Biomedical subjects
Publications and source records attributed to D L Cole.
High-quality oligonucleotides are obtained by selective modification of sequences containing aldehyde apurinic sites with a new chromatographic tag followed by RP-HPLC separation. Hydroxylamine derivative 1 of a water soluble nonionic surfactant modifies oligonucleotides selectively at abasic sites leading to significantly increased retention.
Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.
A triester method for the synthesis of deoxynucleoside phosphorodithioate dimers is described. The phosphorodithioate linkage is introduced using a new dithiophosphorylating reagent DPSE-SP(S)Cl(2)where DPSE = 2-diphenylmethylsilylethyl. This group is removed quickly using tetra-butylammonium fluoride leading to the quantitative formation of phosphorodithioate diesters uncontaminated with the corresponding phosphorothioates. The utility of this group is demonstrated by the synthesis of a penta-decathymidylic acid, [T(PS(2))T(PO(2))](7)T, which contains alternating phosphorodithioate/phosphate diester internucleotide linkages.
Chromatography is an effective tool for obtaining high-purity synthetic oligonucleotides for a variety of end uses, including antisense drug therapy. Reversed-phase and anion-exchange chromatographies are widely used techniques for this application. While selectivity of these techniques can be modified by methods such as ion-pair RP-HPLC or affinity chromatography, these are presently used only at small scales. RP chromatography makes use of terminal hydrophobic-protecting groups to increase retention and selectivity. The main advantages of the RP method are its utility for the purification of a wide variety of modified oligonucleotide structures, its applicability across a range of terminal hydrophobic groups, such as fluorescein, and its ready use from small scale to very large scale with a minimal requirement for process development. AX-HPLC can also give high-purity products at generally higher media capacities. A more extensive method development effort is typically required for the AX-HPLC purification of AO. The AX yield per unit operation can be lower, but the isolated yield of DMT-off desalted oligonucleotide can be equal to or higher than that from RP-HPLC. As additional AO drugs enter and mature in the market, there will be a potential need for ton-scale purification processes. AX provides a way to scale up production on somewhat less expensive equipment with reduced organic solvent requirements.
Liposomes have been recognized as new delivery vehicles for peptide and oligonucleotide drugs, offering effective drug protection and influencing drug distribution from the circulation to tissues. To ensure consistent formulation behavior and drug distribution, the amounts of free and encapsulated active pharmaceutical ingredient in the liposome formulation must be determined. A simple and reliable method has been developed for the determination of free and encapsulated oligonucleotide drugs in liposomes. Capillary electrophoresis in entangled polyacrylamide solution was optimized for the analysis. Liposome samples were treated to release encapsulated oligonucleotide so that total concentration could be determined, and untreated liposomes were applied directly to capillary column to determine the fraction of free oligonucleotides. Recoveries are 96-105% and relative standard deviations are generally 2-3%. Multiple liposome types were analyzed with satisfactory results.
Extensive investigations on the influence of diastereomeric ratios of deoxyribonucleoside phosphoramidites on stereo-reproducibility of solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach indicate that the process is stereoreproducible and under inherent process control.
The purity of a drug substance can influence its toxicity and potency, so impurities must be specifically determined. In the case of synthetic oligodeoxyribonucleotide drugs, however, product complexity makes complete impurity speciation difficult. The goal of the present work was to develop a new analytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide probes were designed, each complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently optimized conditions, only the perfectly sequence-matched oligodeoxyribonucleotide hybridized to each probe, while all other deletion sequences were removed by washing with buffer. The 35S signal intensity of the bound oligodeoxyribonucleotide was proportional to the concentration of each (n-1)mer deletion sequence in the analyte solution. This method has been applied to a number of synthetic phosphorothioate oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n -1)mer deletion sequences present.
It is demonstrated that a solution of dichloroacetic acid in toluene removes dimethoxytrityl groups from the 5'-terminus of an antisense phosphorothioate oligodeoxyribonucleotide (ISIS 5132/CGP69846A) during synthesis on solid support cleanly and efficiently. It is therefore suggested to replace health hazardous dichloromethane which is typically used in oligonucleotide synthesis as solvent for DMTr-removal by toluene.
Oligodeoxyribonucleotide phosphorothioates undergo desulfurization in presence of metal in aqueous ammonia at elevated temperatures. The extent of sulfur loss and chain cleavage have been investigated.
The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.
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