Nihal Tugcu

Purification of an oligonucleotide at high column loading by high affinity, low-molecular-mass displacers.

The development of efficient techniques for large-scale oligonucleotide purification is of great interest due to the increased demand for antisense oligonucleotides as therapeutics as well as their use for target validation and gene functionalization. This paper describes the use of anion-exchange displacement chromatography for the purification of 20-mer phosphorothioate oligonucleotide from its closely related impurities using low-molecular-mass amaranth as the displacer. Experiments were carried out to examine the effect of the feed load on the performance of the displacement chromatography. In contrast to prior work, displacement chromatography was successfully scaled-up to high column loadings while maintaining high purity and yields. Experiments carried out on a Source 15Q column indicated that crude oligonucleotide loading as high as 39.2 mg/ml of column were readily processed, resulting in product recovery of 86% and purity of 92%. These results demonstrate that anion-exchange displacement chromatography can indeed be employed for large-scale oligonucleotide separations at high column loading.

Displacement chromatography of anti-sense oligonucleotide and proteins using saccharin as a non-toxic displacer

In performing displacement chromatography for the purification of biomolecules, one of the biggest challenges has been the selection of the proper high affinity displacer. The displacer not only has to have sufficient dynamic affinity to carry out the displacement but must also have suitable operational properties which will enable a cost effective and simple process. One of these requirements is the non-toxic nature of the displacer, which if satisfied will make displacement chromatography a more attractive tool for biopharmaceutical applications. In this study, a new non-toxic low molecular weight displacer, saccharin, was introduced and characterized for the purification of an oligonucleotide and proteins by anion exchange displacement chromatography. It was demonstrated that saccharin, with only one charge, can indeed displace and purify very highly retained oligonucleotides and proteins. The operating conditions for the displacement experiments were predicted using operating regime plots. The results indicate that saccharin is not only effective as a displacer for isotachic displacements but for selective displacements as well.

Predicting Column Performance in Displacement Chromatography from High Throughput Screening Batch Experiments

A novel high throughput screening (HTS) technique has been recently developed for displacer discovery. In this article, the multicomponent steric mass action (SMA) model is used to determine column performance in displacement chromatography from batch HTS results. The multicomponent isotherm is first used to predict the displacer concentration in the batch HTS experiments without assaying for the displacer. This information is then employed to determine the single component dynamic affinities of the displacer and the protein and to predict displacement efficacy under column conditions. The model is used to predict the column displacement of horse heart cytochrome-C using N-α-benzoyl arginine ethyl ester as the displacer based on batch HTS results.

High throughput screening and quantitative structure efficacy relationship models for designing displacers for antisense oligonucleotide purification in anion-exchange systems

A high throughput screening (HTS) technique was developed for the rapid evaluation of displacers for the purification of antisense oligonucleotides using anion-exchange systems. By employing this technique, a large number of potential displacers with a variety of structural properties were evaluated in parallel, dramatically decreasing the time required for displacer discovery. A database was generated containing molecular descriptors of the screened displacer probes as well as their displacement data obtained from the HTS experiments. A subset of the probe data was used to derive a predictive quantitative structure efficacy relationship (QSER) model using a partial least squares (PLS) approach. The resulting model was well correlated with In addition, the model was shown to predict the molecules not included in the model successfully. The results presented in this paper demonstrate the utility of the HTS technique for the rapid evaluation of displacers for the purification of antisense oligonucleotides. These results applied in concert with the QSER modeling, can aid in the a priori design of high-affinity displacers for anion-exchange systems.