Device and method for concentrating biomolecules

Abstract

A method of concentrating a biomolecule-containing solution (e.g., an oligonucleotide-containing solution) from an initial concentration of about < = 20 mg / mL to a concentration of > = about 200 mg / mL via thin film evaporation (TFE) for high dose / low volume applications is described. Also described are devices for concentrating solutions containing biomolecules, as described herein.

Description

[0001] Incorporation by Reference of Electronically Submitted Sequence Listing The present disclosure is filed with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named “30289_US_PRI,” which was created on February 22, 2024 and is 29.9 kilobytes (kb) in size. The sequence listing information in ST.26 XML format is incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates to chemistry and engineering, and more specifically to devices and methods for concentrating biomolecules, such as single-stranded (ss) or double-stranded (ds) oligonucleotides (i.e., therapeutic oligonucleotides), in solution for use as drug substance (DS) in drug product (DP) manufacturing. BACKGROUND

[0003] Therapeutic oligonucleotides are a relatively new modality for treating and preventing diseases and disorders. Therapeutic oligonucleotide synthesis can involve many upstream and downstream steps, such as synthesis, cleavage and deprotection, purification, and concentration. Of particular interest herein is the concentration of therapeutic oligonucleotides in solution for use as DS in the manufacture of DP or other pharmaceutical compositions.

[0004] Various methods for concentrating solutions containing oligonucleotides are known, such as chromatography, dialysis, evaporation, precipitation, and ultrafiltration / diafiltration (UF / DF). Currently, these methods for concentrating solutions containing oligonucleotides are unable to reliably achieve final oligonucleotide concentrations in solution of greater than about 100 mg / mL. However, there is a need for solutions containing highly concentrated oligonucleotides for formulating and administering to individuals in need thereof with minimal solution volume.

[0005] Accordingly, there is a need for devices and methods for concentrating solutions containing biomolecules, such as solutions containing oligonucleotides, to concentrations > 200 mg / mL. SUMMARY

[0006] To meet this need, the present disclosure first describes a method of concentrating a solution containing oligonucleotides comprising at least the following steps: (a). flowing the solution through an evaporation vessel, wherein the solution has a starting oligonucleotide concentration of < about 20 mg / mL, wherein the evaporation vessel comprises a rotatable doctor blade, a device for heating, and a device for pressurizing, wherein the rotatable doctor blade scrapes the solution into a thin film around the inner surface of the evaporation vessel, and wherein the evaporation vessel can be at a starting temperature and a starting pressure; and (b). passing a gas through the evaporation vessel; (c). condensing and collecting the volatile liquid from the solution in a condenser in fluid communication with the evaporation vessel, wherein the condenser can be at a starting temperature; and (d). repeating steps (a) through (c) until the solution can have a final oligonucleotide concentration of > about 200 mg / mL.

[0007] In some cases, the flow rate of the solution containing the oligonucleotide can be about 0.5 mL / min to about 4.5 mL / min.

[0008] In some cases, the rotating doctor blade can rotate at a speed of about 15 rpm to about 105 rpm.

[0009] In some cases, the starting temperature of the evaporation vessel can be about 35 °C to about 60 °C.

[0010] In some cases, the starting pressure of the evaporation vessel can be about 10 torr to about 65 torr.

[0011] In some cases, the gas can be an inert gas, such as nitrogen, and can be at a starting purge pressure of about 1.5 psi to about 5 psi.

[0012] In some cases, the condenser can be an in-line condenser (i.e., located within the evaporation vessel). In other cases, the condenser can be an off-line condenser (i.e., separate from the evaporation vessel).

[0013] In some cases, the starting temperature of the condenser can be about 0 °C.

[0014] Second, the disclosure describes a method of concentrating a solution containing an oligonucleotide comprising at least the following steps: (a). charging a starting volume of the solution into an evaporation vessel, wherein the solution can have a starting oligonucleotide concentration of < about 20 mg / mL, and wherein the evaporation vessel can be at a starting pressure and a starting temperature; (b). boiling the solution in the evaporation vessel for a period of time; and (c). drawing the water vapor from the evaporation vessel via a condenser, wherein the condenser can be at a starting temperature, such that the solution can have a final oligonucleotide concentration of > about 200 mg / mL.

[0015] In some cases, the starting volume of the solution containing the oligonucleotide is about 1 mL to about 10 mL.

[0016] In some cases, the starting pressure of the evaporation vessel can be about 20 torr to about 30 torr.

[0017] In some cases, the starting temperature of the evaporation vessel can be about 55 °C to about 65 °C.

[0018] In some cases, the period of time can be about 4 minutes to about 4.5 minutes.

[0019] In some cases, the starting temperature of the condenser can be about 0 °C.

[0020] In some cases, the method can further comprise the following steps: (d). transferring the solution having a final oligonucleotide concentration of > about 200 mg / mL from the evaporation vessel to, e.g., a collection vessel.

[0021] In some cases, the method can further comprise the following steps: (e) repeating steps (a) through (d) on a fresh volume of the solution containing oligonucleotides.

[0022] In some cases of the above method, the solution containing oligonucleotides can be a solution containing ss oligonucleotides. In other cases of the above method, the solution containing oligonucleotides can be a solution containing ds oligonucleotides.

[0023] In some cases of the above method, the final oligonucleotide concentration can be about 200 mg / mL to about 400 mg / mL. In other cases of the above method, the final oligonucleotide concentration can be < about 450 mg / mL. In still other cases of the above method, the final oligonucleotide concentration can be < about 500 mg / mL.

[0024] In some cases of the above method, the final volume of the solution containing oligonucleotides can be < about 2 mL.

[0025] In addition, the above method optionally can include a subsequent step of tangential flow filtration (TFF) or lyophilization of the solution for additional concentration and / or purification.

[0026] Third, the present disclosure describes a composition comprising an oligonucleotide, e.g., a ds oligonucleotide, at a concentration of > about 200 mg / mL.

[0027] Alternatively, the present disclosure describes a composition having a concentration of > about 200 mg / mL resulting from the above method.

[0028] In some cases of these compositions, the composition is a solution. In other cases, the composition is a lyophilized powder.

[0029] In some cases of these compositions, especially when a solution, the final volume of the solution can be < about 2 mL.

[0030] Fourth, the present disclosure describes for the first time a device for concentrating a solution containing biomolecules (e.g., a solution containing oligonucleotides) for high dose / low volume administration via intermittent evaporation (IE). The device comprises: (i). an evaporation vessel, wherein the evaporation vessel comprises a means for heating and a first means for pressurization; (ii). a first reservoir in fluid communication with the evaporation vessel, wherein the first reservoir is a feed for unconcentrated solution containing biomolecules; (iii). a second reservoir in fluid communication with the evaporation vessel, wherein the second reservoir is a collector for concentrated solution containing biomolecules; and (iv). a condenser in fluid communication with the evaporation vessel, wherein the condenser comprises a means for cooling and a second means for pressurization.

[0031] An advantage of the methods herein is that they can be used to concentrate ss or ds oligonucleotides.

[0032] An advantage of the methods herein is that they can be used to significantly increase the concentration of oligonucleotides in solution compared to the concentration that can be achieved in a conventional wiped film evaporation (WFE) or TFF setup.

[0033] An advantage of the methods herein is that they do not result in a measurable loss of chemical purity of the oligonucleotides in the solution containing oligonucleotides (i.e., do not degrade the oligonucleotides).

[0034] An advantage of the methods herein is that they result in a reduction in volume for efficient storage and potential high dose requirements.

[0035] An advantage of the methods herein is that they can be combined with other oligonucleotide concentration methods (e.g., TFF or lyophilization) to achieve even higher concentrations thereof. BRIEF DESCRIPTION OF DRAWINGS

[0036] Advantages, effects, features and objects, in addition to those already stated above, will become apparent to those of ordinary skill in the art from the following detailed description, taken in conjunction with the accompanying drawings. Such detailed description, in conjunction with the drawings, discloses only typical embodiments, and is not intended to limit the scope of the disclosure. Figure 1 An exemplary device for concentrating a solution containing oligonucleotides via WFE is shown.

[0037] Figure 2 An exemplary device for concentrating a solution containing oligonucleotides via IE is shown.

[0038] Figure 3A -B shows superimposed denaturing ultra performance liquid chromatography (UPLC) chromatograms of a solution containing oligonucleotides, wherein Figure 3AResults are shown for concentration via TFF only, via TFF followed by WFE concentration, via WFE concentration only, and via WFE concentration followed by WFE (“double-pass WFE”) while Figure 3B Results are shown for concentration via IE.

[0039] Figure 4A -B shows superimposed non-denaturing UPLC chromatograms of solutions containing oligonucleotides, where Figure 4A Results are shown for concentration via TFF only, via TFF followed by WFE concentration, via WFE concentration only, and via “double-pass” WFE concentration while Figure 4B Results are shown for concentration via IE.

[0040] Detailed Description SUMMARY Therapeutic oligonucleotides, especially those based on activating RNA (aRNA), editing RNA (eRNA), inhibitory RNA (iRNA), and messenger RNA (mRNA), are an emerging class of biomolecules. While lyophilization is typically the final step in the preparation of a DS to produce a solid powder, the use of DS solutions of therapeutic oligonucleotides can be advantageous due to their high stability in liquid or frozen solutions and can simplify the production process when preparing a DP solution. For example, the final step of DS preparation can be the removal of any aqueous solution via lyophilization to form a solid powder, while the DP process can conversely include dissolving the DS (as a solid powder / lyophilisate) in water to form a DP. Thus, an alternative to lyophilization is needed to allow for a more seamless transition from DS to DP and to eliminate the costs and time required for lyophilization.

[0041] Using conventional TFF, concentrations of ds oligonucleotide solutions > 100 mg / mL are difficult to achieve due to a decline in permeate flux (which can be due to an increase in osmotic pressure, an increase in viscosity, and / or fouling of the membrane surface). While conventional WFE does not face the same membrane-based challenges as TFF, there can be a problem of gelling or drying of the oligonucleotide in the evaporation vessel (i.e., column), which can occur when conditions favor slow feed flow rates or substantial evaporation in order to achieve very high concentration factors during a single pass through the vessel.

[0042] In contrast, as shown in the Examples below, the "double-pass" WFE method herein and the IE method herein achieve concentrations > about 200 mg / mL. For the IE method, avoiding the use of a "wiper" element of the WFE can help mitigate or inhibit gel and solid formation from over-evaporation of the thin film, allowing for single-pass concentration through the system and avoiding the potential for wiper blade degradation. The devices and methods herein can provide high concentrations of therapeutic oligonucleotides (i.e., > about 200 mg / mL) in low volumes of solution (i.e., < about 2 mL).

[0043] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods, the preferred methods and materials are described.

[0044] In addition, reference to an element by the indefinite article "a" or "an" does not, unless specifically stated otherwise, exclude the possibility that "a" or "an" element is in practice more than one. Thus, the indefinite article "a" or "an" is used herein to mean "at least one" or "one or more."

[0045] Further, the use of "including" and "including, but not limited to," "including" and "including, but not limited to," "include" "includes" and "included" as well as other forms thereof, is not limiting.

[0046] Certain abbreviations used herein are as follows: "ADAR" means adenosine deaminase acting on RNA enzyme; "API" means active pharmaceutical ingredient; "aRNA" means activated RNA; "ASO" means antisense oligonucleotide; "DF" means dilution factor; "DIPEA" means N,N- diisopropylethylamine; "DIW" refers to deionized water; "DNA" refers to deoxyribonucleic acid; "DP" refers to drug product; "ds" refers to double stranded; "DS" refers to drug substance; "DsiRNA" refers to Dicer substrate interfering RNA; "eRNA" refers to edited RNA; "GalNAc" refers to N-acetylgalactosamine; "H2O" refers to water; "HFIP" refers to hexafluoroisopropanol; "hr" refers to hour(s); "iRNA" refers to inhibitory RNA; "IE" refers to intermittent evaporation; "kDa" refers to kilodaltons; "L" refers to liter(s); "MEC" refers to molar extinction coefficient; "mg" refers to milligram(s); "min" refers to minute(s); "mL" refers to milliliter(s); "mol" refers to mole(s); "mRNA" refers to messenger RNA; "miRNA" refers to microRNA; "MW" refers to molecular weight; "MWCO" refers to molecular weight cut off; "PES" refers to polyethersulfone; "PFA" refers to perfluoroalkoxy; "psi" refers to pounds per square inch; "RISC" refers to RNA-induced silencing complex; "RITA" refers to RNA-induced transcriptional activation; "RNA" refers to ribonucleic acid; "rRNA" refers to ribosomal RNA; "rpm" refers to revolutions per minute; "shRNA" refers to short hairpin RNA; "siRNA" refers to small interfering RNA; "SPS" refers to solid phase synthesis; "ss" refers to single stranded; "TFE" refers to thin film evaporation; "TFF" refers to tangential flow filtration; "TMP" refers to transmembrane pressure; "tRNA" refers to transfer RNA; "UPLC" refers to ultra-performance liquid chromatography; "UV" refers to ultraviolet; "V" refers to volume; "W" refers to weight; "WFE" refers to wiped film evaporation.

[0047] Certain definitions used herein are defined as follows: As used herein, "about" means within a statistically meaningful range of one or more values, such as stated concentrations, flow rates, lengths, molecular weights, pH, pressures, sequence similarities, speeds, time ranges, temperatures, volumes, and the like. Such values or ranges can generally be within an order of magnitude of the stated value or range, more typically within 20%, and even more typically within 10%, and even more typically within 5%. The permissible deviation encompassed by "about" will depend on the particular system in question, and can be readily appreciated by one of skill in the art.

[0048] As used herein, "activating RNA" or "aRNA" means a nucleic acid that contains RNA and mediates targeted activation of the promoter of an RNA transcript or other non-coding transcript via the RNA-induced transcriptional activation (RITA) complex pathway. aRNA is typically ds. aRNA activates, increases, modulates, or upregulates expression of a target nucleotide sequence in a cell.

[0049] As used herein, "biomolecule" and the like means a molecule or compound that includes or incorporates an amino acid, a carbohydrate, a lipid, and / or a nucleotide. Examples of biomolecules of interest herein include, but are not limited to, nucleic acids (e.g., oligonucleotides and polynucleotides), peptides, polypeptides, and proteins.

[0050] As used herein, "deoxyribonucleotide" means a nucleotide that has a hydrogen in place of a hydroxyl group at the 2' position of its pentose sugar when compared to a ribonucleotide. A modified deoxyribonucleotide has one or more modifications or substitutions of atoms other than a hydroxyl group at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0051] As used herein, "drug product" or "DP" means the finished product of any therapeutic agent, e.g., a therapeutic oligonucleotide, which is available on the market and ready for use, usually (but not necessarily) in association with one or more other pharmaceutically acceptable ingredients.

[0052] As used herein, "drug substance" or "DS" means the active ingredient (e.g., a therapeutic oligonucleotide) intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body, but does not include intermediates used in the synthesis of such ingredient. DS is also known as active pharmaceutical ingredient (API). DS is used in the manufacture of DP.

[0053] As used herein, "edited RNA" or "eRNA" means a nucleic acid that contains RNA and mediates nucleotide insertion, deletion, and even base substitution within a target nucleotide sequence. RNA editing has been observed in many different types of RNA, e.g., mRNA, microRNA (miRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). RNA editing is enzymatically mediated, either by an exogenously provided adenosine deaminase (ADAR) enzyme that acts on RNA, or by directing an endogenous ADAR to a specific site in a target RNA nucleotide sequence, and typically involves editing at a single nucleotide site by directing the ADAR to that site via a complementary oligonucleotide. eRNA is typically ss.

[0054] As used herein, "inhibitory RNA" or "iRNA" means a nucleic acid that contains RNA and mediates the targeted cleavage of RNA transcripts via RNA interference, for example, through the RNA-induced silencing complex (RISC) pathway. Some iRNAs are ss, while other iRNAs are ds and have a sense strand and an antisense strand, where the sense and antisense strands form a duplex. iRNAs direct the sequence-specific degradation of mRNA via RNA interference. iRNAs attenuate, inhibit, modulate, or reduce the expression of a target nucleotide sequence in a cell. Examples of iRNAs include, but are not limited to, antisense oligonucleotides (ASOs), Dicer substrate interfering RNAs (DsiRNAs), miRNAs, short hairpin RNAs (shRNAs), or small interfering RNAs (siRNAs).

[0055] As used herein, "intermittent evaporation" or "IE" means a concentration process in which a defined amount of solution to be concentrated is charged into a vessel that is heated and under vacuum, and where the concentrated solution is removed before another batch is charged. The concentrated material is collected in a "batch" process here, but the process can also be continuous.

[0056] As used herein, "nucleotide" means an organic compound having a nucleoside (a nucleobase, such as adenine, cytosine, guanine, thymine, or uracil; and a pentose sugar, such as ribose or 2'-deoxyribose) and a phosphate group. Nucleotides can serve as monomeric units of nucleic acid polymers, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0057] As used herein, "oligonucleotide" means a short nucleic acid compound (e.g., a polymer less than about 100 nucleotides in length) that can include deoxyribonucleotides (or modified deoxyribonucleotides), ribonucleotides (or modified ribonucleotides), or both. Likewise, an oligonucleotide can be ss or ds, and thus can or can not have a duplex region.

[0058] As used herein, "synthetic" refers to a nucleic acid or other compound that is artificially synthesized (e.g., using a machine such as a solid-phase nucleic acid synthesizer), or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the nucleic acid or other compound.

[0059] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions at the 2' position of other than hydrogen, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0060] As used herein, "therapeutic oligonucleotide" means an ss or ds nucleic acid having therapeutic applications (i.e., applications in the treatment of disease). Such nucleic acids typically contain one or more modified nucleotide residues or linkages, and can also include targeting ligands and / or delivery vehicles. Examples of therapeutic oligonucleotides include, but are not limited to, aRNA, eRNA, iRNA, and mRNA. Specific examples of therapeutic oligonucleotides include, but are not limited to, ASOs, aptamers, short activating RNAs (saRNAs), siRNAs, miRNAs, and decoys.

[0061] As used herein, "wiped film evaporation" or "WFE" means a concentration process that utilizes the controlled downward flow of a low concentration feed solution (i.e., a solution containing oligonucleotides) along a heated and under reduced pressure column, while a rotating scraper scrapes the solution into a thin film around the inner surface of the column, thereby facilitating the evaporation of volatile liquids (e.g., H2O) in the solution. Also and as used herein, "double-pass WFE" refers to a process in which the same solution containing ds oligonucleotides is subjected to at least two WFEs.

[0062] Apparatus An exemplary WFE apparatus of the present disclosure is shown in Figure 1 Here, a solution containing a low concentration of ss or ds oligonucleotides to be concentrated (not shown) can be placed / stored in a first reservoir (1) that is in fluid communication with an evaporation vessel (2) known in the art for WFE (i.e., including, for example, a heating device and a rotatable scraper). The evaporation vessel (2) can be further in fluid communication with a condenser (3) for removing volatile liquids, a gas purge source (not shown) for mitigating condensation formation within the apparatus, and a second reservoir (4) for collecting the concentrated solution containing oligonucleotides. Vacuum can be applied to the apparatus via a vacuum pump and controller (6). In some cases, the condenser can be an external condenser (as shown in Figure 1 Although an internal condenser can also be used. If an internal condenser is used, an optional collection vessel (5) can be used to collect evaporated water or solvent.

[0063] An exemplary IE apparatus of the present disclosure is shown in Figure 2The solution to be concentrated (not shown) containing ss or ds oligonucleotides can be placed / stored in a first reservoir (1) (e.g., a syringe pump) that is in fluid communication with an evaporation vessel (2) (e.g., a column comprising means for heating the column to a starting temperature and means for pressurizing the column to a starting pressure). The evaporation vessel (2) can be further in fluid communication with a condenser (3) for removing the volatile liquid and a second reservoir (4) for collecting the concentrated solution containing oligonucleotides. A vacuum can be applied to the second reservoir (4) via a vacuum pump and controller (6). The condenser (3) can be in fluid communication with a third reservoir (8) for collecting the concentrated solution containing oligonucleotides. A vacuum can be applied to the third reservoir (8) via a vacuum pump and controller (7). In some cases, the vacuum pump and controller (6) can be the same type as the vacuum pump and controller (7); however, in other cases, the vacuum pump and controller (6) can be a different type than the vacuum pump and controller (7). The apparatus optionally can include a balance (9) for measuring the amount of concentrated solution containing oligonucleotides that has been collected.

[0064] Exemplary apparatuses for heating include, but are not limited to, circulating a heating fluid, such as a gas or a liquid. In some cases, the apparatus for heating can be a glycol / water solution.

[0065] Exemplary apparatuses for pressurizing include, but are not limited to, a vacuum.

[0066] Method The method can include the steps described herein, and the steps can (but need not necessarily) be performed in the order as described. However, other orders are also conceivable. Furthermore, individual or multiple steps can be performed in parallel and / or overlap in time and / or individually or in multiple repeated steps. Furthermore, the method can include additional, unspecified steps.

[0067] Likewise, the oligonucleotides can be prepared by any method known in the art, such as solid phase synthesis (SPS) of individual strands, which, for ds oligonucleotides, can then optionally be subjected to additional steps for purification, solvent exchange, desalting, and concentration, before and / or after annealing into duplexes in water.

[0068] “Double-pass” WFE Briefly, a method of concentrating a solution containing oligonucleotides via a “double-pass” WFE can include the step of (a) flowing the solution through an evaporation vessel having a rotatable doctor blade, wherein the evaporation vessel can be at a starting pressure and a starting temperature, and wherein the rotatable doctor blade scrapes the solution into a thin film around the inner surface of the evaporation vessel.

[0069] In some cases, the oligonucleotides can be ss oligonucleotides. In other cases, the oligonucleotides can be ds oligonucleotides.

[0070] In some cases, the starting oligonucleotide concentration in the solution containing oligonucleotides can be < about 20 mg / mL. In other cases, the starting oligonucleotide concentration can be between about 5 mg / mL to about 20 mg / mL or about 10 mg / mL to about 15 mg / mL. In yet other cases, the starting oligonucleotide concentration can be about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL. In yet other cases, the starting oligonucleotide concentration can be > about 20 mg / mL but < about 30 mg / mL. In some cases, the starting oligonucleotide concentration can be > about 30 mg / mL, so long as the volume is large enough and the viscosity is low enough to flow through the system (e.g., even > about 100 mg / mL where appropriate).

[0071] In some cases, the pH of the solution containing oligonucleotides can be about 6.0 to about 7.0. In other cases, the pH of the solution containing oligonucleotides can be about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0072] In some cases, the solution containing the oligonucleotide can be flowed through the evaporation vessel at a starting flow rate of about 0.5 mL / min to about 4.5 mL / min. In other cases, the starting flow rate can be about 0.6 mL / min to about 4.4 mL / min, about 0.7 mL / min to about 4.3 mL / min, about 0.8 mL / min to about 4.2 mL / min, about 0.9 mL / min to about 4.1 mL / min, about 1.0 mL / min to about 4.0 mL / min, about 1.1 mL / min to about 3.9 mL / min, about 1.2 mL / min to about 3.8 mL / min, about 1.3 mL / min to about 3.7 mL / min, about 1.4 mL / min to about 3.6 mL / min, about 1.5 mL / min to about 3.5 mL / min, about 1.6 mL / min to about 3.4 mL / min, about 1.7 mL / min to about 3.3 mL / min, about 1.8 mL / min to about 3.2 mL / min, about 1.9 mL / min to about 3.1 mL / min, about 2.0 mL / min to about 3.0 mL / min, about 2.1 mL / min to about 2.9 mL / min, about 2.2 mL / min to about 2.8 mL / min, about 2.3 mL / min to about 2.7 mL / min, about 2.4 mL / min to about 2.6 mL / min, or about 2.5 mL / min. In yet other cases, the starting flow rate can be about 0.5 mL / min to about 1.0 mL / min, about 1.0 mL / min to about 1.5 mL / min, about 1.5 mL / min to about 2.0 mL / min, about 2.0 mL / min to about 2.5 mL / min, about 2.5 mL / min to about 3.0 mL / min, about 3.0 mL / min to about 3.5 mL / min, about 3.5 mL / min to about 4.0 mL / min, or about 4.0 mL / min to about 4.5 mL / min.In yet other cases, the starting flow rate can be about 0.5 mL / min, about 0.6 mL / min, about 0.7 mL / min, about 0.8 mL / min, about 0.9 mL / min, about 1.0 mL / min, about 1.1 mL / min, about 1.2 mL / min, about 1.3 mL / min, about 1.4 mL / min, about 1.5 mL / min, about 1.6 mL / min, about 1.7 mL / min, about 1.8 mL / min, about 1.9 mL / min, about 2.0 mL / min, about 2.1 mL / min, about 2.2 mL / min, about 2.3 mL / min, about 2.4 mL / min, about 2.5 mL / min, about 2.6 mL / min, about 2.7 mL / min, about 2.8 mL / min, about 2.9 mL / min, about 3.0 mL / min, about 3.1 mL / min, about 3.2 mL / min, about 3.3 mL / min, about 3.4 mL / min, about 3.5 mL / min, about 3.6 mL / min, about 3.7 mL / min, about 3.8 mL / min, about 3.9 mL / min, about 4.0 mL / min, about 4.1 mL / min, about 4.2 mL / min, about 4.3 mL / min, about 4.4 mL / min, or about 4.5 mL / min.

[0073] In some cases, the flow rate can be fixed (i.e., can remain) during concentration. In other cases, the flow rate can vary (i.e., can increase from the starting flow rate or can decrease from the starting flow rate) during concentration.

[0074] In some cases, the starting pressure of the vaporization vessel can be from about 10 Torr to about 65 Torr. In other cases, the starting pressure can be from about 11 Torr to about 64 Torr, from about 12 Torr to about 63 Torr, from about 13 Torr to about 62 Torr, from about 14 Torr to about 61 Torr, from about 15 Torr to about 60 Torr, from about 16 Torr to about 59 Torr, from about 17 Torr to about 58 Torr, from about 18 Torr to about 57 Torr, from about 19 Torr to about 56 Torr, from about 20 Torr to about 55 Torr, from about 21 Torr to about 54 Torr, from about 22 Torr to about 53 Torr, from about 23 Torr to about 52 Torr, from about 24 Torr to about 51 Torr, from about 25 Torr to about 50 Torr, from about 26 Torr to about 49 Torr, from about 27 Torr to about 48 Torr, from about 28 Torr to about 47 Torr, from about 29 Torr to about 46 Torr, from about 30 Torr to about 45 Torr, from about 31 Torr to about 44 Torr, from about 32 Torr to about 43 Torr, from about 33 Torr to about 42 Torr, from about 34 Torr to about 41 Torr, from about 35 Torr to about 40 Torr, from about 36 Torr to about 39 Torr, or from about 37 Torr to about 38 Torr. In yet other cases, the starting pressure can be from about 10 Torr to about 15 Torr, from about 15 Torr to about 20 Torr, from about 20 Torr to about 25 Torr, from about 25 Torr to about 30 Torr, from about 30 Torr to about 35 Torr, from about 35 Torr to about 40 Torr, from about 40 Torr to about 45 Torr, from about 45 Torr to about 50 Torr, from about 50 Torr to about 55 Torr, from about 55 Torr to about 60 Torr, or from about 60 Torr to about 65 Torr. In yet other cases, the starting pressure can be about 10 Torr, about 11 Torr, about 12 Torr, about 13 Torr, about 14 Torr, about 15 Torr, about 16 Torr, about 17 Torr, about 18 Torr, about 19 Torr, about 20 Torr, about 21 Torr, about 22 Torr, about 23 Torr, about 24 Torr, about 25 Torr, about 26 Torr, about 27 Torr, about 28 Torr, about 29 Torr, about 30 Torr, about 31 Torr, about 32 Torr, about 33 Torr, about 34 Torr, about 35 Torr, about 36 Torr, about 37 Torr, about 38 Torr, about 39 Torr, about 40 Torr, about 41 Torr, about 42 Torr, about 43 Torr, about 44 Torr, about 45 Torr, about 46 Torr, about 47 Torr, about 48 Torr, about 49 Torr, about 50 Torr, about 51 Torr, about 52 Torr, about 53 Torr, about 54 Torr, about 55 Torr, about 56 Torr, about 57 Torr, about 58 Torr, about 59 Torr, about 60 Torr, about 61 Torr, about 62 Torr, about 63 Torr, about 64 Torr, or about 65 Torr.

[0075] In some cases, the pressure of the vaporization vessel can be fixed (i.e., can remain) during concentration. In other cases, the pressure of the vaporization vessel can vary (i.e., can increase from the starting pressure or can decrease from the starting pressure) during concentration.

[0076] In some cases, the starting temperature of the evaporation vessel can be from about 35 °C to about 60 °C. In other cases, the starting temperature can be from about 36 °C to about 59 °C, from about 37 °C to about 58 °C, from about 38 °C to about 57 °C, from about 39 °C to about 56 °C, from about 40 °C to about 55 °C, from about 41 °C to about 54 °C, from about 42 °C to about 53 °C, from about 43 °C to about 52 °C, from about 44 °C to about 51 °C, from about 45 °C to about 50 °C, from about 46 °C to about 49 °C, or from about 47 °C to about 48 °C. In yet other cases, the starting temperature can be from about 35 °C to about 40 °C, from about 40 °C to about 45 °C, from about 45 °C to about 50 °C, from about 50 °C to about 55 °C, or from about 55 °C to about 60 °C. In yet other cases, the starting temperature can be about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, or about 60 °C.

[0077] In some cases, the temperature of the evaporation vessel can be fixed (i.e., can remain) during concentration. In other cases, the temperature of the evaporation vessel can vary (i.e., can increase from the starting temperature or can decrease from the starting temperature) during concentration.

[0078] In some cases, the rotating doctor blade can be rotated at a starting speed of about 15 rpm to about 105 rpm. In other cases, the starting speed can be about 16 rpm to about 104 rpm, about 17 rpm to about 103 rpm, about 18 rpm to about 102 rpm, about 19 rpm to about 101 rpm, about 20 rpm to about 100 rpm, about 21 rpm to about 99 rpm, about 22 rpm to about 98 rpm, about 23 rpm to about 97 rpm, about 24 rpm to about 96 rpm, about 25 rpm to about 95 rpm, about 26 rpm to about 94 rpm, about 27 rpm to about 93 rpm, about 28 rpm to about 92 rpm, about 29 rpm to about 91 rpm, about 30 rpm to about 90 rpm, about 31 rpm to about 89 rpm, about 32 rpm to about 88 rpm, about 33 rpm to about 87 rpm, about 34 rpm to about 86 rpm, about 35 rpm to about 85 rpm, about 36 rpm to about 84 rpm, about 37 rpm to about 83 rpm, about 38 rpm to about 82 rpm, about 39 rpm to about 81 rpm, about 40 rpm to about 80 rpm, about 41 rpm to about 79 rpm, about 42 rpm to about 78 rpm, about 43 rpm to about 77 rpm, about 44 rpm to about 76 rpm, about 45 rpm to about 75 rpm, about 46 rpm to about 74 rpm, about 47 rpm to about 73 rpm, about 48 rpm to about 72 rpm, about 49 rpm to about 71 rpm, about 50 rpm to about 70 rpm, about 51 rpm to about 69 rpm, about 52 rpm to about 68 rpm, about 53 rpm to about 67 rpm, about 54 rpm to about 66 rpm, about 55 rpm to about 65 rpm, about 56 rpm to about 64 rpm, about 57 rpm to about 63 rpm, about 58 rpm to about 62 rpm, about 59 rpm to about 61 rpm, or about 60 rpm.In yet other cases, the starting speed can be from about 15 rpm to about 20 rpm, from about 20 rpm to about 25 rpm, from about 25 rpm to about 30 rpm, from about 30 rpm to about 35 rpm, from about 35 rpm to about 40 rpm, from about 40 rpm to about 45 rpm, from about 45 rpm to about 50 rpm, from about 50 rpm to about 55 rpm, from about 55 rpm to about 60 rpm, from about 60 rpm to about 65 rpm, from about 65 rpm to about 70 rpm, from about 70 rpm to about 75 rpm, from about 75 rpm to about 80 rpm, from about 80 rpm to about 85 rpm, from about 85 rpm to about 90 rpm, from about 90 rpm to about 95 rpm, from about 95 rpm to about 100 rpm, or from about 100 rpm to about 105 rpm. In yet other cases, the starting speed can be about 15 rpm, about 20 rpm, about 25 rpm, about 30 rpm, about 35 rpm, about 40 rpm, about 45 rpm, about 50 rpm, about 55 rpm, about 60 rpm, about 65 rpm, about 70 rpm, about 75 rpm, about 80 rpm, about 85 rpm, about 90 rpm, about 95 rpm, about 100 rpm, or about 105 rpm.

[0079] In some cases, the speed of the rotating doctor blade can be fixed (i.e., can remain) during concentration. In other cases, the speed of the rotating doctor blade can vary (i.e., can increase from a starting speed or can decrease from a starting speed) during concentration.

[0080] The method can further include step (b) of passing a gas (e.g., an inert gas) into the evaporation vessel. In some cases, the gas can be nitrogen. In some cases, the starting purge pressure of the gas can be from about 1.5 psi to about 5.0 psi. In other cases, the starting purge pressure can be from about 1.6 psi to about 4.9 psi, from about 1.7 psi to about 4.8 psi, from about 1.8 psi to about 4.7 psi, from about 1.9 psi to about 4.6 psi, from about 2.0 psi to about 4.5 psi, from about 2.1 psi to about 4.4 psi, from about 2.2 psi to about 4.3 psi, from about 2.3 psi to about 4.2 psi, from about 2.4 psi to about 4.1 psi, from about 2.5 psi to about 4.0 psi, from about 2.6 psi to about 3.9 psi, from about 2.7 psi to about 3.8 psi, from about 2.8 psi to about 3.7 psi, from about 2.9 psi to about 3.6 psi, from about 3.0 psi to about 3.5 psi, from about 3.1 psi to about 3.2 psi, or from about 3.3 psi to about 3.4 psi. In other cases, the starting purge pressure can be from about 1.5 psi to about 2.0 psi, from about 2.0 psi to about 2.5 psi, from about 2.5 psi to about 3.0 psi, from about 3.0 psi to about 3.5 psi, from about 3.5 psi to about 4.0 psi, from about 4.0 psi to about 4.5 psi, or from about 4.5 psi to about 5.0 psi. In yet other cases, the starting purge pressure can be about 1.5 psi, about 1.6 psi, about 1.7 psi, about 1.8 psi, about 1.9 psi, about 2.0 psi, about 2.1 psi, about 2.2 psi, about 2.3 psi, about 2.4 psi, about 2.5 psi, about 2.6 psi, about 2.7 psi, about 2.8 psi, about 2.9 psi, about 3.0 psi, about 3.1 psi, about 3.2 psi, about 3.3 psi, about 3.4 psi, about 3.5 psi, about 3.6 psi, about 3.7 psi, about 3.8 psi, about 3.9 psi, about 4.0 psi, about 4.1 psi, about 4.2 psi, about 4.3 psi, about 4.4 psi, about 4.5 psi, about 4.6 psi, about 4.7 psi, about 4.8 psi, about 4.9 psi, or about 5.0 psi.

[0081] In some cases, the purge pressure can be fixed (i.e., can remain) during concentration. In other cases, the purge pressure can vary (i.e., can increase from the starting pressure or can decrease from the starting pressure) during concentration.

[0082] The method can further comprise step (c) condensing and collecting the volatile liquid from the solution in a condenser in fluid communication with the evaporation vessel, wherein the condenser is at a starting temperature.

[0083] In some cases, the condenser is an in-line condenser (i.e., located within the evaporation vessel). In other cases, the condenser is an off-line condenser (i.e., separate from the evaporation vessel). In some cases, the starting temperature of the condenser is about 0 °C.

[0084] In some cases, the temperature of the condenser can be fixed (i.e., can remain) during concentration. In other cases, the temperature of the condenser can vary (i.e., can increase from the starting temperature or can decrease from the starting temperature) during concentration.

[0085] The method can further comprise step (d) repeating steps (a) through (c) at least one additional time, thereby resulting in a final oligonucleotide concentration of the solution of > about 200 mg / mL. In other cases, the final oligonucleotide concentration can be from about 200 mg / mL to about 400 mg / mL. In yet other cases, the final oligonucleotide concentration can be from about 210 mg / mL to about 390 mg / mL, from about 220 mg / mL to about 380 mg / mL, from about 230 mg / mL to about 370 mg / mL, from about 240 mg / mL to about 360 mg / mL, from about 250 mg / mL to about 350 mg / mL, from about 260 mg / mL to about 340 mg / mL, from about 270 mg / mL to about 330 mg / mL, from about 280 mg / mL to about 320 mg / mL, from about 290 mg / mL to about 310 mg / mL, or about 300 mg / mL. In yet other cases, the final oligonucleotide concentration can be about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL, about 300 mg / mL, about 310 mg / mL, about 320 mg / mL, about 330 mg / mL, about 340 mg / mL, about 350 mg / mL, about 360 mg / mL, about 370 mg / mL, about 380 mg / mL, about 390 mg / mL, or about 400 mg / mL. In still other cases, the final oligonucleotide concentration is < about 450 mg / mL. In yet other cases of the above method, the final oligonucleotide concentration can be < about 500 mg / mL.

[0086] In some cases, the final volume of the solution containing the oligonucleotide can be < about 2 mL.

[0087] “Single-pass” IE Briefly, the method of concentrating a solution containing oligonucleotides via an IE can comprise the step of (a) charging a starting volume of the solution into an evaporation vessel, wherein the evaporation vessel is at a starting pressure and a starting temperature.

[0088] In some cases, the starting oligonucleotide concentration in the solution containing oligonucleotides can be < 20 mg / mL. In other cases, the starting oligonucleotide concentration can be between about 5 mg / mL to about 20 mg / mL or about 10 mg / mL to about 15 mg / mL. In yet other cases, the starting oligonucleotide concentration can be about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL. In yet other cases, the starting oligonucleotide concentration can be > about 20 mg / mL but < about 30 mg / mL. In some cases, the starting oligonucleotide concentration can be > about 30 mg / mL, so long as its volume is large enough and viscosity is low enough to flow through the system (e.g., even > about 100 mg / mL in appropriate cases).

[0089] In some cases, the pH of the solution containing oligonucleotides can be about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0090] In some cases, the starting volume of the solution containing oligonucleotides is about 1 mL to about 10 mL. In other cases, the starting volume can be about 2 mL to about 9 mL, about 3 mL to about 8 mL, about 4 mL to about 7 mL, or about 5 mL to about 6 mL. In yet other cases, the starting volume can be about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL. In even other cases and depending on the size of the evaporation vessel, the starting volume of the solution containing oligonucleotides can be > 10 mL.

[0091] In some cases, the starting pressure of the evaporation vessel can be from about 20 Torr to about 30 Torr. In other cases, the starting pressure can be from about 20 Torr to about 22 Torr, from about 22 Torr to about 24 Torr, from about 24 Torr to about 26 Torr, from about 26 Torr to about 28 Torr, or from about 28 Torr to about 30 Torr. In yet other cases, the starting pressure can be about 20 Torr, about 21 Torr, about 22 Torr, about 23 Torr, about 24 Torr, about 25 Torr, about 26 Torr, about 27 Torr, about 28 Torr, about 29 Torr, or about 30 Torr.

[0092] In some cases, the pressure of the evaporation vessel can be fixed (i.e., can remain) during concentration. In other cases, the pressure of the evaporation vessel can vary (i.e., can increase from the starting pressure or can decrease from the starting pressure) during concentration.

[0093] In some cases, the starting temperature of the evaporation vessel can be from about 55 °C to about 65 °C. In other cases, the starting temperature can be from about 56 °C to about 64 °C, from about 57 °C to about 63 °C, from about 58 °C to about 62 °C, from about 59 °C to about 61 °C, or about 60 °C. In yet other cases, the starting temperature can be from about 55 °C to about 57 °C, from about 57 °C to about 59 °C, from about 59 °C to about 61 °C, from about 61 °C to about 63 °C, or from about 63 °C to about 65 °C. In yet other cases, the starting temperature can be about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, or about 65 °C.

[0094] In some cases, the temperature of the evaporation vessel can be fixed (i.e., can remain) during concentration. In other cases, the temperature of the evaporation vessel can vary (i.e., can increase from the starting temperature or can decrease from the starting temperature) during concentration.

[0095] The method can further comprise step (b) of boiling the solution in the evaporation vessel for a period of time.

[0096] In some cases, the starting period of boiling can be from about 4 minutes to about 4.5 minutes. In other cases, the starting period can be from about 4.1 minutes to about 4.4 minutes, or from about 4.2 minutes to about 4.3 minutes. In yet other cases, the starting period can be from about 4 minutes to 4.1 minutes, from about 4.1 minutes to about 4.2 minutes, from about 4.2 minutes to about 4.3 minutes, from about 4.3 minutes to about 4.4 minutes, or from about 4.4 minutes to about 4.5 minutes. In yet other cases, the starting period can be about 4 minutes, about 4.1 minutes, about 4.2 minutes, about 4.3 minutes, about 4.4 minutes, or about 4.5 minutes, especially about 4.25 minutes.

[0097] The method can further comprise step (c) withdrawing the water vapor from the evaporation vessel via a condenser, wherein the condenser is at a starting temperature, thereby causing the solution to have a final oligonucleotide concentration of > about 200 mg / mL.

[0098] In some cases, the condenser is an in-line condenser (i.e., located within the evaporation vessel). In other cases, the condenser is an off-line condenser (i.e., separate from the evaporation vessel). In some cases, the starting temperature of the condenser is about 0 °C.

[0099] In some cases, the starting temperature of the condenser can be fixed (i.e., can remain) during the concentration. In other cases, the starting temperature of the condenser can vary (i.e., can increase from the starting temperature or can decrease from the starting temperature) during the concentration.

[0100] In some cases, the method can further comprise step (d) transferring the solution having a final oligonucleotide concentration of > about 200 mg / mL from the evaporation vessel to, e.g., a collection vessel.

[0101] In some cases, the method can further comprise step (e) repeating steps (a) through (d) on a fresh starting volume of the oligonucleotide-containing solution.

[0102] In some cases, the oligonucleotide is an ss oligonucleotide. In other cases, the oligonucleotide is a ds oligonucleotide.

[0103] In some cases, the final oligonucleotide concentration in the solution containing oligonucleotides can be > about 200 mg / mL. In other cases, the final oligonucleotide concentration can be from about 200 mg / mL to about 400 mg / mL. In yet other cases, the final oligonucleotide concentration can be from about 210 mg / mL to about 390 mg / mL, from about 220 mg / mL to about 380 mg / mL, from about 230 mg / mL to about 370 mg / mL, from about 240 mg / mL to about 360 mg / mL, from about 250 mg / mL to about 350 mg / mL, from about 260 mg / mL to about 340 mg / mL, from about 270 mg / mL to about 330 mg / mL, from about 280 mg / mL to about 320 mg / mL, from about 290 mg / mL to about 310 mg / mL, or about 300 mg. In yet other cases, the final oligonucleotide concentration can be about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 260 mg / mL, about 270 mg / mL, about 280 mg / mL, about 290 mg / mL, about 300 mg / mL, about 310 mg / mL, about 320 mg / mL, about 330 mg / mL, about 340 mg / mL, about 350 mg / mL, about 360 mg / mL, about 370 mg / mL, about 380 mg / mL, about 390 mg / mL, or about 400 mg / mL. In still other cases, the final oligonucleotide concentration can be < about 450 mg / mL. In yet other cases of the above methods, the final oligonucleotide concentration can be < about 500 mg / mL.

[0104] In some cases, the final volume of the solution containing oligonucleotides can be < about 2 mL. Examples

[0105] The following non-limiting examples are provided for illustrative and not limiting purposes.

[0106] Example 1: Concentration of a solution containing ds oligonucleotides via a combination of TFF and WFE Objective: To evaluate the effect of a combination of TFF and WFE on concentrating a solution containing ds oligonucleotides.

[0107] Method: Oligonucleotide-containing solution: The ds oligonucleotide used was a sodium salt duplex consisting of a 36 nucleotide sense strand containing a GalNAc sugar at positions 28-30 (SEQ ID NO: 3) complexed with a 22 nucleotide antisense strand (SEQ ID NO: 4). The ds oligonucleotide was at a concentration of 20 mg / mL in H2O (determined by UV assay) and fresh aliquots were taken as starting material.

[0108] TFF: TFF was performed on a solution containing 20 mg / mL ds oligonucleotide using a Pendotech® TFF system with a 2 L retentate reservoir, 6.4 mm internal diameter tubing size (Masterflex® 96410-17), retentate and permeate scales (Ohaus), and a Quattroflow® 150 circulation pump (PSG). The membranes used are listed in Table 2. Prior to use, the system and membranes were flushed and equilibrated with H2O and then charged with 765 mL of a solution containing 20 mg / mL ds oligonucleotide in the retentate reservoir, such that the amount of sample was approximately 15.3 g of ds oligonucleotide. Concentration experiments were performed with a feed flow rate of 1 mL / min and a transmembrane pressure (TMP) of 30 psi. When the minimum volume of the retentate vessel was reached, the run was stopped. The primary retentate was collected and then washed with approximately 60 mL and 100 mL of H2O. The system was sterilized by flushing with 0.5 N NaOH for 1 hour and stored in 0.1 N NaOH.

[0109] Table 1: Filters used for TFF of oligonucleotide-containing solutions Filter Name Manufacturer Material Lot # Surface area (m 2 )]]> MWCO (kDa) Centramate T-series Pall Omega PES 31279015R 0.1 3 Centramate T-series Pall Omega PES 31279016R 0.1 3 Centramate T-series Pall Omega PES 31279013R 0.1 3

[0110] WFE: WFE was performed on a 2 inch diameter wiped film (i.e., short path) molecular still (Pope Scientific Inc.) with modifications as shown in Figure 1 The feed funnel was replaced with a 1 L syringe pump (Isco) to allow continuous feed flow, with a metering valve installed in the feed flow path to provide positive back pressure in the syringe pump. The residue collection flask was replaced with a perfluoroalkoxy (PFA) tube (1 / 2 inch outer diameter, 3 / 8 inch inner diameter) connected to a size #35 Ace threaded glass bottle by a Swagelok® hand valve. A length of 1 / 16 inch tubing was inserted into the 1 / 2 inch PFA tube, extending from above the hand valve to the wiped film still body to help liquid flow through the narrow opening at the top of the tube where it connects to the glass adapter. As Figure 1A nitrogen line was attached to reduce condensation formation near the collection tube. The nitrogen line was regulated to 2 psi and connected to a metering valve to regulate the nitrogen flow. The system was connected to a vacuum pump through a vacuum controller. A hot glycol / water bath was circulated in the system to provide heat for evaporation, and another cold glycol / water bath was used to condense the water vapor. There were two configurations for the connection of the heating / cooling liquid to the system: (1) the hot liquid flowed through the jacket of the still, while the cold liquid flowed through the center condensing rod and then through the outer condenser; or (2) the hot liquid flowed through the jacket of the still first and then through the center condensing rod, while the cold liquid flowed through the outer condenser only. Configuration 1 was short-path evaporation, which was suitable for high throughput and low vacuum levels, while configuration 2 was more suitable for better control of the extent of evaporation.

[0111] For the concentration experiments, the solution containing the ds oligonucleotide was loaded into a syringe pump and fed into the device at the set flow rate. The heating jacket temperature was set to 55 °C, the outer condenser temperature was set to 0 °C, the scraper speed was set to 20 rpm, and the 2 psi nitrogen valve was opened to scale 5. The nitrogen purge was adjusted so that there was no water condensation on the arm connected to the residual flask and at the same time still able to maintain the pressure at the target set point. The vacuum pressure and feed flow rate varied slightly between different experiments, with the vacuum pressure values ranging from 20-32 torr and the feed flow rate ranging from 3.4-3.6 mL / min. The values were changed as needed to prevent or mitigate any observed gel formation.

[0112] Density and concentration measurements: Prior to density and concentration measurements, the concentrated sample was filtered with a 0.22 μm filter (Millipore; Burlington, MA, Steriflip 50 mL with 0.22 μm Durapore PVDF membrane).

[0113] Density measurements were recorded at 20 °C using a DMA 4100 M density meter (Anton Paar; Ashland, VA). The density measurements were used to prepare weight dilutions of the solution containing the oligonucleotide for concentration measurements.

[0114] Concentration values were generated using UV assay. Samples were first diluted by weight to approximately 2 mg / mL and then further diluted to 0.02 mg / mL for measurement using a Cary UV-Vis Multicell Peltier instrument. Triplicate measurements were made for each sample using a 1 cm path length cuvette with H2O as the reference. The following equation was used to generate the average concentration value, where Abs is the average absorbance value at 258 nm from the triplicate measurements, V is the volume of the first dilution, density is the measured density of the concentrated solution, MW is the molecular weight of the free acid form of the molecule (20675 g / mol), DF is the dilution factor of the second dilution, W is the sample weight of the concentrated sample, MEC is the molar extinction coefficient (548000 M -1 cm -1 ), path length is 1 cm, and purity was determined by non-denaturing UPLC.

[0115] Denaturing and non-denaturing UPLC purity determinations: Denaturing and non-denaturing UPLC determinations were used to analyze and compare the purity of the oligonucleotides before and after concentration experiments. Samples were diluted to approximately 2 mg / mL for measurement.

[0116] Denaturing determinations were performed using a Waters Acquity UPLC Peptide BEH C18 column (1.7 pm, 2.1 x 100 mm, Part Number 186003686) with a column temperature of 80 °C and detection at 20 Hz, 259 nm. Mobile phase A was 28 mM DIPEA and 100 mM HFIP, and mobile phase B was a 90:10 mixture of methanol:isopropyl alcohol (IPA), with the method gradient listed in Table 2.

[0117] Table 2: Gradient for denaturing UPLC Time (min) Flow rate (mL / min) %A %B Curve 1 Start 0.400 85.0 15.0 Start 2 1.00 0.400 85.0 15.0 6 3 15.00 0.400 79.0 21.0 6 4 15.10 0.400 40.0 60.0 6 5 17.00 0.400 40.0 60.0 6 6 17.10 0.400 85.0 15.0 6 7 20.00 0.400 85.0 15.0 6

[0118] Non-denaturing analysis was performed using a Waters XBridge BEH C18 column (2.5 pm, 2.1 x 50 mm, Part Number 186006029) with a column temperature of 20 °C and detection at 20 Hz, 260 nm. Mobile phase A was 95 mM TEA and 14 mM HFIP, and mobile phase B was a 75:25 mixture of methanol:isopropyl alcohol (IPA), with the method gradient listed in Table 4.

[0119] Table 3: Gradient for non-denaturing UPLC Time (min) Flow rate (mL / min) %A %B Curve 1 Start 0.250 95.0 5.0 Start 2 3.00 0.250 95.0 5.0 6 3 12.00 0.250 67.0 33.0 6 4 13.00 0.250 40.0 60.0 6 5 15.00 0.250 40.0 60.0 6 6 15.10 0.250 95.0 5.0 6 7 19.00 0.250 95.0 5.0 6

[0120] Viscosity measurements: Viscosity measurements were performed on samples collected from additional WFE experiments at various concentrations. For samples below 350 mg / mL, viscosity was measured at 20 °C using an Anton Paar Lovis 2000M viscometer. Measurements were performed using a 1.8 mm capillary and a 1.5 mm steel ball. For samples above 350 mg / mL, viscosity was measured at 20 °C using a RheoSense VROC® Initium viscometer.

[0121] Results: The concentrations of the solutions containing ds oligonucleotides after various treatments are shown in Table 4 below.

[0122] Table 4: Final ds oligonucleotide concentrations of solutions containing 20 mg / mL oligonucleotide after treatment Sample Treatment Final ds oligo concentration (mg / mL) 1 None 20 2 TFF only 64 3 TFF then WFE 280

[0123] Denaturing assay results showed that two major peaks associated with both the sense and antisense single strands of the ds oligonucleotide were observed Figure 3A ). No significant differences in impurities between the starting sample and the concentrated sample were observed. Additionally, non-denaturing assay results showed a major peak associated with the duplex, and both had similar purity levels (about 98.5-98.7 area%, with about 0.8-1 area% residual antisense strand) Figure 4A ). These results indicate that there was no significant change in chemical purity due to the concentration method.

[0124] A double-pass method was required here to achieve a ds oligonucleotide concentration of about 300 mg / mL.

[0125] Example 2: Concentration of a solution containing a ds oligonucleotide via WFE Objective: To evaluate the effect of WFE alone and repeated WFE (“double-pass” WFE) on the concentration of a solution containing a ds oligonucleotide.

[0126] Methods: Solution containing oligonucleotide: The solution containing oligonucleotide was as described in Example 1 above.

[0127] WFE: The WFE was performed as described in Example 1 above; however, in some cases, two WFEs were performed on one sample.

[0128] Density and concentration measurements: Density and concentration measurements were performed as described in Example 1 above.

[0129] Denaturing and non-denaturing UPLC purity measurements: UPLC purity measurements were performed as described in Example 1 above.

[0130] Viscosity measurements: Viscosity measurements were performed as described in Example 1 above.

[0131] Results: The final ds oligonucleotide concentrations of the solutions containing 20 mg / mL oligonucleotide after treatment are shown in Table 5 below.

[0132] Table 5: Final ds oligonucleotide concentrations of solutions containing 20 mg / mL oligonucleotide after treatment Sample Treatment Final ds oligo concentration (mg / mL) 1 None 20 4 WFE only 115 5 WFE then WFE 346

[0133] When starting from intermediate ds oligonucleotide concentrations ranging from about 80 mg / mL to about 115 mg / mL (achieved by single-pass WFE), final ds oligonucleotide concentrations > 300 mg / mL were reliably achieved by "double-pass" WFE.

[0134] Denaturing assay results showed that two major peaks associated with both the sense and anti-sense single strands of the ds oligonucleotide were observed Figure 3A ). No significant differences in impurities between the starting sample and the concentrated sample were observed. In addition, non-denaturing assay results showed a major peak associated with the duplex, and both had similar purity levels (about 98.5-98.7 area%, with about 0.8-1 area% residual anti-sense strand) Figure 4A ). These results indicate that there was no significant change in chemical purity due to the concentration method.

[0135] Example 3: Concentration of a solution containing a ds oligonucleotide via IE Purpose: To evaluate the effect of IE alone on the concentration of a solution containing a ds oligonucleotide.

[0136] Methods: Solution containing oligonucleotide: The solution containing oligonucleotide was as described in Example 1 above.

[0137] IE: The IE was performed using an automated custom rig as shown in Figure 2 . In the concentration experiment, the solution containing oligonucleotide was loaded into a syringe pump (1 L, Isco) and injected into the evaporator vessel at 5 mL per injection. The temperature of the heat transfer solution (ethylene glycol / water) was set to 60 °C and the vacuum was set to 25 torr with water vapor being collected through an external condenser. After a specified boiling time of 4.25 minutes, the concentrated solution was transferred to the collection vessel via a pressure differential created by a 12 second temporary release of the vacuum in the evaporator.

[0138] Density and concentration assay: The density and concentration assay was performed as described in Example 1 above.

[0139] Denaturing and non-denaturing UPLC purity assay: The UPLC purity assay was performed as described in Example 1 above.

[0140] Viscosity assay: The viscosity assay was performed as described in Example 1 above.

[0141] Results: The final ds oligonucleotide concentrations of the solutions containing 20 mg / mL oligonucleotide after treatment are shown in Table 6 below.

[0142] Table 6: Final ds oligonucleotide concentrations of solutions containing 20 mg / mL oligonucleotide after treatment Sample Treatment Final ds oligo concentration (mg / mL) 1 None 20 6 IE only 359* *Due to the small sample volume, an accurate density measurement could not be obtained. The concentration value shown was calculated using the density from sample 5. Using a series of density values generated from the same run, the actual concentration ranged from 353-365 mg / mL.

[0143] The denaturing assay results show that two major peaks associated with both the sense and anti-sense single strands of the ds oligonucleotide were observed Figure 3B ). No significant differences in impurities between the starting sample and the concentrated sample were observed. In addition, the non-denaturing assay results show a major peak associated with the duplex, and both had similar purity levels (about 98.5-98.7 area%, with about 0.8-1 area% residual anti-sense strand) Figure 4B ). These results indicate that there was no significant change in chemical purity due to the concentration method.

[0144] SEQUENCE LISTING The following nucleotide and / or amino acid sequences are referred to in the disclosure above, and are provided below for reference.

[0145] SEQ ID NO: 1 - Synthetic oligonucleotide 1 (36 nt) SEQ ID NO: 2 - Synthetic oligonucleotide 2 (22 nt) SEQ ID NO: 3 - Synthetic oligonucleotide 3 (36 nt) SEQ ID NO: 4 - Synthetic oligonucleotide 4 (22 nt)

Claims

1. A method of concentrating an oligonucleotide in solution, the method comprising the steps of: (a) flowing a solution containing an oligonucleotide through an evaporation vessel, wherein the solution has a starting oligonucleotide concentration of < about 20 mg / mL, wherein the evaporation vessel comprises a rotatable doctor blade, a means for heating, and a means for pressurization, wherein the rotatable doctor blade scrapes the solution into a thin film around the interior surface of the evaporation vessel, and wherein the evaporation vessel is at a starting pressure and a starting temperature; (b) passing a gas through the evaporation vessel; (c) condensing and collecting volatile liquid from the solution in a condenser in fluid communication with the evaporation vessel, wherein the condenser is at a starting temperature; and (d) repeating steps (a) through (c) at least one additional time, such that the solution has a final oligonucleotide concentration of > about 200 mg / mL.

2. The method of claim 1, wherein the solution containing an oligonucleotide has a pH of between about 6 to about 7.

3. The method of claim 1 or claim 2, wherein the oligonucleotide is single stranded (ss).

4. The method of claim 1 or claim 2, wherein the oligonucleotide is double stranded (ds).

5. The method of any one of claims 1 to 4, wherein the solution containing an oligonucleotide is flowed through the evaporation vessel at a starting flow rate of about 0.5 mL / min to about 4.5 mL / min.

6. The method of claim 5, wherein the starting flow rate is about 3.4 mL / min to about 3.6 mL / min.

7. The method of any one of claims 1 to 6, wherein the rotatable doctor blade is rotated at a starting speed of about 15 rpm to about 105 rpm.

8. The method of claim 7, wherein the starting speed is about 20 rpm.

9. The method of any one of claims 1 to 8, wherein the starting pressure of the evaporation vessel is about 10 torr to about 65 torr.

10. The method of claim 9, wherein the starting pressure is about 20 torr to about 32 torr.

11. The method of any one of claims 1 to 10, wherein the starting temperature of the evaporation vessel is about 35 °C to about 60 °C.

12. The method of claim 11, wherein the starting temperature is about 55 °C.

13. The method of any one of claims 1 to 12, wherein the starting purge pressure of the gas is about 1.5 psi to about 5 psi.

14. The method of claim 13, wherein the starting purge pressure is about 2 psi.

15. The method of claim 13 or claim 14, wherein the gas is a nitrogen gas.

16. The method of any one of claims 1 to 15, wherein the starting temperature of the condenser is about 0 °C.

17. The method of any one of claims 1 to 16, wherein steps (a) through (c) are repeated one time.

18. The method of any one of claims 1 to 17, wherein the final oligonucleotide concentration is about 200 mg / mL to about 400 mg / mL.

19. The method of any one of claims 1 to 17, wherein the final oligonucleotide concentration is < about 450 mg / mL.

20. The method of any one of claims 1 to 17, wherein the final oligonucleotide concentration is < about 500 mg / mL.

21. A method of concentrating an oligonucleotide in solution, the method comprising the steps of: (a) charging a starting volume of an oligonucleotide-containing solution into an evaporation vessel, wherein the solution has a starting oligonucleotide concentration of < about 20 mg / mL, and wherein the evaporation vessel is at a starting pressure and a starting temperature; (b) boiling the solution in the evaporation vessel for a starting period of time; and (c) withdrawing water vapor from the evaporation vessel via a condenser, wherein the condenser is at a starting temperature, thereby causing the solution to have a final oligonucleotide concentration of > about 200 mg / mL.

22. The method of claim 21, wherein the pH of the oligonucleotide-containing solution is between about 6 to about 7.

23. The method of claim 21 or claim 22, wherein the oligonucleotide is single-stranded (ss).

24. The method of claim 21 or claim 22, wherein the oligonucleotide is double-stranded (ds).

25. The method of any one of claims 21 to 24, wherein the starting volume of the oligonucleotide-containing solution is about 1 mL to about 10 mL.

26. The method of claim 25, wherein the starting volume is about 5 mL.

27. The method of any one of claims 21 to 26, wherein the starting pressure of the evaporation vessel is about 20 Torr to about 30 Torr.

28. The method of any one of claims 21 to 27, wherein the starting temperature of the evaporation vessel is about 55 °C to about 65 °C.

29. The method of claim 28, wherein the starting temperature is about 60 °C.

30. The method of any one of claims 21 to 29, wherein the starting period of time for boiling is about 4 minutes to about 4.5 minutes.

31. The method of claim 30, wherein the starting period of time is about 4.25 minutes.

32. The method of any one of claims 21 to 31, wherein the starting temperature of the condenser is about 0 °C.

33. The method of any one of claims 21 to 32, further comprising the step of: (d) transferring the solution having a final oligonucleotide concentration of > about 200 mg / mL from the evaporation vessel.

34. The method of any one of claims 21 to 33, further comprising the step of: (e) repeating steps (a) to (d) on a fresh starting volume of an oligonucleotide-containing solution.

35. The method of any one of claims 21 to 33, wherein steps (a) to (d) are repeated continuously. ​ 36. The method of any one of claims 21 to 35, wherein the final oligonucleotide concentration is about 200 mg / mL to about 400 mg / mL.

37. The method of any one of claims 21 to 35, wherein the final oligonucleotide concentration is < about 450 mg / mL.

38. The method of any one of claims 21 to 35, wherein the final oligonucleotide concentration is < about 500 mg / mL.

39. The method of any one of claims 1 to 38, further comprising the step of: subjecting the solution to tangential flow filtration (TFF) or lyophilization for additional concentration and / or purification.

40. An apparatus for concentrating biomolecules in a solution, the apparatus comprising: (i) an evaporation vessel, wherein the evaporation vessel comprises means for heating the evaporation vessel and means for pressurizing the evaporation vessel; (ii) a first reservoir in fluid communication with the evaporation vessel, wherein the first reservoir is a feed for unconcentrated solution containing biomolecules; (iii) a second reservoir in fluid communication with the evaporation vessel, wherein the second reservoir is a collector for concentrated solution containing biomolecules; and (iv) a condenser in fluid communication with the evaporation vessel, wherein the condenser comprises means for cooling the condenser and means for pressurizing the condenser.

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