Sensitive and Robust Capillary Electrophoretic Analysis of Host Cell Residual DNA in Biopharmaceutical Samples

Abstract

Host cell residual DNA (rDNA) is a critical process-related impurity in biopharmaceutical production, involving trace amounts of DNA fragments originating from yeast, bacteria, CHO (Chinese hamster ovary), and other host cells. Monitoring both the size and quantity of rDNA is essential because of potential safety risks, including oncogenicity and immunogenicity, in biological products such as vaccines and other protein therapeutics. Regulatory guidelines from the WHO and U.S. FDA recommend limits of less than 10 ng/dose and below 200 base pairs (bp) for the rDNA content and size in final products. In this work, innovative capillary electrophoresis (CE) technologies that can accurately size rDNA were explored using a single-capillary (BIABooster) CE system and confirmed with a 12-capillary (Femto Pulse) CE system. After optimizing the sample diluent, detection parameters, and hydrodynamic injection volumes, the BIABooster method achieved highly sensitive detection at 200 bp in the linear range of 0.035 ng/mL to 1 ng/mL, with an LOD of 0.035 ng/mL (i.e., 0.0175 ng in 0.5 mL dose, S/N = 3) and an LOQ of 0.065 ng/mL (i.e, 0.0325 ng in 0.5 mL dose, S/N = 10). As a proof of concept, this method was successfully applied to separate rDNA in real biopharmaceutical samples, including yeast-derived in-process intermediate (IPI) samples and CHO-derived final products, showing high sizing accuracy (99.8–104.2%) and robust performance with an intermediate precision of 2.5% RSD from 200 bp to 1000 bp, across preparations, days, analysts, and capillary lots. Compared to conventional methods in rDNA analysis, this method offers advantages including high sensitivity and accuracy, automation, and no need for DNA probe design and amplification steps, resulting in faster method development.