title: Monitoring of RNA clearance in a novel plasmid DNA purification process.
abstract: 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.
authors.affiliation: Department of Biologics Development and Engineering, Merck Research Laboratories, Merck & Co., Inc, P.O. Box 4, West Point, PA 19486-0004, USA. jason_murphy@merck.com
authors.forename: Jason C
authors.initials: JC
authors.lastname: Murphy
authors.forename: Michael A
authors.initials: MA
authors.lastname: Winters
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Watson
authors.forename: John O
authors.initials: JO
authors.lastname: Konz
authors.forename: Sangeetha L
authors.initials: SL
authors.lastname: Sagar
mesh_terms: D001671:Biochemistry / Q000379:methods*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D011232:Chemical Precipitation; D004247:DNA / Q000302:isolation & purification*; D009113:Muramidase / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D012313:RNA / Q000032:analysis*; D012260:Ribonucleases / Q000737:chemistry; D012680:Sensitivity and Specificity; D014462:Ultrafiltration / Q000379:methods
chemical_list: D002593:Cetrimonium Compounds; D012313:RNA; D004247:DNA; D012260:Ribonucleases; D009113:Muramidase; D000077286:Cetrimonium