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.
Filamentous phage M13 is a rod shaped non-lytic bacterial virus. M13 genetic material is used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. The phage has been intensively studied for purposes of phage display and as a delivery vehicle for gene therapy. Phage display was first demonstrated with M13 bacteriophages and the filamentous phage remains a workhorse for this technology. Because of its typical size and rod shape it is considered as a challenging for purification. With large and highly interconnected pores monolithic chromatographic supports are also bridging that problem.
The ability to improve the purification process of M13 and other phages can have a significant impact on the market. By using phages for gene therapy, there will be a decrease in manufacturing time and production costs while enhancing the gene insertion. For phage display, a quicker method for phage purification will allow this powerful tool, which shortens the new drug discovery path and illuminates the basic interactions between different proteins, to be used with higher frequency.
Bacteriophages are used in a broad range of applications, including phage therapy and phage display. With the growing problem of antibiotic resistance leading to untreatable bacterial infections, they are becoming very interesting as antimicrobial agents, not only in medicine, but also in veterinary medicine, food industry and agriculture. Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants.
Here we present efficient single step purification method for a Staphylococcus aureus phage VDX-10 from bacterial lysate on a CIM® QA Disk Monolithic Column (Figure 1). The described method can be used also on a larger scale using a CIM® QA-8 mL Tube Monolithic Column (Figure 2).
Bacteriophages, viruses that infect bacteria, are being used as antibacterial agents, in phage display screening, as gene therapy delivery systems, and for bacteria typing. To use phages in these applications, they must be free of all impurities. A purification and concentration process was recently developed using an ion exchange monolithic column . One of the key challenges faced in phage purification is the monitoring of genomic DNA (gDNA) released to the growth medium which can interfere with the various applications of phages. CIMac™ DEAE Analytical Columns can be used to monitor the fermentation process, evaluate the amount of degraded gDNA to determine the optimal fermentation endpoint and then to efficiently purify the phage particles.