Plasmid DNA Analysis using SEC

Introduction

Within the biopharmaceutical industry, recombinant plasmid DNAs are used as both raw materials and active ingredients in DNA vaccines. GSK is developing several plasmid DNA vaccines, characterised using different methods. Anion exchange-HPLC (AEC) and agarose-gel-electrophoresis (AGE) are used to determine plasmid topology. Light scattering size techniques are used to provide information on molecular weight. Here we present data, for different DNA plasmids analysed using SEC, LS, RI and V. The TDA detector provides information about the DNA plasmids, including molecular weight, which cannot be analysed using techniques such MS due to the large size of the plasmids.

Figure 1: Overlay of UV, DP, RALS and LALS for pDNA.

Figure 2: Plot of UV and Log Molecular weight for pDNA.

Method

The Model 302 TDA was coupled to a Waters Alliance 2690 HPLC. The Waters HPLC UV detector was disconnected, so that the sample flowed from the HPLC into the Model 302 detector. The TDA detectors are connected in series with UV first, LS (90 and 7°), RI then Viscometer.

TD-SEC of DNA

Analysis of DNA was carried out using a Tosoh Biosep DNA-SEC column. The buffer used to elute the DNA was 25 mM Borate with 0.5M sodium chloride pH 9. The column temperature was set at 40°C, and the flow rate was 0.75 ml/min, with UV detection at 260nm. The injection volume was 100 µl.

The DNA samples were run at a concentration of 10ug loading. The dn/dc value used for the DNA calculations was 0.165.

Results

Calculations were carried out using the UV detector as a concentration detector however, the RI was tested and gave comparable results.

Most plasmid DNA comprises of a mixture of three topoisomers: supercoiled (sc), open circle (oc), and linear (lin).

Supercoiled is the most compact form and is expected to be smaller in size than either linear or open circle forms. There is no appreciable size difference between linear and open circle forms. This trend is consistent for all the plasmid DNA constructs that were run.

Conclusions

• The SEC-TDA provides molecular weight data for DNA and proteins that compares well with theoretical calculated values.
• This is consistent for the different constructs, which have a different number of base pairs.
• The hydrodynamic radius data shows that the supercoiled form is smaller in size than the open circle and linear forms of DNA.
• There is also a very small difference in size between the open circle and linear forms of DNA.
• The coupling of TDA to SEC provides a lot of information, which is useful for the characterisation of proteins and DNA.
• The detectors and OmniSEC software allow for the calculation of values of hydrodynamic radius less than 10nm, a clear advantage over MALS.

The OmniSEC software is easy to use, and the data is presented in a format that is easy to understand.

References

• Molloy M.J, et al. Effective and robust plasmid topology analysis and the subsequent characterisation of the plasmid isoforms observed. Nucleic Acid Res., Sep 2004; 32 e129.
• Triple Detector Array (TDA) product brochure, Viscotek (2001-2003)
• Clarke, P.G (Viscotek Europe) The Applications book April 2003 p2-4.