Assessing the impact of nonspecific binding on oligonucleotide bioanalysis.

Aim: Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanalytical methods for more robust analyses. Recent advances toward mitigating sample losses on liquid chromatography (LC) systems have produced recovery advantages for oligonucleotide separations. Results & methodology: LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorganic-organic silica surfaces. Designed to minimize metal-analyte adsorption, these surfaces demonstrated a 73% increase in 25-mer phosphorothioate oligonucleotide recovery using ion-pairing reversed-phase LC versus standard LC surfaces, most particularly upon initial use. Conclusion: Hybrid silica chromatographic surfaces improve the performance, detection limits and reproducibility of oligonucleotide bioanalytical assays.

Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides.

Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP - IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide.

Sieving of proteins in silica colloidal crystals of millimeter dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 µm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.

Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries.

This review presents the need for replacing gels in 2D separations for proteomics, where speed, high-throughput, and the ability to characterize trace level proteins or small samples are the current desires. The theme of the review is isoelectric focusing, which is a valuable tool because it pre-concentrates proteins in addition to separating with high peak capacity. The review traces the technological progress from gel IEF to CIEF to packed capillaries with immobilized gradients for CIEF. Multiple capillary techniques are progressing toward meeting the current desires, providing extremely high sensitivity with regard to concentration and to small samples, integrated automation, and high peak capacity from multiple dimensions of separation. Capillaries with immobilized pH gradients for CIEF are emerging, which will alleviate interference from ampholytes and improve reproducibility in separation times when this valuable technique can be used as one of the dimensions.

Field-free remobilization of proteins after isoelectric focusing in packed capillaries.

Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The capillary dimensions are 100 µm i.d. and 2 cm in length, and the packing is made of 0.9 µm nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II, and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of 2 compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D = 6.1(±0.3) × 10(-8) cm(2)/s for the packed capillary vs D = 28.8(±0.3) × 10(-8) cm(2)/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H = σ(2)/L = 0.64 µm. This limits the resolution to 0.1 pH units for the 2 cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.