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.

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump

“…Moreover, either pDNA or the above mentioned contaminants can be selectively precipitated using e.g. isopropanol [4], cetyltrimethylammonium bromide (CTAB) [5], polyethylene glycol [6], LiCl [7], CsCl [8] or spermidine [9]. Since pDNA molecules are very large (supercoiled plasmids have a size of several thousandÅ in length) [2], the small pores of many traditional chromatographic media are inaccessible for the plasmids, which thus drastically reduces the dynamic binding surface area.…”

Product Specifications and Quality Control

“…The current method of RNA detection is agarose gel electrophoresis. 88 The use of DNAse is another pretreatment strategy that could be used to reduce interference from DNA. Alternative methods to detect RNA are quite similar to the ones described above to detect gDNA and include Northern blot analysis, fl uorescent dye -based technologies, and reverse transcription polymerase chain reaction (RT -PCR).…”

Large‐scale production of endotoxin‐free plasmids for transient expression in mammalian cell culture

“…There are at least two ways for rapid isolation of plasmid from lysate. The first is selective precipitation of plasmid DNA from clarified lysate by CTAB (Lander et al, 2002;Murphy et al, 2005;Tomanee et al, 2004), isopropanol, PEG or ammonium acetate (Wright et al, 2003). Precipitation is cheap and simple, but has a limited scale-up potential.…”