Technical Notes

CIMac™ pDNA-0.3 Analytical Columns (Pores 1.4 μm) were introduced by BIA Separations d.o.o. in 2010. In the following years they were recognised by the biotechnological and analytical community as excellent choice for fast and exact plasmid DNA (pDNA) monitoring and quantification. Due to high sensitivity of the column to small changes in chromatographic conditions we publish this technical note, how to exploit the column’s excellent chromatographic characteristics to the highest possible extent.
A standard testing protocol for the columns is analytical separations of open circular (oc) and supercoiled (sc) pDNA isoforms of a 4.7 kbp large plasmid DNA molecule. The concentration of the pDNA sample used was 23μg/mL. The sample contained between 21 and 22% of the oc pDNA isoform and an exact amount of uracil as unbound tracer. The separation of the isoforms was performed in a linear gradient of NaCl with the temperature of the whole system controlled to 15.0±0.5 °C. The typical experimental conditions together with an example of chromatogram are shown in Figure 1.

Chromatographic applications in diagnostics call for automated systems and high-throughput analyses in order to cope with the large numbers of samples. BIA Separations offers differently modified monoliths (ion exchanging, affinity, hydrophobic…) in 96-well plate format to follow the increasing needs of DIAGNOSTIC laboratories. The example described here is an affinity-based CIM® Protein G 96-monolithic plate (Pores 2 um), which enables efficient and robust capture of different antibodies from complex samples. The application describes the capture of immunoglobulin G (IgG) from human plasma with subsequent IgG glycosylation studies. The stability of the column for at least 70 isolation steps and proof of no cross-contamination are shown.

The stability of QA, DEAE and SO3 CIMac™ Analytical Columns was tested according to the CIP (Cleaning In Place) procedures described in the respective Product Specification Sheets (see Table 1). We compared the separation of a mixture of test proteins, the dynamic binding capacity for BSA and the pressure drop after 50 and 100 CIP procedures with the initial characteristics of the columns.

CIMac™ SO3 Analytical Column is a strong cation exchanger based on a monolithic support. This material provides many advantages over conventional stationary phases for the separation of biomacromolecules, such as: low back-pressure, flow-unaffected chromatographic properties and a high resolution power due to a convective flow transport within open large-diameter (1.5 μm) channels.

One of the key steps in purification of plasmid DNA vaccines for therapeutic use is the separation of supercoiled (SC) and open circular (OC) conformations of plasmid DNA. In order to monitor product quantity and the separation of conformations throughout the downstream process as well as to verify the separation quality, accurate, reliable and user-friendly analytical methods need to be in place.