Merck
  • Home
  • Search Results
  • Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support.

Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support.

Journal of chromatography. A (2014-12-03)
Patrícia Pereira, Ângela Sousa, João A Queiroz, Ana Figueiras, Fani Sousa
ABSTRACT

MicroRNA-based therapeutic applications have fostered a growing interest in the development of microRNAs purification processes in order to obtain the final product with high purity degree, good quality and biologically active. The pre-miR-29 deficiency or overexpression has been associated to a number of clinically important diseases, and its therapeutic application can be considered. Monolithic columns emerged as a new class of chromatographic supports used in the plasmid DNA purification platforms, being an interesting alternative to the conventional particle-based columns. Thus, the current work describes, for the first time, a new affinity chromatography method that combines the high selectivity of agmatine ligands with the versatility of monoliths to specifically and efficiently purify pre-miR-29 from other small RNA species and Rhodovulum sulfidophilum impurities. The effect of different flow rates on pre-miR-29 separation was also evaluated. Moreover, breakthrough experiments were designed to study the effect of different RNA concentrations on the modified monolithic support binding capacity, being verified that the dynamic binding capacity for RNA molecules is dependent of the feed concentration. In order to achieve higher efficiency and selectivity, three different binding and elution strategies based on increased sodium chloride (1.75-3M) or arginine (100mM) and decreased ammonium sulfate (2.4-0M) stepwise gradients are described to purify pre-miR-29. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (97.33 and 94.88%, respectively) as well as purity (75.21 and 90.11%, respectively) were obtained. Moreover, the quality control analysis revealed that the level of impurities (proteins, endotoxins, sRNA) in the final pre-miR-29 sample was negligible. In fact, this new monolithic support arises as a powerful instrument on the microRNA purification to be used in further clinical applications, providing a more rapid and economical purification platform.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sigma 7-9®, ≥99% (titration), crystalline
Sigma-Aldrich
Tromethamine, meets USP testing specifications
Sigma-Aldrich
Ammonium sulfate, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, BioXtra, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, for molecular biology, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, BioUltra, ≥99.0% (T)
Sigma-Aldrich
Agmatine sulfate salt, ≥97%, powder
Sigma-Aldrich
Diethyl pyrocarbonate, 96% (NT)
Supelco
Ammonium sulfate, analytical standard, for Nitrogen Determination According to Kjeldahl Method, traceable to NIST SRM 194
Sigma-Aldrich
Diethyl pyrocarbonate, 96% (NT)
Sigma-Aldrich
Ammonium sulfate, anhydrous, Redi-Dri, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99.0%
Supelco
Tromethamine, pharmaceutical secondary standard, certified reference material
Sigma-Aldrich
Ammonium sulfate, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, ACS reagent, ≥99.0%
SAFC
Tromethamine
Trometamol, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Ammonium-14N2 sulfate, 99.99 atom % 14N
Sigma-Aldrich
Diethyl pyrocarbonate, 96% (NT)
Sigma-Aldrich
Ammonium sulfate, 99.999% trace metals basis
Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Tris(hydroxymethyl)aminomethane, ACS reagent, ≥99.8%
Sigma-Aldrich
Trizma® base, ≥99.9% (titration), crystalline
Sigma-Aldrich
Trizma® base, BioXtra, pH 10.5-12.0 (1 M in H2O), ≥99.9% (titration)
Sigma-Aldrich
Trizma® base, BioPerformance Certified, meets EP, USP testing specifications, suitable for cell culture, ≥99.9% (titration)