PROCESS FOR THE PREPARATION OF HIGH HYDROPHILIC PRODUCTS WITH CONTROLLED HUMIDITY.

Abstract

The invention relates to a new process for preparing highly hydrophilic products with controlled moisture by spray drying in a suitable spray drying equipment that applies specific spray drying parameters.

Description

The invention comprises a process for preparing highly hydrophilic products with controlled moisture content by spray drying in a suitable spray drying unit that applies the following spray drying parameters: RFCC Ln / Lznz / E / YILI N2 inlet temperature (°C) 150 to 300 N2 outlet temperature (°C) 50 to 150 in order to control the water content of the spray-dried, highly hydrophilic product within a range of 1% w / w to 20% w / w. Background of the Invention Highly hydrophilic products such as oligonucleotides are often freeze-dried to obtain a solid form. Due to their high hygroscopicity, freeze-dried powders of highly hydrophilic products tend to be electrostatically charged, have poor free flow, and are therefore difficult to handle. To achieve a certain moisture content and a manageable powder form, freeze-dried powders generally undergo an additional conditioning process where the powder is exposed to Ref. 328708 water vapor in a climatic chamber at a certain temperature and humidity (see WO 2018 / 215391; page 69, line 5). This additional stage is time-consuming and resource-intensive, and is disadvantageous for the large-scale manufacturing of such products. Brief Description of the Invention Therefore, the object of the present invention was to find a process that avoids this additional conditioning stage and allows the production of highly hydrophilic products with controlled humidity. It was discovered that the objective of the invention could be achieved with the process for preparing products of the high hydrophilic type with controlled moisture, characterized in that an aqueous solution of the product of the high hydrophilic type is spray-dried in a suitable spray-drying equipment that applies the following spray-drying parameters: RFCC Ln / Lznz / E / YILI N2 inlet temperature (°C) 150 to 300 N2 outlet temperature (°C) 50 to 150 in order to control the water content of the spray-dried, highly hydrophilic product within a range of 1% w / w to 20% w / w. The following definitions are set forth to illustrate and define the meaning and scope of the various expressions used to describe the invention herein. The term "highly hydrophilic products" refers to products that, due to their molecular structure, exhibit high polarity and, therefore, a high affinity for water (and thus present very high hygroscopicity). Examples of such products are peptides and oligonucleotides. In a preferred embodiment, the high hydrophilicity type product expression represents an oligonucleotide. Brief Description of the Figures FIGURE 1 illustrates the characteristic of a highly hydrophilic oligonucleotide to absorb water, thereby gaining weight in relation to relative humidity compared to a non-hygroscopic compound. Detailed Description of the Invention As used herein, the term oligonucleotide is defined, as commonly understood by a person skilled in the art, as a molecule comprising two or more covalently linked nucleotides. Generally, for use as therapeutically valuable oligonucleotides, oligonucleotides are synthesized with a length of 10 to 40 nucleotides, preferably 10 to 25 nucleotides. Oligonucleotides can consist of optionally modified nucleoside monomers of DNA, RNA or LNA, or combinations thereof. RFCC Ln / ίZΖΠΖ / Β / YΙΛΙ LNA nucleoside monomers are modified nucleosides comprising a linking group (called a biradical or bridge) between C2' and C4' of the ribose sugar ring of a nucleotide. These nucleosides are also referred to as bridge nucleic acid or bicyclic nucleic acid (BNA) in the literature. Optionally, modified, as used herein, refers to nucleosides modified compared to the equivalent DNA, RNA, or LNA nucleoside by the introduction of one or more modifications to the sugar portion or the nucleobase portion. In a preferred embodiment, the modified nucleoside comprises a modified sugar portion and may comprise, for example, one or more 2'-substituted nucleosides and / or one or more LNA nucleosides. The term modified nucleoside may also be used herein interchangeably with the terms nucleoside analogue, modified units, or modified monomers. In general, DNA, RNA or LNA nucleosides are linked by an internucleosidic bond of phosphodiester (P=O) and / or phosphorothioate (P=S) that couples two nucleosides covalently. Consequently, in some oligonucleotides all internucleosidic bonds may consist of a phosphodiester (P=O), in other oligonucleotides all internucleosidic bonds may consist of a RFCC Ln / ίZΖΠZΖ / Β / ΥΙΛΙ phosphorothioate (P=S), or even in other oligonucleotides the sequence of internucleosidic bonds may vary and comprise both a phosphodiester (P=O) and a phosphorothioate (P=S). The nucleobase portions can be indicated by the letter code for each corresponding nucleobase, for example, A, T, G, C, or U, where each letter may optionally include modified nucleobases of equivalent function. For example, in the specified oligonucleotides, the nucleobase portions are described by the uppercase letters A, T, G, and MeC (5-methylcytosine) for the LNA nucleoside and by the lowercase letters a, t, g, c, and MeC for the DNA nucleosides. Modified nucleobases include, but are not limited to, nucleobases that have protecting groups, such as tert-butylphenoxyacetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl, or dimethylformamidine (see Wikipedia). Phosphoramidit-Synthese, https: / / de.wikipedia.org / wiki / Phosphoramidit-Synthese from March 24, 2016). Preferably, the oligonucleotide consists of optionally modified nucleoside monomers of DNA, RNA or LNA or combinations thereof and has a length of 10 to 40, preferably 10 to 25 nucleotides. The oligonucleotide can be a modified 5' amino, meaning that an amino linker is attached to the group RFCC Ln / Lznz / E / YILI of the 5' end of the oligonucleotide. Preferably, the linker is an aliphatic alkyl group of 2 to 12 carbon atoms or an ethylene glycol linker containing 1 to 10 ethylene glycol units. The preferred 5' amino modifier is selected from a C2-12 amino alkyl linker optionally protected by an amino group or an ethylene glycol amino linker containing 1 to 10 ethylene glycol units. The suitable amino protecting groups for the modified 5' amino oligonucleotide are trifluoroacetyl (TEA) or monomethoxytrityl (MMT). In general, the amino linker is introduced by means of a commercially available amino-linking phosphoraamide, such as by means of the Cg-linking phosphoraamides of TEA or MMT, for example, from Sigma Aldrich, or by means of the CE phosphoraamide of the 5' amino modifier TEG (triethylene glycol) from Glen Research. The principles of oligonucleotide synthesis are known in the state of the art (see, for example, Oligonucleotide synthesis; Wikipedia, the free encyclopedia; https: / / en.wikipedia.org / wiki / 01igonucleotide synthesis, from March 15, 2016). Currently, large-scale oligonucleotide synthesis is carried out RFCC Ln / Lznz / E / YILI automatically using computer-controlled synthesizers. In general, oligonucleotide synthesis is a solid-phase synthesis, where the oligonucleotide being assembled is covalently linked, via its 3' end hydroxyl group, to a solid support material and remains attached throughout chain assembly. Suitable supports are commercially available macroporous polystyrene supports, such as GE Healthcare's Primer Support 5G or Kinovate's NittoPhase®HL support. In principle, oligonucleotide synthesis is a step-by-step addition of nucleotide residues to the 5' end of the growing chain until the desired sequence is assembled. In general, each addition is called a synthetic cycle and, in principle, consists of the chemical reactions a1) unlocking the protected hydroxyl group on the solid support, a2) coupling the first nucleoside as activated phosphoramidite with the free hydroxyl group on the solid support, a3) oxidizing or sulfurizing the respective P-bonded nucleoside to form the respective phosphodiester (P=O) or phosphorothioate (P=S); a4) Optionally, terminate all ends RFCC Ln / Lznz / E / YILI unreacted hydroxyl group on the solid support; a5) unlock the 5' hydroxyl group of the first nucleoside attached to the solid support; a6) couple the second nucleoside as activated phosphoramidite to form the respective P-linked dimer; a7) oxidize or sulfonate the respective P-linked dinucleoside to form the respective phosphodiester (P=O) or phosphorothioate (P=S); a8) Optionally, terminate any unreacted 5' hydroxyl group; a9) Repeat steps a5 to a8 until the desired sequence is assembled. Subsequent cleavage of the resin can be performed with concentrated aqueous ammonia. The protecting groups on the phosphate and nucleotide bases are also removed during this cleavage procedure. In another embodiment, the oligonucleotide may comprise cell-directed portions to direct the oligonucleotide to a given receptor, such as the asialglycoprotein receptor (see X.Huang et al, Bioconjugate.Chem. 2017, 28, 283-295). In a preferred embodiment, the cell-directed portion comprises 1 to 3 N-acetylgalactosamine (GalNAc) ligands. RFCC Ln / Lznz / E / YILI Typical GalNAcs comprising cell-directed portions can be selected from: RFCC Ln / ίΖΠΖ / Β / ΥΙΛΙ »* Ή H< >< Ή II III IV or of the portions of the formula RFCC Ln / ίZΖΠΖ / Β / YΙΛΙ V where R2 is hydrogen or a hydroxyl protecting group and n is an integer from 0 to 10, preferably an integer from 0 to 5, more preferably from 1 to 3, but most preferably 2, enantiomers and / or stereoisomers of these. Suitable hydroxy protecting groups are acyl, in particular the C1-12 alkylcarbonyl group, more particularly the C1-6 alkylcarbonyl group which is optionally substituted with alkyl or phenyl of C1-g. Most preferably, it is acetyl, pivaloyl or benzoyl, wherein acetyl is the most preferred hydroxy protecting group. In a preferred embodiment, the GalNAc comprising cell-directed portions can be selected from the V portion of formula. In another preferred embodiment, the oligonucleotide is a GalNAc oligonucleotide conjugate comprising a 5' modified amino oligonucleotide as described above. The purification of products of the high hydrophilicity type follows methods that are generally known to the person skilled in the technique for the type of products. In general, purification comprises chromatography, concentration, and isolation stages, while the chromatography and concentration stages can be applied repeatedly. For oligonucleotides as products of the type of RFCC Ln / Lznz / E / YILI high affinity preferred, suitable purification procedures comprise the sequence of steps of a) chromatography b) concentration c) isolation or, preferably, d) anion exchange chromatography or reversed-phase chromatography e) tangential flow filtration f) lyophilization. The term chromatography includes anion exchange chromatography methods or reversed-phase chromatography and combinations of these. Anion exchange chromatography is based on the competitive interaction of charged ions in the sample solution with the buffer medium. It can be performed using commercially available, conventional anion exchange resins, preferably those functionalized with trimethylammonium. These phase materials can be obtained, for example, from GE Healthcare, Tosoh Bioscience, Bio-Rad, or Merck. Particularly good results were achieved with the TSKgel Super Q-5PW (QAE) anion exchange resin, available from Tosoh Bioscience. Reversed-phase chromatography can be carried out with traditional phase materials available in the RFCC Ln / Lznz / E / YILI commercially available modified silica gel sorbents as the stationary phase and suitable organic solvents, such as acetonitrile, and, if applicable, a buffer. Suitable phase materials of the modified silica gel type can be selected from Kromasil™C18, Kromasil™C8, YMC Triart C18, and YMC Triart C8. Good results were particularly achieved with YMC Triart Prep C8-S. The term concentration includes tangential flow filtration or evaporation methods and combinations of these. In tangential flow or cross-flow filtration, the feed passes through the filter membrane (tangentially) at a positive pressure relative to the permeate side. A portion of the material smaller than the membrane pore size passes through the membrane as permeate or filtrate; the remainder is retained on the feed side of the membrane as retentive material. The principles of tangential flow filtration are also used in nanofiltration, ultrafiltration, diafiltration, and microfiltration processes. Suitable membranes are commercially available, for example, from Merck Millipore under the trade name Pellicon™. Suitable membranes have a molecular weight cutoff (MWCO) of 3 kDa. Merck Millipore Pellicon 2 and 3 membranes with an MWCO of 1 kDa or 3 kDa are preferred. RFCC Ln / ίΖΠΖ / Β / ΥΙΛΙ respectively. As explained above, the process for preparing highly hydrophilic products with controlled moisture is characterized by spray drying an aqueous solution of the highly hydrophilic product with controlled moisture in suitable spray drying equipment applying the following spray drying parameters: RFCC Ln / ίZΖΠΖ / Β / YΙΛΙ N2 inlet temperature (°C) 150 to 300 N2 outlet temperature (°C) 50 to 150 in order to control the water content of the spray-dried, highly hydrophilic product within a range of 1% w / w to 20% w / w. In a preferred mode, the spray drying parameters are: Concentration of the high hydrophilic type product in the feed solution (% w / w) 1 to 50 Concentration of high hydrophilic type product in feed solution (% w / w) 1 to 50 N2 inlet temperature (°C) 180 to 220 N2 outlet temperature (°C) 70 to 100 In a more preferred mode, the spray drying parameters are: Concentration of the high hydrophilic type product in the feed solution (% w / w) 5 to 25 Inlet temperature of N₂ (°C) 180 to 220 Outlet temperature of N₂ (°C) 70 to 100 RFCC Ln / ίZΖΠΖ / Β / YΙΛΙ The process parameters, in general, depend on the spray drying equipment used. However, the ratio of drying gas speed to feed speed and the ratio of gas nozzle speed to feed speed are dimensionless parameters independent of the size of the drying chamber and the type of atomizer. In general, the ratio of drying gas speed to feed speed is therefore selected between 1 and 200, preferably between 10 and 150, and the ratio of gas nozzle speed to feed speed is between 0.5 and 10, preferably between 1 and 8. The spray drying equipment applied for the process of the present invention is a common piece of equipment in the state of the art, comprising a spray chamber with an atomizer and possible downstream equipment, such as a cyclone for collecting the spray-dried powder and a filter unit for cleaning the gas of the remaining particles. The atomizer is designed to finely disperse the feed solution in the form of small droplets in the spray chamber and expose it to the hot gas. In general, the gas is an inert gas, usually nitrogen. In general, the atomizer is selected from a pressure drop nozzle or a two-fluid nozzle or a rotary atomizer. A common spray drying system is commercially available, for example, from Gea (Niro SD Micro), Anhydro (SPX) or Büchi (Büchi Mini). The combination of parameters determines the drying of the dispersed droplets, and ultimately controls the moisture content of the spray-dried product. RFCC Ln / ίZΖΠΖ / Β / YΙΛΙ In a preferred embodiment, a two-fluid nozzle is used. Typical additional drying parameters are: Drying gas speed (kg / h) 10 to 50, preferably 15 to 25 Gas nozzle speed (kg / h) 0.5 to 3, preferably 0.8 to 1.5 Feed speed (g / min) 3 to 18, preferably 7 to 13 The residual water content of the spray-dried high hydrophilic type product can be controlled, preferably, in the range of 5% w / w to 15% w / w, more preferably, in the range of 10% w / w to 15% w / w. The apparent density of the spray-dried high hydrophilic type product can be adjusted in the range of 0.1 g / ml to 0.5 g / ml, preferably in the range of 0.3 g / ml to 0.5 g / ml. As stated above, the products of the high hydrophilicity type are oligonucleotides, preferably oligonucleotides consisting of optionally modified nucleoside monomers of DNA, RNA or LNA or combinations thereof and having a length of 10 to 40, preferably 10 to 25 nucleotides, which are optionally 5' amino modified, and comprising GalNAc comprising a cell-directed portion, as defined above. For illustrative purposes, the following oligonucleotides were selected: GN2-AM-C6-5'-caG*MeC*G*t*a*a*a*g*a*g*a*G*G-3' 5'-T*G*G*c*a*a*g*c*a*t*c*c*T*G*T*a-3' GN2-AM-C6-5'-caC*C*t*a*t*t*t*a*a*c*a*t*c*A*G*A*C-3' where AM-C6 means a C6 amino linker; * means phosphorothioate bridges; A,G,T andMeC (5-methyl cytosine) are LNA nucleoside monomers, ya, t, c, g are DNA nucleoside monomers and GN2 is the GalNAc group portion of formula V above (n = 2; R2 = acetyl). The compounds described herein have the following nucleobase sequences. SEQ ID NO 1: cagcgtaaagagagg ' RFCC Ln / Lznz / E / YILI SEC ID NO 2: tggcaagcatcctgta SEQ ID NO 3: cacctatttaacatcagac. Examples Preparation of GN2-AM-C6-5'-caG*MeC*G*t*a*a*a*g*a*g*a*G*G-3' The title product was prepared in accordance with Example 3B and purified in accordance with Example 4B1 of International Patent Publication WO 2018 / 215391. Example 1 Spray drying of GN2-AM-C6-5'caG*MeC*G*t*a*a*a*g*a*g*a*G*G-3' Thirteen grams of the oligonucleotide of the title were dissolved in 117 grams of water at room temperature. This solution was fed into a Niro SDMICRO™ apparatus (GEA Process Engineering A / S, Soeborg, Denmark) through a two-fluid nozzle in the same direction (0.5 mm, temperature 50 °C, nitrogen atomizer flow rate 0.8 kg / h). The feed was atomized in a hot nitrogen stream (inlet temperature 220 °C) at a liquid feed rate of 10 g / min and a nitrogen flow rate of 15 kg / h. An outlet temperature of approximately 70 °C was obtained. The generated solid was separated from the gas stream by a cyclone connected to the drying chamber into a glass bottle: 11.4 g of solids were collected with a residual water content of 12.3% w / w (isolated yield corrected for water content of 90%) and a density Apparent RFCC Ln / Lznz / Ε / ΥΙΛΙ of 0.45 g / ml. Examples 2 to 31 The following examples were carried out according to example 1, but with a variation of the key parameters. RFCC Ln / Lznz / E / YILI Example No. Solids Content Inlet Temperature Outlet Temperature Drying Gas Velocity Drying Gas Velocity / Feed Velocity Ratio* Gas ​​Nozzle Velocity Gas Nozzle Velocity / Feed Velocity Ratio* Wastewater Yield (run water) % w / w °C °C kg / h - kg / h - % w / w % 2 10 210 100 20 48 1.0 2.4 7.48 31 3 5 210 100 20 42 1.0 2.1 6.84 23 4 5 220 100 20 37 1.5 2.8 4.97 63 5 15 210 90 20 37 1.0 1.9 6.77 71 6 20 210 90 20 39 1.0 2.0 7.63 67 7 20 210 90 20 39 0.8 1.6 6.49 89 8 20 190 80 20 30 1.0 1.5 9.25 75 9 20 190 70 20 35 1.0 1.8 11.14 63 10 20 180 70 20 35 1.0 1.8 8.60 70 11 20 190 70 18 30 1.0 1.7 11.01 63 12 20 200 70 18 27 1.0 1.5 10.65 63 13 20 210 70 18 35 1.0 2.0 11.23 63 14 17.5 200 85 20 28 1.0 1.4 8.67 62 15 25 180 70 25 23 1.2 1.1 9.59 63 16 10 220 70 25 104 1.2 5.0 12.19 60 17 10 180 85 15 21 0.8 1.1 7.73 60 18 10 180 70 25 119 0.8 3.8 10.37 52 19 10 220 100 15 21 1.2 1.7 5.42 47 20 25 220 100 25 139 1.2 6.7 7.43 67 21 25 180 88 15 38 1.2 3.1 5.91 60 22 25 180 70 15 31 0.8 1.7 10.80 65 23 17.5 200 85 20 32 1.0 1.6 5.60 76 24 10 220 100 25 76 0.8 2.4 7.16 74 25 10 180 100 25 83 1.2 4.0 5.70 60 26 25 180 100 25 42 0.8 1.3 5.76 55 27 25 220 70 15 38 1.2 3.1 14.50 52 28 10 180 70 15 14 1.2 1.1 9.93 61 29 25 220 70 25 83 0.8 2.7 13.31 45 30 25 220 100 15 25 0.8 1.3 5.84 98 31 17.5 200 85 20 39 1.0 2.0 8.12 72. *The ratio of drying gas speed / feed speed and the ratio of gas nozzle speed / feed speed are dimensionless parameters independent of the size of the drying chamber and the atomizer. RFCC Ln / Lznz / E / YILI Selected apparent densities: Example Number Wastewater Apparent Density % w / w / ml 1 12.28 0.45 14 8.67 0.40 16 12.19 0.25 28 9.93 0.40 30 5.84 0.40 31 8.12 0.30 Comparative example The title product was prepared according to Example 3B, purified according to Example 4B1, and lyophilized according to Example 4B2 of International Patent Publication WO 2018 / 215391. The material obtained was very hygroscopic, electrostatically charged, non-free-floating, and therefore difficult to control. Therefore, the material was conditioned in a climate-controlled chamber at 21°C and 50% relative humidity until the weight was constant, which was achieved after 48 h. Examples 32 to 34 Examples 32 to 34 were carried out with the following LNAs: • 5'-T*G*G*c*a*a*g*c*a*t*c*c*T*G*T*a-3' (examples 32 and 33) • GN2-AM-C6-5'- RFCC Ln / Lznz / Ε / ΥΙΛΙ caC*C*t*a*t*t*t*a*a*c*a*t*c*A*G*A*C-3' (example 34) according to example 1. Example No. Solids Content Inlet Temperature Outlet Temperature Drying Gas Velocity Drying Gas Velocity / Feed Velocity* Gas ​​Nozzle Velocity Gas Nozzle Velocity / Feed Velocity* Wastewater Yield (run water) % w / w °C °C kg / h kg / h % w / w % 32 10 210 112 5 4 1.0 1.0 4.60 58 33 5 210 112 5 4 1.0 1.0 5.20 65 34 5 210 110 5 4 1.5 1.0 3.20 34 It is hereby stated that, as of this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

Claims

1. A process for preparing highly hydrophilic products with controlled moisture, characterized in that an aqueous solution of the highly hydrophilic product with controlled moisture is spray-dried in a suitable spray-drying equipment applying the following spray-drying parameters: RFCC Ln / Lznz / E / YILI Inlet temperature of N2 (°C) 150 to 300 Outlet temperature of N2 (°C) 50 to 150 in order to control the water content of the spray-dried highly hydrophilic product in a range of 1% w / w to 20% w / w.

2. The process according to claim 1, characterized in that the spray drying parameters are: Concentration of the high hydrophilic type product in the feed solution (% w / w) 1 to 50; N2 inlet temperature (°C) 180 to 220; N2 outlet temperature (°C) 70 to 100 3. The process according to claim 1 or 2, characterized in that the spray drying parameters are: RFCC Ln / Lznz / E / YILI Concentration of the high hydrophilic type product in the feed solution (% w / w) 5 to 25 N2 inlet temperature (°C) 180 to 220 N2 outlet temperature (°C) 70 to 100 4. The process according to any of claims 1 to 3, characterized in that the ratio of the drying gas speed to the feed speed is between 1 and 200, preferably between 10 and 150.

5. The process according to any of claims 1 to 4, characterized in that the ratio of the gas nozzle speed to the feed speed is between 0.5 and 10, preferably between 1 and 8.

6. The process according to any of claims 1 to 5, characterized in that the suitable spray drying equipment comprises a spray chamber and an atomizer selected from a pressure drop nozzle or for two fluids or from a rotary atomizer.

7. The process according to any of claims 1 to 6, characterized in that the suitable spray drying equipment comprises a spray chamber and an atomizer selected from a two-fluid nozzle.

8. The process according to any of claims 1 to 7, characterized in that the following additional spray drying parameters are applied: RFCC Ln / ίZЖРZ� / B / YILI Drying gas speed (kg / h) 10 to 50 Gas nozzle speed (kg / h) 0.5 to 3 9. Process according to claim 8, characterized in that the following spray drying parameters are applied: Drying gas speed (kg / h) 15 to 25 Gas nozzle speed (kg / h) 0.8 to 1.5 10. The process according to any of claims 1 to 9, characterized in that the residual water content of the spray-dried high hydrophilic type product is in the range of 5% w / w to 15% w / w, more preferably in the range of 10% w / w to 15% w / w.

11. The process according to any of claims 1 to 10, characterized in that the apparent density of the spray-dried oligonucleotide is in the range of 0.1 g / ml to 0.5 g / ml, preferably in the range of 0.3 g / ml to 0.5 g / ml.

12. The process in accordance with any of claims 1 to 11, characterized in that the product of the high hydrophilicity type with controlled moisture is an oligonucleotide.

13. The process according to any of claims 1 to 12, characterized in that the oligonucleotide consists of optionally modified nucleoside monomers of DNA, RNA or LNA or combinations thereof and has a length of 10 to 40, preferably 10 to 25 nucleotides.

14. A product of the high hydrophilicity type, characterized in that it can be obtained by the spray drying process in accordance with claims 1 to 13.

15. The product of the high hydrophilicity type according to claim 14, characterized in that it is an oligonucleotide, and the residual water content is in the range of 1% w / w to 20% w / w, preferably in the range of 5% w / w to 15% w / w, more preferably in the range of 10% w / w to 15% w / w.