Plasmid DNA (pDNA) used in vaccination and gene therapy has to meet criteria of highly purity and homogeneity, calling out for efficient, reproducible and scalable purification.
Convective Interaction Media (CIM®) monolith chromatography supports, designed for purification of large molecules and nanoparticles are a matrix of choice for pDNA purification. Monoliths enable high productivity of pDNA downstream processes (DSP) due to high dynamic binding capacity, fast operating flow rates and high resolution due to convection-based mass transfer.
BIA Separations’ HiP2 platform process achieves high purity and homogeneity of plasmid DNA in two chromatography steps. The process starts with optimized E. coli lysis, precipitation of impurities and clarification. The clarified lysate is subjected to chromatography to remove impurities and to isolate the supercoiled plasmid isoform:
- capture step on anion-exchange (AEX) chromatography (CIMmultus™ DEAE) concentrates pDNA and removes majority of host cell proteins (HCP), mRNA, and genomic DNA,
- polishing step on hydrophobic interaction chromatography (HIC) with a high ligand density butyl-modified (CIMmultus™ C4 HLD) monolithic support removes open circular (OC) and linear pDNA isoforms from supercoiled (SC) pDNA.
The process can be used in production of pDNA as raw material or therapeutic product. It is capable of removing the majority of contaminants (RNA, genomic DNA, HCP, endotoxins) and isolate supercoiled pDNA. In addition, the speed of operation allows the process to be completed in a few hours, thus increasing productivity and minimising manufacturing costs. Unique monolith properties facilitate straightforward scalability of the process covering production levels from small scale laboratory to large scale industrial purification of pDNA.
Robustness, reproducibility and product quality are assured with the use of CIMac™ pDNA Analytical Column. Short analysis times can provide analytical data about product quality in near real time. CIMac™ pDNA separates isoforms of plasmid DNA and shows the presence of other impurities. As such it is an ideal tool for in-process samples, as well as final product control.