ABSTRACT
To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2'-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Nucleic Acid Denaturation , RNA/isolation & purification , Endothelial Growth Factors/genetics , Lymphokines/genetics , RNA/chemistry , Spectrophotometry, Ultraviolet , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare sequences from combinatorial libraries of sequence-randomized oligonucleotides (Gold et al., 1995). Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable approach for isolating inhibitors is competitive elution with a molecule that binds the targeted molecule's active or binding site. We have used this approach to isolate antagonists of wheat germ agglutinin (WGA) from a library of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N"-triacetylchitotriose, (GlcNAc)3. The highest affinity aptamers have equilibrium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold improvement relative to (GlcNAc)3, and unlike the carbohydrate, are highly specific. In addition to competing for binding with (GlcNAc)3, aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating that they are able to compete with natural ligands presented on the surfaces of cells. These results illustrate the feasibility of isolating high-affinity, high-specificity antagonists by competitive elution with low molecular weight, relatively low-affinity, and low-specificity small molecules.
Subject(s)
Gene Library , Oligonucleotides/genetics , Wheat Germ Agglutinins/antagonists & inhibitors , Animals , Base Sequence , Binding, Competitive/drug effects , Carbohydrate Metabolism , Cations, Divalent/pharmacology , Cloning, Molecular , Erythrocytes/drug effects , Erythrocytes/metabolism , Hemagglutination/drug effects , Ligands , Molecular Weight , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Oligonucleotides/pharmacology , Sheep , Thermodynamics , Trisaccharides/antagonists & inhibitors , Trisaccharides/metabolism , Wheat Germ Agglutinins/metabolism , Wheat Germ Agglutinins/pharmacologyABSTRACT
A family of sequence-related 2'-aminopyrimidine, 2'-hydroxylpurine aptamers, developed by oligonucleotide-based combinatorial chemistry, SELEX (systematic evolution of ligand by exponential enrichment) technology, binds human nonpancreatic secretory phospholipase A2 (hnps-PLA2) with nanomolar affinities and inhibits enzymatic activity. Aptamer 15, derived from the family, binds hnps-PLA2 with a Kd equal to 1.7 +/- 0.2 nM and, in a standard chromogenic assay of enzymatic activity, inhibits hnps-PLA2 with an IC50 of 4 nM, at a mole fraction of substrate concentration of 4 x 10(-6) and a calculated Ki of 0.14 nM. Aptamer 15 is selective for hnps-PLA2, having a 25- and 2500-fold lower affinity, respectively, for the unrelated proteins human neutrophil elastase and human IgG. Contractions of guinea pig lung pleural strips induced by hnps-PLA2 are abolished by 0.3 microM aptamer 15, whereas contractions induced by arachidonic acid are not altered. The structure that is essential for binding and inhibition appears to be a 40-base hairpin/loop motif with an asymmetrical internal loop. The affinity and activity of the aptamers demonstrate the ability of the SELEX process to isolate antagonists of nonnucleic-acid-binding proteins from vast oligonucleotide combinatorial libraries.
Subject(s)
Enzyme Inhibitors/pharmacology , Oligonucleotides/pharmacology , Phospholipases A/antagonists & inhibitors , Amino Acid Sequence , Animals , Base Sequence , Drug Design , Enzyme Inhibitors/metabolism , Gene Library , Group II Phospholipases A2 , Guinea Pigs , Humans , In Vitro Techniques , Indoles/metabolism , Indoles/pharmacology , Lung/drug effects , Lung/enzymology , Lung/physiology , Male , Molecular Sequence Data , Muscle, Smooth/drug effects , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Phospholipases A/metabolism , Phospholipases A2 , Pleura/drug effects , Pleura/enzymology , Pleura/physiology , RNA/chemistryABSTRACT
Intravenous immunoglobulins (IVIg) purified by cold ethanol fractionation have a very good safety record with regard to the transmission of many viruses. However, a few cases of non-A-non-B hepatitis have been described after intravenous injection of some immunoglobulin preparations. To ensure even higher safety for our IVIg, an additional virus inactivation step, based on pasteurization, was developed. The heating of aqueous IVIg was performed without stabilizer, and at a very low salt concentration (< 1 mM) at acidic pH. No generation of polymer was detected after pasteurization and a significant decrease in the proportion of dimers was observed. Analysis of the secondary structure by circular dichroism showed a very slight change in the secondary structure. The biological properties of the Fc region as well as the Fab region were not affected by the pasteurization. Our method has several advantages: (1) improvement of viral safety; (2) there is no need to add stabilizer which may stabilize viral particles, and (3) the absence of any hypotensive effect and low anticomplementary activity indicates a good clinical tolerance of IgG preparation.
Subject(s)
Hot Temperature , Immunoglobulins, Intravenous/isolation & purification , Sterilization/methods , Animals , Bacteriophage phi X 174/isolation & purification , Bacteriophage phi X 174/physiology , Blood/immunology , Blood/virology , Circular Dichroism , Cold Temperature , Dialysis , Dimerization , Ethanol , Humans , Hydrogen-Ion Concentration , Hypotension/chemically induced , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulins, Intravenous/adverse effects , Immunoglobulins, Intravenous/chemistry , Immunoglobulins, Intravenous/immunology , Protein Denaturation , Protein Structure, Secondary , Rats , Safety , Virus ReplicationABSTRACT
We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not absorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for absorption on thiophilic gels at low and high salt concentration.
