ABSTRACT
Ion pair-reversed phase (IP-RP) HPLC is one of the most widely used methods for the analysis of oligonucleotide impurities. The method is compatible with mass spectrometry and has been used to guide the development of improved synthesis and purification approaches. The ability to detect and characterize impurities depends on the reagents and the IP buffer system employed, as each can directly affect the degree of chromatographic separation and the sensitivity of detection by MS. Previous work in our laboratory has shown that small alkyl amines are suitable IP reagents for the analysis of impurities in phosphate diester oligonucleotides and can be used to differentiate among individual members of composite impurity families. The addition of an alkyl acid often further enhances peak separation, but at the detriment of ion signal. An improved method with increased chromatographic performance and sensitivity of detection is presented here. Improvements were mainly realized through the use of lower concentrations of small alkyl amine (i.e., 5 mM) and acid (0.5 mM) IP reagents, and ammonium bicarbonate (20 mM) as a buffer. The improved capabilities of the new method are demonstrated by separation of the individual components of the composite n - 1 impurity in a set of four production-scale batches of a single oligonucleotide. Addition of the alkyl acid resulted in resolution of most individual n - 1 impurities. The observed enhanced sensitivity of detection allowed multiple reaction monitoring (MRM) experiments, which were used to differentiate among unresolved impurities.
Subject(s)
Amines/chemistry , Chromatography, Reverse-Phase/methods , Drug Contamination , Oligonucleotides , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Oligonucleotides/analysis , Oligonucleotides/chemistryABSTRACT
Safety assessment of drug impurities is a routine part of the drug development process. For oligonucleotide-based drugs, impurities can arise from impurities in starting materials, as by-products of the manufacturing process or from degradation, and are generally structurally similar to the parent oligonucleotide. To study the potential impact of impurities, a representative batch of a 2'-O-methoxyethyl (MOE) antisense oligonucleotide (ASO) was compared to batches of drug that were enriched with nine of the common impurities encountered with the chemical class. Mice were treated for 3 months with weekly subcutaneous injection of 10 or 30 mg/kg. The impurity content of the parent batch was 0.25%-2.5% of total drug substance. The enriched impurity mixtures contained from 3% to 10% of the various impurities. The expected common class effects were observed at the 30 mg/kg/week dose level in hematology, serum chemistry, and histopathology. However, there were no differences between the representative batch of material and those enriched with impurities. Based on these data, common oligonucleotide impurity studies do not appear to contribute to the overall toxicology profile.
Subject(s)
Drug Contamination , Liver/drug effects , Oligonucleotides, Antisense/pharmacology , Animals , Humans , Liver/pathology , Mice , Oligonucleotides, Antisense/therapeutic use , Oligonucleotides, Antisense/toxicityABSTRACT
Antisense oligonucleotides (ASOs) conjugated to triantennary N-acetyl galactosamine (GalNAc) ligands represent an emerging approach to antisense therapy. Our current generation of GalNAc-ASO conjugates link the GalNAc to the 5'-terminus of the ASO. The conjugation reaction can be accomplished using solution-phase or solid-phase techniques. Here we show a direct comparison of a solution-phase and a solid-phase conjugation strategy. The solution-phase approach, using amine-pentafluorophenyl (PFP) ester coupling, is higher yielding and gives material of slightly higher purity, but requires several additional unit operations and longer production time. The solid-phase approach, using a protected GalNAc ligand phosphoramidite, is more expedient, but results in lower yield and purity. Both strategies efficiently deliver conjugated material in excellent purity.
Subject(s)
Acetylgalactosamine/chemistry , Chemistry Techniques, Synthetic/methods , Oligonucleotides, Antisense/chemistry , Solid-Phase Synthesis Techniques/methods , Chromatography, High Pressure Liquid/methods , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry/methods , Molecular Structure , SolutionsABSTRACT
The acetyl capping reaction used throughout solid phase oligonucleotide synthesis is meant to minimize n-1 deletionmer impurities by terminating sequences that fail to couple to a phosphoramidite. However, the reaction is also responsible for the formation of a number of impurities. One capping-related impurity has an additional mass of 98amu from the parent oligonucleotide. The n+98 amu impurity was found to result from modification of an adenine nucleobase. The structure of the impurity was determined by preparation of an oligonucleotide enriched in n+98 amu, enzymatic digestion to individual nucleosides, isolation of the pure nucleoside+98 amu species, crystallization, and X-ray crystallographic analysis. The n+98 amu impurity is an oligonucleotide in which one adenine residue has been converted to 5-amino-4-pyrimidinylimidazole. The mechanism of formation of the impurity was investigated, and a mechanism is proposed.
Subject(s)
Adenine/chemistry , Imidazoles/chemistry , Oligonucleotides/chemical synthesis , Models, Molecular , Molecular Structure , Oligonucleotides/chemistryABSTRACT
The acetyl 'capping' reaction routinely employed during phosphorothioate oligonucleotide synthesis has been implicated in the formation of an impurity species with a mass 41 amu greater than the expected oligonucleotide molecule. The impurity has been found to arise by conversion of a protected guanine nucleobase to N(2)-acetyl-2,6-diaminopurine. A two-part mechanism is proposed consisting of transamidation of the protecting group on guanine and substitution of guanine's O(6) atom.
Subject(s)
2-Aminopurine/analogs & derivatives , Oligonucleotides/chemical synthesis , 2-Aminopurine/chemical synthesis , 2-Aminopurine/chemistry , Molecular Structure , Oligonucleotides/chemistryABSTRACT
A new class of C(2) symmetric bisoxazaborolidinone compounds is described which exhibits bidentate binding to carbonyl substrates. Using the Diels-Alder reaction as a model system, the catalytic activity of these bis-Lewis acids is probed. Reaction rates and selectivities are observed that are higher than the corresponding mono-Lewis acids. This novel system offers a potentially powerful new approach to asymmetric Lewis acid catalysis.
Subject(s)
Boranes/chemical synthesis , Heterocyclic Compounds, 1-Ring/chemistry , Ketones/chemistry , Models, Molecular , Boranes/chemistry , Catalysis , Ligands , Molecular StructureABSTRACT
New achiral sulfamide, phosphoric triamide, and thiophosphoric triamide compounds have been synthesized. Their activity as hydrogen bond catalysts for the Friedel-Crafts and Baylis-Hillman reactions compares favorably with that of a known and active thiourea catalyst. The new compounds were also studied by X-ray crystallography and their solid state structures are described.
