Bioprocess with reduced surface deposits

Abstract

The present disclosure provides (a) a protein and a compound of Formula I: [Case 1] TIFF2023535561000018.tif41170 (In the formula, R 1 -C(=O) is a fatty acid acyl group, and R 2 is H or a substituted or unsubstituted hydrocarbyl group, and X 1 is O or NH, and X 2 is O or NH, n is 0 or an integer of 1 to 5, and R 3 providing an aqueous solution containing a polyalkoxy fatty acyl surfactant of formula II; (b) subjecting the aqueous solution to a bioprocess; The present invention relates to a process including:

Description

[Technical Field]

[0001] This disclosure relates to a method for using polyalkoxy fatty acid acyl surfactants to reduce surface deposits in bioprocesses. [Background technology]

[0002] Biologics, protein-derived drugs, or other bio-derived polymers are rapidly developing as an important type of medicine due to their improved selectivity and reduced side effects compared to conventional small-molecule drugs. However, protein materials are relatively fragile, making the development of bioactive substances that are both therapeutically beneficial and sufficiently stable for processing, distribution, and administration a significant challenge. Surfactants can stabilize and protect proteins in solution by preventing adsorption to interfaces or by forming protective structures in solution. However, surfactants are often unsuitable for many steps in the biologic manufacturing process and are therefore not added until quite late in the process (e.g., final formulation). For example, surfactants can irreversibly adsorb to surfaces, resulting in surface deposits that clog pores / membranes / filters, reduce the surfactant concentration in solution, and limit the surfactant's ability to protect proteins in solution or further downstream, potentially hindering the bioprocess. Furthermore, deposits and clogging can lead to longer cleaning downtimes, potentially reducing process capacity and productivity. In some situations, it is necessary to allow biopharmaceuticals to reach and / or interact with a surface that is not shielded by a surfactant (e.g., a chromatography column or filter). [Overview of the Initiative] [Means for solving the problem]

[0003] This disclosure includes: (a) Proteins and formula I: [ka] (wherein, R [Figure 3C] , [Figure 3B] , [Figure 3A] , , , -C(=O) is a fatty acid acyl group, and R 2 is H or a substituted or unsubstituted hydrocarbyl group, X 1 is O or NH, X 2 is O or NH, n is an integer of 0 or 1 to 5, and R 3 is a polymer group containing polymerized units of Formulas II and III: [Chemical Formula] )(to provide an aqueous solution containing a polyalkoxy fatty acid acyl surfactant), and (b) subjecting this aqueous solution to a bioprocess, to provide a process comprising

[0004] Embodiments are described in the accompanying drawings to facilitate understanding of the concepts presented herein.

Brief Description of the Drawings

[0005] [Figure 1] It shows the aggregation rate of IgG (20 mg / mL) measured by DLS after shaking at room temperature for 24 hours with surfactants having different tail lengths, with the surfactant being 0.03 mg / mL and 0.05 mg / mL in physiological saline. [Figure 2] It is a graph showing the aggregation rate of IgG (20 mg / mL) measured by DLS before shaking, with surfactants having different tail lengths, with the surfactant being 0.03 mg / mL and 0.05 mg / mL in physiological saline. [Figure 3A] It shows a graph of representative DST of six FM1000 derivatives and IgG subjected to the test. [Figure 3B] It shows the ratio of the surface tension decrease due to the first decay to the overall surface tension decrease. [Figure 3C] The surface tension decrease at the first decay is normalized by the characteristic time of the first decay. [Figure 3D] This shows the decrease in surface tension caused by the second damping. [Figure 3E] This shows the characteristic time of the second decay. [Figure 4-1] This shows the QCM-D data. Figure 4A shows the relative adsorption mass of surfactant alone or IgG alone. Figure 4B shows the percentage washed away by surfactant alone or IgG alone. Figure 4C shows the relative amount of adsorbed IgG (in arbitrary units of 100), obtained by first calculating the difference between the adsorption mass of IgG and surfactant and the adsorption mass of surfactant alone, and then normalizing this by the adsorption mass of the IgG-only sample. Figure 4D shows the percentage of the total mass of IgG and surfactant that can be washed away. [Figure 4-2] Continuation of Figure 4-1. [Figure 5-1] This diagram illustrates the adsorption process of IgG and surfactants as tail length increases. In each set of diagrams, the left side shows surfactants and IgG with short tail lengths, the center shows surfactants and IgG with medium tail lengths, and the right side shows surfactants and IgG with long tail lengths. Figure 5A illustrates the initial adsorption (first decay) of the surfactant as revealed by DST. Figure 5B illustrates competitive adsorption as revealed by QCM-D. Figure 5C illustrates the adsorption at equilibrium (second decay) as revealed by DST. Figure 5D illustrates reversible adsorption as revealed by QCM-D. [Figure 5-2] Continuation of Figure 5-1. [Figure 6] This shows the recovery of surfactant after passing through the PVDF filter. Figures 6A and 6B show examples of FM1000 and PM80 chromatograms of samples taken at different points in the filtration process. The weight in mg on the right side of the graph represents the cumulative weight of the filtrate, and the chromatograms shown are those taken until the cumulative weight of the filtrate reached approximately 2000 mg. [Figure 7]This shows the recovery of surfactant after passing through the PES filter. Figures 7A and 7B show examples of FM1000 and PS80 chromatograms of samples taken at different points in the filtration process. The weight in mg on the right side of the graph represents the cumulative weight of the filtrate, and the chromatograms shown are for the portions taken until the cumulative weight of the filtrate reached approximately 2000 mg. [Figure 8] This shows the recovery of surfactants that have passed through a sulfopropyl-modified cross-linked agarose (SP HP) column. Figures 8A and 8B show examples of FM1000 and PS80 chromatograms of samples taken at different elution points. The weight in mg on the right side of the graph represents the cumulative weight of the eluate, and the chromatograms shown are those taken until the cumulative weight of the eluate reached approximately 3000 mg. [Figure 9] This shows the recovery of the surfactant that passed through the Protein A column. Figures 9A and 9B show examples of FM1000 and PS80 chromatograms of samples taken at different elution points. The weight in mg on the right side of the graph represents the cumulative weight of the eluate, and the chromatograms shown are those taken until the cumulative weight of the eluate reached approximately 3000 mg. [Figure 10] This shows the recovery of surfactants that have passed through a quaternary ammonium-modified cross-linked agarose (Q HP) column. Figures 10A and 10B show examples of FM1000 and PS80 chromatograms of samples taken at different elution points. The weight in mg on the right side of the graph represents the cumulative weight of the eluate, and the chromatograms shown are those taken until the cumulative weight of the eluate reached approximately 3000 mg. [Modes for carrying out the invention]

[0006] The above summary and the following detailed description are illustrative and descriptive 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 claims.

[0007] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof are intended to encompass non-exclusive inclusion as well. 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 enumerated or that are inherently present in such process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” means comprehensive or 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).

[0008] Furthermore, the use of "a" or "an" is used to describe the elements and components described herein. 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 the singular also includes the plural unless it is clear that it has a different meaning.

[0009] 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, the materials, methods and examples are illustrative and not intended to be limiting.

[0010] Where a quantity, concentration, or other value or parameter is given as a range, a preferred range, or a list of preferred upper and / or preferred lower limits, this should be understood as specifically disclosing any range formed by any pair of any upper or preferred value of the range and any lower or preferred value of the 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 as including 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.

[0011] The disclosures described herein by illustrative means can be suitably carried out in the absence of any one or more components or limitations not specifically disclosed herein. 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.

[0012] Before discussing the details of the embodiments described later, we will define or clarify some terms.

[0013] The terms "surface" and "interface" are used interchangeably herein.

[0014] The number-average molecular weight is defined as the total weight of a sample divided by the number of molecules contained in the sample.

[0015] 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).

[0016] Polyalkoxy compounds have the structure -(-AO) m The compound comprises one or more groups having the formula -(-AO)- (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 the various -(-AO)- groups may be identical or different from each other.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] When protein molecules are distributed in the form of individual molecules dissolved throughout a continuous liquid medium, the protein is considered to be a solution in the liquid medium (or, equivalently, dissolved in the liquid medium). When the continuous liquid medium contains water in an amount of 60% by weight or more based on the weight of the continuous liquid medium, the protein is considered to be dissolved in water.

[0021] A chemical group is an ionic group if a pH value of 4.5 to 8.5 exists and when in contact with water at that pH value, 50 mol% or more of the chemical group present takes an ionic form.

[0022] A buffering agent is either (i) a compound having the ability to accept a proton to form the conjugate acid of the compound and the pKa of the conjugate acid of the compound is less than 10, or (ii) a compound having the ability to release a proton and the pKa of the compound is greater than 4.

[0023] As used herein, the term "FM1000" means a polyalkoxy fatty acid acyl surfactant of formula I (where R 1 is CH3-(CH2) 11 -CH2-, n is 1, X 1 and X 2 are both NH, R 2 is -CH2(C6H5), and R 3 is a copolymer of PO and EO units end-capped with CH3 having an approximate number average molecular weight of 1000 and a PO to EO ratio of about 3:19). FM1000 has a hydrophobic tail (CH3-(CH2) 11 -CH2-C(=O)) with a length of 14 carbon atoms.

[0024] As used herein, the term "8FM1000" means a FM1000 derivative having a hydrophobic tail with 8 carbon atoms. That is, 8FM1000 has R 1The chemical formula is identical to that of FM1000, except that it is CH3-(CH2)5-CH2-. Similarly, the term "10FM1000" as used herein means an FM1000 derivative having a hydrophobic tail with 10 carbon atoms, i.e., 10FM1000 is R 1 The chemical formula is identical to FM1000 except that it is CH3-(CH2)7-CH2-; the term "12FM1000" as used herein means an FM1000 derivative having a hydrophobic tail with 12 carbon atoms, i.e., 12FM1000 is R 1 The chemical formula is identical to FM1000 except that it is CH3-(CH2)9-CH2-; as used herein, the term "16FM1000" means an FM1000 derivative having a hydrophobic tail with 16 carbon atoms, i.e., 16FM1000 is R 1 is CH3-(CH2) 13 Its chemical formula is identical to FM1000 except that it is -CH2-; as used herein, the term "18FM1000" means an FM1000 derivative having a hydrophobic tail with 18 carbon atoms, i.e., 18FM1000 is R 1 is CH3-(CH2) 15 Except for being -CH2-, its chemical formula is identical to that of FM1000.

[0025] The terms "FM1000" and "14FM1000" are used interchangeably in this specification.

[0026] This disclosure includes (a) proteins and 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 O or NH, and X 2 is O or NH, n is 0 or an integer from 1 to 5, and R 3 Equations II and III: [ka] To provide an aqueous solution containing a polyalkoxy fatty acid acyl surfactant (which is a polymer group containing polymerized units of the same), (b) subjecting this aqueous solution to a bioprocess, It provides a process that includes this.

[0027] As used herein, the term "bioprocess" refers to the downstream portion of a protein bioprocess, which involves processing proteins from an upstream source (e.g., biochemical production or synthesis) to meet purity and quality requirements. A bioprocess includes storage, transport, and purification.

[0028] In some embodiments, the bioprocess is selected from the group consisting of transport, filtration, chromatography, and combinations thereof.

[0029] The aqueous solution provided in step (a) contains a protein dissolved therein (for example, dissolved in water) and a polyalkoxy fatty acid acyl surfactant of formula I. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution in step (a) is 0.001 mg / ml to 1 mg / ml or 0.01 mg / ml to 0.1 mg / ml or 0.01 mg / ml to 0.05 mg / ml based on the total volume of the aqueous solution. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution in step (a) is 1 mg / ml or less or 0.5 mg / ml or less or 0.2 mg / ml or less or 0.1 mg / ml or less or 0.08 mg / ml or less or 0.06 mg / ml or less or 0.05 mg / ml or less based on the total volume of the aqueous solution. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution in step (a) is at least 0.001 mg / ml, or at least 0.002 mg / ml, or at least 0.005 mg / ml, or at least 0.01 mg / ml, or at least 0.02 mg / ml, or at least 0.03 mg / ml, based on the total volume of the aqueous solution.

[0030] In some embodiments, the concentration of protein in the aqueous solution in step (a) is 0.0001 mg / ml to 300 mg / ml or 0.0001 mg / ml to 200 mg / ml or 0.0001 mg / ml to 150 mg / ml or 0.001 mg / ml to 100 mg / ml or 0.01 mg / ml to 100 mg / ml or 0.1 mg / ml to 50 mg / ml or 0.1 mg / ml to 30 mg / ml or 0.1 mg / ml to 10 mg / ml or 10 mg / ml to 30 mg / ml, based on the total volume of the aqueous solution. In some embodiments, the concentration of protein in the aqueous solution in step (a) is 300 mg / ml or less, or 250 mg / ml or less, or 200 mg / ml or less, or 150 mg / ml or less, or 100 mg / ml or less, or 80 mg / ml or less, or 50 mg / ml or less, or 40 mg / ml or less, or 30 mg / ml or less, or 20 mg / ml or less, or 10 mg / ml or less, based on the total volume of the aqueous solution. In some embodiments, the concentration of protein in the aqueous solution in step (a) is at least 0.0001 mg / ml or at least 0.001 mg / ml or at least 0.002 mg / ml or at least 0.005 mg / ml or at least 0.01 mg / ml or at least 0.02 mg / ml or at least 0.05 mg / ml or at least 0.1 mg / ml or at least 0.2 mg / ml or at least 0.5 mg / ml or at least 1 mg / ml or at least 2 mg / ml or at least 5 mg / ml or at least 10 mg / ml, based on the total volume of the aqueous solution. In this disclosure, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I and the concentration of the protein are expressed as a weight / volume ratio (e.g., mg / ml).

[0031] R of the polyalkoxy fatty acid acyl surfactant of formula I 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. Preferably, R 1R is a linear alkyl group having 9 to 22 carbon atoms, or 9 to 18 carbon atoms, or 9 to 16 carbon atoms, or 10 to 17 carbon atoms, or 11 to 17 carbon atoms, or 11 to 15 carbon atoms, or 10 to 14 carbon atoms, or 11 to 13 carbon atoms. In some embodiments, R 1 is CH3-(CH2) 11 It is -CH2- or CH3-(CH2)9-CH2-. In some embodiments, R 1 is CH3-(CH2) 11 It is -CH2-.

[0032] In some embodiments, (when n is not 0), X 1 is NH. In some embodiments, X 2 It is NH.

[0033] In some embodiments, n is 0 or 1, 2, 3, 4, or 5. In some embodiments, n is 0 or 1. In some embodiments, n is 1. In some embodiments, n is 0.

[0034] In some embodiments, n is not 0, R 2 It has 20 or fewer atoms; 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 This represents the side chain of a naturally occurring amino acid.

[0035] In some embodiments, R 3The number-average molecular weight is 600 to 5000 daltons, preferably 800 to 3000 daltons. Preferably, R 3 The group is a statistical copolymer of (II) and (III) or a block copolymer of (II) and (III); more preferably, R 3 The group is a statistical copolymer of (II) and (III). Preferably, -R 3 is structure-R 4 -Has CH3, where R 4 is a polymer group containing polymerization units of structure (II) and structure (III). Preferably, R 4 It does not contain any polymerization units other than structure (II) and structure (III).

[0036] Characterizing the molar ratio of units of structure (II) to units of structure (III) (hereinafter referred to as the "PO / EO ratio") is useful. Preferably, the PO / EO ratio is 0.01:1 to 2:1, more preferably 0.05:1 to 1:1, and particularly 0.1:1 to 0.5:1. As used herein, the term "PO" refers to units of structure (II), and the term "EO" refers to units of structure (III).

[0037] In some embodiments, 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 an approximate number-average molecular weight of about 1000 daltons and a PO to EO ratio of about 3:19.

[0038] In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I does not have an ionic group.

[0039] In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of 12FM1000, FM1000, 16FM1000, 18FM1000 and mixtures thereof. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of 12FM1000, FM1000, 16FM1000 and mixtures thereof. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of 12FM1000, FM1000 and mixtures thereof. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of FM1000, 16FM1000 and mixtures thereof. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is FM1000.

[0040] The polyalkoxy fatty acid acyl surfactant of Formula I can be manufactured by the method disclosed in International Publication No. 2017 / 044366, the entirety of which is incorporated by reference as a part of this Specification for all purposes.

[0041] The polyalkoxy fatty acid acyl surfactant of formula I can be produced by any suitable method. A preferred method is the structure NH2-R 3 A compound having structure V: [ka] Compound and structure VI: [ka] (In the formula, X 3 The process involves reacting a compound selected from compounds of O, S, or NH. Preferred R 1 , X 2 , R 2 , R 3 And n have the same meaning as described above. Preferably, X 3 It is O.

[0042] A more preferred method for producing some embodiments of the polyalkoxy fatty acid acyl surfactant of formula I is as follows. In the first step, a fatty acid amide having a carboxyl group is produced by reacting an acyl chloride with an amino acid, as shown below. [ka]

[0043] Next, in the second step, a fatty acid amide having a carboxyl group is reacted with an amine-terminated polyalkoxy compound as shown below: [ka] (In the formula, PO is structure (II) and EO is structure (III)).

[0044] Preferred proteins included in this disclosure are selected from the group consisting of monoclonal antibodies, growth factors, insulin, immunoglobulins, polyclonal antibodies, antibody-drug conjugates, bispecific antibodies, tripspecific antibodies, hormones, enzymes, polypeptides, peptide fusions, glycosylated proteins, antigens, antigen subunits, and combinations thereof. Preferred proteins have therapeutic efficacy in treating diseases or conditions or functioning as vaccines. Examples of therapeutic proteins include immunoglobulin-G (IgG), 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, pegfilgrastim, secukinumab, streptokinase, cetuximab, omalizumab, ramucirumab, and urokinase. Ze, certolizumab pegol, dupilumab, golimumab, aldesleukin, morglamostim, pegylated interferon α-2b, tislerizumab, follitropin α, gevokizumab, golimumab, spartalizumab, canakinumab, foralumab, valrirumab, nimotuzumab, erythropoietin β, evolocumab, pegargiminase (pegarg iminase), bermekimab, carotuximab, daratumumab, eculizumab, ontuxizumab, adalimumab, camrelizumab, enobrituzumab, interleukin-12, lirirumab, panitumumab, gatipotuzumab, relatrimab, andecaliximab, belimumab, kabilizumab, isacituzumab govitecan(govitecan), monalizumab, pancreatin, pertuzumab, tripalimab, inebilizumab, ofatumumab, pepinemab, cintilimab, alirocumab, milatuzumab, nidanilimab, sotatercept, vedolizumab, bertuzumab, bevacizumab β, isatuximab, orlotamab, tisotumab vedotin, benralizumab, kosiberimab, emuctuzumab, ganitumab, narsoprimab, pidilizumab, sarilumab, trastuzumab emtansine, anetumab tansine, bertilimumab, blinatumomab, gusex These are lucumab, ixekizumab, mepolizumab, obinutuzumab, ubrituximab, alemtuzumab, emibetuzumab, ficratuzumab, ifabotuzumab, mirikizumab, natalizumab, lakosumomab, siltuximab, timigutuzumab, trastuzumab deruxtecan, bimekizumab, brodalumab, cetrerimab, farletuzumab, opinercept, lilonacept, tomzotuximab, urerumab, askrinbakumab, brolucizumab, crazakizumab, kusatuzumab, dalotuzumab, inalumab, itolizumab, and margetuximab. 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 intended. In some embodiments, the protein is an immunoglobulin. In some embodiments, the protein is immunoglobulin G (IgG). In some embodiments, the protein is bovine immunoglobulin G.

[0045] 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), and 2-butanone. In some embodiments, the solvent contains, essentially consists of, or comprises water and organic solvents. In some embodiments, the solvent contains at least 92% by weight, or at least 94% by weight, or at least 96% by weight, or at least 98% by weight, or at least 99% by weight, based on the total weight of the solvent. 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.

[0046] The aqueous solution optionally contains one or more additional components. The additional components are compounds other than water, protein, and polyalkoxy fatty acid acyl surfactant of formula I. Preferred additional components are sugars, sugar alcohols, salts, buffers, amino acids or salts of amino acids, or mixtures thereof. When such additional components are present, preferably the total amount of all additional components is 300 mg / ml or less, or 250 mg / ml or less, or 200 mg / ml or less, or 150 mg / ml or less, or 100 mg / ml or less, or 80 mg / ml or less, or 60 mg / ml or less, or 40 mg / ml or less, or 30 mg / ml or less, or 20 mg / ml or less, or 10 mg / ml or less, based on the total volume of the aqueous solution.

[0047] Preferred sugars to be included in the aqueous solution are sucrose, glucose, mannose, trehalose, maltose, dextrose, dextran, or mixtures thereof. Preferred sugar alcohols to be included in the aqueous solution are sorbitol, mannitol, or xylitol.

[0048] Preferred salts for inclusion in aqueous solutions have cations selected from hydrogen, sodium, potassium, magnesium, calcium, or ammonium, or mixtures thereof. Preferred salts have anions selected from fluoride, chloride, bromide, iodide, phosphoric acid, carboxylic acid, acetic acid, citric acid, or sulfuric acid, or mixtures thereof. Preferred buffers have cations selected from hydrogen, sodium, potassium, magnesium, calcium, or ammonium, or mixtures thereof.

[0049] The amino acids and their salts that are preferred to be included in the aqueous solution are selected from the group consisting of lysine, glycine, proline, arginine, histidine, and mixtures thereof.

[0050] In some embodiments, the aqueous solution is substantially free of other surfactants. As used herein, the term “other surfactants” means surfactants other than the polyalkoxy fatty acid acyl surfactant of formula I. In some embodiments, the other surfactants are selected from the group consisting of polysorbates, poloxamers, and mixtures thereof. In some embodiments, the aqueous solution is substantially free of polysorbate surfactants. In some embodiments, the aqueous solution is substantially free of poloxamer surfactants. In some embodiments, the concentration of the other surfactant in the aqueous solution is 0.01 mg / ml or less, or 0.005 mg / ml or less, or 0.002 mg / ml or less, or 0.001 mg / ml or less, or 0.0005 mg / ml or less, or 0.0002 mg / ml or less, or 0.0001 mg / ml or less, based on the total volume of the aqueous solution. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is the only surfactant present in the aqueous solution.

[0051] In some embodiments, the bioprocess in step (b) is filtration, i.e., the aqueous solution provided in step (a) is filtered in step (b) to produce an aqueous filtrate. In this type of step or process, the aqueous solution is passed through a filter to remove at least some of the contaminating proteins (e.g., host proteins, nucleic acids, protein aggregates, etc.). The proteins and the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution pass through the filter together to produce a filtrate, while the contaminating proteins are retained in the filter.

[0052] In some embodiments, the filter is selected from the group consisting of PVDF filters, PES filters, polypropylene filters, cellulose filters, nylon filters, and combinations thereof. In some embodiments, the filter is a PVDF filter. In some embodiments, the filter is a PES filter. Typically, the filter comprises a separation membrane. As used herein, the term “separation membrane” means a porous membrane used in a filtration process to separate components in an aqueous solution based on their molecular weight or size. As used herein, the term “PVDF filter” means a filter having a separation membrane made of polyvinylidene fluoride (PVDF). As used herein, the term “PES filter” means a filter having a separation membrane made of polyethersulfone (PES). As used herein, the term “polypropylene filter” means a filter having a separation membrane made of polypropylene. As used herein, the term “cellulose filter” means a filter having a separation membrane made of cellulose. As used herein, the term “nylon filter” means a filter having a separation membrane made of nylon.

[0053] In some embodiments, the pore size of the filter or the separation membrane within it is approximately 0.1 μm to approximately 1 μm or approximately 0.1 μm to approximately 0.5 μm. In some embodiments, the pore size of the filter or the separation membrane within it is approximately 0.2 μm. In some embodiments, the filtration process is carried out at room temperature. In some embodiments, the filtration process excludes ultrafiltration and / or diafiltration.

[0054] It has been found that the polyalkoxy fatty acid acyl surfactant of formula I can effectively prevent the absorption or loss of proteins by the separation membrane. Furthermore, the absorption or loss of the polyalkoxy fatty acid acyl surfactant of formula I by the separation membrane is small or minimal. In some embodiments, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% of the total weight of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution supplied to the filter for filtration passes through the filter.

[0055] In some embodiments, the protein passes through the filter at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% of the total weight of the protein supplied to the filter for filtration.

[0056] In some embodiments, the surfactant / protein concentration ratio in the aqueous solution provided in step (a) is substantially equal to the surfactant / protein concentration ratio in the aqueous solution of the filtrate, i.e., the surfactant / protein concentration ratio in the aqueous solution remains substantially unchanged after passing through the filter. In some embodiments, the surfactant / protein concentration ratio in the aqueous solution of the filtrate is within ±5%, ±10%, ±15%, or ±20% of the surfactant / protein concentration ratio in the aqueous solution provided in step (a).

[0057] The polyalkoxy fatty acid acyl surfactant of formula I is a mixture of polymer components with different molecular weights. Typically, the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution provided in step (a) (the polymer components in the mixture and their respective concentrations) is substantially the same as the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution of the filtrate; that is, the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution remains substantially the same even after passing through the filter.

[0058] In some embodiments, the bioprocess in step (b) is chromatography, i.e., the aqueous solution provided in step (a) passes through a chromatography resin (stationary phase) contained in a chromatography column in step (b) so that at least some of the contaminating proteins (e.g., host cell proteins, nucleic acids, protein aggregates, etc.) can be separated from the protein. In some embodiments, the protein is retained in the chromatography column, while the contaminating proteins pass through the chromatography column. In this type of embodiment, after chromatography, a recovery aqueous solution containing a polyalkoxy fatty acid acyl surfactant of formula I can be used to recover or remove the retained protein from the chromatography column. In some embodiments, the recovery aqueous solution is a buffer.

[0059] In some embodiments, the contaminating protein is retained in the chromatography column, while the protein and the polyalkoxy fatty acid acyl surfactant of formula I in aqueous solution pass through the chromatography column. In some embodiments, the chromatography process is carried out at room temperature.

[0060] During the chromatography process, the polyalkoxy fatty acid acyl surfactant of formula I in aqueous solution passes through the chromatography column. The amount of polyalkoxy fatty acid acyl surfactant of formula I absorbed or lost by the chromatography resin is small or minimal. In some embodiments, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% of the total weight of the polyalkoxy fatty acid acyl surfactant of formula I in aqueous solution supplied to the chromatography column passes through the chromatography column.

[0061] Typically, the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution provided in step (a) is substantially the same as the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution (i.e., eluate) that has passed through the chromatography column; that is, the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution remains substantially the same even after passing through the chromatography column.

[0062] A chromatography resin (stationary phase) is contained within the chromatography column. In some embodiments, the chromatography resin is selected from the group consisting of sulfopropyl-modified crosslinked agarose, Protein A, quaternary ammonium-modified crosslinked agarose, hydrophobic interaction chromatography resins, and combinations thereof. Those skilled in the art will understand that Protein A is a 49 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. An example of a hydrophobic interaction chromatography resin is agarose with a butyl substituent. In some embodiments, the chromatography resin is sulfopropyl-modified crosslinked agarose. In some embodiments, the chromatography resin is Protein A. In some embodiments, the chromatography resin is quaternary ammonium-modified crosslinked agarose.

[0063] In some embodiments, the bioprocess is transport, i.e., the bioprocess involves transporting an aqueous solution in a container or through a conduit. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of 12FM1000, FM1000 and mixtures thereof. In some embodiments, the polyalkoxy fatty acid acyl surfactant of formula I is FM1000. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution is about 0.01 mg / ml to about 0.1 mg / ml or about 0.02 mg / ml to about 0.08 mg / ml or about 0.02 mg / ml to about 0.06 mg / ml or about 0.03 mg / ml to about 0.05 mg / ml based on the total volume of the aqueous solution. In some embodiments, the concentration of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution is about 0.03 mg / ml based on the total volume of the aqueous solution.

[0064] It has been found that the polyalkoxy fatty acid acyl surfactant of formula I can effectively reduce the aggregation of proteins in aqueous solutions during transport. In some embodiments, the aqueous solution at the end of transport contains, based on the total weight of proteins in the aqueous solution, 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.

[0065] 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]

[0066] 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.

[0067] Proteins are known to adsorb to interfaces between water, air, oil, and solid surfaces, which often leads to aggregation and denaturation. Furthermore, the violent shaking that frequently occurs during transport can exacerbate this harmful effect. Several methods for stabilizing these therapeutic proteins include the use of pharmaceutical additives such as sugars, salts, amino acids, and surfactants. Surfactants are particularly useful for stabilizing and protecting proteins in solution through two mechanisms: (1) competitive adsorption, where the surfactant overcomes competition with the protein to gain space on the surface where it could denature and aggregate; and (2) preferential association, where the surfactant stabilizes the protein structure by directly interacting with the protein or prevents protein-protein interactions that could cause aggregation.

[0068] While we do not wish to be bound by any particular theory, both of these mechanisms play some role in stabilization, but generally, the first mechanism is considered to be the primary one. Currently available surfactants, such as polysorbate 20 and 80, used for protein stabilization, reduce protein aggregation compared to formulations that do not contain this type of surfactant. When the protein and surfactant solution is kept isothermally at 65°C, 14FM1000 reduces the growth rate of immunoglobulin G (IgG) aggregates more effectively than polysorbate 20 and 80. While we do not wish to be bound by any particular theory, the results with 14FM1000 are thought to be due to its ability to rapidly migrate and adsorb to various interfaces, such as water-air and water-oil interfaces, as demonstrated by dynamic surface tension (DST) measurements.

[0069] To understand the protein-stabilizing ability of surfactants and to identify the structural properties of surfactants that alter their protein-stabilizing effect, six FM1000 derivatives with hydrophobic tail lengths ranging from 8 to 18 carbon atoms were synthesized and tested. Through experiments, it was found that the hydrophobic tail length significantly influences the ability of surfactants to stabilize IgG, a model protein therapeutic agent. The hydrophobic tail length affects the rate of adsorption and the reversibility of adsorption. Hydrophobic tails of medium length, such as the 14-carbon hydrophobic tail (i.e., 14FM1000), reduce surface tension most rapidly and significantly, and exhibit the highest reversibility of adsorption. Such rapid dynamics correlate with the surfactant's ability to stabilize IgG, and 14FM1000 maximizes the effect of minimizing aggregation. This disclosure elucidates the structural and functional relationship between the hydrophobic tail length of surfactants and protein stabilization.

[0070] material Myristoyl chloride, the hydrogen-type strong acid ion exchange resin Amberlite IR-120, and carbonyl diimidazole were purchased from Sigma Aldrich (St. Louis, MO). The cationic (OH-) ion exchange resin Amberlite IRN-78 was purchased from Thermo Fisher Scientific (Waltham, MA). N-hydroxysuccinimide was purchased from Acros Organics (Fair Lawn, NJ). L-phenylalanine was purchased from TCI Chemicals (Portland, OR). Jeffamine M1000 was obtained from Huntsman (The Woodlands, TX). Polysorbate 80 and polysorbate 20 were purchased from Sigma. All chemicals were used in the condition they were received, without further purification.

[0071] Silicone sheets (Dow Corning C6-150) supplied by DuPont were used. Infusion bags were obtained from Baxter Healthcare Corp., cut open, emptied of the saline solution, washed with Milli-Q water, and dried. Polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyethersulfone (PES), and polyethylene (PE) sheets were all obtained from Goodfellow Corp., with a thickness of 0.5 mm and dimensions ranging from 150 × 150 mm to 300 × 300 mm. After cutting them into 3.5 × 1 cm pieces, all surfaces were immersed in various solutions.

[0072] PVDF filters were obtained from Fisher Scientific (Fisherbrand, 33 mm diameter, 0.2 μm), and PES filters were obtained from Millipore Sigma (Millex-GP, 33 mm diameter, 0.22 μm). Chromatographic columns, including sulfopropyl-modified cross-linked agarose columns and Protein A columns (GE Healthcare), were obtained from Cytiva, and all were 1 mL in volume.

[0073] Milli-Q grade water was used. Industrial-grade bovine IgG (immunoglobulin G) was purchased from MP Biomedicals (Santa Ana, CA). Bovine IgG was dissolved in 0.9 wt% physiological saline at a concentration of 40 mg / mL, filtered through a 0.2 μm PVDF filter, and diluted to the corresponding concentration.

[0074] Synthesis of FM1000 derivatives (FM1000 or 14FM1000) having a hydrophobic tail with 14 carbon atoms: Step 1: In a 500 mL round-bottom flask equipped with a stirrer bar, L-phenylalanine (0.0500 mol, 8.26 g), sodium hydroxide (0.0500 mol, 2.00 g) and triethylamine (0.0540 mol, 7.56 mL) were added in DI (deionized) water (250 mL). The mixture was stirred at room temperature (RT) for 1 minute until dissolved. Then, myristoyl chloride (0.0500 mol, 13.6 mL) was slowly added. The reaction mixture was stirred at room temperature (RT) for 1 hour. Then, 5 mL of concentrated HCl was slowly added. The off-white precipitate formed by the addition of the acid was collected by suction filtration, washed with 500 mL of water, and dried overnight. The product was then dissolved in 1500 mL of boiling ethyl acetate and dried over magnesium sulfate. The magnesium sulfate was filtered off, and the ethyl acetate was removed using a rotary evaporator. The product was then dissolved in boiling hexane and slowly cooled in the freezer. The white precipitate formed during this time was collected by suction filtration. Since myristic acid, an impurity, was identified by NMR, the product was dissolved again in boiling hexane and slowly cooled in a freezer. The white precipitate formed during this time was collected by suction filtration. The resulting white powder was dried overnight in a vacuum desiccator (7.9741 g, 43%).

[0075] Step 2: In a 25 mL round-bottom flask equipped with a stirrer bar, n-myristoylphenylalanine (product from Step 1) (0.00100 mol, 0.375 g) and DCM (dichloromethane, 10 mL) were added. The round-bottom flask was stoppered with a septum and purged with N2. Then CDI (1,1'-carbonyldiimidazole, 0.00120 mol, 0.194 g) was added to the reaction mixture, the flask was again stoppered with a septum, and the mixture was again purged with N2. The reaction mixture was then stirred at RT for 4 hours. Next, Jeffamine M1000 (0.00120 mol, 1.17 g) was dissolved and added to the reaction mixture by syringe. The reaction mixture was stirred at RT for 68 hours. Next, the DCM was evaporated using a rotary evaporator, and 150 mL of methanol was added together with the ion exchange resins, Amberlite IRN-78 cationic (OH-) ion exchange resin and Amberlite IR-120 hydrogen-type strong acid ion exchange resin, which had been pre-washed with methanol. This mixture was stirred at RT for 2 hours. The resins were removed by vacuum filtration using frit. Methanol was removed from the solution obtained by vacuum filtration. The product was then dissolved in 400 mL of 10% methanol / DCM and passed through an SiO2 plug. The filtrate was concentrated using a rotary evaporator to obtain a white waxy substance, which was dried overnight in a vacuum oven at 60°C (1.6 g, 49%).

[0076] Shaking test All samples were prepared in 0.9 wt% physiological saline (9 g of NaCl in 1000 mL of Milli-Q water) and contained 20 mg / mL of IgG. A control sample without surfactant was prepared. Other samples containing 0.03 or 0.05 mg / mL of surfactants with different tail lengths were also prepared for the shaking test. The surfactants used were 8FM1000, 10FM1000, 12FM1000, 14FM1000, 16FM1000, and 18FM1000. 8, 10, 12, 14, 16, and 18 represent the tail length (number of carbon atoms) of the surfactant, respectively. The term "tail length" as used herein refers to R 1 This refers to the length of the (CH3-(CH2)) hydrophobic tail. For example, 14FM1000 has a hydrophobic tail length of 14 carbon atoms.11 -CH2-C(=O)).

[0077] Agglutination of IgG protein was induced by stirring. The sample was arranged in a four-component system and shaken at 188 reciprocations / minute on a Thermo reciprocating shaker at room temperature for 24 hours. 0.7 mL of the test sample was placed in each 8 × 43 mm glass vial (Kimble, catalog no. 60831D-843) with a capacity of approximately 1 mL, and the vials were capped with Piercable TPE Lyo Capcluster-96 (Micronic, Aston, PA) stoppers. The vials were arranged in a 96-well configuration in a custom aluminum holder. The effect of the surfactant in preventing agglutination induced by stirring was confirmed by analyzing the shaken samples using dynamic light scattering (DLS) with a Wyatt DynaPro II instrument (Wyatt Technology, Santa Barbara, CA). In addition, the same sample before shaking was analyzed by DLS as a "no-shaking" control (i.e., "0 hours of shaking"). Each well was scanned 5 times, with an acquisition time of 5 seconds per scan. The size and hydrodynamic radius of IgG aggregates were determined by fitting using regularization. Aggregates larger than 10 nm were considered IgG aggregates, and accordingly, assuming a Rayleigh sphere, the intensity percentages were converted to mass percentages and quantified in mass percentage.

[0078] This shaking test was used to understand the ability of surfactants to stabilize proteins, and the shaking was used to accelerate destabilization by constantly agitating the hydrophobic surface. Furthermore, stirring mimics transport conditions that increase IgG aggregation. In Figures 1 and 2, the leftmost bar represents a control sample without surfactant. Figure 1 shows IgG aggregation after 24 hours of shaking, and Figure 2 shows IgG aggregation before shaking. As shown in Figure 2, in the unshaken control, IgG aggregation was essentially 0% at all two concentrations of surfactant tested. Figure 1 shows that 14FM1000 at concentrations of 0.03 mg / ml and 0.05 mg / ml relative to the total volume of the aqueous solution effectively prevents IgG protein aggregation. Furthermore, Figure 1 shows that 12FM1000 at a concentration of 0.05 mg / ml relative to the total volume of the aqueous solution also effectively prevents IgG protein aggregation.

[0079] Generally, a moderate tail length was found to be most effective in preventing IgG aggregation, while shorter or longer tail lengths increased IgG aggregation. When comparing IgG aggregation between surfactant concentrations of 0.03 mg / mL and 0.05 mg / mL, aggregation was almost 0% with 12FM1000 at the higher surfactant concentration (0.05 mg / mL), but some aggregation (1-2%) was observed at the lower concentration of 0.03 mg / mL. Samples using 8FM1000 and 10FM1000 surfactants aggregated heavily (4-7%) at 0.03 mg / mL. At the higher concentration of 0.05 mg / mL, 8FM1000 showed almost the same amount of aggregation as at the lower concentration, but 10FM1000 showed a decrease in aggregation (2-3%). The decrease in IgG aggregation with increasing 10FM1000 concentration is similar to the trend observed when 12FM1000 concentration was increased in the same way. Aggregation was approximately 2-3% for both 16FM1000 and 18FM1000, and there was no significant difference between the two concentrations tested.

[0080] Dynamic surface tension measurement All samples were prepared in 0.9 wt% physiological saline (9 g of NaCl in 1000 mL of Milli-Q water). The surfactants used were 8FM1000, 10FM1000, 12FM1000, 14FM1000, 16FM1000, and 18FM1000, respectively, and the protein was IgG. Seven types of samples were prepared; one contained 10 mg / mL of IgG in 0.9% physiological saline, and the other six samples each contained the respective surfactant at a concentration of 0.05 mg / mL in 0.9% physiological saline.

[0081] Surface tension measurements were performed using a Teclis Tracker pendant drop type surface tension meter (Teclis Scientific, Civrieux d'Azergues, France) via RT (Restorative Time). A 25 ml cuvette was filled with the sample. A bubble was formed using an 18-gauge J-shaped needle. The instrument's feedback control was used to ensure a constant droplet area. Surface tension was determined by fitting the bubble contour to Laplace's equation. The surface tension of the samples was monitored in three sets for 3000 seconds (12 FM 1000, 14 FM 1000, 16 FM 1000, 18 FM 1000, and IgG) or 6000 seconds (8 FM 1000 and 10 FM 1000). Initially, measurements were taken every 0.1 seconds. After 10 seconds, measurements were taken every 1 second.

[0082] Dynamic surface tension (DST) measurements are useful for understanding the kinetics of adsorption and rearrangement of substances at interfaces. The DST at the air / water interface was measured for each surfactant at 0.05 mg / mL and IgG at 10 mg / mL (see Figure 3A). Each DST curve was fitted to a double exponential decay function to qualitatively represent the dynamics of adsorption and rearrangement at the interface (Equation 1). σ(t) is the surface tension at time t, where σ eq θ1 is the surface tension at equilibrium or after infinite time. Furthermore, τ1 is the characteristic time of the faster decay, and θ1 is the decrease in surface tension resulting from this initial decay. τ2 and θ2 correspond to those of the slower decay.

number

[0083] The polyalkoxy fatty acid acyl surfactant of formula I has a hydrophilic head (-R 3 ) and hydrophobic tail (-R 1 It is thought that the ) rearranges and is adsorbed onto the surface. The polyalkoxy fatty acid acyl surfactant of formula I is thought to have initial adsorption and possibly its hydrophilic polymer head (-R 3 Assuming that some form of conformational adjustment occurs due to ) and that two types of surface tension attenuation result from , the first attenuation is thought to correspond to the initial adsorption of the surfactant to the surface (τ1, θ1), and the second attenuation is thought to correspond to the surfactant molecules changing their conformation to the equilibrium orientation (τ2, θ2). Hydrophilic head of polymer and hydrophobic tail of hydrocarbon (-R 1 ) may be adjusting the three-dimensional arrangement.

[0084] 14FM1000 was found to exhibit the highest rate of surface tension reduction during the initial decay (Figure 3B). Compared to 14FM1000, the rate of surface tension reduction due to the initial decay decreases as the tail length increases or decreases. This indicates that the initial adsorption of 14FM1000 is maximized compared to other surfactants due to its tail length. Furthermore, when the initial decay is normalized by the decay time constant, as can be seen in Figure 3C, 14FM1000 reduces surface tension most significantly in the shortest amount of time. In other words, 14FM1000 reaches the surface much faster than others.

[0085] Interestingly, when examining the decrease in surface tension due to the second decay, the opposite trend is observed: those with shorter and longer tails (compared to 14FM1000) both showed the greatest decrease in surface tension during the second decay (see Figure 3D). While we do not wish to be bound by any particular theory, this trend may stem from the fact that longer and shorter tails require a greater change in stereochemistry to reach equilibrium. In the case of longer tails, the hydrophobic tail must rearrange most of it so that it can be adsorbed in its equilibrium orientation. In all surfactants tested, the hydrophilic head may be adsorbed via either the PEO (polyethylene oxide), PPO (polypropylene oxide), or phenylalanine region, thereby significantly reducing surface tension. This adsorption of the hydrophilic head may be more pronounced in shorter tails because the tail itself is less hydrophobic. While we do not wish to be bound by any particular theory, the rapid adsorption of 14FM1000 may be due to the hydrophobic tail being short enough that it does not require significant rearrangement, while at the same time possessing sufficient hydrophobicity to not significantly adsorb PEO, PPO, or phenylalanine at equilibrium. Therefore, the decrease in surface tension due to rearrangement is very small. The characteristic time for the second decay (τ2) decreases as the length of the hydrophobic tail increases (see Figure 3E). While we do not wish to be bound by any particular theory, this is because the more hydrophobic tail has a stronger thermodynamic driving force to rearrange and minimize its (higher) energy. The rearrangement of the polymer's hydrophilic head may also influence this trend regarding the characteristic time for the second decay, and this seems to become more important as the hydrophobicity of the tail decreases (shorter). Because the polymer's hydrophilic head has a larger molecular weight, it takes considerably longer to change its stereochemistry compared to the tail, and the time required for this rearrangement may be influencing the trend of τ2. Figure 3 demonstrates that 14FM1000 has an ideal hydrophobic tail length that rapidly and significantly reduces surface tension in its initial stages.

[0086] The amount and rate at which 14FM1000 adsorbs increases its ability to overcome competition with IgG attempting to adsorb to the surface, ultimately preventing IgG aggregation. Furthermore, combining the DST data and aggregation data, the longer and shorter (compared to 14FM1000) tails indicate that slower adsorption to the surface gives IgG more time to adsorb to the hydrophobic surface and aggregate, resulting in more IgG aggregation. It is thought that the reason 14FM1000 is superior to polysorbate 20 and 80 in terms of its ability to prevent protein aggregation lies in these quick adsorption dynamics. Moreover, if the polyalkoxy fatty acid acyl surfactant of formula I can replace IgG, then surfactants that can reach the surface more quickly will replace more proteins, thereby preventing further aggregation. Interestingly, 8FM1000 is the only surfactant that has a higher surface tension value than IgG at all measurements from approximately 5 seconds onward (see Figure 3A). This suggests that 8FM1000 covers the surface at a rate slower than or about the same rate as IgG, which likely explains why 8FM1000 did not adequately prevent IgG aggregation at either of the concentrations tested.

[0087] Quartz crystal oscillator microbalance combined with dissipative measurement. All samples were prepared in 0.9 wt% physiological saline (9 g of NaCl in 1000 mL of Milli-Q water). The surfactants used were 8FM1000, 10FM1000, 12FM1000, 14FM1000, 16FM1000, and 18FM1000, respectively, and the protein was IgG. The sample solutions were prepared to contain either 0.05 mg / mL of surfactant alone in physiological saline, 1 mg / mL of IgG alone in physiological saline, or a combination of 0.05 mg / mL of surfactant and 1 mg / mL of IgG in physiological saline.

[0088] Measurements were performed using a quartz crystal microbalance with dissipation (QCM-D) equipped with an energy dissipation measurement function, using a QSense Analyzer (Biolin Scientific, Gothenberg, Sweden) with an SiO2-coated quartz crystal (model QSX 303). The sample solution was flowed onto the quartz crystal at a rate of 150 μL / min until equilibrium was reached, and the amount of adsorbed substance was determined. Next, a 0.9 wt% physiological saline solution was flowed onto the quartz crystal at a rate of 150 μL / min until equilibrium was reached, and the amount of surfactant and / or protein that could be washed off after being adsorbed onto the crystal was determined. The change in the third harmonic was monitored, and the relative adsorbed mass was determined according to the Sauerbrey equation, which assumes that the adsorbed mass is proportional to the change in frequency. For solutions containing only surfactant or surfactant and IgG, the relative adsorbed mass was determined by taking the average over the first 10 to 40 minutes after the start. The washing efficiency was determined by calculating the relative adsorption mass before washing with physiological saline and comparing it with the change in average adsorption mass after washing with physiological saline for 40 minutes. Furthermore, the adsorption rate of IgG was calculated by determining the difference between the average adsorption mass over 10 to 40 minutes and the values ​​for each surfactant that did not contain IgG (Figure 4C). All data were normalized to the average adsorption mass when using a sample containing only IgG (100 arbitrary units).

[0089] Using QCM-D, we will monitor the change in the resonant frequency of a silicone-coated quartz crystal oscillator to determine the mass of IgG and surfactant adsorbed on a hydrophobic solid surface. Furthermore, by utilizing washing tests, we will understand whether the adsorption is reversible or irreversible and elucidate how the surfactant and IgG interact with the surface. A sample solution containing only 0.05 mg / mL of surfactant was flowed onto the oscillator surface, and the change in resonant frequency over time was measured. Generally, the longer the tail length of the surfactant, the greater the amount of surfactant adsorbed (see Figure 4A).

[0090] Next, the amount of reversibly desorbable surfactant that could be washed away was measured by flowing a 0.9 wt% physiological saline solution over the oscillator surface. It was found that the longer the tail compared to 14FM1000, the less surfactant could be washed away (see Figure 4B). From 8FM1000 to 14FM1000, the longer the tail, the higher the proportion of surfactant that could be washed away (see Figure 4B). It is thought that surfactants with shorter and longer tails (compared to 14FM1000) are more irreversibly adsorbed due to rearrangement of the surfactant on the surface. This is consistent with the results of DST measurements. It was also found that IgG was the least reversibly adsorbed. Figures 4A and 4B show the QCM-D measurement results for samples containing only surfactant and samples containing only IgG.

[0091] A sample solution containing both IgG and a surfactant was flowed onto the surface of the vibrator. Compared to the case where only the surfactant sample was flowed, the amount of adsorbed IgG is expected to increase because IgG is adsorbed. As shown in Figure 4C, generally, the longer the tail of the surfactant, the less IgG was adsorbed, but in the case of 16FM1000 and 18FM1000, the amount of adsorbed IgG increased slightly. This is probably because, as observed in DST for these samples, the adsorption rate was slower, and IgG was able to overcome the competition with 16FM1000 and 18FM1000 in the early stages of time. In Figure 4C, the relative amount of adsorbed IgG was obtained by first subtracting the adsorbed mass of the surfactant-only sample from the adsorbed mass of the corresponding sample containing both surfactant and IgG, and then dividing this result by the adsorbed mass of the IgG-only sample (i.e., the subtracted result was normalized by the adsorbed mass of the IgG-only sample (100 arbitrary units)). This data is consistent with the conclusions obtained from DST that 14FM1000 has an optimal tail length to prevent IgG adsorption by rapidly adsorbing and / or to replace already adsorbed IgG before irreversible adsorption occurs. Furthermore, when IgG and surfactant were washed away together, the inventors confirmed that the reversible adsorption of the 14FM1000 surfactant was maximized compared to those with longer or shorter tail lengths, as surfactants with longer tail lengths or IgG (when surfactants with shorter tail lengths were used) were irreversibly adsorbed (see Figure 4D).

[0092] Shaking tests and DST and QCM-D experiments demonstrated that the hydrophobic tail length of each surfactant, which is thought to affect the ability to prevent IgG adsorption and subsequent aggregation, influences the rate, amount, and reversibility of surfactant adsorption (see Figure 5). Regarding initial adsorption of surfactants, short tails such as 8FM1000 have minimal force toward the surface and therefore hardly adsorb at all. Furthermore, longer tails such as 18FM1000 adsorb slowly during the initial decay, thus adsorbing IgG as well. Surfactants with tails of intermediate length, such as 14FM1000, exhibit fast and strong initial adsorption (see Figure 5A). Because surfactant 14FM1000, with its intermediate tail length, can adsorb quickly, it can overcome competition with IgG for surface adsorption and significantly reduce surface tension without rearrangement. Surfactants with shorter tails (e.g., 8FM1000) do not adsorb as strongly, perhaps because they need to rearrange their stereochemistry to reduce surface tension. Therefore, IgG can overcome the competition with these and begin to aggregate on the surface. In contrast, 18FM1000 and other longer tails have a strong force toward the surface and overcome the competition with IgG, but some IgG has already aggregated before enough 18FM1000 can reach it (see Figure 5B). While we do not wish to be bound by any particular theory, the hydrophobic tail of 8FM1000 is not sufficiently hydrophobic, and therefore other parts of the surfactant, such as phenylalanine, PPO, or PEO units, are likely to be adsorbed as well. Also, the hydrophobic tail of 18FM1000 may change its stereochemistry, so the longer tail will aggregate effectively on the surface. This leads to equilibrium adsorption that affects reversibility on the surface (see Figure 5C). Finally, as shown in the saline wash in QCM-D, the adsorption of 14FM1000 is more reversible (see Figure 5D). While we do not wish to be bound by any particular theory, this is thought to be related to the small decrease in surface tension during the second decay, suggesting that there is a smaller change in stereochemistry that allows 14FM1000 to adhere more strongly to the surface.On the other hand, surfactants such as 8FM1000 and 18FM1000, as well as IgG, adsorb more irreversibly in an attempt to stabilize them. Furthermore, washing with physiological saline simulates changes in surface area due to arbitrary movements or shaking during transport, suggesting that 14FM1000 is superior in protecting IgG from aggregation on newly formed surfaces and preventing adhesion to other transient surfaces.

[0093] 14FM1000 was found to adsorb quite rapidly to surfaces, thereby preventing the adsorption and consequent aggregation of IgG. 14FM1000 had the fastest initial adsorption rate compared to other surfactants tested. Surfactants with short tails adsorbed slowly and did not adsorb significantly to surfaces, allowing IgG adsorption. Similarly, surfactants with long tails also adsorbed slowly, enabling IgG adsorption and aggregation, although their equilibrium adsorption was strong. Furthermore, 14FM1000 exhibited the highest reversibility of adsorption, and therefore likely possessed a high ability to rapidly desorb and adsorb to transient surfaces, thus protecting IgG and preventing aggregation on each new hydrophobic surface. Understanding the relationship between surfactant structure and activity related to protein stabilization is useful for designing surfactants that improve the stability and efficacy of protein therapeutics.

[0094] contact angle measurement To investigate the protein adhesion prevention activity of 10FM1000, 14FM1000 (FM1000), and 18FM1000, contact angle measurements were performed on various polymer surfaces and compared with polysorbate 80 (PS80) and polysorbate 20 (PS20). In bioprocesses, biopharmaceuticals are exposed to many polymer surfaces (tubes, filters, storage containers, etc.), and the biopharmaceuticals may be adsorbed there. This not only leads to the loss of expensive materials and increases the risk of biopharmaceutical aggregation, but can also disrupt the structure and function of the therapeutic agent. Surfactants can prevent adhesion to surfaces through fast kinetics at the interface. (Wang W. Protein aggregation and its inhibition in biopharmaceutics, Int J Pharm 2005 Jan.31;289(1-2):1-30).

[0095] In contact angle measurements, surface hydrophilicity was evaluated by measuring the angle between a water droplet and the surface beneath it. A higher angle indicates higher surface hydrophobicity, while a lower angle indicates a greater affinity between the hydrophilic surface and water. Different surfaces, selected to represent various bioprocess materials, were immersed in saline solution containing immunoglobulin G (IgG), saline solution, or a mixture of surfactant and IgG in saline solution. The saline solution used herein is a 0.9 wt% saline solution (9 g of NaCl in 1000 mL of Milli-Q water). The surface behaved closer to its original state in the control saline solution, and the contact angle tended to be higher. However, when immersed in IgG alone (in saline solution), a hydrophilic coating formed, and the contact angle measurement decreased. The contact angle values ​​obtained for saline solutions containing different concentrations of surfactant (0.001 to 0.1 mg / mL) fell between the saline control values ​​and the IgG control values. Values ​​closer to the physiological saline control indicate that the surfactant has the ability to prevent protein adhesion, while values ​​closer to the IgG control indicate that it failed to prevent protein adsorption to the surface. Partial protein adhesion was observed at intermediate surfactant concentrations. The activity of 14FM1000 was compared with derivatives having shorter or longer hydrophobic tails (10FM1000 and 18FM1000) and polysorbates.

[0096] All surfactant solutions were prepared in physiological saline. A 2 mg / mL stock solution was prepared by dissolving 20-40 mg of surfactant in 10-20 mL of physiological saline. All of these were stirred at 60°C until the surfactant was completely dissolved. The solutions were then allowed to return to room temperature before further use. The IgG stock solution was similarly prepared in physiological saline, usually at 40 mg / mL (6-9 g in 150-225 mL of physiological saline), and vigorously stirred to dissolve the protein. All IgG solutions were filtered through a 0.2 μm polyethersulfone filter (PES, ThermoFisher), diluted, and then used to obtain the final formulation.

[0097] IgG control, physiological saline control, and physiological saline solution containing IgG (20 mg / mL) and surfactant (surfactant concentration range 0.001-0.1 mg / mL) were prepared in vials to a total volume of 15 mL. All final solutions were prepared in physiological saline. Small pieces with different surfaces were immersed in the solution at room temperature for 24 hours and then dried under nitrogen. Contact angle measurements were performed by dropping water droplets (3 μL × 4-6 drops) onto the surface placed between the camera and brightfield of an Ossila instrument. Images of stationary droplets were captured using Ossila software (v.1.1.02). Analysis to extract the average contact angle was also performed using Ossila software (v.3.0.6). For each surface, the average value of 4-6 drops was calculated using JMP software (v.15). The results are shown in Tables 1-4.

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] [Table 4]

[0102] Notes to Tables 1-4: (1) All solutions except the physiological saline control contained 20 mg / mL of IgG. (2) The physiological saline control was 0.9% physiological saline containing neither IgG nor surfactants. (3) The IgG control was 0.9% physiological saline containing 20 mg / mL of IgG, without surfactants. (4) PVC refers to polyvinyl chloride. (5) PES, PE, PVDF, PVC, silicone, and PTFE refer to polymer materials on the surface.

[0103] Measurement of loss on filter and chromatography column surfaces In downstream processing, biopharmaceuticals undergo a variety of purification steps. These consist of numerous chromatography columns and filters, where increased interactions can lead to protein adsorption or aggregation (Li et al., Protein Instability at Interfaces During Drug Product Development—Fundamental Understanding, Evaluation, and Mitigation. AAPS series, Springer 2021. ISSN 2210-7371). Surfactants such as polysorbate can help stabilize these proteins, but they are usually adsorbed onto the surface and therefore added during post-processing of the formulation (Zhou et al., Non-specific binding and saturation of Polysorbate-20 with aseptic filter membranes for drug substance and drug product during mAb production. Journal of Membrane Science 2008, 325(2), 735-741; Mahler et al., Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters. Journal of Pharmaceutical Sciences 2010, 99(6), 2620-2627). To address this problem, a solution has been proposed that involves pre-saturating the filter membrane with a surfactant. However, this is not always feasible because the amount of buffer held in the filter could lead to dilution of the protein product.Therefore, this precaution may require flushing the solution containing both the surfactant and the product vigorously to prevent the latter from being diluted (Mahler et al., Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters. Journal of Pharmaceutical Sciences 2010, 99(6), 2620-2627), which would result in the loss of an expensive product and a decrease in yield.

[0104] Therefore, to ensure that surfactants function effectively in bioprocesses, it is desirable to identify molecules that do not easily adhere to filter and column materials. Another important factor is to investigate the integrity of the surfactants, ensuring they retain their activity after contact with the purification surface. Here, surfactant solutions were rapidly flowed through widely used filters (PVDF, PES) and columns (sulfopropyl-modified cross-linked agarose, Protein A, and quaternary ammonium-modified cross-linked agarose), and the filtrates and eluates were analyzed by liquid chromatography. The elution of 14FM1000 and its derivatives with shorter and longer tails was compared with the elution of polysorbate 80 (PS80). Analysis of the polysorbate 80 chromatogram revealed that some was recovered initially, but there was no consistency regarding subsequent complete recovery.

[0105] In the surface loss test, a 1 mg / mL surfactant stock solution was prepared in water, and this was diluted with milliQ water to prepare a 0.03 mg / mL (30 ppm) solution. Syringe (Becton Dickinson, BD Luer-Lok) TMThe solution was passed through a filter or chromatography column using 3 mL or 10 mL syringes. Since surfactant residue may remain on the syringe surface, all syringes were pre-washed with a 0.03 mg / mL surfactant solution in an amount three times the volume of the syringe. After washing, the syringes were filled with unused surfactant solution, and approximately 100-200 mg of the solution was injected into a vial (12 × 32 mm, Thermo Scientific) containing a low-volume insert (Thermo Scientific). The first sample was always collected directly from the syringe without passing through a filter or column to serve as a control for comparison with the subsequent filtrate. In the filter test, a needle (BD 21G, 0.8 mm × 50 mm) was attached to the syringe to facilitate the delivery of the solution to the bottom of the insert. After collecting the control, the needle was purged with air to remove any remaining solution, and then attached to the filter outlet. Furthermore, the filtrate was collected in vials at intervals of approximately 100-200 mg, and the weight of the vials was measured before and after collection to accurately determine the weight of the solution. In the column experiment, the column was pre-washed with approximately 15 column volumes of water to remove the storage solution and prepare it for use. Next, a syringe containing 0.03 mg / mL of surfactant solution, which had been pre-washed (rinsed with surfactant solution similar to the filter), was attached to the column, and elution was performed using a vertically mounted syringe pump (Kd Scientific) set to deliver the solution at the recommended rate of 1 mL / min. Samples were collected at the same intervals as in the filter test, and the weight of the solution was accurately determined.

[0106] The surfactant was quantified using a high-performance liquid chromatography (HPLC) system (Vanquish, Thermo Scientific) equipped with a charged particle detector (CAD) controlled by Chromeleon software (v 7.3, Thermo Scientific). All samples were packed into an autosampler chamber set to 20°C. Acclaim TMSeparation was performed using a Surfactant Column (Thermo Scientific, 3 × 150 mm, particle size 3 μm) and a mobile phase containing 10 mM ammonium acetate (LC-MS grade, Sigma Aldrich) and acetonitrile (JTBaker) fixed at pH 5. The elution rate was set to 0.6 mL / min, starting with 90% aqueous mobile phase, then increasing to 95% acetonitrile, with the two solutions transitioning over 2 minutes. The surfactant eluted in the presence of a highly organic mobile phase. A calibration curve was created by measuring stock solutions with surfactant concentrations ranging from 0.06 mg / mL to 0.0005 mg / mL using the same method, and this curve was used to quantify the surfactant from the filtrate. The Milli-Q water signal was subtracted from the chromatograms of all samples. All analyses were performed using Chromeleon software, followed by calculations in Excel and JMP v.15. The results are shown in Figures 6-9.

[0107] Figure 6 shows the loss of surfactants when passed through a PVDF filter. The peak for FM1000 did not change significantly, while PS80 appeared as four peaks. Of these, the two on the right were present immediately after passing through the filter, but the two on the left changed considerably during filtration (see Figure 6B). This indicates that some components of the PS80 composition (represented by the two peaks on the right) are not adsorbed by the PVDF filter, while other components of the PS80 composition (represented by the two peaks on the left) are adsorbed and lost by the PVDF filter. Therefore, the composition and properties of PS80 change during filtration. In contrast, FM1000 passed through the PVDF filter with a uniform peak (see Figure 6A). This indicates that the composition and properties of FM1000 do not change during filtration.

[0108] Figure 7 shows the loss of surfactants when passed through a PES filter. The peak for FM1000 did not change significantly, while PS80 appeared as four peaks. Of these, the two on the right were present immediately after passing through the filter, but the two on the left changed considerably during filtration (see Figure 7B). This indicates that some components of the PS80 composition (represented by the two peaks on the right) are not adsorbed by the PES filter, while other components of the PS80 composition (represented by the two peaks on the left) are adsorbed and lost by the PES filter. Therefore, the composition and properties of PS80 change during filtration. In contrast, FM1000 passed through the PES filter with a uniform peak (see Figure 7A). This indicates that the composition and properties of FM1000 do not change during filtration.

[0109] Figure 8 shows the loss of surfactants when passed through a sulfopropyl-modified cross-linked agarose (SP HP) chromatography column. The FM1000 peak did not change significantly, while PS80 appeared as four peaks, the two on the right of which were present much earlier than the two on the left after passing through the column (see Figure 8B). This indicates that some components of the PS80 composition (represented by the two peaks on the right) are not adsorbed onto the sulfopropyl-modified cross-linked agarose column, while other components of the PS80 composition (represented by the two peaks on the left) are adsorbed and lost. Therefore, the composition and properties of PS80 change during chromatography. In contrast, FM1000 passed through the sulfopropyl-modified cross-linked agarose column with uniform peaks (see Figure 8A). This indicates that the composition and properties of FM1000 do not change during chromatography.

[0110] Figure 9 shows the loss of surfactants when passed through a Protein A chromatography column. The FM1000 peak did not change significantly, while PS80 appeared as four peaks, the two on the right of which were present much earlier than the two on the left after passing through the column (see Figure 9B). This indicates that some components of the PS80 composition (represented by the two peaks on the right) are not adsorbed onto the Protein A column, while other components of the PS80 composition (represented by the two peaks on the left) are adsorbed onto the Protein A column and lost. Therefore, the composition and properties of PS80 change during chromatography. In contrast, FM1000 passed through the Protein A column with uniform peaks (see Figure 9A). This indicates that the composition and properties of FM1000 do not change during chromatography.

[0111] Figure 10 shows the loss of surfactants when passed through a quaternary ammonium-modified cross-linked agarose (Q HP) chromatography column. The FM1000 peak did not change significantly, while PS80 appeared as four peaks, the two on the right of which were present much earlier than the two on the left after passing through the column (see Figure 10B). This indicates that some components of the PS80 composition (represented by the two peaks on the right) are not adsorbed onto the quaternary ammonium-modified cross-linked agarose column, while other components of the PS80 composition (represented by the two peaks on the left) are adsorbed and lost. Therefore, the composition and properties of PS80 change during chromatography. In contrast, FM1000 passed through the quaternary ammonium-modified cross-linked agarose column with uniform peaks (see Figure 10A). This indicates that the composition and properties of FM1000 do not change during chromatography.

[0112] Analysis was performed to determine the volume of surfactant solution required to reach a surfactant concentration of 90 wt%. Specifically, the cumulative amount of surfactant contained in the filtrate / eluate was analyzed to determine at what point (in terms of filtrate / eluate volume) the cumulative amount of surfactant (contained in the surfactant solution) reached 90 wt% of the total amount of surfactant supplied to the filter / column (contained in the surfactant solution). The results are summarized in Table 5.

[0113] The top row of Table 5 shows the type of surfactant, and the left column shows the type of filter or chromatography column. The amounts in the table represent the volume (in mL) of filtrate / eluate required to reach 90 ± 1 wt% of the surfactant recovered from the filtrate / eluate. A larger volume indicates a longer time required to recover the surfactant from the filtrate / eluate. The volume values ​​in Table 5 were determined by integrating the HPLC-CAD peaks of each component in the filtrate / eluate sample, and then dividing by the amount in the sample that was not filtered or passed through the column (set to 100%). The volume value recorded is the first sample that reached the threshold of 90 wt% for each operation.

[0114] [Table 5]

[0115] 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.

[0116] In this specification described above, the concepts have been explained 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.

[0117] 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 critically important, necessary, or essential features of any or all of the claims.

[0118] For clarity, it should be understood that certain features described herein in relation to separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features described in relation to a single embodiment may be provided separately or in any partial combination.

Claims

1. (a) Protein and 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 O or NH, X 2 is O or NH, n is 0 or an integer from 1 to 5, and R 3 Equations II and III: 【Chemistry 2】 To provide an aqueous solution containing a polyalkoxy fatty acid acyl surfactant (which is a polymer group containing polymerized units of the same), (b) (i) A step of filtering the aqueous solution, wherein the protein and the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution pass through the filter together to produce a filtrate, or (ii) The step of passing the aqueous solution through a chromatography column. The total weight of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution supplied to the filter or chromatography column is at least 60%, or at least 70%, or at least 75% of the polyalkoxy fatty acid acyl surfactant of formula I. Applying the aqueous solution to a bioprocess in which %, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 98%, or at least 99%, passes through the filter or chromatography column, A process that includes this.

2. The process according to claim 1, wherein the polyalkoxy fatty acid acyl surfactant of formula I is selected from the group consisting of polyalkoxy fatty acid acyl surfactants and mixtures thereof, wherein n in formula I is 1, both X1 and X2 are NH, R2 is -CH2(C6H5), and R3 is a copolymer of CH3-terminated PO and EO units having an approximate number average molecular weight of 1000 and a PO to EO ratio of about 3:19, and R1 is selected from the group consisting of polyalkoxy fatty acid acyl surfactants selected from CH3-(CH2)9-CH2-, CH3-(CH2)11-CH2-, CH3-(CH2)13-CH2-, or CH3-(CH2)15-CH2-.

3. The process according to claim 1, wherein the polyalkoxy fatty acid acyl surfactant is a polyalkoxy fatty acid acyl surfactant of formula I, which is a copolymer of CH3-terminated PO and EO units, where n is 1, X1 and X2 are both NH, R2 is -CH2(C6H5), R1 is CH3-(CH2)11-CH2-, and R3 has an approximate number average molecular weight of 1000 and a PO to EO ratio of about 3:

19.

4. The process according to claim 1, comprising the step of (b)(i) filtering the aqueous solution, wherein the polyalkoxy fatty acid acyl surfactant is a copolymer of CH3-terminated PO and EO units, where n is 1, X1 and X2 are both NH, R2 is -CH2(C6H5), and R3 has an approximate number average molecular weight of 1000 and a PO to EO ratio of about 3:19, and R1 is selected from the group consisting of polyalkoxy fatty acid acyl surfactants of formula I selected from CH3-(CH2)11-CH2-, CH3-(CH2)13-CH2-, or CH3-(CH2)15-CH2- and mixtures thereof.

5. The process according to claim 1, comprising the step of (b)(i) filtering the aqueous solution, wherein the filter is selected from the group consisting of PVDF filters, PES filters, polypropylene filters, cellulose filters, nylon filters and combinations thereof.

6. The process according to claim 5, wherein the filter is a PVDF filter or a PES filter.

7. The process according to claim 1, comprising step (b)(i), wherein the surfactant / protein concentration ratio in the aqueous solution provided in (a) is substantially equal to the surfactant / protein concentration ratio in the aqueous solution of the filtrate after passing through the filter.

8. The process according to claim 1, comprising the step of (b)(ii) passing the aqueous solution through a chromatography column, wherein the chromatography resin is selected from the group consisting of sulfopropyl-modified crosslinked agarose, Protein A, quaternary ammonium-modified crosslinked agarose, hydrophobic interaction chromatography resins and combinations thereof.

9. The chromatography resin is sulfopropyl-modified crosslinked agarose, Protein The process according to claim 8, wherein the material is A or quaternary ammonium-modified crosslinked agarose.

10. The process according to claim 1, wherein the composition of the polyalkoxy fatty acid acyl surfactant of formula I in the aqueous solution remains substantially the same even after passing through the chromatography column.

11. Use of a polyalkoxy fatty acid acyl surfactant as defined in any one of claims 1 to 4 for reducing surface deposits in a bioprocess.

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