Enhancing Plasmid DNA Quality Through Robust Topology Measurement

Plasmid DNA is a fundamental starting material for cell and gene therapy products, where quality attributes, such as topology, directly impact production. DNA topology measures the distribution of DNA isoforms, including supercoiled, open circular, linear, and other isoforms. Common analytical methods for topology assessment include agarose gel electrophoresis (AGE), capillary electrophoresis with laser-induced fluorescence (CE-LIF), and anion exchange chromatography (AEX). Topology assessment remains challenging due to method-dependent bias, limited harmonization, and a lack of well-characterized reference materials.

The United States Pharmacopeia (USP) has developed plasmid DNA analytical reference materials to assist in the characterization of plasmid DNA. These reference materials range in size from 4.1 kb – 8.8 kb to align with the different plasmid sizes used during production. The plasmids were predominantly supercoiled but also contained other isoforms to capture capabilities of each analytical method. Herein, we will share data from a multi-laboratory study and highlight differences between the three techniques.

These reference materials provide a standardized tool for improving topology assessment, supporting method development, qualification, and cross-platform comparability. Combined with USP documentary standards, they help enable more consistent and reliable testing practices, advancing the overall quality and characterization of plasmid DNA used in therapeutic manufacturing.

Presented by Dr. Anthony Blaszczyk, Principal Scientist in the Global Biologics Department at U.S. Pharmacopeia

Dr. Anthony Blaszczyk is a Principal Scientist in the Global Biologics Department at U.S. Pharmacopeia (USP), where he leads initiatives within the Pipeline Development group to advance standards for Cell and Gene Therapy (CGT). His work focuses on the development of both documentary and physical standards that support product quality and innovation. He plays a pivotal role in USP’s efforts to meet evolving industry needs through collaboration with scientific experts and stakeholders across the biopharmaceutical sector. Prior to joining USP, Anthony earned his Ph.D. in Biochemistry from Penn State University. He previously managed an Analytical Development team at Catalent’s Gaithersburg and Rockville sites, where he specialized in method development, qualification, and method transfer to support AAV-based gene therapy production.

Followed by a live question and answer session