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WO2023067038A1 - Procédé de purification d'oligonucléotides - Google Patents

Procédé de purification d'oligonucléotides Download PDF

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Publication number
WO2023067038A1
WO2023067038A1 PCT/EP2022/079156 EP2022079156W WO2023067038A1 WO 2023067038 A1 WO2023067038 A1 WO 2023067038A1 EP 2022079156 W EP2022079156 W EP 2022079156W WO 2023067038 A1 WO2023067038 A1 WO 2023067038A1
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WIPO (PCT)
Prior art keywords
oligonucleotide
process according
solution
solid support
desalting
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PCT/EP2022/079156
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English (en)
Inventor
Dennis Jul HANSEN
Inna LARSEN APPELDORFF
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Roche Innovation Center Copenhagen A/S
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Publication of WO2023067038A1 publication Critical patent/WO2023067038A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation

Definitions

  • the present invention relates to processes for preparing and purifying oligonucleotides.
  • the process of the invention is ideally suited for small-scale and intermediate-scale production and is significantly more cost and/or time effective than methods used in the art to date.
  • oligonucleotide synthesis The principles of oligonucleotide synthesis are well known in the art (see e.g., Oligonucleotide synthesis; Wikipedia; https://en.wikipedia.org/wiki/Oligonucleotide synthesis, of March 15, 2016). Larger scale oligonucleotide synthesis nowadays is carried automatically using computer-controlled synthesizers. Oligonucleotides which are typically prepared via solid phase synthesis, after cleavage from the resin, still contain a significant amount of impurities. For standard monomers of a 15- to 20-mer length the API purity is at best in the range of 70 to 80%. For chemically modified monomers or for longer sequences, the API content is typically even lower.
  • Processes which require filtration and evaporation steps may require substantial both operation and waiting time.
  • the process of the present invention is a more efficient process compared to other methods used to date.
  • This invention relates to a process for purification oligonucleotides from a crude aqueous mixture comprising said oligonucleotide and a solid support, said process comprising the step of: a. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide.
  • a process for providing a purified oligonucleotide, said process comprising the steps of: a. synthesising said oligonucleotide on a solid support; b. cleaving the oligonucleotide from said solid support to provide a first solution comprising said oligonucleotide and said solid support; c. deprotecting said oligonucleotide while in said first solution, to provide a second solution comprising deprotected oligonucleotide and solid support; d.
  • Figure 1 illustrates a conventional process for oligonucleotide purification (open arrows to the right), and the process according to the invention (shaded arrows to the left).
  • Scale of oligonucleotide synthesis When referring to the scale of oligonucleotide synthesis we refer to the molar amount of oligonucleotide product present in the crude mixture. The process according to the present invention is applicable across a broad scale range of oligonucleotide amount.
  • the scale is greater than 1 pmol, such as greater than 5 pmol, such as greater than 10 pmol, such as greater than 100 pmol, such as greater than 200 pmol, such as greater than 500 pmol, such as greater than 1000 pmol (1 mmol), such as greater than 2 mmol, such as greater than 5 mmol, such as greater than 10 mmol, such as greater than 50 mmol or greater than 100 mmol or greater than 200 mmol.
  • a preferred scale is between 10 pmol and 100 pmol. Particularly preferred is a scale of around 20 pmol.
  • the purified oligonucleotide product obtained from the method of the invention may, in some embodiments, be at least about 75% pure, such as at least about 80% pure, such as at least about 85% pure, such as at least about 90% pure, such as at least about 95% pure. Purity of the oligonucleotide may be determined using standard assays known in the art, such as HPLC, LC-MS, or UPLC.
  • a process for providing a purified oligonucleotide, said process comprising the steps of: a. synthesising said oligonucleotide on a solid support; b. cleaving the oligonucleotide from said solid support to provide a first solution comprising said oligonucleotide and said solid support; c. deprotecting said oligonucleotide while in said first solution, to provide a second solution comprising deprotected oligonucleotide and solid support; d.
  • a solvent preferable an aqueous solution
  • diluting the second solution comprising said deprotected oligonucleotide and said solid support with a solvent, preferable an aqueous solution to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support, e. desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide, and f. evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.
  • oligonucleotides described herein may be prepared using standard solid phase oligonucleotide synthesis. Suitable methodology will be familiar to the skilled artisan (see, for example, Sanghvi et al, 1999, Chemical synthesis and purification of phosphorothioate antisense oligonucleotides, in "Manual of Antisense Methodology" (G.
  • the oligonucleotide may be prepared using a solid phase synthesizer such as, for example, MerMade 12 or 192 synthesiser from Biosearch, an ABI-type bench synthesizer, a Millipore 8800 DNA synthesizer or a Cytiva Oligopilot or OligoProcess synthesizer.
  • the scale of oligonucleotide synthesis may be varied by selection of the appropriate oligonucleotide synthesizer, for example for lOOmmol scale synthesis an Oligo Process (Cytiva, formerly GE Healthcare) may be used.
  • the oligonucleotide synthesis in principle is a stepwise addition of nucleotide residues to the 5 '-terminus of the growing chain until the desired sequence is assembled.
  • the oligonucleotides are prepared using phosphoramidite coupling chemistry of 5’- protected nucleotides. More preferably, the 5'-protecting group is a 4,4'-dimethoxytrityl (DMT) protecting group. Other protecting groups useful in oligonucleotide synthesis are also suitable and will be familiar to the skilled person. Oligonucleotides
  • oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleotides.
  • the oligonucleotides may consist of optionally modified DNA, RNA or LNA nucleoside monomers or combinations thereof.
  • the oligonucleotide may comprise at least one modified nucleotide, such as at least one Locked Nucleic Acid (LNA).
  • LNA Locked Nucleic Acid
  • “Modified” as used herein refers to nucleosides modified as compared to the equivalent DNA, RNA or LNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase moiety.
  • the modified nucleoside comprises a modified sugar moiety, and may for example comprise one or more 2' substituted nucleosides and/or one or more LNA nucleosides.
  • modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified "units" or modified “monomers”.
  • LNA nucleoside monomers are modified nucleosides which comprise a linker group (referred to as a biradical or a bridge) between C2' and C4' of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • a linker group referred to as a biradical or a bridge
  • BNA bicyclic nucleic acid
  • modified nucleoside units are 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'- amino-DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2'MOE RNA unit.
  • 2'fluoro-DNA refers to a DNA analogue with a substitution to fluorine at the 2' position (2'F).
  • 2'fluoro-DNA is a preferred form of 2'fluoro-nucleotide.
  • 2'-deoxy-2'- fluoro-arabinonucleic acid (FANA) is another example.
  • the nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties are described with capital letters A, T, G and Me C (5-methyl cytosine) for LNA nucleoside and with small letters a, t, g, c and Me c for DNA nucleosides.
  • Modified nucleobases include but are not limited to nucleobases carrying protecting groups such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl or dimethylformamidino (see Wikipedia, https://en.wikipedia.org/wiki/Nucleoside_phosphoramidite).
  • oligonucleotides prepared by the process of the invention are single stranded oligonucleotides which optionally comprise one or more nucleotide analogues, such as LNA, which form part of, or the entire contiguous nucleotide sequence of the oligonucleotide.
  • nucleotide analogues such as LNA
  • the oligonucleotide prepared by the process of the invention is 100% complementary to a miRNA sequence, such as a human microRNA sequence, or one of the microRNA sequences referred to in WO 2009043353.
  • the oligonucleotide is single stranded, this refers to the situation where the oligonucleotide is in the absence of a complementary oligonucleotide, i.e., it is not a double stranded oligonucleotide complex, such as an siRNA. It will be recognised that once purified according to the present invention, the oligonucleotide may be hybridised with other oligonucleotides which may be complementary to part of or all of the oligonucleotide prepared according to the present invention, to form, for example, a siRNA.
  • High affinity nucleotide analogues are nucleotide analogues which result in oligonucleotides having a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This may be determined by measuring the melting temperature of the duplex (Tm).
  • the oligonucleotide of the invention is a gapmer.
  • a gapmer oligonucleotide is an oligonucleotide which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B (B), wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as from 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A (A) and C (C) respectively.
  • the oligonucleotide is a LNA gapmer oligonucleotide (i.e. having a gapmer structure in which at least one nucleotide is LNA).
  • LNA Locked Nucleic Acid
  • the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (A) (5' region) consists of or comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region B (B) consists of or comprises at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (C) (3'region) consists of or comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region D (D), when present consists of or comprises 1, 2 or 3 nucleo
  • region A consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • LNA units such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • region B consists of or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably from 4- 12 DNA units, more preferably from 6-10 DNA units, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.
  • region A consist of 3 or 4 nucleotide analogues, such as LNA
  • region B consists of 7, 8, 9 or 10 DNA units
  • region C consists of 3 or 4 nucleotide analogues, such as LNA.
  • Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8- 3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.
  • the oligonucleotide of the invention is a "mixmer"
  • a mixmer is an oligonucleotide comprising both DNA and LNA. Chemical modifications in a mixmer may be mixed in almost random order. Typically, a mixmer consists of alternating short stretches of LNA and DNA.
  • a mixmer according to the invention may comprise several other nucleotide analogues at the same time e.g. DNA-inverted, 2'OMe, 2'F, DNA, in a single oligonucleotide sequence.
  • the oligonucleotide of the invention consists of a single type of sugar moiety e.g. 100% DNA sequence or 100% 2'MOE RNA.
  • the length of a nucleotide molecule corresponds to the number of monomer units, i.e. nucleotides, irrespective as to whether those monomer units are nucleotides or nucleotide analogues.
  • monomer and unit are used interchangeably herein.
  • the process of the present invention is suitable for purifying oligonucleotides which are for example, 40 nucleotide units in length or less, such as 30 nucleotide units in length or less, more preferably 6 to 25 nucleoside units in length.
  • the oligonucleotide has a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • oligonucleotides are typically synthesized as 5 to 40 nucleotides, preferably 10 to 25 nucleotides in length.
  • the present invention is particularly suitable for the purification of oligonucleotides, for example, consisting of 14 to 25 nucleotide units.
  • the process of the present invention is particularly suitable for purifying oligonucleotides, in which at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of the nucleotide units of the oligonucleotide are DNA nucleotide units.
  • the oligonucleotide is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 75 %, preferably at least 80 %, preferably at least 85%, preferably at least 90%, preferably at least 95%, or 100% of the nucleotides are Locked Nucleic Acid (LNA) nucleotide units.
  • LNA Locked Nucleic Acid
  • the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleotide units.
  • at least 80% (for example, such as 85%, 90%, 95%, or 100%) of the internucleotide bonds in the oligonucleotide of the invention are phosphorothioate bonds.
  • At least 80% (for example, such as 85%, 90%, 95%, or 100%) of the internucleotide bonds in the oligonucleotide of the invention are phosphodiester bonds.
  • the oligonucleotide is cleaved from the solid phase and the protecting groups are removed, e.g. using standard techniques which are well known in the art.
  • steps of cleaving and deprotection are carried out essentially simultaneously, to provide a solution comprising deprotected oligonucleotide and solid support.
  • the steps of cleaving and deprotection may for example be carried out via treatment (of the solid-supported oligonucleotide) with a basic aqueous solution, preferably an aqueous ammonia solution.
  • the steps of cleaving and deprotection are carried out via treatment with an aqueous ammonia solution, and wherein said process does not comprise a step of ammonia evaporation between prior to a desalting step.
  • ammonia evaporation is one of the most time-consuming steps (e.g. overnight).
  • the realisation by the present inventors that ammonia evaporation can be avoided runs counter to prevailing understanding, and allows the process to proceed directly to subsequent steps of dilution and desalting, while maintaining good levels of purity in the oligonucleotide product.
  • the second solution comprising said deprotected oligonucleotide and said solid support is diluted with a solvent, preferable an aqueous solution, to provide a crude aqueous mixture comprising said deprotected oligonucleotide and said solid support.
  • the crude oligonucleotide is dissolved in a metal salt solution, such as saline (NaCI solution).
  • a metal salt solution such as saline (NaCI solution).
  • the crude aqueous mixture may comprise at least one metal salt.
  • the metal salt solution is a buffer which is suitable for HPLC.
  • the pH of the crude aqueous oligonucleotide solution is adjusted with a base, for example, aqueous sodium hydroxide solution.
  • the pH is preferably adjusted to about 7 to about 8.
  • the pH is adjusted by the addition of an aqueous solution of sodium hydroxide (for example, using a 10 mM NaOH solution).
  • a process for purifying an oligonucleotide from a crude aqueous mixture comprising said oligonucleotide and a solid support, said process comprising the step of: desalting said crude aqueous mixture by means of size exclusion chromatography, while simultaneously removing at least a portion of said solid support, to provide a desalted solution comprising said oligonucleotide.
  • the crude aqueous mixture obtained e.g. from the synthesis, cleavage, deprotection and dilution steps, is typically an unpurified product from oligonucleotide synthesis which typically comprises the oligonucleotide product as well as truncated versions of the oligonucleotide, deletion fragments as well as cleaved protection groups.
  • the term "desalting” refers to a process by which impurities, such as inorganic salts, are removed from a mixture.
  • the oligonucleotide is either dissolved in an electrolyte solution, or is dissolved in a suitable solvent, and an electrolyte is subsequently added to the oligonucleotide solution.
  • Water is typically used as the solvent.
  • the electrolyte may, for example, be a metal salt, such as a sodium or potassium salt, such as a sodium or potassium halogen salt, such as KCI, NaCI, or NaBr.
  • the metal salt acts as a counter ion for the oligonucleotide anion.
  • the pH of the oligonucleotide solution may be adjusted to a pH of 7 or above, such as a pH of between about 7 to about pH 8.
  • the suitable pH of the oligonucleotide solution may be achieved by using a basic solvent to dissolve the oligonucleotide, or by adjusting the pH of the oligonucleotide solution to a pH of 7 or higher.
  • the step of desalting suitably removes at least ammonium, sodium and chloride ions from said crude aqueous mixture.
  • the desalting step takes place on a size exclusion chromatography column.
  • Suitable desalting columns are e.g. those sold under the "Cytiva HiPrep" brand.
  • One example of a suitable size exclusion chromatography column comprises optionally-functionalised cross-linked dextran, known as Sephadex.
  • the desalting step takes place on a single size exclusion chromatography column.
  • This is enabled by appropriately adjusting the liquid volumes used in the process of the present invention. For instance, the volume of the aqueous ammonia solution and I or the volume and I or concentration of the aqueous sodium chloride solution can be adjusted, so that the final volume of the solution does not exceed the capacity of one column.
  • This lower volume can thus be purified using a single column, instead of 2 or more columns in series, which saves time and material.
  • Experiments have shown that by reducing the number of columns from two to one (i.e. going from method 2a to 2b, below) the purification time was also reduced by approx. 50%.
  • the desalting step takes place using a size exclusion chromatography column equipped with an inlet filter.
  • the inlet filters can be end-of-line filters for tubing for single use, HPLC solvent filters or exchangeable suction frits made of porous PTFE, for example BOLA Suction Filters.
  • the next step in the process is evaporating solvent from said desalted solution so as to provide said purified oligonucleotide.
  • evaporating solvent means that at least a portion of a solvent is removed.
  • “Lyophilisation” requires evaporation of solvent to dryness, typically producing a powder or crystalline solid.
  • the purification process is typically followed by quality control, QC.
  • oligonucleotides from the insoluble resin and evaporation of concentrated aqueous ammonia to yield crude oligonucleotide are both time-demanding steps that can take up to 24 hours depending on the instrument used.
  • the oligonucleotide is concentrated or evaporated to be tightly packed material, it can be very difficult to dissolve it again in order to continue with further purification.
  • the dissolution step can take several hours and require assistive device such as heating baths in order to provide complete solution.
  • Each compound A, B, C was synthesized in three batches (1, 2, 3) in order to take each sequence through three different purification process to compare the yield.
  • each compound was treated with 20 ml of concentrated aqueous ammonia and 60 C for at least 5 to 24 hours for cleavage and deprotection. After this point, each sequence was taken through different purification methods:
  • oligonucleotide separation was done using sample filtration columns e.g. Kinesis TELOS.
  • the filter was also washed using approx. 10 ml 50 % ethanol in water.
  • the filtrate containing oligonucleotide, ammonia and ethanol was evaporated using SpeedVac Vacuum Concentrator to give a crude oligonucleotide sample.
  • the solution was loaded into LC system with two columns in series, for instance using two HiPrep 26/10 Desalting columns.
  • the non-uniform suspension with oligonucleotide and the insoluble resin suspended in concentrated aqueous ammonia was used as is for the following steps.
  • CHNSO was determined by combustion analysis.

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Abstract

La présente invention concerne un procédé de purification d'oligonucléotides à partir d'un mélange comprenant ledit oligonucléotide et un support solide, le procédé de l'invention consistant à dessaler le mélange par chromatographie par exclusion de taille dans laquelle simultanément au dessalage, au moins une partie du support solide est éliminée par filtration.
PCT/EP2022/079156 2021-10-22 2022-10-20 Procédé de purification d'oligonucléotides WO2023067038A1 (fr)

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EP21204146.1 2021-10-22

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026540A1 (fr) * 1994-03-11 1997-07-24 Barrskogen, Inc. Procede de minifiltration et appareil correspondant
WO2009043353A2 (fr) 2007-10-04 2009-04-09 Santaris Pharma A/S Oligonucléotides micromir
WO2012010711A1 (fr) 2010-07-23 2012-01-26 Santaris Pharma A/S Procédé
WO2020083898A1 (fr) 2018-10-24 2020-04-30 F. Hoffmann-La Roche Ag Procédé de purification d'oligonucléotides
WO2020173845A1 (fr) 2019-02-26 2020-09-03 Roche Innovation Center Copenhagen A/S Procédé de formulation d'oligonucléotides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026540A1 (fr) * 1994-03-11 1997-07-24 Barrskogen, Inc. Procede de minifiltration et appareil correspondant
WO2009043353A2 (fr) 2007-10-04 2009-04-09 Santaris Pharma A/S Oligonucléotides micromir
WO2012010711A1 (fr) 2010-07-23 2012-01-26 Santaris Pharma A/S Procédé
WO2020083898A1 (fr) 2018-10-24 2020-04-30 F. Hoffmann-La Roche Ag Procédé de purification d'oligonucléotides
WO2020173845A1 (fr) 2019-02-26 2020-09-03 Roche Innovation Center Copenhagen A/S Procédé de formulation d'oligonucléotides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAPALDI, D.C.SCOZZARI, A.N.: "Antisense Drug Technology", 2008, CRC PRESS, article "Manufacturing and Analytical Processes for 2'-0-(2-Methoxyethyl)-Modified Oligonucleotides", pages: 401 - 434
DESHMUKJ: "Handbook of Bioseparations", vol. 2, 2000, article "Large Scale Chromatographic Purification of Oligonucleotides", pages: 511 - 534
SANGHVI ET AL.: "Manual of Antisense Methodology", 1999, KLUWER ACADEMIC PUBLISHERS, article "Chemical synthesis and purification of phosphorothioate antisense oligonucleotides", pages: 2 - 23

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