Monitoring of RNA Clearance in a Novel Plasmid DNA Purification Process

• Published in: Biotechnology progress Vol. 21; no. 4; pp. 1213 - 1219
• Main Authors: Murphy, Jason C., Winters, Michael A., Watson, Matthew P., Konz, John O., Sagar, Sangeetha L.
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.07.2005; American Institute of Chemical Engineers
• Subjects: alcohols; Biochemistry - methods; Biological and medical sciences; Biotechnology; Cetrimonium Compounds - chemistry; Chemical Precipitation; DNA; DNA - isolation & purification; Fluorescent indicators; Fundamental and applied biological sciences. Psychology; Gel electrophoresis; High-performance liquid chromatography; Impurities; Muramidase - chemistry; Muramidase - genetics; Plasmids; Plasmids - chemistry; Plasmids - genetics; Precipitation; Purification; Q1; Ribonucleases - chemistry; RNA; RNA - analysis; Sensitivity and Specificity; Spermidine; Ultrafiltration - methods; Vaccines; Plasmid; Purification; RNA; Operating process; DNA; Monitoring
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp050033o

As the field of plasmid DNA‐based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale‐up. Residual host‐cell RNA is a major impurity in current large‐scale separation processes for the production of clinical‐grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye‐based assays. However, these methodologies are difficult to employ as in‐process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate‐based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale‐up of a novel, non‐chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.