Plasmid DNA

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.

CIM Convective Interaction Media® 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.

Sartorius BIA Separations’ HiP2 platform process achieves high purity and homogeneity of pDNA in two chromatography steps. 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, host cell proteins (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.

See the Instruction Manual and suggested protocol on how to use our Plasmid Process Pack.

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 HCP, mRNA, and genomic DNA,
Preparative chromatogram of capture step on CIMmultus DEAE 8 mL.
Preparative chromatogram of capture step on CIMmultus DEAE 8 mL.
  • 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.
Preparative chromatogram of polishing step on CIMmultus C4 HLD 1 mL.
Preparative chromatogram of polishing step on CIMmultus C4 HLD 1 mL.

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 pDNA and shows the presence of other impurities. As such it is an ideal tool for in-process samples, as well as final product control.

Chromatogram on CIMac pDNA analytical column with PATfix LC system.
Chromatogram on CIMac pDNA analytical column with PATfix LC system.

Monitor and Optimize Your Process With pDNA-Specific Analytics

PATfix pDNA Analytical Platform combines an at-line LC system with the monolith technology and pre-validated methods. Specifically set for non-chromatography expert to run analytics in process lab in only 5-15 minutes.

  1. Purity and content tracking during all process steps
  2. Single run separation of process related impurities (RNA, protein) and product related impurities (separation of key pDNA isoforoms sc/oc/lin)

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