The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.
Sample displacement chromatography exploits the different relative binding affinities of components in a sample mixture to achieve accummulation of a desired substance on the column before elution. In pharmaceutical applications, requirements for purity and efficacy of plasmid DNA (pDNA) as a therapeutic product are stringent. The separation of linear, supercoiled (sc) and open-circular (oc) pDNA isoforms has already been established on CIM® butyl (C4 HLD) monolithic columns at preprative scale. This process requires high concentration of ammonium sulphate for loading which increases the overall production requirements. Competing adsorption in sample displacement chromatography utilises the binding capacity of the chromatographic resin more efficiently and increases productivity of the chromatographic step.
This application note investigates three monolithic chromatographic supports with different hydrophobicities regarding their applicability for sample displacement of pDNA. CIMac™ C4 HLD (butyl, high ligand density) as a commercial product and pyridine and histamine as custom immobilised columns are compared.
Plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy and often one or more chromatographic steps are used in the downstream process. High ligand density butyl-modified chromatographic monolith (CIMmultus™ C4 HLD, part of CIMmultus™ HiP² Plasmid Process Pack™ 1-1, product number 100.0011-2) is currently used in a polishing step of a pDNA purification process (1), is mainly used for separation of supercoiled (sc) pDNA separation from open circular (oc) and linear pDNA isoforms as well as for removal of remaining gDNA and RNA.
This application note presents a comparison of two different polishing processes employing monoliths, namely bind-elute (BE) and the more recently described (2) sample displacement purification (SDP).
DNA immunization can potentially induce both, humoral and cellular immune responses, and thus comprises an attractive approach for the development of an effective vaccine against HCV. The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2.
In order to satisfy the high demanding plasmids consumption for clinical trials, the downstream process was improved to reach the quantities need it for clinical trials.
As the demand for plasmid DNA (pDNA) based gene therapy and vaccines increases, large scale, cost effective, and reproducible pDNA production will be required. The key to success is a real time in-process control method that ensures a high percentage of supercoiled pDNA in the final product. CIMac™ pDNA Analytical Column allows the monitoring of degradation products (open circular and linear pDNA), the removal of impurities (RNA), and ensures that each production step is yielding the amount of supercoiled pDNA anticipated.