Chromatographic Separation of Circular Plasmid DNA Topoisomers by HPLC: The quinine carbamate CSPs were originally designed for enantiomer separations. However, it was recently discovered that these
silica-based stationary phases with quinine carbamate ligands behave like mixedmode reversed-phase/weak anion-exchangers (18)
and are potentially useful in other applications as well. Such recently reported applications include among others the separations
of hydrophilic peptides with closely related structures including isomers (19). Even more notable, quinine carbamate ligands
attached to silica gel have also been demonstrated to be powerful chemoaffinity materials for the chromatographic separation
of plasmid topoisomers (20–21).
Table 2: The SFC gradient programme.
Plasmid DNA is a popular vector in biotechnology for gene transfer into mammalian cells and is currently being clinically
tested for use in human gene therapy. Quality guidelines state that for human use, plasmid DNA should be in its supercoiled
isoform, the so-called covalently closed circular (ccc) form. Native ccc form consists of a distribution of individual topological
isomers (topoisomers) which differ only in the linking number, that is, by the degree of supercoiling. If a single strand
break (nick) occurs in a supercoiled species, the torsional strain is released and an open-circular (oc) form is obtained.
The oc form differs significantly from the ccc form in its hydrodynamic radius and is regarded as an impurity in plasmid DNA
(pDNA) samples. Therefore, within the pharmaceutical industry there is a demand for powerful and selective stationary phases,
which allow the separation of the two plasmid topologies for quality control. QN AX, along with other quinine-derived CSPs,
are able to selectively interact with different supercoiled topoisomers of circular pDNA and allow simultaneous separation
of oc form (Figure 3). The peak with the asterisk in Figure 3 represents the oc form which is less strongly bound to the quinine
carbamate stationary phase (21). The Gaussian distribution of peaks observed later is the ccc form eluting, with each individual
peak constituting a single topoisomer. Adjacent peaks differ in the linking number by a single supercoil. Resolving supercoiling
is of significance in various biochemical studies into DNA topology. Furthermore, quinine carbamate stationary phases might
also be a useful tool for in-process analysis and quality control in the biotechnological production of pDNA biopharmaceuticals
to monitor topoisomer patterns as another process parameter (20).
Figure 5: Straightforward method transfer from gradient to isocratic. (a-b) N-Benzoyl-DL-phenylalanine (Compound 12), (c-d)
3-oxo-1-Indancarboxylic acid (Compound 5); Gradient elution: (a) and (c); Modifier % in the isocratic runs: (b) 45%, (d) 30%.
SFC Applications: SFC applications using columns packed with the weak anion-exchanger CSPs have not been investigated until very recently.
Although several research groups — mainly in private companies — have undertaken some successful tests on these columns, the
efforts focused on specific applications and no systematic investigation was performed. Moreover, the results obtained have
not been published due to confidentiality reasons.
In this context, studies of the main parameters to be considered in the SFC mode with anion-exchange selectors have been carried
out recently (22-23). The different elements will be discussed in this section.
Figure 6: Dependence of SFC results on percentage of formic acid (FA). Column: QN AX, Modifier: MeOH/NH4OOCH 100/0.35 v/m + FA.
In HPLC, methanol has been demonstrated to be a versatile mobile phase on QN AX and QD AX columns. Owing to its pronounced
protic properties, methanol provides efficient salvation of all the ionized species involved in the ion exchange equilibria.
Often used as a single solvent in HPLC, the eluotropic strength of methanol can be adjusted by the concentration of the counter-ion
(acidic additives) and by the ionic strength (acidic and salt additives).
The same principles can be applied in SFC in terms of the choice for the bulk modifier and the acid salt additives. The investigations
performed showed that formic acid (FA) and NH4OOCH added into methanol were leading to remarkable results when used as the modifier of SF-CO2.