| Popis: |
peer-reviewed Antisense technology is a nucleic acid-based approach capable of down regulating the expression of specific genes. Several antisense drugs have or will shortly gain approval for medical use. Phosphorothioate oligonucleotides (PS oligonucleotides), in which one of the non-bridging oxygen atoms of the internucleotide phosphodiester linkage is replaced by a sulfur atom is one of the most extensively used backbone modifications of antisense oligonucleotides. Therefore, they are the focus of this study. Their unique manufacturing routes and bioactivity raises hitherto, unaddressed environmental issues. The manufacture and use of antisense oligonucleotides at industrial scale could result in its unintentional release to waste streams and hence into the environment. This research aims to determine the environmental issues associated with the production lifecycle of an antisense drug and to develop effective treatment processes to degrade/remove antisense drugs from specific manufacturing waste streams. The composition of waste streams generated from each process step of the synthesis was identified and simulated using SuperPro Designer?? 6.0 flow sheets. This study highlighted that large quantities of chemical wastes, most specifically, significant volumes of acetonitrile (2037 L) and toluene (1018 L) are produced during the synthesis of crude 20-mer PS oligonucleotide at 1.5 kg scale. It is predominantly during downstream processing, in particular purification, that there is a potential for loss of synthetic nucleic acid material during which it is estimated that upwards of 50% of synthetic oligonucleotide (750 g) is lost to waste streams. In this study, the main strategies adapted for the degradation and removal of antisense oligonucleotides include physical heat treatment, acidic and basic pH at ambient room temperature and in combination with elevated temperature (Chapter Three), chemical treatment with soft metal ions (silver nitrate) and oxidising agents (iodine, potassium permanganate, potassium dichromate, sodium hypochlorite, peracetic acid, hydrogen peroxide and Virkon??) (Chapter Four) and enzymatic treatment with commercially available nucleases (Chapter Five). Analysis was undertaken both qualitatively and quantitatively using Polyacrylamide Gel Electrophoresis and Ion-Pair Reversed-Phase chromatography. Based upon the concentration of reagents required, treatment duration, economic considerations and potential environmental impacts, six optimum treatments were chosen. These include treatment with low pH (HCl, 0.5 M) at 40??C, low pH (H2SO4, 0.5 M) at 40??C, iodine (25 mM), potassium dichromate (0.125 M), nuclease P1 (5 U/ml) and steam sterilisation for their efficacy in a simulated waste stream environment. Steam sterilisation was ineffective at degrading the PS oligonucleotides in the waste stream solution. Treatment with nuclease P1 was the most effective treatment process degrading the PS oligonucleotide by 96.9%. The next most effective treatment method was low pH with HCl at 40??C, low pH with H2SO4 at 40??C, acidic potassium dichromate and iodine degrading the PS oligonucleotide in simulated waste stream conditions by 92.9, 92.4, 88.3 and 86.7%, respectively. The application of low pH with HCl at 40??C or nuclease P1 presented as the most effective treatment methods of degrading the PS oligonucleotides in the simulated purification waste stream and demonstrated their suitability as the most technically feasible and eco-friendly methods of treatment. These methodologies may potentially be used to treat PS oligonucleotide containing waste streams, rendering them free of active drug product. EPA |
