Protein bioprocesses

Abstract

This invention provides a method for stabilizing proteins in aqueous solutions during protein purification and / or concentration. [Solution] (a) Protein and general formula (I): JPEG2026062713000015.jpg46170 The method comprises the steps of: (a) providing an aqueous solution containing a polyalkoxy fatty acid acyl surfactant; (b) contacting the aqueous solution with a separation membrane; and (c) subjecting the aqueous solution to a diafiltration step and / or an ultrafiltration step to produce a retention solution product which is an aqueous solution containing protein, wherein the compound of formula (I) reduces protein aggregation in steps (a) to (c) of the method, and the compound of formula (I) passes through the separation membrane in step (c).

Description

Technical Field

[0001] Background Field of Disclosure The present disclosure relates to a method for stabilizing a protein in an aqueous solution during protein purification and / or concentration, particularly during ultrafiltration and / or diafiltration procedures.

Background Art

[0002] Description of Related Art Biologics, which are pharmaceuticals derived from proteins or other biopolymers, are rapidly developing as an important field of medicine. Since protein substances have relatively easily destructible properties, the development of bioactive substances that are beneficial for treatment and have sufficient stability to withstand processing, distribution, and administration remains a considerable challenge. Manufacturers and formulators of proteins, which are biological drugs, use various processing techniques for purification and formulation to convert biologics into their final dosage forms. Among such techniques are ultrafiltration (UF) and diafiltration (DF) procedures, which are commonly used for protein purification, concentration, or buffer exchange. However, when ultrafiltration and diafiltration are performed, protein denaturation, aggregation, or precipitation may also occur due to shear stress during ultrafiltration or diafiltration operations, contact between the protein and the interface, or high concentration of the protein on the surface of the filtration membrane.

[0003] Surfactants are generally used in the final formulations of protein-based biologics for the purpose of protecting the biologics from various destabilizing forces such as interactions with interfaces and shear. Such forces also exist at earlier stages in the development of biologics (such as during upstream and downstream processing), but the use of surfactants is restricted because it is difficult for surfactants to interact with processing equipment (e.g., membrane fouling) and to reliably remove surfactants during protein filtration, buffer exchange, and concentration.

[0004] Polysorbate 80 is the most commonly used surfactant in biological products. (Callahan, Stanley, and Li, JOURNAL OF PHARMACEUTICAL SCIENCES 103:862-869, 2014) It has been shown that polysorbate 80 has the ability to stabilize proteins in ultrafiltration and diafiltration processes, and it has also been stated that polysorbate 80 becomes concentrated and does not effectively pass through the ultrafilter and diafiltration.

[0005] The website (https: / / www.researchgate.net / post / How_to_add_TWEEN_80_after_protein_ultrafiltration_diafiltration) states that Tween 80 (polysorbate 80) should be added after diafiltration because it is adsorbed to the membrane.

[0006] U.S. Patent Application Publication 20130195888A1 teaches and illustrates the use of polysorbate 80 at a concentration of 0.1 mg / mL in a dialysis filtration process. While the protein is protected from aggregation, the change in polysorbate 80 content during the ultrafiltration concentration step has not been evaluated.

[0007] Lei et al, Biotechnol. Prog., 2013, Vol. 29, No. 6, showed that the composition and concentration of polysorbate 20 change when an ultrafiltration membrane with a 30 kDa (molecular weight cutoff) is used. Regardless of whether the concentration of polysorbate 20 is above or below the critical micelle concentration, polysorbate 20 is retained in the membrane.

[0008] Poloxamer 188 is commonly used as a stabilizer for cells producing biopharmaceuticals in upstream bioprocessing. However, poloxamer is removed during Protein A chromatography upstream of ultrafiltration and dialysis filtration processes, and therefore does not exist to stabilize biopharmaceuticals during downstream processing (such as ultrafiltration and dialysis filtration). An example of this can be found in Xu, et al. Bioprocess Biosyst Eng (2017) 40:1317-1326. It is shown. [Overview of the Initiative] [Means for solving the problem]

[0009] Summary of Disclosure This disclosure provides a method for stabilizing proteins in aqueous solution during protein purification and / or concentration. The method involves (a) a protein and general formula I: [ka] (In the formula, R 1 -C(=O) is a fatty acid acyl group, R 2 is H or a substituted or unsubstituted hydrocarbyl group, X 1 is S, O, or NH, and X 2 is S, O, or NH, n is 0 or an integer from 1 to 5, and R 3 General formulas II and III: [ka] A method comprising the steps of: (a) providing an aqueous solution containing a polyalkoxy fatty acid acyl surfactant (which is a polymer group containing polymerization units); (b) contacting the aqueous solution with a separation membrane; and (c) subjecting the aqueous solution to a diafiltration step to reduce the concentration of soluble low molecular weight components or to introduce one or more soluble low molecular weight components therein, and / or subjecting the aqueous solution to an ultrafiltration step to concentrate the protein in order to produce a retention solution product which is an aqueous solution containing protein, wherein the compound of formula I reduces protein aggregation in steps (a) to (c) of the method, and the compound of formula I passes through the separation membrane in step (c). [Modes for carrying out the invention]

[0010] Detailed explanation The above summary and the following detailed description are illustrative and descriptive only and do not limit the invention as defined in the attached claims. Other features and advantages of any one or more embodiments will become apparent from the following detailed description and the claims.

[0011] The terms "comprises", "comprising", and "include" as used herein are not applicable. The words "(includes)", "including", "has", "having", or any other variation thereof are intended to encompass non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of components is not necessarily limited to these components alone, and may include other components that are not expressly listed or that are inherently present in such process, method, article, or apparatus. Furthermore, unless the opposite is expressly stated, "or" means comprehensive or not, not exclusive or. For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0012] Furthermore, the use of "one (a, an)" refers to the components and ingredients described herein. This is used for the purpose of [details omitted]. This is done solely for convenience and to give a general sense of the scope of the invention. This description should be interpreted as encompassing one or at least one, and singular also includes plural unless it is clear that it has a different meaning.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. In case of any conflict, including definitions, this specification shall prevail. Similar or equivalent methods and materials may be used in carrying out or testing embodiments of the present invention, but preferred methods and materials are described below. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0014] Where a quantity, concentration, or other value or parameter is given as a range, preferred range, or list of preferred upper and / or preferred lower limits, this should be understood to specifically disclose any range formed by any pair of an upper or preferred value of any range and a lower or preferred value of any range, regardless of whether the range is disclosed separately. Where a range of a number is described herein, unless otherwise specified, that range is intended to include its endpoint and all integers and fractions within that range. For example, where the range “1 to 10” is described, this described range should be interpreted to include the ranges “1 to 8”, “3 to 10”, “2 to 7”, “1.5 to 6”, “3.4 to 7.8”, “1 to 2 and 7 to 10”, “2 to 4 and 6 to 9”, “1 to 3.6 and 7.2 to 8.9”, “1 to 5 and 10”, “2 and 8 to 10”, “1.5 to 4 and 8”, and similar ranges.

[0015] In this specification, compositions and methods are described using the term “comprising” various components or steps, but unless otherwise specified, these compositions and methods may also “consist essentially of” or “consist of” such various components or steps.

[0016] Before discussing the details of the embodiments described later, we will define or clarify some terms.

[0017] As used herein, the term “aqueous solution” means a solution in which the solvent contains water in an amount of at least 90% by weight, based on the total weight of the solvent. In some embodiments, the solvent further comprises organic solvents such as acetone, ethanol, DMSO (dimethyl sulfoxide), 2-butanone, ethyl caprylate, and ethyl laurate. In some embodiments, the solvent contains, essentially consists of, or comprises water and an organic solvent. In some embodiments, the solvent contains water in an amount of at least 92% by weight, based on the total weight of the solvent. It contains %, or at least 94% by weight, or at least 96% by weight, or at least 98% by weight, or at least 99% by weight. In some embodiments, the solvent is essentially water or consists of water. In some embodiments, the solvent is water. In some embodiments, the aqueous solution is substantially free of organic solvents. In some embodiments, the liquid medium of the aqueous solution is essentially water or consists of water.

[0018] As used herein, the term "ultrafiltration" refers to the process of passing a protein solution through a semipermeable membrane (i.e., a separation membrane) that allows the solvent and low molecular weight components such as dissolved salts and sugars to pass through (permeate) while retaining the protein (retaining solution). This concentrates the protein solution.

[0019] As used herein, the term "diafiltration" refers to a filtration and solvent exchange process in which a protein solution is filtered using a semipermeable membrane (i.e., a separation membrane) that allows some of the solvent and dissolved low molecular weight components such as salts and sugars to pass through (permeate) while retaining the protein (retaining solution). The lost solvent (i.e., the solvent that has passed through the separation membrane) is replaced with a new solvent that optionally contains new low molecular weight components dissolved therein, and the resulting new solution is again subjected to filtration using a semipermeable membrane (i.e., a separation membrane) that allows the solvent and dissolved low molecular weight components such as salts and sugars to pass through (permeate) while retaining the protein (retaining solution).

[0020] Dialysis filtration can be performed continuously or batch-wise. In some embodiments, dialysis filtration is performed continuously, with new solvent being continuously added to the holding solution at a rate equal to the rate at which the permeate is generated. In such embodiments, it is possible to reduce the concentration of low molecular weight components, such as salts and sugars, dissolved in the protein solution without substantially changing the protein concentration in the solution. In some embodiments, the new solvent contains low molecular weight components, such as salts and sugars, that are different from the low molecular weight components that were present in the original protein solution. In such embodiments, one or more low molecular weight components can be introduced into the protein solution using dialysis filtration.

[0021] As used herein, the term “separation membrane” means a porous membrane or filter used in ultrafiltration (UF) or diafiltration (DF) to separate components in an aqueous solution based on their molecular weight or size. Molecules larger than the membrane’s pores, such as proteins, are retained, while smaller molecular weight compounds pass through.

[0022] As used herein, the term “surfactant / protein concentration ratio” means the ratio of the concentration of the polyalkoxy fatty acid acyl surfactant of formula I to the concentration of protein in an aqueous solution. In this disclosure, the concentrations of the polyalkoxy fatty acid acyl surfactant of formula I and the protein are expressed in weight / volume ratio (e.g., mg / ml).

[0023] Polyalkoxy compounds have the structure -(-AO) m The compound comprises one or more groups having the formula -(wherein m is 3 or greater, and A is an unsubstituted alkyl group). The A group may be linear, branched, cyclic, or a combination thereof. The various A groups in each of the -(-AO)- groups may be identical or different from each other.

[0024] A fatty acid compound is a compound containing one or more fatty acid groups. A fatty acid group is a group containing eight or more carbon atoms, each of which is bonded to one or more other carbon atoms of the group. A polyalkoxy fatty acid compound is a compound that is both a polyalkoxy compound and a fatty acid compound.

[0025] The number-average molecular weight is defined as the total weight of a sample divided by the number of molecules contained in the sample. .

[0026] A hydrocarbyl group is a group containing a hydrogen atom and a carbon atom. An unsubstituted hydrocarbyl group contains only a hydrogen atom and a carbon atom. A substituted hydrocarbyl group contains one or more substituents that include one or more atoms other than hydrogen and carbon.

[0027] Proteins are polymers whose polymerization units are amino acids. Amino acids are linked to each other by peptide bonds. Proteins contain 20 or more polymerization units of one or more types of amino acid residues. The term protein encompasses not only linear polypeptide chains but also more complex structures that include polypeptide chains.

[0028] If the protein molecules are distributed throughout a continuous liquid medium in the form of individual molecules dissolved in the medium, the protein is considered to be a solution in the liquid medium (or, synonymously, dissolved in the liquid medium). If the continuous liquid medium contains 60% by weight or more of water based on the weight of the continuous liquid medium, the protein is considered to be dissolved in water.

[0029] When a chemical group is present in a pH range of 4.5 to 8.5, and comes into contact with water at that pH, more than 50 mole% of the chemical group will take on an ionic form, thus becoming an ionic group.

[0030] A buffer is either (i) a compound having the ability to accept a proton to form a conjugate acid of the compound, where the pKa of the conjugate acid of the compound is less than 10, or (ii) a compound having the ability to release a proton, where the pKa of the compound is greater than 4.

[0031] Surprisingly, as disclosed in International Publication No. 2017 / 044367, which is incorporated herein by reference in its entirety for all purposes, certain types of surfactant compounds that have already been found to stabilize proteins in aqueous formulations can be efficiently and effectively removed from protein solutions when the protein solutions are subjected to ultrafiltration and / or diafiltration processes. Thereby, it becomes possible to use this type of surfactant in the upstream stages of protein production (rather than in the formulation stage itself), where they can reduce protein aggregation and particle formation that occur during purification and / or concentration of protein solutions, thereby improving protein yields. Unlike polysorbates, this type of surfactant can be easily filtered through a separation membrane even at concentrations that still protect the protein, thus reducing the problem of protein denaturation and aggregation during filtration.

[0032] Thus, the present disclosure provides a method for stabilizing a protein in an aqueous solution during purification and / or concentration of the protein. The method comprises (a) a protein and a general formula I:

Chemical formula

Chemical formula

[0033] It has been found that the polyalkoxy fatty acid acyl surfactant of formula I exhibits a lower critical micelle concentration (the concentration above which the surfactant spontaneously aggregates into micelles) than conventional surfactants such as polysorbate and poloxamer 188. See JSKatz et al., Mol.Pharmaceutics 2019, 16, pp. 282-291. The lower critical micelle concentration is a result of this. This indicates a stronger driving force for aggregation, which can be interpreted as faster stabilization of the interface. While we do not wish to be bound by any particular theory, the lower critical micelle concentration may explain the finding that the polyalkoxy fatty acid acyl surfactant of formula I approaches the surface equilibrium one to two orders of magnitude faster than conventional surfactants, overcoming proteins at the interface and thus reducing the likelihood of protein aggregation.

[0034] The aqueous solution provided in step (a) contains proteins and a polyalkoxy fatty acid acyl surfactant of general formula I dissolved therein (e.g., dissolved in water). Optionally, the aqueous solution further contains low molecular weight components such as sugars, sugar alcohols, salts, buffers, amino acids, salts of amino acids, and mixtures thereof. The low molecular weight components are also dissolved in the aqueous solution. If low molecular weight components are present, the total amount of all low molecular weight components is preferably not greater than 300 mg / ml.

[0035] Preferred sugars are selected from the group consisting of sucrose, glucose, mannose, trehalose, maltose, dextrose, dextran, and mixtures thereof. Preferred sugar alcohols are selected from the group consisting of sorbitol, mannitol, xylitol, and mixtures thereof. Preferred salts have cations selected from the group consisting of hydrogen, sodium, potassium, magnesium, calcium, ammonium, and mixtures thereof. Preferred salts have anions selected from fluoride salts, chloride salts, bromide salts, iodide salts, phosphates, carboxylates, acetates, citrates, sulfates, and mixtures thereof. Preferred buffers have cations selected from the group consisting of hydrogen, sodium, potassium, magnesium, calcium, ammonium, and mixtures thereof. Preferred amino acids are selected from the group consisting of lysine, glycine, proline, arginine, histidine, and mixtures thereof.

[0036] In step (c), the aqueous solution provided in step (a) is subjected to a diafiltration step and / or an ultrafiltration step. In the diafiltration step, at least a portion The solvent and the polyalkoxy fatty acid acyl surfactant of formula I pass through the separation membrane. As a result, the amount or concentration of the compound of formula I in the resulting retaining solution (aqueous solution containing protein) is reduced. In the diafiltration step, at least some of the low molecular weight components dissolved in the aqueous solution may also pass through the separation membrane. As a result, the amount or concentration of the low molecular weight components in the resulting retaining solution is lower than the amount or concentration initially present in the aqueous solution provided in step (a). In the diafiltration step, the aqueous solution containing protein (e.g., the one provided in step (a)) is purified by removing at least some of the compound of formula I and undesirable low molecular weight components from the aqueous solution.

[0037] During diafiltration, the solvent and low molecular weight components (permeate) removed from the protein aqueous solution (retention solution) are replaced with a new solvent. In some embodiments, the new solvent contains one or more new low molecular weight components dissolved therein, and these one or more new low molecular weight components are introduced into the protein aqueous solution. The new solvent and the one or more new low molecular weight components dissolved therein may independently be the same as or different from the solvent and low molecular weight components initially present in the aqueous solution provided in step (a). In some embodiments, the retention solution product produced at the end of diafiltration contains a reduced concentration of formula I polyalkoxy fatty acid acyl surfactant. In some embodiments, the protein concentration in the retention solution product produced at the end of diafiltration is substantially equal to the protein concentration in the aqueous solution provided in step (a). In some embodiments, the protein concentration in the retention solution product produced at the end of diafiltration is within ±5%, ±10%, or ±15% of the protein concentration in the aqueous solution provided in step (a).

[0038] In the ultrafiltration step, some of the solvent and the polyalkoxy fatty acid acyl surfactant of formula I pass through the separation membrane. As a result, the protein concentration in the resulting retention solution product (an aqueous solution containing protein) is higher than the protein concentration in the aqueous solution provided in step (a), i.e., the protein in the aqueous solution is concentrated. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the resulting retention solution product remains substantially equal to its concentration in the aqueous solution provided in step (a).

[0039] In some embodiments, step (c) includes, essentially consists of, or comprises both a sequentially performed diafiltration step and an ultrafiltration step. In some embodiments, step (c) includes, essentially consists of, or comprises a diafiltration step followed by an ultrafiltration step, i.e., the retained fluid produced by diafiltration is subjected to ultrafiltration. In some embodiments, step (c) includes, essentially consists of, or comprises an ultrafiltration step, and diafiltration is not performed in step (c). In some embodiments, step (c) includes, essentially consists of, or comprises a diafiltration step, and no separate ultrafiltration step is performed in step (c).

[0040] The retention solution product produced at the end of step (c) is an aqueous solution containing the protein dissolved therein. In some embodiments, the protein concentration in the aqueous solution of the retention solution product is 0.01 mg / ml to 700 mg / ml, preferably 5 mg / ml to 300 mg / ml. In some embodiments, the aqueous solution of the retention solution product is substantially free of the polyalkoxy fatty acid acyl surfactant of formula I. In some embodiments, the aqueous solution product of the retention solution product further contains the polyalkoxy fatty acid acyl surfactant of formula I dissolved therein. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution of the retention solution product is 1 mg / ml or less, or 0.1 mg / ml or less, or 0.05 mg / ml or less, or 0.01 mg / ml or less, or 0.005 mg / ml or less, or 0.001 mg / ml or less.

[0041] In some embodiments, the aqueous solution of the retention solution product produced at the end of step (c) contains, based on the total weight of protein in the aqueous solution of the retention solution product, at least 80% by weight of monomeric protein, or at least 85% by weight of monomeric protein, or at least 90% by weight of monomeric protein, or at least 92% by weight of monomeric protein, or at least 94% by weight of monomeric protein, or at least 96% by weight of monomeric protein, or at least 98% by weight of monomeric protein, or at least 99% by weight of monomeric protein.

[0042] In some embodiments, the surfactant / protein concentration ratio in the aqueous solution of the retention liquid product produced at the end of step (c) is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0043] In some embodiments, the concentration of the compound of formula I in the aqueous solution provided in step (a) is about 0.001 mg / ml to about 5 mg / ml, preferably about 0.005 mg / ml to about 1 mg / ml, preferably about 0.01 mg / ml to about 0.5 mg / ml, more preferably about 0.01 mg / ml to about 0.1 mg / ml, and more preferably about 0.01 mg / ml to about 0.05 mg / ml.

[0044] In some embodiments, the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, the concentration of the compound of formula I in the aqueous solution provided in step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml or approximately 0.01 mg / ml to approximately 0.05 mg / ml, the aqueous solution in the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0045] In some embodiments, the concentration of protein in the aqueous solution of step (a) is 0.0001 mg / ml to 150 mg / ml, preferably 0.1 mg / ml to 50 mg / ml, and more preferably 1 mg / ml to 20 mg / ml.

[0046] In some embodiments, step (c) includes an ultrafiltration step, where the concentration of protein in the aqueous solution after ultrafiltration in step c) is 0.01 mg / ml to 700 mg / ml, preferably 5 mg / ml to 300 mg / ml.

[0047] In some embodiments, the molecular weight cutoff of the separation membrane is approximately 10 kDa to approximately 100 kDa, meaning that proteins larger than the molecular weight cutoff can be retained (in the retention solution), while smaller proteins and other molecules can pass through the separation membrane to form a permeate. It is preferable to minimize protein loss across the separation membrane. Therefore, the separation membrane should be selected to have a molecular weight cutoff that does not exceed one-third of the molecular weight of the protein to be retained. If the molecular weight cutoff is in the range of approximately 30 kDa to approximately 50 kDa, it can play a role in retaining most of the large proteins, such as antibodies used in biopharmaceuticals. In some embodiments, the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa.

[0048] In some embodiments, the molecular weight cutoff of the separation membrane is approximately 100 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.005 mg / ml to approximately 1 mg / ml, or approximately 0.05 mg / ml to approximately 1 mg / ml, or approximately 0.01 mg / ml to approximately 0.5 mg / ml, or approximately 0.01 mg / ml to approximately 0.1 mg / ml, or approximately 0.01 mg / ml to approximately 0.05 mg / ml.

[0049] In some embodiments, the molecular weight cutoff of the separation membrane is approximately 100 kDa, the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.05 mg / ml to approximately 1 mg / ml, the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0050] In some embodiments, the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml, or approximately 0.01 mg / ml to approximately 0.05 mg / ml, or approximately 0.001 mg / ml to approximately 0.025 mg / ml, or approximately 0.001 mg / ml to approximately 0.01 mg / ml.

[0051] In some embodiments, step (c) includes, essentially consists of, or consists of an ultrafiltration step, in which no dialysis filtration is performed before or after ultrafiltration, the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.001 mg / ml to approximately 0.025 mg / ml, preferably approximately 0.001 mg / ml to approximately 0.01 mg / ml.

[0052] In some embodiments, step (c) includes, essentially consists of, or consists of an ultrafiltration step, in which no dialysis filtration is performed before or after ultrafiltration in step (c), the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.001 mg / ml to approximately 0.025 mg / ml or approximately 0.001 mg / ml to approximately 0.01 mg / ml, the aqueous solution of the retention solution product contains monomeric proteins, and the total weight of the proteins in the aqueous solution of the retention solution product Based on this, it contains at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, and the surfactant / protein concentration ratio in the aqueous solution of the retaining liquid product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0053] In some embodiments, step (c) includes, essentially consists of, or consists of a dialysfiltration step followed by an ultrafiltration step, the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml or approximately 0.01 mg / ml to approximately 0.05 mg / ml, the aqueous solution of the retaining solution product contains monomeric proteins, and the retaining solution product The aqueous solution contains at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution, and the surfactant / protein concentration ratio in the aqueous solution of the retaining liquid product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0054] In some embodiments, step (c) includes, essentially consists of, or comprises a diafiltration step, in which ultrafiltration is not performed before or after diafiltration in step (c), the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml.

[0055] In some embodiments, step (c) includes, essentially consists of, or comprises a diafiltration step, wherein ultrafiltration is not performed before or after diafiltration in step (c), the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml, and the aqueous solution of the retention solution product contains monomeric proteins based on the total weight of proteins in the aqueous solution of the retention solution product, at least 8 The product contains 0% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, and the surfactant / protein concentration ratio in the aqueous solution of the retaining liquid product produced at the end of step (c) is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0056] In the polyalkoxy fatty acid acyl compound of formula I, R 1 is preferably a substituted or unsubstituted aliphatic group. Among substituted aliphatic groups, a preferred substituent is hydroxyl. More preferably, R 1 is an unsubstituted aliphatic group; more preferably, R 1 is an unsubstituted alkyl group. In some embodiments, R of the compound of formula I 1 C 9~22 It is a linear alkyl, that is, R 1 R is a linear alkyl group having 9 to 22 carbon atoms. In some embodiments, R of the compound of formula I is used. 1 C 10~18 It is a linear alkyl group. In some embodiments, the R of the compound of formula I 1 C 10~16 It is a linear alkyl group. In some embodiments, the R of the compound of formula I 1 C 11~15 It is a linear alkyl group.

[0057] Preferably (if n is not 0), X 1 is O or NH. More preferably, X 1 is NH. Preferably, X 2 is O or NH. More preferably, X 2 It is NH.

[0058] n is 0, or 1, 2, 3, 4, or 5. Preferably, n is 0 or 1. More preferably, n is 1.

[0059] Preferably, R 2 It has 20 or fewer atoms; more preferably 15 or fewer atoms. Preferably, R 2 If R is not hydrogen, 2 It contains one or more carbon atoms. Preferably, R 2 is hydrogen or an unsubstituted hydrocarbon group; more preferably, R 2 is hydrogen, an unsubstituted alkyl group, or an alkyl group whose sole substituent is an unsubstituted aromatic hydrocarbon group. Among unsubstituted alkyl groups, methyl is preferred. Among alkyl groups whose sole substituent is an unsubstituted aromatic hydrocarbon group, -CH2-(C6H5) is preferred, where -(C6H5 ) is a benzene ring. Preferably, R 2 represents the side chain of a naturally occurring amino acid. In some embodiments, n is 1, and R of the compound of formula I. 2 R represents the side chain of a naturally occurring amino acid. In some embodiments, R of the compound of formula I is used. 2 H, unsubstituted C 1~4 It is alkyl or -CH2-(C6H5).

[0060] In some embodiments, the R of compound I 3 The number-average molecular weight is 600 to 5000 daltons, preferably 800 to 3000 daltons. In some embodiments, R 3 The group is either a statistical copolymer of structural (II) and structural (III) units or a block copolymer of structural (II) and structural (III) units. Preferably, R 3The group is a statistical copolymer of structural (II) and structural (III) units.

[0061] Preferably, -R 3 is structure-R 4 -CH3(wherein, R 4 (This is a polymer group that contains, essentially consists of, or comprises polymer units of structure (II) and structure (III). Preferably, R 4 It does not contain any polymerization units other than structure (II) and structure (III).

[0062] In some embodiments, R 1 R is a linear, unsubstituted alkyl group having 10 to 16 carbon atoms. 2 R is selected from the group consisting of hydrogen, methyl, and -CH2-(C6H5) (where -(C6H5) is a benzene ring), 3 The number-average molecular weight is 800-3000.

[0063] It is useful to characterize the molar ratio of units of structure (II) to units of structure (III) (hereinafter referred to as the "PO / EO ratio"). PO is structure (II), and EO is structure (III). In some embodiments, the PO / EO ratio is in the range of 0.01:1 to 2:1, or 0.05:1 to 1:1, or 0.1:1 to 0.5:1.

[0064] In a particularly preferred embodiment of the compound of formula (I), R 1 is CH3-(CH2) 11 -CH2-, n is 1, X 1 and X 2 Both are NH, and R 2 It is CH2(C6H5), and R 3 This is a copolymer of CH3-terminated PO and EO units, having a number-average molecular weight of approximately 1000 daltons and a PO / EO ratio of approximately 3:19. Such a compound of formula (I) is referred to as FM1000 in the examples herein.

[0065] In another preferred embodiment of the compound of formula (I), R 1 is CH3-(CH2) 11 -CH2-, n is 0, X 2 NH is R 3 It is a copolymer of CH3-ended PO and EO units with a number-average molecular weight of approximately 1000 daltons and a PO / EO ratio of approximately 3:19.

[0066] Preferably, the compound of formula (I) does not have an ionic group.

[0067] The compound of formula (I) can be prepared by the method disclosed in International Publication No. 2017 / 044366, the entirety of which is incorporated herein by reference for all purposes as a part of this specification.

[0068] Proteins preferred for use in the methods of this disclosure can be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody-drug conjugates, bispecific antibodies, tripspecific antibodies, growth factors, insulin, immunoglobulins, peptide hormones, enzymes, polypeptides, fusion proteins, glycosylated proteins, antigens, antigen subunits, and combinations thereof.

[0069] Preferred proteins are those that treat diseases or conditions, or function as vaccines. It has therapeutic efficacy. Examples of therapeutic proteins include immunoglobulin-g, adalimumab, interferon-alpha, bevacizumab, human growth hormone, rituximab, human serum albumin, insulin, erythropoietin-alpha, pembrolizumab, etanercept, filgrastim, nivolumab, trastuzumab, durvalumab, interleukin-2, infliximab, chorionic gonadotropin, avelumab, denosumab, ranibizumab, aflibercept, tremelimumab, factor VIII, interferon-beta, ipilimumab, atezolizumab, abatacept, tocilizumab, ustekinumab, and peguf. Filgrastim, secukinumab, streptokinase, cetuximab, omalizumab, ramucirumab, urokinase, certolizumab pegol, dupilumab, golimumab, aldesleukin, morglamostim, pegylated interferon α-2b, tislerizumab, follitropin α, gevokisumab, golimumab, spartalizumab, canakinumab, foralumab, varlilumab, nimotuzumab, erythropoietin β, evolocumab, pegargiminase, bermekimab, carotuximab, daratumumab, eculizumab, on Tuxizumab, adalimumab, camrelizumab, enobrituzumab, interleukin-12, lirirumab, panitumumab, gatipotuzumab, relatrimab, Andecaliximab, belimumab, kabilizumab, isactuzumab govitecan, monalizumab, pancreatin, partuzumab, tripalimab, rice Vilizumab, ofatumumab, pepinemab, cintilimab, alirocumab, milatuzumab, nidanilimab, sotatercept, vedolizumab, bertuzumab, beva Cizumab β, isatuximab, orlotamab, tisotumab vedotin, ben Larizumab, Kosiberimab, Emactuzumab, Ganitumab, Narsoprimab, Pidilizumab, Sarilumab, Trastuzumab Emtansine, Anetumab Labutansine, Vertilimumab, Blinatumomab, Guselkumab, Ixekizumab, Mepolizumab, Obinutuzumab, Ublituximab, Alemtuzumab, Emibetuzumab, Ficlatuzumab, Ifabotuzumab, Mirikizumab, Natalizumab, Lacosumomab, Siltuximab, Timigutuzumab, Trastuzumab Deruxtecan, Bimekizumab, Brodalumab, Se Trelimab, Faretuzumab, Opinercept, Rilonacept, Tomzotuximab, Urelumab, Ascrinbakumab, Brolucizumab, Clazakizumab cusatuzumab, dalotuzumab, inalumab, itolizumab, and margherita This is tuximab. Proteins that can be used as medical diagnostic agents, or that have beneficial effects in food compositions, or that can be incorporated into cleaning compositions or coating formulations are also being considered.

[0070] Many aspects and embodiments have been described above, but these are merely illustrative and not limiting. After reading this specification, those skilled in the art will understand that other aspects and embodiments are possible without departing from the scope of the invention. [Examples]

[0071] Examples The concepts described herein will be further illustrated in the following examples, but the examples will not limit the scope of the present invention as defined in the claims.

[0072] material Unless otherwise specified, all materials are sourced from Sigma-Aldrich or Fisher, and furthermore It was used without purification. Jeffamine M-1000 was provided by Huntsman. Polysorbate 80 (PS80) was Sigma-Aldrich's "tested according to Ph.Eur." grade, product number 59924. This was used to minimize oxidation. To minimize spoilage, it was stored under nitrogen headspace. Industrial-grade bovine IgG (immunoglobulin G) was purchased from MP Biomedicals (Santa Ana, CA).

[0073] FM1000 is a surfactant compound of formula (I), where R 1 is CH3-(CH2) 11 -CH2-, n is 1, X 1 and X 2 Both are NH, and R 2 R3 is -CH2(C6H5), where -(C6H5) is a benzene ring, and R3 is a copolymer of CH3-terminated PO and EO units with a number-average molecular weight of approximately 1000 daltons and a PO / EO ratio of approximately 3:19. FM1000 was prepared as reported in International Publication No. 2017 / 044366. Briefly, myristoyl chloride was amidated with phenylalanine in water in the presence of sodium hydroxide and triethylamine. The resulting suspension was acidified to pH 2 with concentrated hydrochloric acid and filtered. The filtered powder was then recrystallized on hexane. Myristoylphenylalanine was amidated by melt condensation with Jeffamine M-1000. The crude FM1000 product was dissolved in methanol and stirred on DuPont Amberlite® IRN 77 and IRN 78 ion exchange resins. The substance was removed. The final product was dried under vacuum.

[0074] Example 1: Maintaining FM1000 during centrifugal filtration. A 1 mg / ml deionized aqueous solution of FM1000 or a 0.9% physiological saline solution was prepared. Half of the sample was taken directly from the refrigerator, and the other half was heated to 60°C for a short time before centrifugation. 2 ml of each solution was placed in an Amicon Ultra-4 centrifuge tube and centrifuged at 3000 rpm for 2 minutes. The molecular weight cutoff (MWCO) of the filter (separation membrane) was set to 30, 50, or 100 kDa. The FM1000 concentration of the permeate was measured by HPLC (high-performance liquid chromatography) analysis. The results are shown in Table 1.

[0075] [Table 1]

[0076] Under all test conditions, FM1000 was not detected in the permeate after passing through a 30 or 50 kDa MWCO filter. These data indicate that FM1000, at a concentration of 1 mg / ml, could effectively pass through a 100 kDa MWCO separation membrane, but could not effectively pass through a 30 or 50 kDa MWCO separation membrane.

[0077] Examples 2-5 In Examples 2-5, 200 ml of aqueous solution containing IgG and FM1000 at a concentration of 1 mg / ml was prepared in the reservoir of a standard ultrafiltration (UF) apparatus. In the case of diafiltration (DF), the main reservoir contained the same aqueous solution (containing IgG and FM1000 at a concentration of 1 mg / ml) as the aqueous solution in the ultrafiltration reservoir. The second reservoir contained a replacement aqueous solution, which was identical to the aqueous solution in the main reservoir except that it did not contain FM1000 and IgG. The replacement aqueous solution was withdrawn and flowed into the main reservoir at the same rate at which the aqueous solution in the main reservoir was filtered and discarded. That is, the replacement aqueous solution was withdrawn and flowed into the main reservoir at the same rate at which the permeate was formed. In this way, the protein concentration in the aqueous solution was kept basically constant during diafiltration. All preparative solutions for analysis were taken from the ultrafiltration reservoir or the main reservoir of the diafiltration apparatus. The filter (separation membrane) is a Pall Minimate Capsule with an MWCO of 30 kDa, and the flow rate is 120 I set it to ml / min.

[0078] Example 2 (comparative): UF of aqueous solutions containing 0.05 mg / ml of FM1000 and 1 mg / ml of IgG. A 200 ml aqueous solution (0.9% physiological saline) containing 1 mg / ml IgG and 0.05 mg / ml FM1000 was applied to a UV / Vis filter only and concentrated to approximately 5 ml (volume of retaining solution). The MWCO of the separation membrane was set to 30 kDa. Samples were taken at various points in the concentration process (i.e., when the retaining solution reached various volumes), and the IgG concentration was analyzed by UV / Vis (A280 nm, 10-fold dilution), and the FM1000 concentration by HPLC (A210 nm). The results are shown in Table 2.

[0079] [Table 2]

[0080] These data indicate that FM1000 at a concentration of 0.05 mg / ml cannot effectively pass through a 30 kDa MWCO separation membrane in the ultrafiltration process.

[0081] Example 3: DF / UF of aqueous solutions containing 0.05 mg / ml of FM1000 and 1 mg / ml of IgG. In Example 3, 200 ml of an aqueous solution containing 1 mg / ml of IgG and 0.05 mg / ml of FM1000 was applied to the DF, and then to the UF. That is, the aqueous solution of the retaining liquid product generated at the end of the DF was applied to the UF. The 200 ml of aqueous solution (applied to the DF) was either a 0.9% physiological saline solution or a 25 mM (millimole concentration) pH 6.5 histidine buffer solution. When the 200 ml of aqueous solution (applied to the DF) was a 0.9% physiological saline solution (containing IgG and FM1000), the replacement aqueous solution for the second reservoir was a pure 0.9% physiological saline solution that did not contain IgG and FM1000. If the 200 ml aqueous solution (attached to DF) is a 25 mM pH 6.5 histidine buffer solution (containing IgG and FM1000), the replacement aqueous solution for the second reservoir was a pure 25 mM pH 6.5 histidine buffer solution that did not contain IgG or FM1000.

[0082] During DF, 200 ml of 0.9% physiological saline solution (containing IgG and FM1000) in the main reservoir was replaced by diafiltration with 1.6 liters of pure 0.9% physiological saline solution (not containing IgG and FM1000), and 200 ml of 25 mM pH 6.5 histidine buffer solution (containing IgG and FM1000) was replaced by diafiltration with 1.0 liter of pure 25 mM pH 6.5 histidine buffer (not containing IgG and FM1000). At the end of DF, each aqueous solution of the retaining solution product was subjected to further UR (Ultraviolet Infiltration) to concentrate the IgG protein contained therein, thereby reducing the retaining solution volume to approximately 25 ml.

[0083] In Example 3, a separation membrane with an MWCO of 30 kDa was used for DF and UF. Samples were taken at various time points in the DF and UF processes, and the IgG concentration was analyzed by UV / Vis (A280 nm, 10-fold dilution), and the FM1000 concentration was analyzed by HPLC (A210 nm). The results are shown in Table 3. The "Saline HPLC" column shows the HPLC signal intensity (indicating FM1000 concentration) at various time points in the DF and UF processes of a 0.9% saline solution. The "Saline UV / Vis" column shows the UV / Vis signal intensity (indicating IgG concentration) at various time points in the DF and UF processes of a 0.9% saline solution. The "Histidine HPLC" column shows the HPLC signal intensity at various time points in the DF and UF processes of a 25 mM histidine buffer solution. The "Histidine UV / Vis" column shows the UV / Vis signal intensity (indicating IgG concentration) at various time points in the DF and UF processes of a 25 mM histidine buffer solution.

[0084] [Table 3]

[0085] These data demonstrate that FM1000 at a concentration of 0.05 mg / ml can effectively remove 30 kDa MWCO in a process using a separation membrane for both DF and UF, including a dialysis filtration step followed by an ultrafiltration step. These data also demonstrate that FM1000 at a concentration of 0.05 mg / ml can effectively pass through the separation membrane for 30 kDa MWCO in the dialysis filtration process.

[0086] Example 4: DF / UF of aqueous solutions containing 0.1 mg / ml of FM1000 and 1 mg / ml of IgG. In Example 4, 200 ml of a 0.9% saline solution containing 1 mg / ml of IgG and 0.1 mg / ml of FM1000 was subjected to a DF (diodermectomy) and then to a UF (ultrafiltration) membrane. The initial concentration of FM1000 in the 200 ml aqueous solution was set to 0.1 mg / ml. In this process, only the 0.9% saline solution was used / tested. In the diafiltration, 1.5 liters of pure 0.9% saline solution (without IgG and FM1000) was replaced by the DF, and in the UF, the volume of the retaining solution was reduced to approximately 50 ml. The DF / UF process was carried out in the same manner as in Example 3. In Example 4, a separation membrane with an MWCO of 30 kDa was used for both the DF and UF. The results are shown in Table 4.

[0087] [Table 4]

[0088] These data showed that when MWCO used a 30 kDa separation membrane, FM1000 at a concentration of 0.1 mg / ml was partially removed from the aqueous solution (retention solution) by slow dialysfiltration. Subsequent ultrafiltration by MWCO using a 30 kDa separation membrane concentrated both IgG and FM1000, but the FM1000 / protein concentration ratio in 50 ml of aqueous solution of the retention solution product was much lower than that in 200 ml of 0.9% physiological saline solution.

[0089] Example 5: UF of an aqueous solution containing 0.01 mg / ml of FM1000 and 1 mg / ml of IgG. A 200 ml aqueous solution (0.9% physiological saline) containing 1 mg / ml IgG and 0.01 mg / ml FM1000 was applied to a UV / Vis filter only and concentrated to approximately 2 ml (volume of retained solution). The MWCO of the separation membrane was set to 30 kDa. Samples were taken at various points in the concentration process (i.e., when the retained solution reached various volumes), and the IgG concentration was analyzed by UV / Vis (A280 nm, 10-fold dilution), and the FM1000 concentration by HPLC (A210 nm). The results are shown in Table 5.

[0090] [Table 5]

[0091] <LLD means below the detection limit. These data indicate that the protein IgG was concentrated. On the other hand, FM1000 was not detected at all, indicating that it was not concentrated in UF. Therefore, from these data, it was shown that FM1000 at a concentration of 0.01 mg / ml can effectively pass through a separation membrane with a MWCO of 30 kDa in the ultrafiltration process.

[0092] Example 6: Effect of FM1000 and polysorbate 80 (both at a concentration of 0.01 mg / ml) on the stability of the protein cetuximab in aqueous solution Cetuximab is commercially available under the trade name Erbitux® and was obtained from a pharmacy. This was formulated as a 2 mg / ml aqueous solution (pH 7.2) in 10 mM phosphate buffer and 145 mM sodium chloride.

[0093] The Erbitux® solution (cetuximab 2 mg / ml) was reformulated to add FM1000 or polysorbate 80 to this solution so that the target cetuximab concentration was achieved. By diluting the Erbitux® solution, 1.5 mg / ml cetuximab samples were prepared. By centrifugal filtration of the Erbitux® solution to concentrate it to 10 mg / ml and then diluting it to 7.5 mg / ml, 7.5 mg / ml cetuximab samples were prepared. The surfactant (FM1000 or polysorbate 80) concentration in the cetuximab samples was set to 0.01 mg / ml. Surfactant was not added to Samples 9 and 10. The cetuximab samples were either shaken (after shaking) or not shaken (before shaking) prior to analysis. Inside a 5 ml serum vial from Wheaton ​​​3.0 ml of each shaking sample was added. The sample used before shaking was the portion removed to reduce the shaking sample volume to 3 ml. The shaking samples were shaken overnight in a reciprocating shaker at 150 reciprocations / minute.

[0094] The particles present in the sample solution (indicating the aggregation of the protein cetuximab) were analyzed in terms of particle quantity (number of particles) and particle size (expressed as particle radius). The results are shown in Table 6. To quantify particles invisible to the naked eye, microflow imaging was performed using a Biotechne Microflow Imaging instrument. Particle counting was performed using software. The process was automated. Size reduction was performed using the Agilent 1260 Bioinert liquid chromatography system. Dechromatography was performed, followed by UV detection. An Agilent AdvanceBio 300A SEC column was used, and phosphate buffer was used as the buffer flow. UV absorbance was measured using a Molecular Devices M3 plate reader. Dynamic light scattering (DSC) was measured using a Wyatt DynaPro II. did.

[0095] [Table 6]

[0096] In Table 6, Samp means sample, CTX means cetuximab, Conc means concentration, and Surf means surfactant. These data show that when the amount of surfactant is 0.01 mg / ml, FM1000 can reduce the aggregation of the protein cetuximab far more significantly than polysorbate 80.

[0097] Please note that not all of the actions described above are necessary in the summary or examples, some of the actions may not be necessary, and one or more additional actions may be performed in addition to those described. Furthermore, the order in which the actions are listed does not necessarily indicate the order in which they are performed.

[0098] In this specification described above, concepts have been described in relation to specific embodiments. However, those skilled in the art will understand that various modifications and alterations can be made without departing from the scope of the invention as set forth in the following claims. Therefore, this specification should be considered illustrative rather than restrictive, and all such modifications are intended to be included within the scope of the invention.

[0099] Benefits, other advantages, and solutions to problems have been described above in relation to specific embodiments. However, benefits, advantages, solutions to problems, and any features that may produce or make more prominent any benefits, advantages, or solutions should not be construed as any or all of the claims to be critically important, necessary, or essential features.

[0100] For clarity, it should be fully understood that certain features described herein in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features described in relation to a single embodiment may also be provided individually or in any partial combination.

[0101] Embodiment For further explanation, further non-limiting embodiments of this disclosure are shown below.

[0102] For example, Embodiment 1 is a method for stabilizing proteins in aqueous solution during protein purification and / or concentration. This method involves (a) a protein and general formula I: [ka] (In the formula, R 1 -C(=O) is a fatty acid acyl group, R 2 is H or a substituted or unsubstituted hydrocarbyl group, X 1 is S, O, or NH, and X 2is S, O, or NH, n is 0 or an integer from 1 to 5, and R 3 General formulas II and III: [ka] A method comprising the steps of: (a) providing an aqueous solution containing a polyalkoxy fatty acid acyl surfactant (which is a polymer group containing polymerization units); (b) contacting the aqueous solution with a separation membrane; and (c) subjecting the aqueous solution to a diafiltration step to reduce the concentration of soluble low molecular weight components or to introduce one or more soluble low molecular weight components therein, and / or subjecting the aqueous solution to an ultrafiltration step to concentrate the protein in order to produce a retention solution product which is an aqueous solution containing protein, wherein the compound of formula I reduces protein aggregation in steps (a) to (c) of the method, and the compound of formula I passes through the separation membrane in step (c).

[0103] Embodiment 2 is the method according to Embodiment 1, wherein the aqueous solution in the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0104] Embodiment 3 is a method according to any one of the prior embodiments, wherein step (c) includes both a sequentially performed diafiltration step and an ultrafiltration step.

[0105] Embodiment 4 is an aqueous solution provided in step (a) in which the concentration of compound I is about 0.001 mg / ml to about 5 mg / ml, preferably about 0.005 mg / ml to about 1 mg / ml, preferably about 0.01 mg / ml to about 0.5 mg / ml, more preferably about 0.01 This method is based on one of the prior embodiments, wherein the concentration is mg / ml to approximately 0.1 mg / ml, more preferably approximately 0.01 mg / ml to approximately 0.05 mg / ml.

[0106] Embodiment 5 is the method of any one of the preceding embodiments, wherein the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, the concentration of the compound of formula I in the aqueous solution provided in step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml or approximately 0.01 mg / ml to approximately 0.05 mg / ml, the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99% compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0107] Embodiment 6 is a method according to any one of the preceding embodiments, wherein the concentration of protein in the aqueous solution in step (a) is 0.0001 mg / ml to 150 mg / ml, preferably 0.1 mg / ml to 50 mg / ml, and more preferably 1 mg / ml to 20 mg / ml.

[0108] Embodiment 7 is a method according to any one of the preceding embodiments, wherein step (c) includes an ultrafiltration step, and the concentration of protein in the aqueous solution after ultrafiltration in step c) is 0.01 mg / ml to 700 mg / ml, preferably 5 mg / ml to 300 mg / ml.

[0109] Embodiment 8 is the method according to any one of Embodiments 1 to 4 and 6 to 7, wherein the molecular weight cutoff of the separation membrane is about 10 kDa to about 100 kDa, preferably about 30 kDa to about 50 kDa.

[0110] Embodiment 9 is a method according to any one of Embodiments 1 to 4 and 6 to 8, wherein the molecular weight cutoff of the separation membrane is approximately 100 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.005 mg / ml to approximately 1 mg / ml, or approximately 0.05 mg / ml to approximately 1 mg / ml, or approximately 0.01 mg / ml to approximately 0.5 mg / ml, or approximately 0.01 mg / ml to approximately 0.1 mg / ml, or approximately 0.01 mg / ml to approximately 0.05 mg / ml.

[0111] Embodiment 10 is the method according to Embodiment 9, wherein the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0112] Embodiment 11 is a method according to any one of Embodiments 1 to 3 and 6 to 7, wherein the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml.

[0113] Embodiment 12 is such that step (c) includes, essentially consists of, or consists of an ultrafiltration step, and in step (c), ultrafiltration The method is described in any one of Embodiments 1 to 2 and 6, wherein no dialysis filtration is performed before or after step (a), the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.001 mg / ml to approximately 0.025 mg / ml, preferably approximately 0.001 mg / ml to approximately 0.01 mg / ml.

[0114] Embodiment 13 is the method of Embodiment 12, wherein the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0115] Embodiment 14 is the method according to any one of Embodiments 1 to 2 and 6, wherein step (c) includes, essentially consists of, or consists of a dialysfiltration step followed by an ultrafiltration step, the fractional molecular weight of the separation membrane is about 30 kDa to about 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is about 0.01 mg / ml to about 0.1 mg / ml or about 0.01 mg / ml to about 0.05 mg / ml.

[0116] Embodiment 15 is the method of Embodiment 14, wherein the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0117] Embodiment 16 is the method according to any one of Embodiments 1 to 2 and 6, wherein step (c) includes a diafiltration step, is essentially derived from a diafiltration step, or consists of a diafiltration step, in which ultrafiltration is not performed before or after diafiltration in step (c), the molecular weight cutoff of the separation membrane is approximately 30 kDa to approximately 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is approximately 0.01 mg / ml to approximately 0.1 mg / ml.

[0118] Embodiment 17 is the method of Embodiment 16, wherein the aqueous solution of the retention solution product contains monomeric protein in an amount of at least 80% by weight, or at least 85% by weight, or at least 90% by weight, or at least 95% by weight, based on the total weight of protein in the aqueous solution of the retention solution product, and the surfactant / protein concentration ratio in the aqueous solution of the retention solution product produced at the end of step (c) is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, compared to the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0119] Embodiment 18 is a compound of formula I in which R 1 C 9~22This method is a linear alkyl group and is the method described in any one of the preceding embodiments.

[0120] Embodiment 19 is a compound of formula I in which R 1 C 11~15 This is the method according to Embodiment 18, wherein the alkyl group is linear.

[0121] Embodiment 20 is a compound of formula I in which X 2 This is a method according to any one of the prior embodiments, which is NH.

[0122] Embodiment 21 is a method according to any one of the preceding embodiments, wherein in the compound of formula I, n is 1, 2, 3, 4, or 5, preferably 1.

[0123] Embodiment 22 is a compound of formula I in which X 1 This is a method according to any one of the prior embodiments, which is NH.

[0124] Embodiment 23 is a compound of formula I in which n is 1 and R 2 This is a method according to one of the preceding embodiments, representing the side chain of a naturally occurring amino acid.

[0125] Embodiment 24 is a compound of formula I in which R 2 H, unsubstituted C 1~4 The method is one of any two embodiments 1 to 22, wherein the group is alkyl or -CH2-(C6H5).

[0126] Embodiment 25 is a compound of formula I in which R 3 The number-average molecular weight is 600 to 5000 daltons, preferably 800 to 3000 daltons, according to the method of any one of the prior embodiments.

[0127] Embodiment 26 is a compound of formula I in which R 1 R is a linear, unsubstituted alkyl group having 10 to 16 carbon atoms. 2R is selected from the group consisting of hydrogen, methyl, and -CH2-(C6H5) (where -(C6H5) is a benzene ring), 3 The number average molecular weight is 800 to 3000, and the method is according to any one of embodiments 1 to 17 and 20 to 22.

[0128] Embodiment 27 is a method according to any one of the preceding embodiments, wherein the ratio of propylene oxide (PO) units to ethylene oxide (EO) units in the compound of formula I is in the range of 0.01:1 to 2:1, preferably in the range of 0.05:1 to 1:1, and more preferably in the range of 0.1:1 to 0.5:1.

[0129] Embodiment 28 is a compound of formula I in which R 1 is CH3-(CH2) 11 -CH2-, n is 1, X 1 and X 2 Both are NH, and R 2 It is -CH2(C6H5), and R 3 The present invention relates to any one of Embodiments 1 to 17, wherein the copolymer is a CH3-terminated copolymer of PO and EO units, having a number-average molecular weight of approximately 1000 daltons and a PO / EO ratio of approximately 3:19.

[0130] Embodiment 29 is a compound of formula I in which R 1 is CH3-(CH2) 11 -CH2-, n is 0, X 2 NH is R 3 The present invention relates to any one of Embodiments 1 to 17, wherein the copolymer is a CH3-terminated copolymer of PO and EO units, having a number-average molecular weight of approximately 1000 daltons and a PO / EO ratio of approximately 3:19.

[0131] Example 30 is a method according to any one of the preceding embodiments, wherein the protein is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody-drug conjugates, bispecific antibodies, tripspecific antibodies, growth factors, insulin, immunoglobulins, peptide hormones, enzymes, polypeptides, fusion proteins, glycosylated proteins, antigens, antigen subunits, and combinations thereof.

[0132] Embodiment 31 is a method according to any one of the preceding embodiments, wherein the aqueous solution of step (a) further comprises a sugar selected from the group consisting of sucrose, glucose, mannose, trehalose, maltose, dextrose, dextran, and mixtures thereof. ru.

[0133] Embodiment 32 is the method according to any one of Embodiments 1 to 30, wherein the aqueous solution of step (a) further comprises a sugar alcohol selected from the group consisting of sorbitol, mannitol, xylitol, and mixtures thereof.

[0134] Embodiment 33 is the method of any one of the preceding embodiments, wherein the aqueous solution of step (a) further comprises a salt having a cation selected from the group consisting of hydrogen, sodium, potassium, magnesium, calcium, ammonium, and mixtures thereof, and the salt having an anion selected from the group consisting of fluoride, chloride, bromide, iodide, phosphoric acid, carbonic acid, acetic acid, citric acid, sulfuric acid, and mixtures thereof.

[0135] Embodiment 34 is a method according to any one of the prior embodiments, wherein the aqueous solution of step (a) further comprises naturally occurring amino acids.

[0136] Embodiment 35 is the method according to Embodiment 34, wherein the amino acid is selected from the group consisting of lysine, glycine, proline, arginine, histidine, and mixtures thereof.

Claims

1. A method for stabilizing a protein in an aqueous solution during the purification and / or concentration of the protein, comprising: (a) Proteins and general formula I: 【Chemistry 1】 (In the formula, R １ -C (=O) is a fatty acid acyl group, R ２ is H or a substituted or unsubstituted hydrocarbyl group, X １ is S, O, or NH, and X ２ is S, O, or NH, where n is 0 or an integer from 1 to 5, and R ３ General formulas II and III: 【Chemistry 2】 The steps include providing an aqueous solution containing a polyalkoxy fatty acid acyl surfactant (which is a polymer group containing polymerization units), (b) The step of bringing the aqueous solution into contact with the separation membrane, (c) A method comprising the steps of: (c) subjecting the aqueous solution to a diafiltration step to reduce the concentration of soluble low molecular weight components or to introduce one or more soluble low molecular weight components therein, and / or subjecting the aqueous solution to an ultrafiltration step to concentrate the protein in order to produce a retention solution product which is an aqueous solution containing the protein, A method wherein the compound of formula I reduces the aggregation of the protein in steps (a) to (c) of the method, and the compound of formula I passes through the separation membrane in step (c).

2. The method according to claim 1, wherein the surfactant / protein concentration ratio in the aqueous solution of the retaining liquid product generated at the end of step (c) is at least 50% lower than the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

3. The method according to claim 1 or 2, wherein the concentration of the compound of formula I in the aqueous solution provided in step (a) is about 0.001 mg / ml to about 5 mg / ml.

4. The method according to claim 1 or 2, wherein the molecular weight cutoff of the separation membrane is about 100 kDa, and the concentration of the compound of formula I in the aqueous solution in step (a) is about 0.005 mg / ml to about 1 mg / ml.

5. The method according to claim 1 or 2, wherein the molecular weight cutoff of the separation membrane is about 30 kDa to about 50 kDa, and the concentration of the compound of formula I in the aqueous solution in step (a) is about 0.01 mg / ml to about 0.1 mg / ml.

6. The method according to claim 1 or 2, wherein step (c) includes an ultrafiltration step, in which no dialysis filtration is performed before or after the ultrafiltration, the fractional molecular weight of the separation membrane is about 30 kDa to about 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is about 0.001 mg / ml to about 0.025 mg / ml.

7. The method according to claim 1 or 2, wherein step (c) comprises a dialysis filtration step followed by an ultrafiltration step, the fractional molecular weight of the separation membrane being about 30 kDa to about 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) being about 0.01 mg / ml to about 0.1 mg / ml.

8. The method according to claim 1 or 2, wherein step (c) comprises a dialysis filtration step, in which ultrafiltration is performed neither before nor after the dialysis filtration, the fractional molecular weight of the separation membrane is about 30 kDa to about 50 kDa, and the concentration of the compound of formula I in the aqueous solution of step (a) is about 0.01 mg / ml to about 0.1 mg / ml.

9. In the compound of formula I, X １ and X ２ The method according to any one of claims 1 to 8, wherein both are NH.

10. The method according to any one of claims 1 to 9, wherein in the compound of formula I, n is 1.

11. In the compound of formula I, R １ is a linear unsubstituted alkyl group having 10 to 16 carbon atoms, and R ２ is selected from the group consisting of hydrogen, methyl, and -CH ２ -(C ６ H ５ )(where -(C ６ H ５ ) is a benzene ring), and R ３ has a number average molecular weight of 800 to 3000, the method according to any one of claims 1 to 10.

12. In the compound of formula I, R １ CH ３ - (CH ２ ) １１ -CH ２ - and n is 1, X １ and X ２ Both are NH, and R ２ is, -CH ２ - (C ６ H ５ ) and R ３ CH has a number-average molecular weight of approximately 1000 daltons and a PO:EO ratio of approximately 3:

19. ３ The method according to any one of claims 1 to 8, wherein the copolymer is of PO and EO units with terminal encapsulation.

13. In the compound of formula I, R １ CH ３ - (CH ２ ) １１ -CH ２ - and n is 0, X ２ NH and R ３ CH has a number-average molecular weight of approximately 1000 daltons and a PO:EO ratio of approximately 3:

19. ３ The method according to any one of claims 1 to 8, wherein the copolymer is of PO and EO units with terminal encapsulation.

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