Self-displacement

When purifying oligonucleotides it is particularly useful to use sample self displacement chromatography since the required component of the mixture is generally the later eluting moiety. With this approach the column loading is increased to such a point that the more strongly retained component displaces the... 

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

The self-displacement approach to purification of a synthetic peptide containing 25 amino acids is shown below[51]. The analytical HPLC trace (Figure 5.1) shows the impurity peak (A), which is displaced by the main component (B) in the demonstration. 

Self-displacement will operate with almost any mode of chromatography including reversed phase as demonstrated above, normal phase, chiral and ion-exchange. The following procedures provide a generic approach to development of a... 

D. Displacement Chromatography and Sample Self-Displacement Chromatography... 

Additional results from AX purification of DMT-off synthetic oligonucleotides are discussed in the following section, based on the principles of displacement and sample self-displacement chromatography. 

Figure 9 shows an example of sample self-displacement chromatographic purification of DMT-off crude phosphorothioate oligonucleotide ISIS 2302... 

FIGURE 10 Purification of a 8 mer phosphorothioate on AX with sample self-displacement chromatography. A 8 mer phosphorothioate. DMT-off crude was purified on an AX column (Q Hyper D F, BioSepra Corp., Marlborough, MA). Buffer A 50 mM NaOH buffer B 50 mM NaOH + 2.5 M KCI column 50 X 100 mm Waters AP-5 column flow rate 35 mL / min (107 cm / hr). Gradients for the two purification rounds are shown in the chromatograms. A pool for the first purification rounds was collected, the conductivity reduced by dilution with water and injected for the second purification. Feed purity by CGE 77%. Purity of the product pool after second cycle 98% by CGE. Purity by AX feed =71%, product pool after second cycle = 99.5%. 

If a compound is the major component in a mixture, the production rate increases if the impurities are eluted first. However, it is preferable for the product to be eluted prior to any closely retained impurity if a compound is not the main component in the mixture. Linder self-displacement conditions, the product can actually be separated in a more concentrated solution than in a touching-band separation, especially if a later-retained impurity is in high concentration. The production rate in this case can be improved by an increase in loading, despite some decrease in recovery. 

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