibodies. The assay thus requires multiple repetitive liquid handling steps including sample and reagent addition to a 96-well plate and cell pellet resuspension. To address these limitations, sample prep was automated on a Tecan EVO configured with an Agilent Vspin centrifuge for cell washing and two Inheco TEC heating blocks for cell wall digestion. An Excel template with Visual Basic code was used to create workbooks and generate liquid handling worklists to drive the reformatting of samples from tubes into 96-well plates. These worklists also determine and execute the optimal dilutions for cell wall digestion based on OD results. This automated workflow has increased sample throughput enabling faster turnaround time for data, more in-depth screening, and the improved design of fermentation studies to provide a greater understanding of protein expression during fermentation.