Ultrafiltration Method

Abstract

The present invention relates to a method for the advantageous use of osmolytes to ultrafilter molecules of interest and uses related thereto. In particular, the present invention provides a method and use for concentrating molecules of interest by ultrafiltration.

Description

[Technical Field]

[0001] The present invention relates to the field of ultrafiltration of molecules of interest. The method of the present invention utilizes osmotically active (non-permeable) molecules ("osmolytes") on the permeate side of an ultrafiltration membrane. The use of osmolytes has the advantage of, among other things, increasing the flux through the ultrafiltration membrane, e.g., improving the concentration of a solution containing a molecule of interest. [Background technology]

[0002] Ultrafiltration is an important technological process that uses semipermeable membranes. Like related separation techniques such as microfiltration, ultrafiltration membranes can retain the (large) molecules of interest in the retentate, while the permeate contains low-molecular-weight molecules (e.g., salts, organic solvents, or other low-molecular-weight by-products) and water molecules that have passed through the membrane.

[0003] Therefore, in principle, there are two main target sites: the retentate side of the ultrafiltration membrane and the permeate side of the ultrafiltration membrane. Furthermore, depending on the respective goal to be achieved, the permeate (e.g., in water clarification) or the retentate (e.g., in concentration of a target molecule) is the target fraction.

[0004] Generally, ultrafiltration is widely used for macromolecules having a molecular weight of about 1 kDa to about 1000 kDa. As can be easily understood by those skilled in the art, ultrafiltration is specifically intended for the purification and / or concentration of target molecules, more particularly for the purification and / or concentration of target molecules that can be used as active pharmaceutical ingredients (APIs), and therefore also for the purification and / or concentration of target molecules, particularly in the preparation of pharmaceutical products / drugs.

[0005] Other medical applications of ultrafiltration include hemodialysis. Industrial applications of ultrafiltration include food and beverage processing and wastewater treatment. Twardowski et al. (1983) Artificial Organs 7(4), 420-427 reported the use of polyanions and glucose as osmotic agents in simulated peritoneal dialysis. However, the authors of this paper were not interested in concentrating solutions containing compounds of interest (e.g., proteins or oligonucleotides). Therefore, the paper does not teach or suggest water removal. Rather, the authors were interested in investigating the reversible binding of ions to polymers in response to applied pressure.

[0006] Specific applications of ultrafiltration include the transfer of a molecule of interest into another buffer / solution (e.g., diluting an old buffer by continuously adding a flow of new buffer through an ultrafiltration membrane), and the separation of a molecule of interest from (undesirable) lower molecular weight molecules by passing it through an ultrafiltration membrane (e.g., in a desalting process).

[0007] In general, there are two main ways to perform ultrafiltration: direct filtration and tangential filtration, also known as tangential flow filtration.

[0008] More specifically, for example, "diafiltration," as a specific ultrafiltration method, is a method that involves removing permeable components such as salts, organic solvents, and other undesirable molecules from a solution. Ultrafiltration / diafiltration ("UF / DF") is a common variant of ultrafiltration. Diafiltration uses so-called dilution and exchange buffers, which are (continuously) mixed with the retentate and (re)introduced into the ultrafiltration process.

[0009] A further important application of ultrafiltration is the concentration of macromolecules, such as molecules of interest, which may be required and / or desirable, for example, in purifying molecules of interest, and in particular in the preparation of pharmaceutical products / drugs.

[0010] Exemplary important target molecules for which ultrafiltration is widely used include polypeptides / proteins, plasmids, mRNA, and oligonucleotides. German Patent Publication No. 10 2015 108 501 (A1) discloses a method for producing a concentrate solution by tangential flow filtration, in which a high concentration of salt is added to the permeate solution. According to this document, the salt is diffusible through the ultrafiltration membrane and thus mixed with the retentate solution. The salt used as an osmolyte is unable to bind water and therefore has no water absorption properties. Furthermore, German Patent Publication No. 10 2015 108 501 (A1) does not suggest concentrating nucleic acids such as oligonucleotides.

[0011] Notably, in the preparation of pharmaceutical products, e.g., therapeutic drugs, oligonucleotides are commonly provided as lyophilized solids that are reconstituted into the final aqueous formulation. One of the reasons for the use of such lyophilization processes is that lyophilized products are often considered more stable than aqueous solutions of oligonucleotides, although aqueous solutions are now generally considered to be sufficiently stable (see Muslehiddinoglu et al.). In either case, the oligonucleotide solution is typically desalted and concentrated by ultrafiltration / diafiltration (UF / DF) prior to lyophilization.

[0012] One specific reason active pharmaceutical ingredients, such as oligonucleotide active pharmaceutical ingredients, are provided as solids is that ultrafiltration often does not deliver the oligonucleotide at a concentration (e.g., >150 mg / ml) suitable for the final drug formulation. Furthermore, the final concentration also impacts lyophilization capacity, which can be an obstacle when manufacturing very large batch volumes.

[0013] Therefore, a high final concentration of oligonucleotides after ultrafiltration or UF / DF is generally considered desirable, and therefore there is a general need in the art for methods that can provide solutions containing highly concentrated molecules of interest.

[0014] According to the literature, typical antisense oligonucleotides can be prepared using standard UF / DF processes to achieve final concentrations of 40-100 mg / mL, depending on the sequence and composition of the oligonucleotide (see Muslehiddinoglu et al.). Experimental data from this application indicates that maximum concentrations of approximately 60-90 mg / mL are typically achievable for various oligonucleotides.

[0015] In any case, as already mentioned above, such concentrations are usually not high enough to prepare the resulting liquid formulation as a pharmaceutical product, and for this reason, lyophilization is usually required.Therefore, there is a wide need in the art to provide an improved ultrafiltration method that can overcome the above limitations, for example, by facilitating the production of highly concentrated solutions of target molecules by ultrafiltration alone, without the need for ultrafiltration.

[0016] More specifically, with regard to ultrafiltration technology, important parameters describing the ultrafiltration process include feed pressure, retentate pressure, permeate pressure, differential pressure, and transmembrane pressure. All of these parameters can be continuously recorded by the ultrafiltration system (if required). The feed pressure is primarily determined by the feed flow and the viscosity of the solution being pumped, while the retentate and permeate pressures can be controlled by adjustable valves. The differential pressure, Δp, is defined as: Δp = supply pressure - holding pressure The driving force for mass transfer through an ultrafiltration membrane, the transmembrane pressure ("TMP"), may be calculated by the following formula: TMP = (feed pressure + retentate pressure) / 2 - permeate pressure Since the sum of the retentate flow rate (recycle flow rate) and the permeate flow rate (flux) is equal to the feed flow rate, the flux through the ultrafiltration membrane can be controlled by increasing the feed flow rate or decreasing the retentate flow rate (increasing the retentate pressure).

[0017] Typical process control modes in ultrafiltration are TMP control mode or flux control mode.

[0018] During the ultrafiltration process, the viscosity of the solution being pumped typically increases, causing an increase in the apparent concentration of components on the membrane surface. If the feed flow rate is kept constant and the retentate valve is not opened to offset the increased viscosity, the feed pressure will increase until it reaches a critical point and / or fouling occurs, causing ultrafiltration to stop. However, if the feed flow rate is automatically reduced or the retentate valve is opened, the flux will decrease to the point where no further flux through the ultrafiltration membrane can be observed.

[0019] For these reasons, it may be impossible to concentrate an oligonucleotide solution beyond a certain limit using the classical UF / DF method, and therefore there is a need for an improved ultrafiltration method that can overcome such limitations.

[0020] The present invention addresses all of the above objectives and needs by providing solutions as defined in the claims. Summary of the Invention [Means for solving the problem]

[0021] In particular, the present invention provides a novel approach that utilizes the principles of osmosis, such as osmotic flow generated by "osmotically active molecules." As used herein, the term "osmotically active molecules" is used interchangeably with "osmolytes."

[0022] More particularly, the present invention relates to a method for the advantageous use of osmolytes to ultrafilter a molecule of interest and uses related thereto. The present invention in particular provides a method and use for concentrating a molecule of interest by ultrafiltration.

[0023] Examples of osmolytes include, but are not limited to, polymers, preferably selected from the group consisting of polyethylene glycol (PEG), polyacrylic acid (PAA), polyvinylpyrrolidone, carboxymethylcellulose, polyvinyl alcohol, and mixtures thereof. The above limitations can be overcome by providing an osmolyte on the permeate side of the ultrafiltration membrane.

[0024] The present inventors have discovered that the principles of osmosis can be advantageously utilized in the ultrafiltration of target molecules. Specifically, by providing an osmolyte on the permeate side of an ultrafiltration membrane, the flux through the membrane can be increased. In this manner, for example, methods for concentrating target molecules using ultrafiltration can be improved, resulting in significantly higher concentrations of target molecules compared to standard ultrafiltration techniques.

[0025] The present invention generally provides the subject matter defined in the claims.

[0026] Furthermore, the present invention particularly relates to the subject matter defined in the following paragraphs [1] to

[0152] . [1] A method for ultrafiltration of a solution containing a target molecule, comprising: contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane; A method wherein the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane. [2] A method for ultrafiltration of a solution containing a target molecule, comprising: 1. A method comprising the step of increasing the osmolality of the permeate side of an ultrafiltration membrane relative to the retentate side of the ultrafiltration membrane by providing an osmolyte on the permeate side of the ultrafiltration membrane having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane. [3] A method for ultrafiltration of a solution containing a target molecule, comprising: A method comprising the step of improving ultrafiltration by providing an osmolyte on the permeate side of an ultrafiltration membrane having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane. [4] A method for ultrafiltration of a solution containing a target molecule, comprising: generating an osmotic flow through the ultrafiltration membrane; The method of generating the osmotic flow by providing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane to the permeate side of the ultrafiltration membrane. [5] A method for improving ultrafiltration of a solution containing a molecule of interest, comprising: A method comprising the step of contacting the permeate side of an ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane, thereby increasing the flux through the ultrafiltration membrane. [6] An ultrafiltration method for preparing a concentrated solution of a target molecule from a starting solution of the target molecule, comprising: contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane; A method wherein the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane. [7] An ultrafiltration method for preparing a concentrated solution of a target molecule from a starting solution of the target molecule, comprising: A method comprising a step of concentrating the solution by providing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane to the permeate side of the ultrafiltration membrane. [8] The method according to any one of the preceding items, wherein the method is a method for concentrating a solution containing a target molecule by ultrafiltration, and the ultrafiltration is tangential flow filtration (TFF) or cross-flow filtration (CFF). [9] The method of any one of the preceding claims, wherein the target molecule is concentrated to a concentration of at least 100 mg / ml, particularly to a concentration of at least 150 mg / ml.

[10] The method of any one of the preceding claims, wherein the target molecule is concentrated to a concentration of at least 200 mg / ml, particularly to a concentration of at least 250 mg / ml.

[11] The method of any one of the preceding claims, wherein the target molecule is concentrated to a concentration of at least 300 mg / ml, particularly to a concentration of at least 350 mg / ml.

[12] The method of any one of the preceding claims, wherein the target molecule is concentrated to a concentration of at least 400 mg / ml.

[13] The method according to any one of the preceding claims, wherein the polynucleotide as the target molecule is concentrated to a concentration of up to 500 mg / ml, and / or the polypeptide as the target molecule is concentrated to a concentration of up to 1000 mg / ml.

[14] The method of any one of the preceding items, wherein the target molecule is i) an optionally modified oligonucleotide, or ii) an optionally modified polypeptide.

[15] The method of any one of the preceding claims, wherein the target molecule is an oligonucleotide, which may be modified or unmodified.

[16] The method according to item

[15] , wherein the optionally modified oligonucleotide is selected from the group consisting of DNA, RNA, and a mixture thereof (e.g., heteroduplex), in particular, the DNA and / or RNA may be single-stranded or double-stranded, and in particular, the RNA is selected from the group consisting of RNAzyme, siRNA, DsiRNA, shRNA, miRNA, anti-miRNA, mRNA, rRNA, tRNA, and sgRNA.

[17] The method according to item

[15] or

[16] , wherein the oligonucleotide is selected from the group consisting of optionally modified DNA, optionally modified RNA, and optionally modified mixtures thereof (e.g., optionally modified heteroduplexes).

[18] The method according to item

[15] or

[16] , wherein the oligonucleotide is an optionally modified ssDNA.

[19] The method according to item

[15] or

[16] , wherein the oligonucleotide is an optionally modified dsDNA.

[20] The method according to item

[15] or

[16] , wherein the oligonucleotide is an optionally modified ssRNA.

[21] The method according to item

[15] or

[16] , wherein the oligonucleotide is an optionally modified dsRNA.

[22] The method of item

[15] or

[16] , wherein the oligonucleotide is selected from the group consisting of an optionally modified siRNA, an optionally modified DsiRNA, an optionally modified shRNA, an optionally modified miRNA, an optionally modified anti-miRNA, an optionally modified mRNA, an optionally modified rRNA, an optionally modified tRNA, and an optionally modified sgRNA.

[23] The method according to item

[15] or

[16] , wherein the oligonucleotide is selected from the group consisting of an optionally modified plasmid, an optionally modified aptamer, a DNAzyme, an RNAzyme, and an optionally modified antisense oligonucleotide.

[24] The method according to any one of items

[15] to

[23] , wherein the length of the optionally modified oligonucleotide is 10 to 6000 nucleotides, preferably 10 to 300 nucleotides, particularly 14 to 130 nucleotides, for example, 16 to 110 nucleotides.

[25] The method according to any one of items

[15] to

[23] , wherein the molecular weight of the optionally modified oligonucleotide is 3 to 90 kDa, particularly 4 to 39 kDa, and particularly 5 to 33 kDa.

[26] The method according to any one of items

[15] to

[25] , wherein the oligonucleotide is a mixture of optionally modified oligonucleotides, for example, a double-stranded oligonucleotide that is a mixture of two single-stranded oligonucleotides, or a mixture of various oligonucleotides.

[27] The method according to any one of items

[15] to

[25] , wherein the optionally modified oligonucleotide is a mixture of any of various optionally modified oligonucleotides according to any one of items

[17] to

[23] .

[28] The method according to any one of items [1] to

[14] , wherein the target molecule is a polypeptide, and the polypeptide may be modified or unmodified.

[29] The method according to

[28] , wherein the target molecule is an optionally modified polypeptide, and the optionally modified polypeptide is selected from the group consisting of an enzyme, a cell receptor ligand, a protein ligand, a signaling protein, a cytokine, an antibody, an antibody fragment, an antibody domain, a diabody, an scFv fragment, an sc(Fv)2 fragment, and a chimeric antibody.

[30] The method according to item

[28] or

[29] , wherein the polypeptide is an enzyme which may be modified.

[31] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified cell receptor ligand.

[32] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified cellular protein ligand.

[33] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified signal transduction protein.

[34] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified cytokine.

[35] The method according to item

[28] or

[29] , wherein the polypeptide is an antibody which may be modified.

[36] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified antibody fragment or an optionally modified antibody domain.

[37] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified scFv fragment or an optionally modified sc(Fv)2 fragment.

[38] The method according to item

[28] or

[29] , wherein the polypeptide is an optionally modified chimeric antibody.

[39] The method according to any one of items

[28] to

[38] , wherein the polypeptide comprises 90 to 1800 amino acid residues, particularly 135 to 1800 amino acid residues, and particularly 135 to 1350 amino acid residues.

[40] The method according to any one of items

[28] to

[39] , wherein the molecular weight of the polypeptide is 10 to 200 kDa, particularly 15 to 200 kDa, and particularly 15 to 150 kDa.

[41] The method according to any one of items

[28] to

[40] , wherein the polypeptide is a mixture of polypeptides.

[42] The method according to any one of items

[28] to

[41] , wherein the polypeptide is a mixture of any of the various polypeptides according to any one of items

[30] to

[38] .

[43] The method according to any one of items [1] to

[42] , wherein the molecular weight of the target molecule is 10 to 200 kDa.

[44] The method according to any one of items [1] to

[43] , wherein the target molecule is modified.

[45] The method according to any one of items [1] to

[27] , wherein the target molecule is a modified oligonucleotide containing one or more modifications selected from the group consisting of methylation, carboxymethylation, acetylation, methoxyaminomethylation, methoxycarbonylation, thiolation, adenylation, polyadenylation, base modification, backbone modification, sugar chain modification (particularly glycosylation), linker, lipid, peptide, and dye.

[46] wherein the one or more modifications are independently selected from a base modification, a backbone modification, a sugar chain modification, and other modifications; i) the base modification is hypoxanthine, inosine, 8-oxoadenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5 -chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), and any combination thereof; ii) The backbone modification may preferably be selected from the group consisting of various modified forms of phosphodiesters present in DNA or RNA, preferably phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylphosphonothioate, methylphosphate, methylphosphorothioate, ethylphosphate, ethylphosphorothioate, boranophos phosphate, boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, methylboranophosphonate, methylboranophosphonothioate, and derivatives thereof; phosphoramidite; phosphoramidate; N3'→P5' phosphoramidate; phosphorodiamidate; phosphorothiodiamidate; sulfamate; dimethylene sulfoxide; sulfonate; triazole; oxalyl; carbamate; methyleneimino (MMI); thioacetamido nucleic acid (TANA); derivatives thereof; and any combination thereof, iii) The sugar chain modification is 2'-O-modified RNA, for example, 2'-O-alkyl or 2'-O-(substituted) alkyl, for example, 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl; 2'-deoxy(DNA); 2'-O-(haloalkoxy)methyl (Arai K. et al. Bioorg. Med. Chem. 2011, 21, 6285), e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, e.g., 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo, e.g., 2'-F, FANA (2'-F arabinosyl nucleic acid); carba- and aza-sugar modifications; 3'-O-alkyl, e.g., 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl; and derivatives thereof; other sugar modifications such as "bridged" or "bicyclic" nucleic acids (BNA), e.g., For example, it may be selected from the group consisting of locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo-DNA; unlocked nucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); morpholino (e.g., those contained in PMO, PPMO, PMOPlus, PMO-X); and derivatives thereof and any combination thereof; and / or iv) the other modifications may be selected from the group consisting of peptide-base nucleic acids (PNAs), boron-modified PNAs, pyrrolidine-cyclic oxypeptide nucleic acids (POPNAs), glycol nucleic acids or glycerol nucleic acids (GNAs), threose nucleic acids (TNAs), acyclic threoninol nucleic acids (aTNAs), oligonucleotides incorporating bases and backbones, pyrrolidine-amide oligonucleotides (POMs), linkers (e.g., propane, hexane, or polyethylene glycol), branching agents (e.g., symmetric or asymmetric branching agents), lipids (e.g., palmitate, stearate), peptide- or dye-modified oligonucleotides, cholesterol- or GalNAc-modified oligonucleotides, and derivatives thereof, and any combination thereof; The method according to any one of items [1] to

[27] and

[45] .

[47] The method according to any one of items [1] to

[27] , wherein the target molecule is a modified polypeptide containing one or more modifications selected from the group consisting of phosphorylation, disulfide bond, glycosylation, acetylation, amidation, γ-carboxyglutamic acid, hydroxylation, methylation, sulfation, lipids (particularly lipids selected from myristate ester, palmitate ester, farnesyl, geranylgeranyl, GPI anchor, and N-acyldiglyceride), linker, dye, and signal sequence.

[48] ​​The method according to item

[47] , wherein the modification is selected from the group consisting of a linker, a lipid, a sugar chain, and a dye.

[49] The method according to any one of items [1] to

[48] , wherein the target molecule is selected from the group consisting of a research compound, a commercially available compound, an active pharmaceutical ingredient (API), an adjuvant, and an additive.

[50] The method according to any one of items [1] to

[49] , wherein the target molecule is an active pharmaceutical ingredient (API).

[51] The method according to any one of paragraphs [1] to

[49] , which is a method for preparing an active pharmaceutical ingredient (API) for incorporation into a pharmaceutical product and / or a method for concentrating an active pharmaceutical ingredient (API) for incorporation into a pharmaceutical product.

[52] The method according to any one of items [1] to

[51] , which does not involve freeze-drying.

[53] The method according to any one of items [1] to

[52] , wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 150 kDa, particularly 1 to 100 kDa, particularly 1 to 30 kDa, and particularly 1 to 10 kDa.

[54] The method according to item

[53] , wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 100 kDa.

[55] The method according to item

[53] or

[54] , wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 30 kDa.

[56] The method according to any one of items

[53] to

[55] , wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 10 kDa.

[57] The method according to any one of items [1] to

[56] , wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 7 kDa, for example, 1 to 5 kDa, particularly 1 to 3 kDa, for example, about 2 kDa.

[58] A P / R ratio of at least 1.25, particularly at least 1.5, particularly at least 2.0, particularly at least 3.0, at least 5.0, at least 10.0, or at least 20.0; P is the osmolality of the solution on the permeate side of the ultrafiltration membrane; R is the osmolality of the solution on the retentate side of the ultrafiltration membrane; The method according to any one of items [1] to

[57] .

[59] In the step of contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte, the P / R ratio is at least 1.25, particularly at least 1.5, particularly at least 2.0, particularly at least 3.0, at least 5.0, at least 10.0, or at least 20.0; P is the osmolality of the solution on the permeate side of the ultrafiltration membrane; R is the osmolality of the solution on the retentate side of the ultrafiltration membrane; The method according to any one of items [1], [5], [6] and [8] to

[58] .

[60] Providing an osmolality of the permeate side solution such that the P / R ratio is at least 1.25, particularly at least 1.5, particularly at least 2.0, particularly at least 3.0, at least 5.0, at least 10.0, or at least 20.0; P is the osmolality of the solution on the permeate side of the ultrafiltration membrane; R is the osmolality of the solution on the retentate side of the ultrafiltration membrane; The method according to any one of items [2] to [5], [7] to

[57] and

[58] .

[61] The method according to any one of items

[58] to

[60] , wherein the P / R ratio is at least 1.25.

[62] The method according to any one of items

[58] to

[61] , wherein the P / R ratio is at least 2.

[63] The method according to any one of items

[58] to

[62] , wherein the P / R ratio is at least 5.

[64] The method according to any one of items

[58] to

[63] , wherein the P / R ratio is at least 10.

[65] The method according to any one of items

[58] to

[64] , wherein the P / R ratio is at least 20.

[66] The method according to any one of items

[58] to

[65] , wherein the P / R ratio is at least 1.25 throughout the method.

[67] The method according to any one of items

[58] to

[66] , wherein the P / R ratio is at least 1.5 throughout the method, particularly at least 2.0, particularly at least 3.0, at least 5.0, at least 10.0, or at least 20.0.

[68] The method according to any one of items [1] to

[67] , wherein the osmolality of the solution on the permeate side of the ultrafiltration membrane after the small molecules that have passed through the ultrafiltration membrane reach equilibrium is higher than the osmolality on the retentate side of the ultrafiltration membrane.

[69] The method according to any one of items [1] to

[68] , wherein the osmolyte is provided in the form of a starting solution containing the osmolyte.

[70] an S1 / S2 ratio of at least 1.25, in particular at least 1.5, in particular at least 2.0, in particular at least 3.0, at least 5.0, at least 10.0, at least 20.0, or at least 30.0; S1 is the osmolality of the starting solution containing the osmolyte; S2 is the osmolality of the starting solution containing the target molecule used in the method; The method described in item

[69] .

[71] The method according to item

[69] or

[70] , wherein the S1 / S2 ratio is at least 2.

[72] The method according to any one of items

[69] to

[71] , wherein the S1 / S2 ratio is at least 5.

[73] The method according to any one of items

[69] to

[72] , wherein the S1 / S2 ratio is at least 10.

[74] The method according to any one of items

[69] to

[73] , wherein the S1 / S2 ratio is at least 30.

[75] The method according to any one of items [1] to

[74] , wherein the osmolality of the retentate side is calculated taking into account all osmolytes contained in each solution, and the osmolality of the permeate side is calculated taking into account only the osmolytes having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[76] The method according to any one of items [1] to

[74] , wherein each osmolality is calculated taking into account only osmotically active molecules (including the target molecule) in each solution that have a molecular weight greater than the MWCO of the ultrafiltration membrane.

[77] The method according to any one of items [1] to

[76] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a concentration of at least 12.5 mM, particularly at least 25 mM, particularly at least 40 mM, particularly at least 50 mM.

[78] The method according to item

[77] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a concentration of at least 25 mM.

[79] The method according to item

[77] or

[78] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a concentration of at least 40 mM.

[80] The method according to any one of items

[77] to

[79] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a concentration of at least 50 mM.

[81] The method according to any one of items

[68] to

[80] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a molar concentration at least twice that of the starting solution containing the target molecule.

[82] The method according to item

[81] , wherein the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a molar concentration at least 5 times the molar concentration of the starting solution containing the target molecule.

[83] The method according to any one of items [1] to

[82] , comprising, as one or more preceding additional steps, a step of ultrafiltration of the target molecule, in particular a step of concentrating the target molecule.

[84] The method of paragraph

[83] , wherein the one or more further steps do not include using an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[85] The method according to any one of items [1], [5], [6] and [8] to

[84] , wherein, before the step of contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte, one or more further steps are performed, such as ultrafiltration of the target molecule, particularly a step of concentrating the target molecule.

[86] The method of paragraph

[85] , wherein the one or more further steps do not include using an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[87] The method according to any one of items [1], [5], [6] and [8] to

[86] , wherein the step of contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte is repeated at least once.

[88] The method of paragraph

[87] , wherein each repetition includes using a new starting solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[89] The method according to any one of items [2] to [5] and [7] to

[84] , wherein, before providing the osmolyte, one or more steps include ultrafiltration of the target molecule, particularly a step of concentrating the target molecule.

[90] The method according to paragraph

[89] , wherein the one or more steps do not include using an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[91] The method according to any one of items [2] to [5], [7] to

[84] ,

[89] and

[90] , wherein the provision of the osmolyte is repeated at least once.

[92] The method of paragraph

[91] , wherein each repetition includes using a new starting solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[93] The method of any one of the preceding claims, wherein the method does not include a step that does not involve providing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane, and in particular does not include an ultrafiltration step that does not involve providing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[94] The method according to any one of the preceding claims, wherein the starting solution containing the target molecule used in the method has previously been subjected to a concentration step, which concentration step is preferably selected from i) a precipitation step, and ii) an ultrafiltration method that does not involve using an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[95] The method of any one of the preceding claims, wherein any one or more of the steps are repeated.

[96] The method of any one of the preceding clauses, comprising circulating or passing a solution containing the osmolyte on the permeate side of the ultrafiltration membrane, preferably comprising circulating a solution containing the osmolyte on the permeate side of the ultrafiltration membrane.

[97] The method according to item

[96] , wherein the circulating step includes using a pump, and the circulating step may further include using a reservoir, the reservoir already containing the starting solution containing the osmolyte and forming part of the circulation.

[98] The method of any one of the preceding claims, wherein the phrase "contacting the permeate side of the ultrafiltration membrane with a solution" is replaced with the phrase "combining the permeate with a solution."

[99] The method of any one of the preceding claims, comprising the use of one or more pumps.

[0100] Item 10. The method of any one of the preceding items, comprising rinsing the retentate side of the ultrafiltration membrane to increase the yield of the target molecule.

[0101] Item 10. The method of any one of the preceding items, wherein the osmolyte is selected from the group consisting of polyethylene glycol (PEG), polyacrylic acid (PAA) sodium salt, polyvinylpyrrolidone, carboxymethylcellulose, polyvinyl alcohol, (poly)ethylene oxide / (poly)propylene oxide copolymer, and mixtures thereof, in particular, the osmolyte is PEG or (poly)ethylene oxide / (poly)propylene oxide copolymer.

[0102] Item 10. The method of any one of the preceding items, wherein the average molecular weight of the osmolyte is 1,000 to 10,000,000 g / mol, in particular 1,000 to 2,000,000 g / mol, more particularly 2,000 to 200,000 g / mol.

[0103] Item 10. The method according to any one of the preceding items, wherein the average molecular weight of the osmolyte is 2,000 to 60,000 g / mol, in particular 2,000 to 20,000 g / mol, for example 4,000 to 10,000 g / mol.

[0104] The method according to any one of items [1] to

[0103] , wherein the concentration of the osmolyte (e.g., the PEG) is at least 12.5 mM, particularly at least 20 mM, particularly at least 25 mM, particularly at least 40 mM, and particularly at least 50 mM.

[0105] The method according to any one of items [1] to

[0104] , wherein the molecular weight of the osmolyte is at least 1.2 times, particularly at least 1.25 times, particularly at least 1.3 times, particularly at least 1.5 times, particularly at least 2.0 times, more particularly at least 3.0 times, for example at least 4.0 times the MWCO of the ultrafiltration membrane.

[0106] The method according to any one of items [1] to

[0105] , wherein the osmolyte does not substantially pass through the ultrafiltration membrane, specifically, the amount of the osmolyte that passes through the ultrafiltration membrane is less than 10%, preferably less than 5%, particularly less than 2%, particularly less than 1%, more particularly less than 0.1%, more particularly less than 0.01%, and more particularly less than 0.001%.

[0107] The method according to any one of items [1] to

[0106] , wherein the target molecules do not substantially pass through the ultrafiltration membrane, specifically, the amount of the target molecules that passes through the ultrafiltration membrane is less than 10%, preferably less than 5%, particularly less than 2%, particularly less than 1%, more particularly less than 0.1%, more particularly less than 0.01%, and even more particularly less than 0.001%.

[0108] i) a method for enhancing the concentration of a solution containing said target molecule; ii) a method for improving the efficiency of said ultrafiltration, in particular a method for improving the concentration achieved by said ultrafiltration; iii) enhancing osmotic flow through said ultrafiltration membrane; iv) a method for removing solvent molecules (particularly solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing the target molecule; v) a method for obtaining a high final concentration of the target molecule; vi) a method for preparing a compound selected from the group consisting of a research compound, a commercially available compound, an active pharmaceutical ingredient (API), an adjuvant, and an additive; vii) methods for preparing active pharmaceutical ingredients (APIs), in particular methods for preparing pharmaceutical products; viii) Ultrafiltration / Diafiltration methods; ix) concentrating the solution containing the molecule of interest to a concentration of at least 150 mg / ml, in particular at least 200 mg / ml, in particular at least 250 mg / ml, in particular at least 300 mg / ml, in particular at least 350 mg / ml, in particular at least 400 mg / ml; x) methods to avoid freeze-drying; xi) methods for improving the efficiency of subsequent freeze-drying; and / or xii) A method for enhancing the flux through said ultrafiltration membrane. Item 10. The method of any one of the preceding items, wherein:

[0109] The method described in item

[0108] , which is a method for enhancing the concentration of a solution containing the target molecule.

[0110] The method described in item

[0108] or

[0109] , which is a method for improving the efficiency of ultrafiltration.

[0111] The method according to any one of items

[0108] to

[0110] , which is a method for enhancing osmotic flow through the ultrafiltration membrane.

[0112] The method according to any one of items

[0108] to

[0111] , which is a method for removing solvent molecules (particularly, solvent molecules selected from water, organic solvents, and mixtures thereof) from a solution containing the target molecule.

[0113] The method according to any one of items

[0108] to

[0112] , which is a method for obtaining the target molecule at a high final concentration.

[0114] The method according to any one of items

[0108] to

[0113] , which is a method for preparing a compound selected from the group consisting of a research compound, a commercially available compound, an active pharmaceutical ingredient (API), an adjuvant, and an additive, in particular a method for preparing an active pharmaceutical ingredient (API).

[0115] The method according to any one of items

[0108] to

[0114] , which is a method for preparing a pharmaceutical product.

[0116] The method according to any one of items

[0108] to

[0115] , which is a tangential flow filtration method, in particular an ultrafiltration / diafiltration method.

[0117] The method according to any one of items

[0108] to

[0116] , wherein the solution containing the target molecule is concentrated to a concentration of at least 150 mg / ml, particularly at least 200 mg / ml, particularly at least 250 mg / ml, particularly at least 300 mg / ml, particularly at least 350 mg / ml, particularly at least 400 mg / ml.

[0118] The method according to any one of items

[0108] to

[0117] , which is a method for avoiding freeze-drying or a method for improving the efficiency of freeze-drying as a subsequent step.

[0119] The method according to any one of items

[0108] to

[0118] , which is a method for enhancing the flux through the ultrafiltration membrane.

[0120] 1. Use of an osmolyte in a method for ultrafiltration of a solution containing a molecule of interest, comprising: the method comprising contacting a solution containing the osmolyte with the permeate side of an ultrafiltration membrane; the osmolyte has a molecular weight greater than the MWCO of the ultrafiltration membrane; a solution on the permeate side of the ultrafiltration membrane having a higher osmolality than a solution on the retentate side of the ultrafiltration membrane; In particular, the use is further defined by any one of items [1] to

[0119] .

[0121] The use according to item

[0120] , wherein the ultrafiltration method further has the features according to any one of items [1], [5], [6], [8] to

[88] and

[93] to

[0119] .

[0122] Use of an osmolyte to improve a method for ultrafiltration of a solution containing a molecule of interest, comprising: the osmolyte has a molecular weight greater than the MWCO of the ultrafiltration membrane; the osmolyte is provided on the permeate side of the ultrafiltration membrane to improve ultrafiltration; In particular, the use is further defined by any one of items [1] to

[0119] .

[0123] The use according to item

[0122] , wherein the ultrafiltration method further has the features according to any one of items [2] to [5], [7] to

[84] , and

[89] to

[0119] .

[0124] The use according to any one of items

[0120] to

[0123] , wherein the ultrafiltration method is a method of concentrating a solution containing a target molecule by ultrafiltration.

[0125] The use, i) use to enhance the concentration of a solution containing said target molecule; ii) the use for improving the efficiency of said ultrafiltration; iii) Use for enhancing osmotic flow through said ultrafiltration membrane; iv) Use for removing solvent molecules (particularly solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing the target molecule; v) Use to obtain a high final concentration of the target molecule; vi) Use for preparing a compound selected from the group consisting of a research compound, a commercially available compound, an active pharmaceutical ingredient (API), an adjuvant, and an additive; vii) Use for preparing active pharmaceutical ingredients (API), in particular for preparing medicinal products; viii) Use for performing ultrafiltration / diafiltration of the target molecule; ix) use for concentrating a solution containing said target molecule to a concentration of at least 150 mg / ml, in particular to a concentration of at least 200 mg / ml, in particular to a concentration of at least 250 mg / ml, in particular to a concentration of at least 300 mg / ml, in particular to a concentration of at least 350 mg / ml, in particular to a concentration of at least 400 mg / ml; x) Use to avoid freeze-drying, xi) Use to improve the efficiency of subsequent freeze-drying, and / or xii) Use for enhancing the flux through said ultrafiltration membrane. The use according to any one of items

[0120] to

[0124] ,

[0126] The use described in item

[0125] , which is used to enhance the concentration of a solution containing the target molecule.

[0127] The use described in item

[0125] or

[0126] , which is used to improve the efficiency of ultrafiltration.

[0128] The method described in item

[0125] or

[0126] , wherein the use is for enhancing osmotic flow through the ultrafiltration membrane.

[0129] The use described in item

[0125] or

[0126] , which is for removing solvent molecules (particularly solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing the target molecule.

[0130] The use described in item

[0125] or

[0126] , which is used to obtain the target molecule at a high final concentration.

[0131] The use described in paragraph

[0125] or

[0126] , which is for preparing a compound selected from the group consisting of research compounds, commercially available compounds, active pharmaceutical ingredients (APIs), adjuvants, and additives.

[0132] The use according to paragraph

[0125] or

[0126] , which is for preparing an active pharmaceutical ingredient, in particular for preparing a pharmaceutical product.

[0133] The use described in paragraphs

[0125] or

[0126] , which is used in an ultrafiltration / diafiltration method.

[0134] The use described in item

[0125] or

[0126] , which is a use for concentrating a solution containing the target molecule to a concentration of at least 150 mg / ml, particularly at least 200 mg / ml, particularly at least 250 mg / ml, particularly at least 300 mg / ml, particularly at least 350 mg / ml, particularly at least 400 mg / ml.

[0135] The use described in paragraphs

[0125] or

[0126] , which is a use for avoiding freeze-drying or a use for improving the efficiency of freeze-drying as a subsequent step.

[0136] The use described in paragraphs

[0125] or

[0126] for enhancing the flux through the ultrafiltration membrane.

[0137] An ultrafiltration device for filtering, in particular concentrating, a solution containing a molecule of interest, i) contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane to increase the flux through the ultrafiltration membrane; and / or ii) combining the ultrafiltration permeate with a solution containing osmotically active molecules having a molecular weight greater than the MWCO of the ultrafiltration membrane, and circulating (or allowing to circulate) the resulting solution on the permeate side of the ultrafiltration membrane; An ultrafiltration device comprising:

[0138] Item 0137. An ultrafiltration apparatus according to item 0137, wherein the permeate is not returned to the retentate side of the ultrafiltration membrane by any of the means.

[0139] An ultrafiltration device according to item

[0137] or

[0138] , wherein the means includes a pump and a reservoir for a starting solution containing the osmolyte.

[0140] i) configured to operate in a flow-through mode in which the retentate is not recirculated on the retentate side of the ultrafiltration membrane; or ii) configured to operate in a recirculation mode in which the retentate is recirculated through the retentate side of the ultrafiltration membrane; Preferably, it is configured to operate in the recirculation mode described in ii) above. The ultrafiltration device according to any one of items

[0137] to

[0139] .

[0141] i) configured to operate in a pass-through mode in which the permeate is not recirculated on the permeate side of the ultrafiltration membrane; or ii) configured to operate in a recirculation mode in which the permeate is recirculated through the permeate side of the ultrafiltration membrane; Preferably, it is configured to operate in the recirculation mode described in ii) above. The ultrafiltration device according to any one of items

[0137] to

[0140] .

[0142] The ultrafiltration device according to any one of paragraphs

[0137] to

[0140] , comprising a starting solution containing the osmolyte, in particular, the device and / or the starting solution is further defined by any one of the preceding paragraphs.

[0143] (a) ultrafiltrating the solution containing the target molecule by applying a pressure difference to obtain a concentrated solution containing the target molecule; (b) ultrafiltering the concentrated solution containing the target molecule while contacting the permeate side of the ultrafiltration membrane with the solution containing an osmolyte having a molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane. The method according to any one of items [1] to

[0119] , comprising:

[0144] The method according to item

[0143] , wherein step (b) includes applying a pressure differential.

[0145] The method according to item

[0143] or

[0144] , wherein the differential pressure in step (a) and / or step (b) is in the range of 0.1 bar to 1.0 bar.

[0146] The method according to any one of items

[0143] to

[0145] , wherein the differential pressure in step (a) and / or step (b) is in the range of 0.3 bar to 0.7 bar.

[0147] The method according to any one of items

[0143] to

[0146] , wherein in step (a), the transmembrane pressure difference reaches a range of 2 to 4.

[0148] The method according to any one of items

[0143] to

[0147] , wherein in step (a), the transmembrane pressure difference reaches a range of 2.5 to 3.5.

[0149] The method according to any one of items

[0143] to

[0148] , wherein in step (a), the permeate side of the ultrafiltration membrane is not in contact with a solution containing an osmolyte having a molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane.

[0150] Item 11. The method, use or apparatus of any one of the preceding items, wherein the osmolyte is a hygroscopic polymer.

[0151] Item 11. The method, use or device according to any one of the preceding items, wherein the osmolyte is capable of absorbing water.

[0152] Item 11. The method, use or apparatus according to any one of the preceding items, wherein the osmolyte is capable of binding water. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is a schematic diagram of an exemplary experimental setup according to the present invention for recycling osmolyte solutions, showing only the main circulation elements. [Figure 2] FIG. 1 is a schematic diagram more specifically illustrating an exemplary experimental setup for recycling osmolyte solutions. [Figure 3] 1 is an exemplary record of retentate weight (WIRC2100 [g]) during PEG-assisted concentration. The weight of the retentate (solution) was monitored over time during the concentration run according to the method of the present invention. [Figure 4] FIG. 1 is a schematic diagram more particularly illustrating an exemplary experimental setup utilizing the concept of passing through an osmolyte (rather than recycling the osmolyte (see FIGS. 1 and 2)). [Figure 5] The graph shows the change in oligonucleotide concentration over time as measured by taking samples from the retentate solution at the indicated times (Example 7). The vertical line in the graph indicates the end of concentration step 1. After concentration step 1, the experimental setup was modified to load a 40% (w / w) PEG solution onto the permeate side of the ultrafiltration membrane. DETAILED DESCRIPTION OF THE INVENTION

[0028] In a first aspect, the present invention provides a method for ultrafiltration of a solution containing a molecule of interest, comprising the steps of: contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane; wherein the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane.

[0029] In a second aspect, the present invention provides a method for ultrafiltration of a solution containing a molecule of interest, comprising: providing an osmolyte on the permeate side of an ultrafiltration membrane having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane, thereby increasing the osmolality of the permeate side of the ultrafiltration membrane relative to the retentate side of the ultrafiltration membrane.

[0030] In a third aspect, the present invention provides a method for ultrafiltration of a solution containing a molecule of interest, comprising the steps of: The present invention relates to a method for improving ultrafiltration by providing an osmolyte on the permeate side of an ultrafiltration membrane having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane.

[0031] In a fourth aspect, the present invention provides a method for ultrafiltration of a solution containing a molecule of interest, comprising: generating an osmotic flow through the ultrafiltration membrane; wherein the osmotic flow is generated by providing an osmolyte on the permeate side of the ultrafiltration membrane having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane.

[0032] Generally, in a preferred embodiment herein, the method according to any one of the preceding aspects is a method for concentrating a solution containing a molecule of interest by ultrafiltration.

[0033] Thus, in a further aspect (fifth aspect), the present invention provides a method for preparing a concentrated solution of a molecule of interest from a starting solution of said molecule of interest using ultrafiltration, comprising the steps of: contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane; wherein the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane.

[0034] Similarly, in a further aspect (sixth aspect), the present invention provides a method for preparing a concentrated solution of a molecule of interest from a starting solution of said molecule of interest using ultrafiltration, comprising the steps of: The present invention relates to a method comprising the step of concentrating an osmolyte having a molecular weight or average molecular weight greater than the MWCO of an ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[0035] With respect to the method of the present invention, "ultrafiltration methods" are generally well known to those skilled in the art and are well known in the art, as mentioned at the beginning of this specification. In the present invention, the term "ultrafiltration method" is used interchangeably with "ultrafiltration method." Ultrafiltration methods of the present invention include direct filtration and tangential filtration, with tangential filtration being preferred in the context of the present invention. Ultrafiltration methods of the present invention also include, but are not limited to, ultrafiltration / diafiltration, which is a variant of ultrafiltration also referred to herein as "UF / DF."

[0036] Generally, there are two main methods for performing ultrafiltration: direct filtration (also called "dead-end filtration") and tangential flow filtration ("TFF"). Tangential flow filtration is often referred to as cross-flow filtration ("CFF"). With this in mind, and for purposes described in the detailed description of the present invention, the terms "TFF" and "CFF" may be used interchangeably herein.

[0037] In either case, tangential flow filtration (TFF) is the preferred ultrafiltration method in the present invention.

[0038] The central component in any ultrafiltration device is the ultrafiltration membrane, which is not particularly limited herein and may take a wide variety of forms, shapes, and types. In any case, the "ultrafiltration membrane" herein is characterized as a semipermeable membrane with two sides, a "retentate side" and a "permeate side." As used herein, the "retentate side of the ultrafiltration membrane" is the side on which the target molecules are retained.

[0039] As used herein, "flux through an ultrafiltration membrane" refers to the passage of small molecules through the ultrafiltration membrane, and these small molecules are smaller than the pore size of the ultrafiltration membrane. The term "flux through an ultrafiltration membrane" includes the flux of water molecules and low molecular weight compounds. Examples of low molecular weight compounds include, but are not limited to, salt molecules and organic solvent molecules. In the method of the present invention, this flux can be promoted by the pressure commonly used in ultrafiltration and also by the use of osmolytes.

[0040] In any event, in certain embodiments herein, the ultrafiltration methods of the present invention are carried out without the application of additional transmembrane pressure (TMP).

[0041] Ultrafiltration may further include the use of pressure to generate and maintain a flux through the ultrafiltration membrane. Thus, in another general embodiment herein, the ultrafiltration method of the present invention is further carried out with the application of transmembrane pressure (TMP). In certain embodiments, TMP may be applied throughout the entire method of the present invention, while in other embodiments, TMP is applied only during part of the method of the present invention.

[0042] Furthermore, when tangential flow filtration (TFF) is used in accordance with preferred embodiments of the present invention, the retentate flows in a direction parallel to the ultrafiltration membrane, which, as will be readily appreciated by those skilled in the art, can, for example, improve efficiency and significantly reduce clogging of the ultrafiltration membrane.

[0043] In any event, all such methods for performing ultrafiltration are generally well known to those skilled in the art, as described elsewhere herein.

[0044] Generally, the term "osmolality" is used herein in the sense understood in the art and may be used interchangeably with "osmolality." Typically, the term "osmolality" is used herein with reference to a particular solution, such as a solution contacting an ultrafiltration membrane on the retentate side or a solution contacting an ultrafiltration membrane on the permeate side. Thus, when "a solution on the permeate side of an ultrafiltration membrane has a higher osmolality than a solution on the retentate side of the ultrafiltration membrane" (e.g.,), such osmolality is believed to generate osmotic flow through the ultrafiltration membrane, which contributes to the aforementioned flux.

[0045] Generally, one of ordinary skill in the art can readily determine whether a solution on the permeate side of an ultrafiltration membrane has a higher osmolality than a solution on the retentate side of the ultrafiltration membrane.

[0046] Osmolality can be easily calculated by determining the number of solute particles per liter of solvent. For example, in a 1 mol / L NaCl solution, the NaCl dissolves and the resulting Na + Cations and Cl - The presence of anions gives an osmolality of 2 osmol / L.

[0047] As a different unit from osmolality, osmolality is calculated by determining the number of solute particles per kg of solvent. Therefore, osmolality is independent of the temperature of each solution. In another general embodiment described herein, the term "osmolality" is replaced with the term "osmolality."

[0048] Both osmolality and osmolality are standard measures of the concentration of dissolved particles in a solution, based solely on the number of solute particles and independent of the properties, size, and shape of each molecule. In principle, osmotic flow occurs across an ultrafiltration membrane from the side of an aqueous solution with a lower osmolality to the side with a higher osmolality / osmolality, and this osmotic flow continues until equilibrium is reached. Ultrafiltration membranes are (selectively) permeable, allowing only small molecules (e.g., ions and other small molecules) to pass through, which can affect osmolality.

[0049] The osmolality of a given aqueous solution may be determined, for example, by comparative measurements of the relationship between the freezing point of pure water and the freezing point of the aqueous solution of interest. The freezing point of water is 0°C, whereas the freezing point of a solution with a salt concentration of 1 Osmol / kg is -1.858°C (this phenomenon is also called "freezing point depression"). In one embodiment, the osmolality of a given aqueous solution is determined by the freezing point depression method of the 36th Revision of the United States Pharmacopeia. <785> In another embodiment, osmolality (or osmolality) is measured as described in Section 2.2.35 of the European Pharmacopoeia, Revision 7.3.

[0050] In either case, the solution on the permeate side of the ultrafiltration membrane need not have a higher osmolality than the solution on the retentate side of the ultrafiltration membrane throughout the method of the invention. Rather, in certain preferred embodiments of the methods and uses of the invention, "the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane" once there is no net flow of small molecules through the ultrafiltration membrane, e.g., in the absence of osmolyte. In either case, depending on the particular starting solution used, the initial osmolality of the retentate side of the ultrafiltration membrane may be higher than the permeate side.

[0051] Furthermore, in the context of the present invention, the term "osmolyte" may be used interchangeably herein with the term "osmotically active molecule" and generally refers to a molecule that contributes to the osmolality of a solution. For the most part of this specification (and even when a "molecule of interest" in the strict sense also acts as an osmolyte), the term "osmolyte" refers to an osmolyte present on the permeate side of an ultrafiltration membrane, as can be readily understood from the context of this specification.

[0052] In either case, as one skilled in the art will readily appreciate, an osmolyte having a molecular weight or average molecular weight greater than the MWCO ("molecular weight cut-off") of the ultrafiltration membrane is osmotically active in that it is suitable for generating osmotic flow onto the osmolyte molecular side of the ultrafiltration membrane.

[0053] Without wishing to be bound by any theory, it is believed that the higher the concentration of osmolyte and the lower the concentration of another osmotically active molecule on the other side of the ultrafiltration membrane (the retentate side), the greater the osmotic flow. Thus, the greater the concentration difference between the target molecule and the osmolyte, the greater the osmotic flow, especially at the point where small molecules no longer pass through the ultrafiltration membrane.

[0054] Additionally, terms such as "improving ultrafiltration" and "improving ultrafiltration" are used interchangeably herein and may refer to various types of improvements, such as increasing the flux through an ultrafiltration membrane, increasing the concentration of a target molecule, and / or improving the separation of undesired molecules (those that can pass through an ultrafiltration membrane) from a solution containing the target molecule.

[0055] As will be further appreciated by those skilled in the art upon perusal of the claims and detailed description of the present invention with respect to the methods of the present invention, the osmotic phenomenon utilized in the present invention requires the presence of multiple molecules, such as multiple "osmolytes" or multiple "osmotically active molecules," and a solution of target molecules typically contains multiple target molecules. Thus, when such a target molecule is referred to in the singular, the singular target molecule always encompasses the plural target molecules, unless expressly stated otherwise.

[0056] As used herein, the expression that an osmolyte "has a molecular weight greater than the MWCO of an ultrafiltration membrane" and similar terms can be easily understood by those skilled in the art, and "MWCO" is an abbreviation that indicates the value of the "molecular weight cut-off" of an ultrafiltration membrane. Both the molecular weight cut-off and the MWCO are preferably expressed in kDa for easy comparison.

[0057] As will be readily appreciated by those skilled in the art, the MWCO preferably indicates the minimum molecular weight of molecules that can be retained by the membrane to at least 90%.

[0058] According to preferred embodiments herein, the molecular weight of the osmolyte is at least 1.2 times, particularly at least 1.25 times, particularly at least 1.3 times, particularly at least 1.5 times, particularly at least 2.0 times, more particularly at least 3.0 times, for example at least 4.0 times, the MWCO of the ultrafiltration membrane.

[0059] In certain other general embodiments herein, the osmolyte has an "average molecular weight" greater than the MWCO of the ultrafiltration membrane, and in more specific embodiments, this "average molecular weight" is the same as the "molecular weight" described herein. In particular, when the osmolyte is a polymer, the average molecular weight of the polymer may be considered to be the molecular weight.

[0060] In correspondingly preferred embodiments herein, the average molecular weight of the osmolyte is at least 1.3 times, particularly at least 1.35 times, particularly at least 1.4 times, particularly at least 1.5 times, particularly at least 2.0 times, more particularly at least 3.0 times, for example at least 4.0 times, the MWCO of the ultrafiltration membrane.

[0061] Furthermore, in another series of specific embodiments of the present invention, particularly those aimed at retaining the target molecule and the osmolyte on the desired side of the ultrafiltration membrane, it is preferable to use a MWCO that is significantly smaller than the size of each molecule, for example, 5 / 8, 1 / 2, 1 / 4, 1 / 5, 1 / 6, 1 / 7, or even 1 / 10 of the size of each molecule.

[0062] In particular, in certain embodiments, the MWCO is selected to correspond to 5 / 8 of the molecular weight value of the osmolyte. Preferably, the MWCO is selected to correspond to 1 / 2 of the molecular weight value of the osmolyte. In further non-limiting embodiments, the MWCO is selected to correspond to 1 / 4 of the molecular weight value of the osmolyte. In further non-limiting embodiments, the MWCO is selected to correspond to 1 / 5 of the molecular weight value of the osmolyte. In further non-limiting embodiments, the MWCO is selected to correspond to 1 / 6 of the molecular weight value of the osmolyte, etc.

[0063] Similarly, the MWCO is preferably selected to correspond to 1 / 2 the molecular weight value of the molecule of interest. In a further non-limiting embodiment, the MWCO is selected to correspond to 1 / 4 the molecular weight value of the molecule of interest. In a further non-limiting embodiment, the MWCO is selected to correspond to 1 / 5 the molecular weight value of the molecule of interest. In a further non-limiting embodiment, the MWCO is selected to correspond to 1 / 6 the molecular weight value of the molecule of interest, etc.

[0064] In the context of the present invention, the ultrafiltration method may generally be used to concentrate a solution containing a molecule of interest, and the phrase "method for concentrating a solution containing a molecule of interest by ultrafiltration" and corresponding expressions indicate that the initial concentration of the molecule of interest is increased by said method.

[0065] In certain embodiments, the methods of the present invention can concentrate a target molecule to a concentration of at least 100 mg / ml. Preferably, the methods of the present invention can concentrate a target molecule to a concentration of at least 150 mg / ml. In further preferred embodiments, the methods of the present invention can concentrate a target molecule to a concentration of at least 200 mg / ml. In certain embodiments, the methods of the present invention can concentrate a target molecule to a concentration of at least 250 mg / ml, for example, at least 300 mg / ml. In certain embodiments, the methods of the present invention can concentrate a target molecule to a concentration of at least 350 mg / ml, for example, at least 400 mg / ml.

[0066] In further particular embodiments, particularly in the context of a polypeptide, the method of the invention allows for the concentration of the molecule of interest to a concentration of at least 500 mg / ml, preferably at least 750 mg / ml, and for example up to 1000 mg / ml.

[0067] As one of skill in the art will readily appreciate, the absolute value achievable will depend on the particular molecule of interest, with higher absolute values ​​typically being easier to achieve for polypeptides than for polynucleotides.

[0068] However, in preferred embodiments herein, the methods of the present invention are characterized by the ability to concentrate a target molecule to a concentration significantly higher than that achievable by the same type of ultrafiltration method (preferably TFF or CFF) without the use of an osmolyte; these concentration differences may be referred to as "improved concentration," and this improvement may be expressed as a specific percentage value. In certain embodiments, the methods of the present invention achieve an improvement in concentration of at least 10% (in other words, the concentration achieved with an osmolyte is at least 10% higher than that achieved without the use of an osmolyte). In further preferred embodiments, this improvement is at least 15%, preferably at least 20%, and preferably at least 25%, for example, at least 30%, or even at least 50%. In other words, each embodiment may be described in terms such as "improving ultrafiltration by at least 10%, at least 15%, or the like."

[0069] As used herein, the term "starting solution" in the context of a solution containing a molecule of interest or a solution containing an osmolyte refers to the respective solution initially used in the respective methods, as will be readily understood by those skilled in the art. Furthermore, as will be readily understood by those skilled in the art, the methods of the present invention may or may not include additional steps (such as an additional ultrafiltration step or other steps) depending on the embodiment. Thus, the "starting solution" refers to the respective initial solution used in the respective embodiments, regardless of any prior treatment, for example, of the solution containing the molecule of interest. Furthermore, as will be readily understood, the ultrafiltration method of the present invention is a continuous process using a flux passing through the ultrafiltration membrane. Therefore, the composition of each starting solution typically does not remain constant as it undergoes the ultrafiltration process. In the case of a starting solution containing a molecule of interest, the content of the starting solution may change, for example, depending on the (additional) solution introduced to the retentate side of the ultrafiltration membrane and / or may change due to the circulation of the solution on the retentate side of the ultrafiltration membrane. Similarly, for the osmolyte-containing starting solution, the contents of the starting solution are expected to change as the starting solution is combined with the resulting eluate and / or as the solution is circulated on the permeate side of the ultrafiltration membrane. However, for the osmolyte-containing starting solution, the composition of the starting solution may be kept approximately constant by using the starting solution in such a large volume that dilution is negligible for purposes of the method of the present invention.

[0070] Furthermore, as described elsewhere herein, in another embodiment herein, the solution on the permeate side of the ultrafiltration membrane is not circulated. Instead, in a flow-through mode, the solution on the permeate side of the ultrafiltration membrane contacts the ultrafiltration membrane. As will be appreciated by those skilled in the art, in a variation of the flow-through mode, a single starting solution containing osmolyte continuously contacts the ultrafiltration membrane throughout the process of the present invention.

[0071] Generally, as will be appreciated by those skilled in the art, neither the characteristics nor the size of a molecule of interest in the context of the present invention are particularly limited, and those skilled in the art will appreciate the types of molecules that may generally be subjected to ultrafiltration and the respective purposes for which ultrafiltration may be performed on such molecules. That is, generally, as used herein, the molecular weight of a molecule of interest is preferably in the range of 0.5 kDa to 1,000 kDa, and the molecular weight of a molecule of interest may be 1,500 kDa or less, or even 1,800 kDa or less (e.g., for an mRNA of about 6,000 nucleotides in length).

[0072] Generally, the term "molecule of interest" and similar terms may be used herein interchangeably with the term "compound of interest," and should be interpreted accordingly, with further embodiments contemplated in which the term "molecule" is replaced by the term "compound."

[0073] In a particularly preferred embodiment, the molecular weight of the target molecule herein is 2 kDa to 200 kDa, for example, 10 kDa to 150 kDa.

[0074] Generally, as used herein, a molecule of interest may be modified or unmodified, and therefore in a general set of embodiments, the molecule of interest is unmodified.

[0075] In another set of general embodiments, the molecule of interest is modified.

[0076] Modifications that can be added to each target molecule can be easily understood by those skilled in the art and are not particularly limited.

[0077] According to a preferred, non-limiting embodiment of the method of the present invention, the molecule of interest is an oligonucleotide or a polypeptide, which may be modified as described above.

[0078] Generally, in the present invention, modified molecules may be biologically modified (e.g., modified in a cell in vivo), chemically modified, enzymatically modified, or chemo-enzymatically modified.

[0079] In a preferred embodiment of the present invention, the modified molecules described herein are chemically modified molecules.

[0080] Therefore, in a first series of particularly preferred embodiments, the target molecule in the context of the present invention is oligonucleotide.As mentioned above, this oligonucleotide can generally be modified or unmodified.Therefore, this specification also contemplates the specific embodiment of the oligonucleotide described below, in which each oligonucleotide is modified.Preferred modification methods are described elsewhere in this specification.

[0081] Oligonucleotides are well known to those skilled in the art as DNA oligomers or RNA oligomers, and have a wide range of technical applications in many fields.Oligonucleotides can be biologically synthesized and / or modified (can be synthesized and / or modified in cells in vivo), chemically synthesized and / or modified, enzymatically synthesized and / or modified, or chemoenzymatically synthesized and / or modified, as desired.Specifically, the oligonucleotides used in the context of the present invention can be suitable for medical use.Usually, the oligonucleotides herein can be relatively short.

[0082] In either case, in certain overall embodiments, the molecule of interest is ssDNA (single-stranded DNA). In other embodiments, the molecule of interest is dsDNA (double-stranded DNA). Similarly, in certain overall embodiments, the molecule of interest is ssRNA (single-stranded RNA). In other embodiments, the molecule of interest is dsRNA (double-stranded RNA). In further overall embodiments, the molecule of interest is a heteroduplex consisting of ssRNA and ssDNA.

[0083] In yet a further embodiment, the molecule of interest is a ribozyme, commonly known as an "RNA enzyme" or "RNAzyme," ie, an RNA molecule that catalyzes a specific biochemical reaction.

[0084] Furthermore, in a further embodiment, the molecule of interest is a "DNAzyme."

[0085] In certain embodiments, the molecule of interest is an siRNA (small interfering RNA), also known as a "short interfering RNA" or "silencing RNA." siRNAs are typically double-stranded RNAs and can typically be used to silence the expression of target genes via RNA interference. siRNAs may be modified or unmodified, and may or may not contain non-RNA components such as DNA, LNA, or GNA. In a non-limiting exemplary embodiment, the siRNA is a double-stranded RNA 20-24 base pairs in length. Generally, as used herein, particularly in the case of siRNAs, the entire siRNA may be subjected to the methods of the present invention, or each individual strand may be subjected to the methods of the present invention separately.

[0086] In another embodiment, the molecule of interest is a DsiRNA ("Dicer substrate short interfering RNA"), which is well known to those skilled in the art as a Dicer substrate that is cleaved / processed to yield siRNA.

[0087] In another embodiment, the molecule of interest is shRNA ("short hairpin RNA"), also known as "small hairpin RNA". shRNA is generally known as an artificial RNA molecule that contains a hairpin loop, and can be used to silence the expression of target genes through RNA interference.

[0088] In a further embodiment, the molecule of interest is a miRNA (microRNA), which is known as a single-stranded, short, non-coding RNA that can function, for example, in RNA silencing and post-transcriptional regulation of gene expression.

[0089] Furthermore, according to another embodiment, the molecule of interest is an anti-miRNA, which may be used to antagonize and / or neutralize the function of the miRNA.

[0090] In a further embodiment, the molecule of interest is mRNA (messenger RNA), which is known as a short, single-stranded coding RNA that has recently become particularly important in connection with, for example, mRNA vaccines.

[0091] In a further embodiment, the molecule of interest is rRNA (ribosomal RNA).

[0092] In a further embodiment, the molecule of interest is a tRNA (transfer RNA).

[0093] In a further embodiment, the molecule of interest is a sgRNA (single guide RNA).

[0094] In certain embodiments, the molecule of interest is a plasmid.

[0095] In another preferred embodiment, the target molecule is an antisense oligonucleotide, commonly known as a single-stranded DNA or RNA, which is complementary to a selected sequence and can target a specific complementary sequence.

[0096] According to a generally preferred embodiment, the antisense oligonucleotides may be modified, in certain embodiments, the antisense oligonucleotides are modified with or present as 2'OMe, 2'MOE, LNA, constrained ethyl nucleic acid (cEt), thioate, or so-called Gapmers (i.e., mixed forms of DNA and modifications).

[0097] In some non-limiting embodiments, the length of the oligonucleotides described herein is 10-100 nucleotides, particularly 15-50 nucleotides, and particularly 20-30 nucleotides.

[0098] In a particular embodiment herein, the molecule of interest is an oligonucleotide comprising 10 to 300 nucleotides, particularly 14 to 130 nucleotides, and particularly 16 to 110 nucleotides.

[0099] Therefore, in certain embodiments herein, the target molecule is an oligonucleotide having a molecular weight of 3 to 90 kDa, particularly an oligonucleotide having a molecular weight of 4 to 39 kDa, and particularly an oligonucleotide having a molecular weight of 5 to 33 kDa.

[0100] In a further embodiment herein, the target molecule is an oligonucleotide containing 10 to 6000 nucleotides (for example, when the target molecule is mRNA).

[0101] Specific examples of embodiments of (therapeutic) oligonucleotides that may be advantageously used in the methods of the present invention include, but are not limited to, fomivirsen (Vitravene), pegaptanib sodium (Macugen), mipomersen (Kynamro), defibrotide (Defiterio), eteplirsen (Exondys 51), nusinersen (Spinraza), hepatitis B surface antigen (Heplisav-B), patisiran (Onpattro), inotersen (Tegsedi), voranesorsen (Waylivra), givosiran (Giblar), and golodirsen (Vyondys 53). In this list, the names in parentheses are the names of the therapeutic agents, and the INN name is shown before the therapeutic agent name.

[0102] Calculation methods for calculating the molecular weight of an oligonucleotide having a given sequence are readily available (eg, online) to those skilled in the art.

[0103] In the embodiment of the present invention, oligonucleotide can generally be a mixture of oligonucleotides.In one embodiment, oligonucleotide is a mixture of any of the various oligonucleotides defined herein.Therefore, in another embodiment, oligonucleotide is a double-stranded oligonucleotide that is a mixture of two specific oligonucleotides, and can also be a mixture of three or more oligonucleotides.In another non-limiting embodiment, oligonucleotide can be a mixture of the same kind of oligonucleotides defined herein, for example, a mixture of various RNAs.

[0104] In certain embodiments herein, the molecule of interest is an oligonucleotide containing one or more modifications selected from the group consisting of methylation, carboxymethylation, acetylation, methoxyaminomethylation, methoxycarbonylation, thiolation, adenylation, polyadenylation, base or backbone modifications (e.g., modifications in which the oligonucleotide is selected from GNA, TNA, and FNA), sugar chain modifications (e.g., modifications with GalNAc), linkers, lipids, peptides, and dyes.

[0105] Thus, the modified oligonucleotides described herein may further comprise components other than nucleotides, such as "artificial bases" attached to a chemical backbone that is different from that of normal nucleotides. Thus, in certain embodiments, the (modified) oligonucleotide in the context of the present invention is selected from the group consisting of GNA, TNA and FNA.

[0106] In a particularly preferred embodiment, the one or more modifications are independently selected from a base modification, a backbone modification, a sugar modification and other modifications.

[0107] In the present invention, the base modifications include hypoxanthine, inosine, 8-oxoadenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, and 5-(C2-C6)-alkynylcytosine. , 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), and any combination thereof.

[0108] In the present invention, the backbone modification may be selected from the group consisting of various modified forms of phosphodiesters present in DNA or RNA, preferably phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylphosphonothioate, methylphosphate, methylphosphorothioate, ethylphosphate, ethylphosphorothioate, boranophosphate, Preferably, the group is selected from boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, methylboranophosphonate, methylboranophosphonothioate, and derivatives thereof; phosphoramidate; phosphoramidate; N3'→P5' phosphoramidate; phosphorodiamidate; phosphorothiodiamidate; sulfamate; dimethylene sulfoxide; sulfonate; triazole; oxalyl; carbamate; methyleneimino (MMI); thioacetamido nucleic acid (TANA); derivatives thereof; and any combination thereof.

[0109] In the present invention, sugar chain modification refers to a modified form of a ribosyl moiety, for example, 2'-O-modified RNA, such as 2'-O-alkyl or 2'-O-(substituted) alkyl, for example, 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl; 2'-deoxy(DNA); 2'-O-(haloalkoxy)methyl (Arai K. et al. Bioorg. Med. Chem. 2011, 21, 6285), e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, e.g., 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo, e.g., 2'-F, FANA (2'-F arabinosyl nucleic acid); carba- and aza-sugar modifications; 3'-O-alkyl, e.g., 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl; and derivatives thereof; other sugar modifications such as "bridged" or "bicyclic" nucleic acids (BNA), For example, it is preferably selected from the group consisting of locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo-DNA; unlocked nucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); morpholino (e.g., those contained in PMO, PPMO, PMOPlus, PMO-X); and derivatives thereof and any combination thereof.

[0110] Other possible modifications may be selected from the group consisting of peptide-base nucleic acids (PNAs), boron-modified PNAs, pyrrolidine-cyclic oxypeptide nucleic acids (POPNAs), glycol or glycerol nucleic acids (GNAs), threose nucleic acids (TNAs), acyclic threoninol nucleic acids (aTNAs), oligonucleotides incorporating bases and backbones, pyrrolidine-amide oligonucleotides (POMs), linkers (e.g., propane, hexane, or polyethylene glycol), branching agents (e.g., symmetric or asymmetric branching agents), lipids (e.g., palmitate, stearate), peptide- or dye-modified oligonucleotides, cholesterol- or GalNAc-modified oligonucleotides, and derivatives thereof and any combination thereof.

[0111] In another set of preferred embodiments described herein, the molecule of interest is a polypeptide.

[0112] As disclosed above, the polypeptides herein generally may also be modified or unmodified, and therefore further embodiments of the specific embodiments relating to the polypeptides described below, in which each polypeptide is modified, are also contemplated herein.

[0113] Generally, a polypeptide is well known to those skilled in the art as a chain of amino acids linked via peptide bonds. In the context of the present invention, a polypeptide is defined as having a length of at least 10 residues, preferably at least 20 residues (in certain embodiments, at least 50 residues). In the present invention, "polypeptide" preferably encompasses "protein."

[0114] Generally, the polypeptide is not limited herein in any way, as the method of the present invention is generally expected to work for a wide variety of polypeptides, but preferably the polypeptide is one that is soluble in water or an organic mixture thereof.

[0115] In the context of the general embodiment of the present invention, the polypeptide is a protein, which is preferably selected from the group consisting of an enzyme, a cell receptor ligand, a protein ligand, a signaling protein, a cytokine, an antibody, an antibody fragment, an antibody domain, a diabody, an scFv fragment, an sc(Fv)2 fragment, and a chimeric antibody.

[0116] All such polypeptides are well known to those skilled in the art, and their meaning in the context of the present invention is the same as their ordinary meaning in the art.

[0117] Thus, in certain embodiments, the molecule of interest is a protein.

[0118] In certain embodiments, the molecule of interest is an enzyme.

[0119] In another embodiment, the molecule of interest is a cell receptor ligand.

[0120] In another embodiment, the molecule of interest is a protein ligand.

[0121] In another embodiment, the molecule of interest is a signaling protein.

[0122] In another embodiment, the molecule of interest is a cytokine.

[0123] In a further embodiment, the molecule of interest is an antibody.

[0124] In another related embodiment, the molecule of interest is an antibody fragment.

[0125] In certain embodiments, the molecule of interest is an antibody domain.

[0126] In certain embodiments, the molecule of interest is an scFv fragment.

[0127] In a specific embodiment, the molecule of interest is an sc(Fv)2 fragment.

[0128] In a further embodiment, the molecule of interest is a diabody (commonly known as a combination of two scFvs linked by a short linker peptide).

[0129] In a further embodiment, the molecule of interest is a chimeric antibody.

[0130] Thus, in certain non-limiting embodiments herein, the polypeptides described herein contain 90 to 1800 amino acid residues, for example, 110 to 1800 amino acids, particularly 135 to 1800 amino acid residues, and particularly 135 to 1350 amino acids.

[0131] Similarly, in some non-limiting embodiments herein, the molecular weight of the polypeptides described herein is 10 to 200 kDa, particularly 15 to 200 kDa, and particularly 15 to 150 kDa.

[0132] As with polypeptides, calculation methods for calculating the molecular weight of an oligonucleotide having a given sequence are readily available to those skilled in the art (eg, online).

[0133] In a general embodiment of the invention, the polypeptide may be a mixture of polypeptides, for example a mixture of any of the various polypeptides defined herein above, or alternatively, the polypeptide may be a polypeptide mixture of the same type of polypeptide as defined herein, for example a mixture of different scFv fragments.

[0134] In a specific embodiment of the present specification, the target molecule is a polypeptide containing one or more modifications selected from the group consisting of phosphorylation, disulfide bond, glycosylation, acetylation, amidation, γ-carboxyglutamic acid, hydroxylation, methylation, sulfation, lipid (particularly, a lipid selected from myristate, palmitate, farnesyl, geranylgeranyl, GPI anchor, and N-acyldiglyceride), linker, dye, and signal sequence. The modification is preferably selected from a sugar chain, lipid, linker, and dye.

[0135] Generally, the ultrafiltration membrane used in ultrafiltration herein is not particularly limited in its type, size, molecular weight cutoff, etc., and may basically be freely selected by a person skilled in the art based on the desired specific application.

[0136] However, in preferred embodiments herein, the molecular weight cut-off (also referred to herein as "MWCO") of the ultrafiltration membrane is in the range of 1 to 100 kDa, preferably in the range of 1 to 30 kDa, and particularly in the range of 1 to 10 kDa. In further specific embodiments, depending on the particular application as will be readily apparent to those skilled in the art, the molecular weight cut-off of the ultrafiltration membrane is in the range of 1 to 7 kDa, particularly in the range of 1 to 5 kDa, particularly in the range of 1 to 3 kDa, and particularly about 2 kDa.

[0137] In the present invention, the difference in osmolality between the retentate and permeate sides generally provides the driving force to increase the flux through the ultrafiltration membrane.

[0138] Thus, without wishing to be bound by any theory, there are at least two main driving forces that cause flux through an ultrafiltration membrane: the aforementioned driving force caused by the osmolality provided by the osmolyte, and the pressure exerted by the ultrafiltration (preferably TFF or CFF) itself.

[0139] In preferred embodiments, the osmolality of the solution on the permeate side of the ultrafiltration membrane is at least 1.25 times, particularly at least 1.5 times, particularly at least 2.0 times, particularly at least 3.0 times, at least 5.0 times, at least 10.0 times, or at least 20.0 times the osmolality of the solution on the retentate side of the ultrafiltration membrane.

[0140] Thus, when the method of the present invention is defined by the step of contacting a solution containing an osmolyte with the permeate side of an ultrafiltration membrane, the osmolality of the solution on the permeate side of the ultrafiltration membrane is at least 1.25 times, particularly at least 1.5 times, particularly at least 2.0 times, and particularly at least 3.0 times the osmolality of the solution on the retentate side of the ultrafiltration membrane.

[0141] Similarly, when the method of the present invention is defined as including a step of providing an osmolyte on the permeate side, the osmolality of the solution on the permeate side obtained by the method of the present invention is preferably at least 1.25 times, particularly preferably at least 1.5 times, particularly preferably at least 2.0 times, particularly preferably at least 3.0 times, preferably at least 5.0 times, preferably at least 10.0 times, or preferably at least 20.0 times the osmolality of the solution on the retentate side of the ultrafiltration membrane.

[0142] Thus, generally, in certain embodiments, the osmolality of the permeate side is at least 1.25 times the osmolality of the retentate side. In further such embodiments, the osmolality of the permeate side is at least 1.5 times the osmolality of the retentate side. In further such preferred embodiments, the osmolality of the permeate side is at least 2.0 times the osmolality of the retentate side, e.g., at least 2.5 times the osmolality of the retentate side, or even at least 3.0 times, at least 5.0 times, at least 10.0 times, or at least 20.0 times the osmolality of the retentate side.

[0143] As will be appreciated by those skilled in the art, the osmolality of the solution on the permeate side of the ultrafiltration membrane will vary throughout the process of the present invention due to the permeate passing through the ultrafiltration membrane and dilution of the osmolyte.

[0144] However, in certain embodiments herein, the osmolality of the solution on the permeate side of the ultrafiltration membrane remains at least at the factor specified herein throughout the method of the present invention.

[0145] In another embodiment, the method of the present invention is carried out until the osmolality of the solution on the permeate side of the ultrafiltration membrane matches the osmolality on the retentate side of the ultrafiltration membrane.

[0146] In a further preferred embodiment, the method of the present invention is carried out until a specific concentration is reached. This "specific concentration" may also be referred to as a "desired concentration." This "specific concentration" may depend on the specific purpose of a particular method and may be tested, for example, by taking a sample and measuring the OD value, etc.

[0147] Similarly, in certain embodiments herein, the osmolality of the solution on the permeate side of the ultrafiltration membrane is not less than a factor previously set forth herein throughout the method of the present invention.

[0148] In the method of the present invention, the osmolyte may be defined as being provided in the form of a starting solution comprising said osmolyte.

[0149] Thus, in each embodiment, the osmolality of the starting solution may be defined as being at least 1.5 times, particularly at least 1.75 times, particularly at least 2.0 times, particularly at least 3.0 times, or at least 5.0 times, at least 10.0 times, at least 20.0 times, or at least 30.0 times the osmolality of the starting solution comprising the molecule of interest used in the method of the invention.

[0150] Thus, in certain embodiments, the osmolality of the starting solution containing the osmolyte is at least 1.5 times that of the starting solution containing the molecule of interest used in the methods of the present invention, e.g., at least 1.75 times that of the starting solution containing the molecule of interest used in the methods of the present invention. Specifically, the osmolality of the starting solution containing the osmotically active molecule is at least 2.0 times that of the starting solution containing the molecule of interest used in the methods of the present invention, e.g., at least 2.5 times that of the starting solution containing the molecule of interest used in the methods of the present invention. More specifically, the osmolality of the starting solution containing the osmolyte is at least 3.0 times, at least 5.0 times, at least 10.0 times, at least 20.0 times, or at least 30.0 times that of the starting solution containing the molecule of interest used in the methods of the present invention.

[0151] Generally, in the set of embodiments described herein, the retentate side osmolality is calculated taking into account all osmolytes contained in each solution, and the permeate side osmolality is calculated taking into account only osmolytes having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[0152] In another set of preferred embodiments herein, each osmolality (i.e., both the retentate side osmolality and the permeate side osmolality) is calculated taking into account only osmolytes in each solution that have a molecular weight greater than the MWCO of the ultrafiltration membrane.

[0153] In any case, in certain preferred embodiments, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution at a concentration of at least 12.5 mM, particularly at least 25 mM, particularly at least 40 mM, and particularly at least 50 mM.

[0154] That is, in certain embodiments, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution with a concentration of at least 25 mM. In certain embodiments, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution with a concentration of at least 40 mM. In certain embodiments, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution with a concentration of at least 50 mM.

[0155] Alternatively, in a particularly preferred embodiment of the present invention, the difference in osmolality is defined by referring to the molar concentrations of the starting solutions on both sides of the ultrafiltration membrane. That is, in a specific embodiment, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a molar concentration at least twice that of the starting solution containing the target molecule. In another preferred embodiment, the osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane is provided in the form of a starting solution having a molar concentration at least five times that of the starting solution containing the target molecule.

[0156] In the method of the present invention as defined herein above, the method comprises, as one or more preceding steps, a step of ultrafiltration of the target molecule, in particular a step of concentrating the target molecule. The one or more additional steps may not include using an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane. In other words, before carrying out the method of the present invention, i.e., before using the osmolyte, one or more (conventional) ultrafiltration steps may be carried out. In other words, a solution containing the target molecule may be subjected to standard ultrafiltration, and the solution containing the target molecule obtained by this ultrafiltration may then be used as the starting solution containing the target molecule in the method of the present invention.

[0157] Therefore, in certain embodiments, before the step of contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte, one or more additional steps are performed: ultrafiltration of the target molecule, particularly a step of concentrating the target molecule. Preferably, this one or more additional steps include concentration with the aid of TMP. This one or more additional steps may not include using an osmolyte with a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane. Similarly, in each embodiment of these methods of the present invention in which an osmolyte is provided, one or more steps are performed before providing the osmolyte: ultrafiltration of the target molecule, particularly a step of concentrating the target molecule. This one or more steps may not include using an osmolyte with a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane. Therefore, in either case, the method of the present invention may be characterized in that the starting solution containing the target molecule used in the method of the present invention has previously been subjected to a concentration step (e.g., a concentration step selected from a precipitation step and an ultrafiltration method), preferably an ultrafiltration method that does not involve the use of an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane on the permeate side of the ultrafiltration membrane.

[0158] In the above-mentioned embodiments, typically, such one or more additional steps may be carried out in a separate ultrafiltration device (preferably a TFF device or a CFF device), or may be carried out in the same ultrafiltration device as the essential steps of the method of the present invention. In other words, such one or more preceding steps may be carried out in the same ultrafiltration device in which the other steps defining each method of the present invention are carried out. Alternatively, such one or more preceding steps may be carried out in a separate ultrafiltration device before being fed to the ultrafiltration device used in the other steps defining each method of the present invention.

[0159] In another series of general embodiments herein, the method of the present invention does not include a step that does not include providing an osmolyte on the permeate side of the ultrafiltration membrane having a molecular weight greater than the MWCO of the ultrafiltration membrane. In another series of general embodiments herein, the method of the present invention does not include an ultrafiltration step that does not include providing an osmolyte on the permeate side of the ultrafiltration membrane having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[0160] Alternatively, such a prior ultrafiltration step, in particular the prior ultrafiltration steps described herein above, may be incorporated as part of the method of the present invention.

[0161] Furthermore, as will be appreciated by those skilled in the art, the step of contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte in the methods of the present invention may be repeated at least once. In other words, each method of the present invention may include multiple such steps. Similarly, in each embodiment of such methods of the present invention defined by providing an osmolyte, providing the osmolyte may be repeated at least once. In other words, each method of the present invention may include multiple such steps. Thus, in any case, the methods of the present invention defined herein generally may be repeated to further improve the results. In certain embodiments, each repetition involves using a new starting solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane.

[0162] According to more general embodiments of the method of the present invention, any one or more of the above steps may be repeated.

[0163] Generally, the method of the present invention is described herein as comprising: i) passing the retentate side solution through the retentate side of the ultrafiltration membrane without recirculating it through the retentate side of the ultrafiltration membrane; or ii) (preferably) recirculating the retentate side of the ultrafiltration membrane by recirculating the retentate side solution on the retentate side of the ultrafiltration membrane. may also include:

[0164] Similarly, the method of the present invention comprises: i) passing the permeate side solution through the permeate side of the ultrafiltration membrane without recirculating it through the permeate side of the ultrafiltration membrane; or ii) (preferably) recycling the permeate side of the ultrafiltration membrane by recycling the permeate side solution on the permeate side of the ultrafiltration membrane. may also include:

[0165] Thus, in certain embodiments throughout the present specification, an osmolyte-containing solution is used by a passing method, in which the osmolyte-containing solution is passed from one reservoir to another. An exemplary and specific embodiment for achieving this is shown in FIG. 4. In certain embodiments, this passing occurs by gravity. Alternatively, the osmolyte-containing solution may be pumped from one reservoir to another, for example, using a pump. Thus, in embodiments without circulation, one or more pumps may be used (i.e., the osmolyte is pumped parallel to the ultrafiltration membrane by a pump, but is not recirculated). Alternatively, as will be readily apparent to those skilled in the art, pumping parallel to the ultrafiltration membrane may be achieved without the use of a pump or the like.

[0166] Furthermore, as will be appreciated by those skilled in the art, the present invention may advantageously involve circulating an osmolyte-containing solution on the permeate side of the ultrafiltration membrane. Thus, in a further preferred overall embodiment of the present invention, the method of the present invention includes circulating an osmolyte-containing solution on the permeate side of the ultrafiltration membrane. Exemplary (specific) embodiments for achieving this are shown in Figures 1 and 2.

[0167] In certain embodiments, the circulating step includes using a pump, and the circulating step may further include using a reservoir, which already contains a starting solution including an osmolyte and forms part of the circulation.

[0168] In certain embodiments, the methods of the present invention include the use of one or more pumps. In one particular embodiment, the methods of the present invention include the use of a pump on the retentate side of the ultrafiltration membrane (which may be, for example, a feed pump, and in certain embodiments, is a recirculation pump), and the use of a pump on the permeate side of the ultrafiltration membrane (which in certain embodiments is a recirculation pump). In certain preferred embodiments, the pump is a peristaltic pump.

[0169] In either case, in some embodiments, the methods of the present invention may be carried out by using an ultrafiltration device that circulates on both sides of an ultrafiltration membrane at a given time, and may be carried out until an equilibrium state is reached where the concentration of the target molecule no longer increases.

[0170] In a preferred embodiment, the method of the present invention is carried out until a particular concentration (or desired concentration) is reached, which may be a concentration depending on the particular purpose of the method and may be monitored, for example, by taking samples and making measurements.

[0171] Further embodiments of the present invention are provided by replacing the phrase "contacting the solution with the permeate side of the ultrafiltration membrane" in any of the embodiments described herein with the phrase "combining the permeate with the solution," and each specific embodiment is similar to the above.

[0172] As will be appreciated by those skilled in the art, the overall recovery of a target molecule by ultrafiltration may be improved by rinsing the retentate side of the ultrafiltration membrane and combining the resulting solution with the retentate. While such a procedure may reduce the final concentration of the target molecule, those skilled in the art can readily determine whether or not to use such measures depending on the circumstances and the particular goals to be achieved. Thus, in a further embodiment of the method of the present invention, the method further comprises the step of rinsing the retentate side of the ultrafiltration membrane to increase the yield of the target molecule.

[0173] Furthermore, with regard to osmolytes, it is preferred that the osmolyte is a polymer. In another preferred embodiment, the osmolyte is hygroscopic and water-soluble. Most preferably, the osmolyte is a hygroscopic and water-soluble polymer. Preferred examples of suitable osmolytes include, but are not limited to, polyethylene glycol (PEG), polyacrylic acid sodium salt, polyvinylpyrrolidone, carboxymethylcellulose, polyvinyl alcohol, (poly)ethylene oxide / (poly)propylene oxide copolymer, and mixtures thereof.

[0174] In the above particularly preferred embodiments, the osmolyte is polyethylene glycol (PEG) or a (poly)ethylene oxide / (poly)propylene oxide copolymer. Without wishing to be bound by any theory, PEG is particularly useful for achieving the above purpose because it has (high) solubility in water despite its (large) molecular weight. In some specific embodiments, the PEG is mPEG.

[0175] In certain preferred embodiments, the average molecular weight of the osmolyte (e.g., PEG) is 1,000 to 10,000,000 g / mol, particularly 1,000 to 2,000,000 g / mol, more particularly 2,000 to 200,000 g / mol, particularly 2,000 to 60,000 g / mol, and especially 2,000 to 20,000 g / mol.

[0176] In some non-limiting specific embodiments, the average molecular weight of the osmolyte (e.g., PEG) is 6,000 to 12,500 g / mol. In more specific embodiments, the average molecular weight of the osmolyte (e.g., PEG) is 7,000 to 9,000 g / mol. In certain embodiments herein, the average molecular weight of the PEG is approximately 8,000 g / mol. In preparing an osmolyte (e.g., PEG) having a certain average molecular weight, the exact composition is typically determined by combining osmolyte molecules of various sizes to achieve the average molecular weight.

[0177] In general embodiments herein, as already mentioned above, the osmolyte (e.g., PEG) has a molecular weight greater than the MWCO of the ultrafiltration membrane. In another preferred specific embodiment herein, the osmolyte (e.g., PEG) has an average molecular weight greater than the MWCO of the ultrafiltration membrane.

[0178] Also, in particularly preferred embodiments of both of the above aspects, the osmolytes used in the methods described herein do not substantially pass through ultrafiltration membranes.

[0179] As used herein, the expression (and similar terms) that a molecule (e.g., a molecule of interest in the context of the present invention and / or an osmolyte in the context of the present invention) "passes through the ultrafiltration membrane" essentially means that the molecule passes through the ultrafiltration membrane due to its size. Typically, molecules smaller than the size of the pores of the ultrafiltration membrane pass through the ultrafiltration membrane. Similarly, typically, molecules larger than the size of the pores of the ultrafiltration membrane do not pass through the ultrafiltration membrane.

[0180] In other words, it can be said that molecules having a molecular weight smaller than the MWCO of the ultrafiltration membrane pass through the membrane, and similarly, it can be said that molecules having a molecular weight larger than the MWCO of the ultrafiltration membrane do not pass through the membrane.

[0181] In any case, in preferred embodiments herein, the molecular weight or average molecular weight of the osmolyte (used in combination with the MWCO of a particular ultrafiltration membrane) is selected so that the osmolyte does not substantially pass through the ultrafiltration membrane.In certain preferred embodiments herein, the amount of osmolyte that passes through the ultrafiltration membrane is less than 10%, preferably less than 5%, particularly less than 2%, particularly less than 1%, more particularly less than 0.1%, more particularly less than 0.01%, for example less than 0.001%.

[0182] Similarly, in certain preferred embodiments herein, the target molecule does not substantially pass through the ultrafiltration membrane. However, depending on the application, the target molecule not passing through the ultrafiltration membrane is often less important than the osmolyte not passing through the ultrafiltration membrane. Thus, in certain embodiments, the target molecule passing through the ultrafiltration membrane is less than 20%, preferably less than 10%, particularly less than 5%, particularly less than 2%, more particularly less than 1%, more particularly less than 0.1%, and more particularly less than 0.01%.

[0183] In either case, one skilled in the art can readily determine whether a particular method achieves each desired goal by testing whether the molecule can pass through the ultrafiltration membrane, for example, by detecting the molecule using an antibody. Furthermore, one skilled in the art can readily determine and adjust the degree of retention of each molecule depending on the particular application to be implemented by the methods and uses of the present invention.

[0184] Without wishing to be bound by any theory, it is believed that, as is evident from the examples of the present invention, a higher concentration of PEG improves the results of the method of the present invention. Thus, in preferred embodiments herein, the concentration of osmolyte (e.g., the PEG molecule) is at least 20 mM, particularly at least 25 mM, particularly at least 40 mM, and particularly at least 50 mM.

[0185] Generally, the methods of the present invention may be characterized by any of the following items, as they are believed to be suitable for a variety of purposes and associated with a variety of advantages. Importantly, the following items are generally not exclusive, and certain preferred embodiments of the present invention may be characterized by any number or combination of the following items. Accordingly, such combinations are expressly considered to be included within the scope of embodiments of the present invention, even if the combination is not literally set forth below. Thus, any method of the present invention may include: ·Methods for concentrating compounds for research purposes; · Methods for concentrating commercially available compounds; - a method for concentrating an adjuvant or additive; · A method for concentrating active pharmaceutical ingredients (APIs) for incorporation into pharmaceutical products; - Methods that do not involve freeze-drying, How to avoid freeze-drying, A method for improving the yield of a molecule of interest (which may also improve the efficiency of the subsequent freeze-drying step), A method for enhancing the concentration of a solution containing a molecule of interest, ·Methods for improving the efficiency of ultrafiltration; A method for enhancing osmotic flow through an ultrafiltration membrane; A method for removing solvent molecules (in particular solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing target molecules, -How to obtain a high final concentration of the target molecule, · a method for preparing an active pharmaceutical ingredient; Methods for preparing pharmaceutical products, · Ultrafiltration / Diafiltration ("UF / DF") methods, a method for concentrating a solution containing the molecule of interest to a concentration of at least 150 mg / ml, in particular to a concentration of at least 200 mg / ml, in particular to a concentration of at least 300 mg / ml, and / or ·Methods for enhancing flux through ultrafiltration membranes may be.

[0186] In certain embodiments, the method of the present invention is a method for preparing an active pharmaceutical ingredient (API) for incorporation into a pharmaceutical product.

[0187] In certain embodiments, the method of the present invention is a method for concentrating an active pharmaceutical ingredient (API) for incorporation into a pharmaceutical product.

[0188] In certain embodiments, the method of the present invention is a freeze-drying-free method.

[0189] In certain embodiments, the methods of the present invention are suitable for avoiding freeze-drying.

[0190] In another specific embodiment, the method of the present invention is suitable for improving the results of freeze-drying as a subsequent step. That is, when a highly concentrated target molecule sample is obtained using the method of the present invention and then freeze-dried to produce a final product, the highly concentrated sample obtained before freeze-drying has the advantage of enabling a very large amount of final product to be obtained without changing the type of freeze-drying. As a specific example, if a product can be produced at a concentration three times higher than when the method of the present invention is not used, three times the amount of final product can be obtained in the subsequent freeze-drying step compared to when the method of the present invention is not used. As those skilled in the art will appreciate, despite the extremely high efficiency or yield achieved, the energy required for freeze-drying itself remains the same.

[0191] Thus, the methods and uses of the present invention have the additional advantage that subsequent freeze-drying can be significantly improved without having a significant adverse effect on the energy required for freeze-drying.

[0192] In certain embodiments, the method of the present invention is a method for enhancing the concentration of a solution containing a molecule of interest.

[0193] In certain embodiments, the method of the present invention is a method for improving the efficiency of ultrafiltration.

[0194] In certain embodiments, the method of the present invention is a method for enhancing osmotic flow through an ultrafiltration membrane. In particularly preferred embodiments herein, the method of the present invention is a method for enhancing flux through an ultrafiltration membrane.

[0195] In certain embodiments, the method of the present invention is a method for removing solvent molecules (particularly solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing a molecule of interest.

[0196] As one skilled in the art will readily appreciate, other small molecules, such as salts, may also be removed from the solution containing the molecule of interest. This may or may not be desirable in certain circumstances. In either case, if retention of a particular small molecule is desired, one skilled in the art can readily replenish such molecules, for example, by using a respective solution.

[0197] In certain embodiments, the methods of the invention are methods for obtaining a molecule of interest at a high final concentration, hi a preferred embodiment, the methods of the invention are methods for obtaining a molecule of interest at a higher final concentration than would be achievable in a comparable method that does not use the osmolyte.

[0198] In certain embodiments, the method of the present invention is a method for preparing an active pharmaceutical ingredient.

[0199] In certain embodiments, the method of the present invention is a method for preparing a medicament.

[0200] In certain embodiments, the process of the present invention is an ultrafiltration / diafiltration ("UF / DF") process.

[0201] In a particular embodiment, the method of the invention is a method for concentrating a solution containing a molecule of interest to a concentration of at least 150 mg / ml, in particular to a concentration of at least 200 mg / ml, in particular to a concentration of at least 300 mg / ml.

[0202] In certain embodiments, the methods of the present invention are methods for avoiding freeze-drying.

[0203] In certain embodiments, the method of the present invention is a method for enhancing flux through an ultrafiltration membrane.

[0204] In a further aspect (i.e., a seventh aspect) herein, there is provided the use of an osmolyte in a method for ultrafiltration of a solution containing a molecule of interest, comprising the steps of: the method comprising contacting a solution containing the osmolyte with the permeate side of an ultrafiltration membrane; the osmolyte has a molecular weight greater than the MWCO of the ultrafiltration membrane; a solution on the permeate side of the ultrafiltration membrane having a higher osmolality than a solution on the retentate side of the ultrafiltration membrane; Use is provided.

[0205] In a preferred embodiment of said use, further characteristics of the ultrafiltration method corresponding to this use are defined above.

[0206] In yet another aspect (i.e., an eighth aspect) herein, there is provided a method for improving a method of ultrafiltration of a solution containing a molecule of interest, comprising the steps of: the osmolyte has a molecular weight greater than the MWCO of the ultrafiltration membrane; The osmolyte is provided on the permeate side of the ultrafiltration membrane to improve ultrafiltration. Use is provided.

[0207] In a preferred embodiment of said use, further characteristics of the ultrafiltration method corresponding to this use are defined above.

[0208] Furthermore, in a preferred embodiment of any of the above uses of the present invention, the ultrafiltration method is a method for concentrating a solution containing a target molecule by ultrafiltration.

[0209] Generally, as with the methods of the invention outlined above, the uses of the invention are believed to be suitable for a variety of purposes and associated with a variety of advantages. Thus, as with the methods of the invention defined above, any use of the invention may be, for example, Use to enhance the concentration of a solution containing a molecule of interest, Use to improve the efficiency of ultrafiltration, Use to enhance osmotic flow through ultrafiltration membranes, Use for removing solvent molecules (in particular solvent molecules selected from water, organic solvents or mixtures thereof) from a solution containing a molecule of interest, Use to obtain high final concentrations of target molecules, Use for preparing active pharmaceutical ingredients, Use for preparing medicinal products, Use for ultrafiltration / diafiltration of target molecules, Use for concentrating a solution containing a molecule of interest to a concentration of at least 150 mg / ml, in particular to a concentration of at least 200 mg / ml, in particular to a concentration of at least 300 mg / ml, Use to avoid freeze-drying, Use to improve the yield of a molecule of interest (possibly to improve the efficiency of subsequent lyophilization), and / or Use to enhance flux through ultrafiltration membranes may be.

[0210] Thus, in a particular embodiment, the use of the present invention is to enhance the concentration of a solution containing a molecule of interest.

[0211] In a particular embodiment, the use of the present invention is to improve the efficiency of ultrafiltration.

[0212] In a particular embodiment, the use of the present invention is to enhance osmotic flow through an ultrafiltration membrane.

[0213] In a particular embodiment, the use of the present invention is for removing solvent molecules (particularly solvent molecules selected from water, organic solvents and mixtures thereof) from a solution containing a molecule of interest.

[0214] In a particular embodiment, the use of the present invention is to obtain a high final concentration of a molecule of interest.

[0215] In a particular embodiment, the use of the present invention is for the preparation of an active pharmaceutical ingredient.

[0216] In a particular embodiment, the use of the present invention is for preparing a medicament.

[0217] In a particular embodiment, the use of the present invention is for carrying out ultrafiltration / diafiltration of a molecule of interest.

[0218] In a particular embodiment, the use of the present invention makes it possible to concentrate a solution containing a molecule of interest to a concentration of at least 150 mg / ml, in particular to a concentration of at least 200 mg / ml, in particular to a concentration of at least 300 mg / ml.

[0219] In a particular embodiment, the use of the present invention is to avoid lyophilization.

[0220] In a particular embodiment, the use of the present invention is to enhance the flux through an ultrafiltration membrane.

[0221] In a further embodiment (i.e., a ninth embodiment) of the present invention, there is provided an ultrafiltration device for filtering, in particular concentrating, a solution containing a molecule of interest, comprising: i) contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane to increase the flux through the ultrafiltration membrane; and / or ii) combining the ultrafiltration permeate with a solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane, and circulating (or allowing to circulate) the resulting solution on the permeate side of the ultrafiltration membrane; An ultrafiltration device is provided, comprising:

[0222] Thus, in a particular embodiment of the ninth aspect, there is provided an ultrafiltration device for filtering, in particular concentrating, a solution containing a molecule of interest, comprising: a means for contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane to increase the flux through the ultrafiltration membrane; and optionally including means for circulating the resulting solution on the permeate side of the ultrafiltration membrane. An ultrafiltration device is provided.

[0223] In another embodiment of the ninth aspect, there is provided an ultrafiltration device for filtering, in particular concentrating, a solution containing a molecule of interest, comprising: means for combining the ultrafiltration permeate with a solution containing an osmolyte having a molecular weight greater than the MWCO of said ultrafiltration membrane, and means for circulating (or allowing to circulate) the resulting solution on the permeate side of said ultrafiltration membrane; The present invention provides an ultrafiltration device comprising:

[0224] Furthermore, in each combination of embodiments of the ninth aspect, there is provided an ultrafiltration device for filtering, in particular concentrating, a solution containing a molecule of interest, comprising: a means for contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane to increase the flux through the ultrafiltration membrane; means for combining the ultrafiltration permeate with a solution containing an osmolyte having a molecular weight greater than the MWCO of the ultrafiltration membrane, and means for circulating (or allowing to circulate) the resulting solution on the permeate side of the ultrafiltration membrane; An ultrafiltration device is provided, comprising:

[0225] In said embodiment, preferably none of said means may return permeate to the retentate side of the ultrafiltration membrane.

[0226] In a further embodiment of the ultrafiltration device of the invention, the circulation means comprises a pump (preferably a peristaltic pump) and a reservoir for the starting solution comprising the osmolyte.

[0227] Generally, as used herein, the ultrafiltration device of the present invention comprises: i) may be configured to operate in a flow-through mode in which the retentate is not recirculated on the retentate side of the ultrafiltration membrane; or ii) may be configured to operate in a recirculation mode in which the retentate is recirculated through the retentate side of the ultrafiltration membrane; It is preferably configured to operate in the recirculation mode described in ii) above.

[0228] Generally, as used herein, the ultrafiltration device of the present invention comprises: i) may be configured to operate in a pass-through mode in which the permeate is not recirculated on the permeate side of the ultrafiltration membrane; or ii) may be configured to operate in a recirculation mode in which the permeate is recirculated through the permeate side of the ultrafiltration membrane; It is preferably configured to operate in the recirculation mode described in ii) above.

[0229] In a further embodiment of the ultrafiltration device of the present invention, the ultrafiltration device of the present invention comprises a starting solution comprising said osmolyte.

[0230] In a further embodiment, the ultrafiltration device of the present invention is further defined according to any of the methods of the present invention defined above.

[0231] In a further embodiment, said starting solution is further defined as described for the method of the invention defined above. [Example]

[0232] The present invention will now be further described by reference to the following examples. It is important to note that the examples described herein are intended to further illustrate the invention and are not intended to limit the invention in any way.

[0233] Example 1 An exemplary experimental procedure for the PEG8000-assisted concentration of an oligonucleotide solution is detailed below. 1) The following materials and equipment were used. Lyophilized modified 23-mer oligonucleotide (molecular weight: 7631 g / mol, calculated extinction coefficient: 245.174 mM) -1 cm -1 ) ·Ultra pure water Polyethylene glycol 8000 Sartoflow Smart CF System Sartocon Slice Hydrosart cassette (filtration area: 0.1 m) 2 , MWCO: 2kD) Sartocon Slice 200 benchtop peristaltic pump (Sartorius) ·UV spectrophotometer HDPE bottles

[0234] 2) Preparation of oligonucleotide solution 23 g of the oligonucleotide solution was dissolved in 1081 mL of ultrapure water. The concentration was determined by absorbance measurement (1000-fold dilution) and found to be 492 OD / mL (15.3 mg / mL). A total of 531.228 OD (16.53 g, the amount calculated from the extinction coefficient) of oligonucleotide solution was used in the experiment.

[0235] 3) Preparation of 40% (w / w) PEG 8000 solution 200 g of a 40% (w / w) PEG 8000 solution was prepared by mixing 80 g of PEG 8000 with 120 g of water.

[0236] 4) Concentration of oligonucleotide solution 1080.6 mL of the oligonucleotide solution was pumped (retentate valve open) into the Sartoflow Smart CF system and concentrated in four steps (a-d) according to the following parameters:

[0237] Step a) First concentration step A new 1 L HDPE permeate bottle was prepared and weighed empty (tare weight: 77 g). The differential pressure (Δp = feed pressure - retentate pressure) was set to 0.5 bar, and the retentate valve was kept closed until permeate flow was visible. The pump flow rate at this stage was automatically adjusted by the Sartoflow Smart CF system to meet the differential pressure criterion. This process was continued until the remaining retentate volume was maintained at approximately 500 mL.

[0238] After step a), the permeate valve was closed and the permeate bottle was weighed. The total weight of the permeate bottle was 712 g, which corresponds to a total net weight of 634 g (634 mL) of solution. Therefore, the calculated volume of oligonucleotide loading solution after this step was 446 mL.

[0239] Step b) Second concentration step A new 250 mL HDPE bottle was weighed empty (tare weight: 27 g) and placed on the permeate side of the ultrafiltration membrane. The cross-flow setting was maintained the same as in step a) until the remaining retentate volume was maintained at approximately 330 mL. After step b), the pump was stopped and the permeate bottle was weighed. The total weight of the permeate bottle was 171 g, which corresponds to a total net weight of 144 g (144 mL) of water. Therefore, the calculated volume of oligonucleotide retentate solution after this step was 302 mL.

[0240] Step c) Third concentration step (TMP=3 bar) A new 250 mL HDPE bottle was weighed empty (tare weight: 27 g) and placed on the permeate side of the ultrafiltration membrane. The retentate valve was closed and the TMP was further increased to reach approximately 3 bar. This step was monitored, as the pump did not automatically stop above 3 bar. This step continued until no visible permeate flow was observed at a maximum TMP of 3 bar. The pump was stopped and the permeate bottle was weighed. The total weight of the permeate bottle was 163 g, corresponding to a total net weight of 136 g (136 mL) of water. Therefore, the calculated volume of oligonucleotide solution after this step was 165 mL.

[0241] Step d) Fourth concentration step (TMP=4 bar) A new 250 mL HDPE bottle was weighed empty (tare weight: 27 g) and placed on the permeate side of the ultrafiltration membrane. The maximum feed pressure in the Sartoflow Smart system is 4 bar. The retentate valve was further closed to gradually increase the feed pressure. After reaching the maximum feed pressure, the sample could not be further concentrated. After reaching 4 bar, the pump was stopped and the permeate bottle was weighed. The total weight of the permeate bottle was 60 g, which corresponds to a total net weight of 33 g (33 mL) of water. Therefore, the calculated volume of oligonucleotide solution after this step was 132 mL.

[0242] After step d), the absorbance of all permeate fractions was measured to determine the total amount of oligonucleotide lost due to breakthrough. The OD values ​​were as follows: Permeate fraction 1:880 OD Permeate fraction 2: 2.770 OD Permeate fraction 3: 7.943 OD Permeate fraction 4: 6.019 OD Total loss during concentration step: 17.612 OD (approximately 3%) Amount of oligonucleotide remaining in retentate: 513617 OD Calculated oligonucleotide concentration in retentate: 3879 OD / mL = 121 mg / mL

[0243] 5) Osmotic pressure-assisted concentration of oligonucleotide solutions An empty 500 mL glass bottle was weighed (tare weight: 396 g) and filled with 207 g of a 40% (w / w) PEG 8000 solution. This PEG solution was connected to the permeate side of the ultrafiltration membrane via silicone tubing. A peristaltic pump was used to circulate the PEG solution through the permeate side of the ultrafiltration membrane. The retentate valve was fully opened, and the oligonucleotide solution was pumped without applying retentate pressure to the ultrafiltration membrane. Osmotic pressure generated a permeate flow, removing water from the oligonucleotide solution. Pumping continued until no further significant flow through the ultrafiltration membrane was observed (approximately 2.5 hours). The pump was stopped, and the permeate bottle was weighed. The total weight of the permeate bottle was 685 g, corresponding to a total net gain of 80.9 g (80.9 mL) of water on the permeate side. Therefore, the calculated volume of oligonucleotide solution in the retentate after this step was 52 mL.

[0244] The absorbance value of the PEG solution was measured to determine the amount of oligonucleotide lost from the retentate due to breakthrough. The absorbance value was corrected against a similarly diluted PEG solution as a blank measurement. PEG solution breakthrough: 12.834 OD Total loss in the first concentration step and PEG-assisted concentration: 30.444 OD (approximately 6%) Total loss during PEG-assisted concentration: 12.832 OD (approximately 3%) Calculated amount of oligonucleotide in the retaining solution: 500.784 OD Calculated concentration of oligonucleotide in retentate: 9.724 OD / mL = 303 mg / mL Overall concentration of oligonucleotide solution using osmotic pressure: 303 [mg / ml] / 121 [mg / ml] × 100 [%] = 250 [%]

[0245] Example 2a and Example 2b - Final concentration of retentate depending on PEG concentration Similar to the experiment described in Example 1, two additional experiments were performed using either a 20% w / w PEG solution (Example 2a) or a 60% w / w PEG solution (Example 2b).

[0246] In each additional experiment, the same experimental setup was used (total volume: 519,959 OD), and the initial concentration was performed as in Example 1. During this initial concentration, very little loss of oligonucleotide was observed due to breakthrough into the permeate. The concentrate concentration at the end of the initial concentration was estimated to be 2920.3 OD / mL, equivalent to 90.9 mg / mL. From the volume of the permeate, the retentate volume was calculated to be 177.5 mL. During the PEG-assisted concentration step, the retentate volume decreased by only approximately 40 mL, significantly less than the retentate volume decrease observed in Example 1, resulting in only a 1.3-fold increase in concentration.

[0247] In the experiment using a 60% w / w PEG 8000 solution, the first concentration run was performed as in Examples 1 and 2a (total amount used: 507111 OD). This first concentration run resulted in very little loss of oligonucleotide due to breakthrough into the permeate. The estimated concentrate concentration was very similar to the results in Example 4a (2894.7 OD / mL, equivalent to 90.1 mg / mL). The retentate volume was calculated to be 174 mL from the volume of the permeate. During the PEG-assisted concentration step, the retentate volume decreased by approximately 110 mL. This corresponds to a 2.8-fold increase in concentration, very similar to the results observed in Example 1.

[0248] The above experiments showed that the concentration of the PEG solution can affect the final concentration of the oligonucleotide solution: the lower the PEG concentration, the lower the final concentration of the oligonucleotide can be.

[0249] Example 3 - PEG-assisted concentration using different membrane types 0.1m 2PEG-assisted concentration was performed according to Example 1 using a Sartocon Slice Hydrosart cassette. In this Example 3, a different type of membrane is tested. The experimental setup consisted of a 0.1 m PEG membrane with a 1 kDa molecular weight cut-off. 2 The experiment was the same as in Example 1, except that a polyethersulfone (PESU) membrane was used. The first concentration step was performed as in Example 1, except that the TMP reached 2 bar. This first concentration step observed a larger breakthrough of oligonucleotide into the permeate than that observed in Example 1 (approximately 13%). The remaining amount of oligonucleotide was calculated to be 449641 OD, which was estimated to be a concentration of approximately 100-120 mg / mL, similar to the concentration range observed with the Hydrosart membrane used in the previous experiment. After the first concentration step, the experimental setup was modified and the experiment was continued as in Example 1, with a 40% w / w PEG solution circulating on the permeate side of the ultrafiltration membrane. The generated osmotic pressure removed water from the retentate side. During this PEG-assisted concentration step, the retentate volume decreased from approximately 125-130 mL to approximately 40-60 mL, corresponding to an approximately 2-3-fold increase in the concentration of the oligonucleotide solution.

[0250] These results demonstrate that PEG-assisted concentration can be performed with different membrane types and different molecular weight cutoffs, achieving similar final concentrations.

[0251] Example 4a and Example 4b - Enrichment of another oligonucleotide sample with the aid of PEG To demonstrate the general applicability of PEG-assisted enrichment, two other oligonucleotide samples were further tested following the experimental setup outlined in Example 1.

[0252] In Example 4a, a fully thiolated 24-mer DNA oligonucleotide was used. The initial concentration step was performed in the same manner as in Example 1. Approximately 5% of the initial 548402 OD was lost due to breakthrough into the permeate. The remaining amount of oligonucleotide after loss into the permeate was calculated in the same manner as in Example 1. From this calculation, the maximum achievable concentration in the retentate was estimated to be 3430 OD / mL, which was equivalent to a concentration of 123 mg / mL of this oligonucleotide. From the volume of the permeate solution, the retentate volume was calculated to be 140 mL. After PEG-assisted concentration according to the principles described in Example 1, the retentate volume was reduced to 51 mL, which was calculated to be equivalent to an approximately 2.8-fold increase in concentration.

[0253] In Example 4b, a fully thiolated 20-mer MOE / DNA oligonucleotide was used. The initial concentration step was performed as in Example 1, except that the concentration was stopped after the TMP reached 3 bar. Approximately 1% of the initial 548595 OD was lost due to breakthrough to the permeate. The maximum achievable concentration in the retentate was estimated to be approximately 1900 OD / mL (80 mg / mL). From the volume of the permeate, the retentate volume was calculated to be approximately 285 mL. After PEG-assisted concentration according to the principles described in Example 1, the retentate volume was reduced from approximately 285 mL to 155 mL, which was calculated to equate to an approximately 1.8-fold increase in concentration.

[0254] These examples demonstrate that the principles outlined in Example 1 can be applied to other oligonucleotides.

[0255] Examples 5a and 5b - Concentration of Oligonucleotides with the Aid of Other Hygroscopic, Highly Water-Soluble Polymers This series of experiments demonstrates that other hygroscopic, highly water-soluble polymers can also assist in osmotic ultrafiltration according to the principles outlined in Example 1.

[0256] In Example 5a, 261.6 g (201 mL) of an aqueous polyacrylic acid polymer solution (45% wt. aqueous solution) was used. A first concentration step without polymer was performed using the same oligonucleotide according to the procedure described in Example 1. Approximately 3.5% of the initial 554768 OD was lost due to breakthrough into the permeate. The remaining amount of oligonucleotide after loss into the permeate was calculated as in Example 1. From this calculation, the maximum achievable concentration in the retentate in this experiment was estimated to be 4384 OD / mL, which was equivalent to a concentration of 136 mg / mL. After polyacrylic acid-assisted concentration according to the principles described in Example 1, the volume of the retentate was reduced from approximately 122 mL to 58 mL, which was calculated to be equivalent to a 2.1-fold increase in concentration.

[0257] In Example 5b, 200 g of a 40% (w / w) aqueous solution of ethylene oxide / propylene oxide copolymer was used. A first concentration step without polymer was performed using the same oligonucleotide according to the procedure described in Example 1. Approximately 8% of the initial 502027 OD was lost due to breakthrough into the permeate. The remaining amount of oligonucleotide after loss into the permeate was calculated as in Example 1. From this calculation, the maximum achievable concentration in the retentate in this experiment was estimated to be 3064 OD / mL, which is equivalent to a concentration of 95 mg / mL. After concentration using ethylene oxide / propylene oxide copolymer-assisted techniques according to the principles described in Example 1, the retentate volume was reduced from approximately 151 mL to approximately 30-40 mL, which was calculated to be equivalent to a 3.8- to 5-fold increase in concentration.

[0258] These experiments indicated that other highly water-soluble polymers of similar molecular size would also be suitable as osmotically active molecules to remove water from the retentate side and aid in concentrating the oligonucleotide solution.

[0259] Example 6 - PEG-assisted enrichment of a non-oligonucleotide biomolecule (protein lysozyme) This experiment demonstrates that PEG-assisted osmotic ultrafiltration, following the principles outlined in Example 1, can be used to concentrate proteins and other biomolecules to higher final concentrations than conventional UF / DF.

[0260] Lysozyme was chosen as a model protein. Lysozyme is a highly water-soluble protein. To determine the concentration of lysozyme in aqueous solution, the absorbance at 280 nm was measured. The extinction coefficient was 38940 L·mol -1 cm -1 The concentration was calculated taking into account the molar weight of 14,400 g / mol.

[0261] In this concentration experiment, a 20.4 g / L solution was prepared (whole solution OD: 110692). This protein solution was first concentrated by conventional UF / DF in the following steps as in Example 1. a) Concentration to approximately 985 mL at a differential pressure of 0.5 bar b) Concentration to approximately 600 mL at a differential pressure of 0.5 bar c) Concentration of the retentate at a differential pressure of 0.5 bar until the TMP reaches 3 bar. d) Concentration of the retentate with a differential pressure of 0.5 bar until a maximum feed pressure of 4 bar is reached

[0262] As in Example 1, permeate fractions were collected at each stage and their absorbance was measured. Unlike the experiments using oligonucleotides, the loss of lysozyme due to breakthrough was completely negligible (<0.2%). The remaining solution volume was calculated from the difference between the initial solution weight and the total volume of permeate collected, and it was estimated that a retentate concentration of up to 220 mg / mL could be achieved.

[0263] After this first concentration step, the experimental setup was modified similarly to that described in Example 1. The permeate side was charged with 224 g of a 40% (w / w) PEG solution. Similar to the method described in Example 1, the lysozyme solution was further concentrated against this PEG solution. This second concentration step reduced the retentate volume from approximately 186 mL to approximately 120 mL, corresponding to a 1.5- to 1.6-fold increase in protein concentration (approximately 340 mg / mL).

[0264] Example 7 - Sampling during the concentration process In this experiment, samples were taken from the retentate solution at regular intervals during the initial and PEG-assisted concentration steps according to the method described in Example 1. This method allows for the determination of how the concentration of the oligonucleotide solution changes throughout the experiment.

[0265] In this experiment, a partially thiolated 23-mer 2'-F oligonucleotide or 2'-OMe oligonucleotide was selected. For this concentration experiment, a 17.3 g / L solution was prepared (OD of the total solution: 550-595). This oligonucleotide solution was concentrated by standard UF / DF in the following four steps, as in Example 1. a) Concentration up to 500 mL with a differential pressure of 0.5 bar b) Concentration to 333 mL at a differential pressure of 0.5 bar c) Concentration of the retentate at a differential pressure of 0.5 bar until the TMP reaches 3 bar. d) Concentration of the retentate with a differential pressure of 0.5 bar until a maximum feed pressure of 4 bar is reached

[0266] At each stage, samples were taken from the retentate at regular intervals and the concentration of the oligonucleotide solution was determined by measuring UV absorbance, after which the sampled solution was returned to the retentate for comparison with experiments in which no samples were taken.

[0267] A sample taken at the end of step d showed that a maximum concentration of 80 mg / mL was achievable. From the volume of the permeate solution, the volume of the retentate was calculated to be 183 mL.

[0268] After this first concentration step, the experimental setup was modified as described in Example 1. The permeate side was charged with 199 g of a 40% (w / w) PEG solution. Similar to the method described in Example 1, the oligonucleotide solution was further concentrated against this PEG solution until no further permeate flow was observed. Sampling of the retentate for concentration determination was performed as in the first concentration step. After 5 hours of concentration, the retentate volume decreased from 183 mL to 71.5 mL, corresponding to a 2.5-fold increase in concentration. The maximum concentration measured by sampling was 165 mg / mL.

[0269] In another example using the same oligonucleotide as described in Example 1, the concentration measured after the first concentration step was 84 mg / mL. The maximum concentration of oligonucleotide measured in the retentate after PEG-assisted concentration was 208 mg / mL (a 2.5-fold increase based on OD).

[0270] List of References (1)Muslehiddinoglu et al., Technical Considerations for Use of Oligonucleotide Solution API, Nucleic Acid Therapeutics 2020, 30, 189-197 (2)Twardowski et al. (1983) Artificial Organs 7(4), 420-427 (3) German Patent Publication No. 10 2015 108 501 (A1)

Claims

1. A method for concentrating a solution containing a target molecule by ultrafiltration, comprising: contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane; A method wherein the solution on the permeate side of the ultrafiltration membrane has a higher osmolality than the solution on the retentate side of the ultrafiltration membrane.

2. 10. The method of claim 1, wherein the ultrafiltration is tangential flow filtration.

3. (a) ultrafiltrating the solution containing the target molecule by applying a pressure difference to obtain a concentrated solution containing the target molecule; and (b) ultrafiltrating the concentrated solution containing the target molecule while contacting the solution containing the osmolyte with the permeate side of the ultrafiltration membrane; 3. The method of claim 1 or 2, comprising:

4. The method of claim 3 , wherein step (b) comprises applying a pressure differential.

5. 5. The method according to claim 3, wherein the pressure difference in step (a) and / or step (b) is in the range of 0.1 bar to 1.0 bar.

6. 6. The method according to claim 3, wherein the pressure difference in step (a) and / or step (b) is in the range of 0.3 bar to 0.7 bar.

7. The method according to any one of claims 3 to 6, wherein the transmembrane pressure in step (a) and / or step (b) reaches a range of 2 to 4.

8. The method according to any one of claims 3 to 7, wherein the transmembrane pressure in step (a) and / or step (b) reaches a range of 2.5 to 3.

5.

9. The method according to any one of claims 3 to 8, wherein in step (a), the permeate side of the ultrafiltration membrane is not substantially contacted with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane.

10. 10. The method of any one of the preceding claims, wherein the osmolyte is a water-soluble and hygroscopic polymer.

11. 10. The method of any one of the preceding claims, wherein the osmolyte is selected from the group consisting of polyethylene glycol (PEG), polyacrylic acid (PAA) sodium salt, polyvinylpyrrolidone, carboxymethylcellulose, polyvinyl alcohol, (poly)ethylene oxide / (poly)propylene oxide copolymer, and combinations thereof.

12. 10. The method of any one of the preceding claims, wherein the osmolyte is polyethylene glycol (PEG) having an average molecular weight of 1,000 to 10,000,000 g / mol.

13. 10. The method of any one of the preceding claims, comprising concentrating the molecule of interest to a concentration of at least 100 mg / ml.

14. 10. The method of any one of the preceding claims, wherein the molecule of interest is an oligonucleotide.

15. 10. The method of any one of the preceding claims, wherein the MWCO of the ultrafiltration membrane is in the range of 1 to 100 kDa.

16. 10. The method of any one of the preceding claims, wherein the osmolality of the solution on the permeate side of the ultrafiltration membrane is at least 1.25 times the osmolality of the solution on the retentate side of the ultrafiltration membrane.

17. An ultrafiltration device for filtering a solution containing a target molecule, in particular for concentrating a solution containing a target molecule, comprising: i) contacting the permeate side of the ultrafiltration membrane with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the molecular weight cut-off (MWCO) of the ultrafiltration membrane to increase the flux through the ultrafiltration membrane; and / or ii) combining the ultrafiltration permeate with a solution containing an osmolyte having a molecular weight or average molecular weight greater than the MWCO of the ultrafiltration membrane, the resulting solution optionally being circulated on the permeate side of the ultrafiltration membrane; Including, none of the means returns the permeate to the retentate side of the ultrafiltration membrane; the circulation means includes a pump and a reservoir for a starting solution containing the osmolyte; the device includes a starting solution containing the osmolyte, the starting solution having a higher osmolality than a starting solution containing the target molecule to be ultrafiltered in the device; Device.

18. 12. The method of claim 11, wherein the osmolyte is a (poly)ethylene oxide / (poly)propylene oxide copolymer.

19. 19. The method of any one of claims 1 to 16 and 18, comprising obtaining a concentrated solution containing the target molecule at a concentration of at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml, at least 250 mg / ml, or at least 300 mg / ml.

20. 20. The method of claim 19, wherein the molecule of interest is a nucleic acid, preferably an oligonucleotide.

21. 21. The method of claim 20, wherein the concentrated solution is obtained without lyophilization.

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