PROTEIN SAMPLE

FIELD OF THE INVENTION

[0001] The present disclosure provides a method of determining the concentration of a polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a second surfactant to the composition, wherein the concentration of the second surfactant in the composition is below the critical micelle concentration (CMC) of the second surfactant, and wherein the second surfactant has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate in the supernatant using a fluorescent micelle assay.

BACKGROUND

[0002] Traditional protein purification methods can involve the use of surfactants. Surfactants are amphiphilic molecules which form micelles once the critical micelle concentration (CMC) of the surfactant is reached. Surfactants aid in the dissolution of the cell membrane, freeing membrane-bound or cytosolic protein products and allowing further protein purification steps to isolate a protein product. Surfactants are also used to stabilize the purified protein products. For therapeutic proteins in drug products, accurate measurement of the final concentration of surfactants in the drug product is often required, especially if the drug product is intended for parenteral injection into a subject.

[0003] The co-precipitation of certain surfactants such as polysorbates with therapeutic proteins can make an accurate determination of the surfactant concentration difficult. Inaccurate quantification of surfactants may result in undesirable concentrations of polysorbates in the final drug product. Inaccurate quantification may also result in undetected degradation of polysorbates, which might be caused by the susceptibility of polysorbates towards auto oxidation and/or hydrolysis. SUMMARY OF THE INVENTION

[0004] In some embodiments, the present disclosure provides a method of determining the concentration of polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a second surfactant to the composition, wherein the concentration of the second surfactant in the composition is below the critical micelle concentration (CMC) of the second surfactant, and wherein the second surfactant has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the high-CMC surfactant in the supernatant using a fluorescent micelle assay.

[0005] In some embodiments, the precipitating comprises centrifuging the composition.

[0006] In some embodiments, the precipitating comprises combining a Cl to C6 organic solvent with the composition.

[0007] In some embodiments, the precipitating comprises adding the Cl to C6 organic solvent to the composition.

[0008] In some embodiments, the precipitating comprises combining acetonitrile, acetone, butanone, pentanone, hexanone, methanol, or ethanol with the composition.

[0009] In some embodiments, the precipitating comprises combining acetone with the composition.

[0010] In some embodiments, the organic solvent is combined with the composition before the centrifuging.

[0011] In some embodiments, the organic solvent is combined with the composition for less than 10 minutes before the centrifugation.

[0012] In some embodiments, the ratio of the organic solvent to the composition is about 1:10 to

10:1. [0013] In some embodiments, the ratio of the organic solvent to the composition is about 1:4 to 4:1.

[0014] In some embodiments, the ratio of organic solvent to the composition is about 1 : 1 to 4: 1.

[0015] In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or combinations thereof.

[0016] In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80.

[0017] In some embodiments, the composition comprises about 0.0001% to about 0.1% (w/v) polysorbate.

[0018] In some embodiments, the composition comprises about 0.001% to about 0.01% (w/v) polysorbate.

[0019] In some embodiments, the supernatant is separated from the precipitate before determining the concentration of the polysorbate in the supernatant.

[0020] In some embodiments, the CMC mass value of the second surfactant is at least 20% greater than the CMC mass value of the polysorbate.

[0021] In some embodiments, the CMC mass value of the second surfactant is at least 50% greater than the CMC mass value of the polysorbate.

[0022] In some embodiments, the CMC mass value of the second surfactant is at least 100% greater than the CMC mass value of the polysorbate.

[0023] In some embodiments, the CMC mass value of the second surfactant is at least 4-fold greater than the CMC mass value of the polysorbate.

[0024] In some embodiments, the CMC mass value of the second surfactant is at least 10-fold greater than the CMC mass value of the polysorbate.

[0025] In some embodiments, the second surfactant is a poloxamer. [0026] In some embodiments, the poloxamer has a molecular weight of 1600 Da to 25000 Da.

[0027] In some embodiments, the poloxamer comprises 5 to 250 polyethyleneoxide subunits.

[0028] In some embodiments, the poloxamer comprises 10 to 80 polyethyleneoxide subunits.

[0029] In some embodiments, the poloxamer comprises 10 to 80 polypropylene oxide subunits.

[0030] In some embodiments, the poloxamer comprises 20 to 60 polypropylene oxide subunits.

[0031] In some embodiments, the poloxamer is Pluronic® L10, Pluronic® L35, Pluronic® F38, Pluronic® L42, Pluronic® L43, Pluronic® L44, Pluronic® L61, Pluronic® L62, Pluronic® L64, Pluronic® L65, Pluronic® F68, Pluronic® F77, Pluronic® L81, Pluronic® P84, Pluronic® P85, Pluronic® F87, Pluronic® F88, Pluronic® L92, Pluronic® F98, Pluronic® L101, Pluronic®P103, Pluronic® P104, Pluronic® P105, Pluronic® F108, Pluronic® L121, Pluronic® 122, Pluronic® PI 23, Pluronic® FI 27, Kolliphor® EL, Kolliphor® PI 88, Kolliphor® P407, Synperonics® PR/P84 or combinations thereof.

[0032] In some embodiments, the poloxamer is Kolliphor® PI 88.

[0033] In some embodiments, the second surfactant is a polyoxyethylene alkyl ether.

[0034] In some embodiments, the second surfactant is polyoxyethylene (23) lauryl ether.

[0035] In some embodiments, the second surfactant has a concentration of about 0.005% to 0.5% (w/v) in the composition.

[0036] In some embodiments, the protein is a therapeutic protein.

[0037] In some embodiments, the protein is an antibody.

[0038] In some embodiments, the concentration of the protein in the composition is about 1 mg/mL to about 300 mg/mL. [0039] In some embodiments, the fluorescent micelle assay uses N-phenyl-l-naphtylamine (NPN), 4,4'-dianilino-l,l'-binaphthyl-5,5'-8 disulfonic acid dipotassium salt, or Vybrant® Dil.

[0040] In some embodiments, the fluorescent micelle assay uses a fluorescent detector, and wherein the fluorescent detector was set to an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0041] In some embodiments, the present disclosure provides a method of determining the concentration of a polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a poloxamer to the composition, wherein the concentration of the poloxamer is below the critical micelle concentration (CMC) of the poloxamer, and wherein the poloxamer has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate in the supernatant using an fluorescent micelle assay.

[0042] In some embodiments, the present disclosure provides a method for determining the concentration of a polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a polyoxyethylene alkyl ether to the composition, wherein the concentration of the polyoxyethylene alky ether is below the critical micelle concentration (CMC) of the polyoxyethylene alkyl ether, and wherein the polyoxyethylene alkyl ether has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate in the supernatant using a fluorescent micelle assay.

[0043] In some embodiments, the precipitating comprises combining acetone with the composition. BRIEF DESCRIPTION OF THE FIGURES

[0044] FIG. 1 represents the calibration curves for polysorbate 80 in drug substance and polysorbate 80 in water.

[0045] FIG. 2 represents the calibration curves for polysorbate 80 in drug substance and polysorbate 80 in water (PP).

[0046] FIG. 3 represents the calibration curves for water and water containing poloxamer.

[0047] FIG. 4 represents the calibration curves in water and water containing Brij .

[0048] FIG 5. represents comparison of peak heights of standards containing 0.0100% polysorbate 80, or 0.0100% polysorbate 80 containing Brij or Poloxamer.

[0049] FIG 6. represents the calibration curves for polysorbate 80 in drug substance protein precipitate poloxamer and polysorbate 80 in water with poloxamer.

[0050] FIG. 7 represents calibration curves for polysorbate 80 in drug substance protein precipitate poloxamer and polysorbate 80 in water with poloxamer.

[0051] FIG. 8 represents calibration curves for polysorbate in drug substance with poloxamer and polysorbate in water with poloxamer.

[0052] FIG 9. represents calibration curves for polysorbate in drug substance with poloxamer and polysorbate in water with poloxamer.

[0053] FIG. 10 is an example of a chromatogram, overlay of standard at 0.01% (w/v) PS90 and blank.

[0054] FIG. 11 represents the calibration curves for polysorbate 20 in drug substance and polysorbate 20 in water (acetone precipitation) with no poloxamer.

[0055] FIG. 12 represents the calibration curves for polysorbate 20 in drug substance and polysorbate 20 in water (acetone precipitation) with addition of poloxamer. DETAILED DESCRIPTION OF THE INVENTION

[0056] The present disclosure relates to methods of determining the concentration of a polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a second surfactant to the composition, wherein the concentration of the second surfactant in the composition is below the critical micelle concentration (CMC) of the second surfactant, and wherein the second surfactant has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate surfactant in the supernatant using a fluorescent micelle assay.

[0057] As used herein, “a” or “an” may mean one or more. As used herein, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein, “another” or “a further” may mean at least a second or more.

[0058] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the method/device being employed to determine the value, or the variation that exists among the study subjects. Typically, the term “about” is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or higher variability, depending on the situation. In some embodiments, one of skill in the art will understand the level of variability indicated by the term “about,” due to the context in which it is used herein. It should also be understood that use of the term “about” also includes the specifically recited value.

[0059] The use of the term “or” in the claims is used to mean “and/or,” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

[0060] As used herein, the terms “comprising” (and any variant or form of comprising, such as “comprise” and “comprises”), “having” (and any variant or form of having, such as “have” and “has”), “including” (and any variant or form of including, such as “includes” and “include”) or “containing” (and any variant or form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any composition (e.g., formulation) or method of the present disclosure. Furthermore, compositions (e.g., formulations) of the present disclosure can be used to achieve methods of the present disclosure.

[0061] The use of the term “for example” and its corresponding abbreviation “e.g.” (whether italicized or not) means that the specific terms recited are representative examples and embodiments of the disclosure that are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[0062] As used herein, “between” is a range inclusive of the ends of the range. For example, a number between x and y explicitly includes the numbers x and y, and any numbers that fall within x and y.

[0063] Surfactants are compounds which decrease the surface tension between two liquids, generally comprised of one or more hydrophilic groups and one or more hydrophobic groups. Classified by their hydrophilic “head” groups, surfactants may be non-ionic (no charged groups in its hydrophilic “head”), anionic (net negatively charged), cationic (net positively charged), or zwitterionic (positive and negatively charged). Surfactants are commonly used in protein purification methods and aid in dissolving cell membranes and stabilizing proteins. Surfactants form micelles once the critical micelle concentration (CMC) of the surfactant is reached, depending on the solvent, temperature, and pressure. A micelle is a supramolecular assembly of surfactant molecules dispersed in a liquid colloid wherein the hydrophilic “head” regions of the surfactant molecules interact with the liquid solvent and the hydrophobic “tail” regions of the surfactant are sequestered in the center or core of the micelle.

[0064] The present disclosure provides a method of determining the concentration of a polysorbate in a composition, e.g., a composition comprising a protein. In some embodiments, the composition comprises a therapeutic protein. In some embodiments, the composition is a drug product.

[0065] The present disclosure provides a method of reducing the coprecipitation of a polysorbate and a protein, e.g., a therapeutic protein, e.g., an antibody, in the composition by adding a second surfactant to the composition. Since the polysorbate does not coprecipitate (or co precipitation is greatly reduced), and the second surfactant is not detected using the FMA analysis, the disclosure a method of more accurately determining the concentration of the polysorbate in the composition.

[0066] As used herein, a polysorbate is a non-ionic surfactant comprised of polyethoxylated esters of 3,6-sorbitan. In some embodiments, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the polysorbate is polysorbate 20 and polysorbate 80.

[0067] As used herein, a second surfactant is a surfactant different from the polysorbate. In some embodiments, the second surfactant has a CMC mass value higher than the polysorbate mass value.

[0068] As used herein, a “CMC mass value” is defined as the molecular weight of the surfactant multiplied by the critical micelle concentration (CMC) of the surfactant, with units of mg/ml of surfactant.

[0069] In some embodiments, the CMC mass value of the second surfactant is greater than 0.1 mg/mL, greater than 0.2 mg/mL, greater than 0.3 mg/mL, greater than 0.4 mg/mL, greater than 0.5 mg/mL, greater than 0.6 mg/mL, greater than 0.7 mg/mL, greater than 0.8 mg/mL, greater than 0.9 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 1.0 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, or 10 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 0.1 mg/mL and less than 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, or 10 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 1 mg/mL and less than 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, or 10 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 20 mg/mL, 40 mg/mL, 60 mg/mL, 80 mg/mL, or 100 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 150 mg/mL, 200 mg/mL, or 300 mg/mL. In some embodiments, the CMC mass value of the second surfactant is greater than 400 mg/mL, 500 mg/mL, 600 mg/mL, 800 mg/mL, or 1000 mg/mL.

[0070] In some embodiments, the CMC mass value of the second surfactant is at least 10% greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 20% greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 50% greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 100% greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 200%, 300%, 400%, or 500% greater than the CMC mass value of the polysorbate.

[0071] In some embodiments the CMC mass value of the second surfactant is at least 2-fold greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 4-fold greater, at least 6-fold greater, or at least 8-fold greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 10-fold greater than the CMC mass value of the polysorbate. In some embodiments, the CMC mass value of the second surfactant is at least 20-fold greater than the CMC mass value of the polysorbate.

[0072] One of skill in the art can appreciate that CMC (and therefore CMC mass value) of a particular surfactant is dependent on several factors, e.g., temperature, pressure, and (sometimes strongly) on the presence and concentration of other surface active substances and electrolytes. Micelles only form above critical micelle concentration. Thus, as used herein, the values for CMC for a given surfactant are determined under standard conditions, e.g., room temperature, one atmosphere pressure, in the presence of water without other electrolytes and surface active substances. Generally, the accepted CMC and CMC mass values known in the art are known and appreciated by the skilled artisan.

[0073] In some embodiments, the second surfactant is a poloxamer. Poloxamers are non-ionic tri block linear copolymers of poly(ethylene oxide) and polypropylene oxide). In some embodiments, the poloxamer can be represented by the formula

[0074] wherein a can be 2-130 and b can be 10 to 70. In some embodiments, the poloxamer has a molecular weight of 1600 Da to 25000 Da, e.g., 2000 Da to 20,000 Da, 4000 Da to 16000 Da, 6000 Da to 14000 Da, or 8000 Da to Da. In some embodiments, the poloxamer is 2000 Da to 15,000 Da, 2000 Da to 10,000 Da, or 2000 Da to 5000 Da. In some embodiments, the poloxamer is 5000 Da to 25,000 Da, 10,000 Da to 25,000 Da, 15,000 Da to 25,000 Da, or 20,000 Da to 25,000 Da.

[0075] In some embodiments, the poloxamer comprises about 5 to 250 polyethyleneoxide subunits, e.g., about 10 to 220 polyethyleneoxide subunits, about 25 to 200 polyethyleneoxide subunits, or about 50 to 150 polyethyleneoxide subunits. In some embodiments, the poloxamer comprises about 10 to 80 polyethyleneoxide subunits. In some embodiments, the poloxamer comprises about 1 to 80 polypropylene oxide subunits, e.g., about 2 to 70 polypropylene oxide subunits, about 5 to 60 polypropylene oxide subunits, about 10 to 50 polypropylene oxide subunits, about 15 to 50 polypropylene oxide subunits, or about 20 to 40 polypropylene oxide subunits. In some embodiments, the poloxamer comprises 20 to 60 polypropylene oxide subunits. One of skill in the art can appreciate that the subunit count can be an average or approximation, and that minor variations of chain length may exists in the poloxamer. Likewise, the skilled artisan can appreciate that two or more poloxamers can be combined, e.g., mixed, and that would still be envisioned and encompassed by the present disclosure.

[0076] In some embodiments, the poloxamer is Pluronic® L10, Pluronic® L35, Pluronic® F38, Pluronic® L42, Pluronic® L43, Pluronic® L44, Pluronic® L61, Pluronic® L62, Pluronic® L64, Pluronic® L65, Pluronic® F68, Pluronic® F77, Pluronic® L81, Pluronic® P84, Pluronic® P85, Pluronic® F87, Pluronic® F88, Pluronic® L92, Pluronic® F98, Pluronic® L101, Pluronic®P103, Pluronic® P104, Pluronic® P105, Pluronic® F108, Pluronic® L121, Pluronic® 122, Pluronic® PI 23, Pluronic® FI 27, Kolliphor® EL, Kolliphor® PI 88, Kolliphor® P407, Synperonics® PR/P84or combinations thereof.

[0077] In some embodiments, the second surfactant is a polyoxyethylene alkyl ether. In some embodiments, the second surfactant is polyoxyethylene (4-40) alkyl ether. In some embodiments, the second surfactant is a polyoxyethylene (4-30) C6-C24 alkyl ether. In some embodiments, the second surfactant is a polyoxyethylene (15-25) C6-C24 alkyl ether. In some embodiments, the second surfactant is a polyoxyethylene (15-25) C8-C18 alkyl ether. In some embodiments, the second surfactant is a Brij® surfactant, e.g., Brij® L23, Brij® L4, Brij® 58, Brij® 020, SP Brij® C2 MB AL-SO-(SG), Brij® SI 00, Brij® 010, Brij® 93, Brij® CIO, Brij® S20, Brij® S2 MBAL, or Brij® 35. In some embodiments, the second surfactant is polyoxyethylene (23) lauryl ether, i.e., Brij-35.

[0078] The present disclosure provides a method for determining the concentration of a polysorbate surfactant in a composition. The polysorbate can be present in any concentration suitable for determination using any known fluorescence micelle assay (FMA). In some embodiments, the composition comprises about 0.0001% to about 1% (w/v) high-CMC surfactant, or about 0.0001% to about 0.1% (w/v) polysorbate. In some embodiments, the composition comprises about 0.001% to about 0.01% (w/v) polysorbate. In some embodiments, the composition comprises about 0.01% to about 1.0% (w/v) polysorbate.

[0079] In some embodiments, the second surfactant has a concentration of about 0.001% to about 1% (w/v), about 0.005% to about 0.5% (w/v), about 0.001% to about 0.1% (w/v), about 0.005% to about 0.1% (w/v), about 0.01% to about 0.1% (w/v) in the composition. In some embodiments, the second surfactant has a concentration of about 1% to about 10% (w/v) in the composition. In some embodiments, the second surfactant has a concentration of 0.001, 0.01, 0.1, or 1.0% (w/v) in the composition. [0080] The compositions of the present disclosure comprise a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein can be used in a vaccine. In some embodiments, the protein is an antibody, a nanobody, a single-domain antibody, an enzyme, a peptide, or any other macromolecule comprised of amino acids known in the art. In some embodiments, the term “protein” can refer to a protein fragment, e.g., a partial protein. In some embodiments, the term protein can refer to a modified protein, e.g., a protein that has been glycosylated. In some embodiments, the term protein can refer to a mutated protein, e.g., a genetically engineered protein comprising one or more mutations not commonly found in nature. In some embodiments, the protein is an isolated protein, e.g., a protein which has been substantially purified from other proteins, lipids, nucleic acids and other cellular material from which is was produced. In some embodiments, the composition can comprise a single type of protein, e.g., a monoclonal antibody. In some embodiments, the composition can comprise more than one type of protein, e.g., proteins with variability in their amino acid sequences, e.g., a population of polyclonal antibodies. In some embodiments, the composition comprising more than one type of protein can be different proteins, e.g., an antibody and an enzyme.

[0081] The composition can comprise various concentrations of the protein. In some embodiments, the concentration of the protein in the composition is about 0.001% to about 75% (w/v), about 0.01% to about 50% (w/v), about 0.1% to about 35% (w/v), about 1% to about 20% (w/v), or about 0.1% to about 10% (w/v). In some embodiments, the concentration of the protein in the composition is about 10% to about 20%. In some embodiments the concentration of the protein in the composition is about 20% to about 50%. In some embodiments, the concentration of the protein in the composition is about 50% to about 75%.

[0082] The method of the present disclosure comprises precipitating the protein in the composition to form a precipitate and a supernatant. The terms precipitate and supernatant refer to their commonly understood definition known to the skilled artisan, generally as the solid phase and liquid phase, respectively. Precipitation can occur using any methods known in the art, such as but not limited to centrifugation, isoelectric precipitation using tricholoroacetic acid (TCA), precipitation using an organic solvent, e.g., acetonitrile precipitation, acetone precipitation, TCA-acetone precipitation, or phenol precipitation,, ammonium acetate/methanol precipitation, methanol chloroform precipitation, salt-induced precipitation, or lypophilization. In some embodiments, the precipitation comprises using an organic solvent. In some embodiments, the precipitation uses acetone.

[0083] In some embodiments, the precipitating comprises centrifuging the composition. The centrifuging may occur at any relative centrifugal force, revolutions per minute, temperature or pressure known in the art.

[0084] In some embodiments, the precipitating comprises combining a Cl to C6 organic solvent with the composition. In some embodiments, the precipitating comprises adding the Cl to C6 organic solvent to the composition. The combining may take place in any order, e.g., adding the composition and the solvent simultaneously, adding the composition to the solvent, or adding the solvent to the composition. A Cl to C6 organic solvent includes but it is not limited to any organic solvent with 1 to 6 carbon atoms, such as acetic acid, acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, di ethylene glycol, diethyl ether, di ethylene glycol dimethyl ether, 1,2-dimethoxy ethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, hexanone, hexamethylphosphoramide, hexamethylphosphorous triamide, hexane, methanol, methyl t-butyl ether, methylene chloride, N-methyl-2-pyrrolidinone, nitrom ethane, pentane, pentanone, petroleum ether, 1 -propanol, 2-propanol, pyridine, tetrahydrofuran, and toluene.

[0085] In some embodiments, the precipitating comprises combining acetonitrile, acetone, butanone, pentanone, hexanone, ethanol and/or methanol with the composition. In some embodiments, the precipitating comprises combining acetone with the composition. In some embodiments, the precipitating comprises adding acetone to the composition. In some embodiments, the precipitating comprises adding the composition to acetone.

[0086] In some embodiments, the organic solvent is combined with the composition before the centrifuging. The organic solvent may be combined with the composition for any length of time before centrifuging, e.g., for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, or 60 minutes before centrifuging. In some embodiments, the organic solvent is combined with the composition for less than 10 minutes before the centrifugation.

[0087] Various ratios of organic solvent and composition can be used to perform the precipitation. In some embodiments, the ratio of organic solvent to the composition is about 1 : 10 to 10: 1. In some embodiments, the ratio of organic solvent to the composition is about 1:4 to 4:1. In some embodiments, the ratio of organic solvent to the composition is about 1:1 to 4:1. In some embodiments, the ratio of organic solvent to the composition is about 1 :20 to 20: 1.

[0088] In some embodiments, after the precipitation, the supernatant is separated from the precipitate before determining the concentration of the polysorbate in the supernatant. The separation of the supernatant from the precipitate can occur immediately before determining the concentration, 1 minute to 60 minutes, 2 to 45 minutes, 5 to 30 minutes, or 5 to 10 minutes before determining the concentration In some embodiments, the separation of the supernatant from the precipitate can occur immediately before determining the concentration, 20 to 60 minutes before determining the concentration, or up to 24 hours before determining the concentration of the polysorbate in the supernatant.

[0089] The present disclosure provides for a new and improved method of determining the concentration of a polysorbate in a composition comprising a protein. Traditionally, the surfactant level in a composition comprising a protein was determined using a fluorescent micelle assay (FMA). In traditional FMA, the protein in a composition, e.g., a drug product, needed to be removed prior determining the surfactant concentration in the composition. Traditionally, to remove the protein in the composition, the protein was precipitated. The concentration of the surfactant in the resulting supernatant was then determined by adding a dye that was fluorescent when present inside a micelle, and less fluorescent when outside a micelle at a given excitation/emission wavelength. However, in the traditional method, the surfactant often co-precipitates with the protein, thereby the resulting supernatant comprises less than the original concentration of surfactant present in the original composition, and the measured concentration of the high-CMC surfactant is less than the true value. The present disclosure overcomes the inaccurate and inconsistent determination of the concentration of surfactant in the composition, by recognizing that co precipitation of traditional surfactants (i.e., polysorbates) used in FMA can be reduced by the additional of a second type of surfactant, i.e., a poloxamer, to the composition before the precipitation. In some embodiments, the second surfactant reduces the co-precipitation of the polysorbate during precipitation, thereby increasing the accuracy when quantifying the polysorbate concentration. The second surfactant does not interfere with the FMA method, because at the concentration below the CMC, no micelles are formed.

[0090] In some embodiments, the FMA is combined with chromatography, e.g., liquid chromatography (LC), e.g., high performance liquid chromatography (HPLC). Thus, in some embodiment, the concentration of the polysorbate in the supernatant is determined by FMA, e.g, LC-FMA, HPLC -FMA, etc.

[0091] Thus, in some embodiments, the disclosure provides a method of determining the concentration of a polysorbate in the supernatant using FMA. In some embodiments, the disclosure provides a method of determining the concentration of the polysorbate surfactant in the supernatant using an LC-FMA. In some embodiments, the disclosure provides a method of determining the concentration of the polysorbate in the supernatant using an HPLC-FMA.

[0092] The FMA relies on the micelle forming ability of the surfactant, and a fluorescent dye, e.g., the hydrophobic dye N-phenyl-l-napthylamine (NPN). The fluorescent dye, e.g., NPN, produces higher fluorescence at a specific excitation and emission wavelength when it is incorporated into the hydrophobic interior of a micelle. The amount of generated fluorescent signal from an individual sample may be compared with a calibration curve and the signal can be extrapolated to determine the concentration of the polysorbate surfactant. Additional methods of determining the concentration of the polysorbate surfactant in the supernatant are known in the art.

[0093] In some embodiments, the fluorescent micelle assay uses N-phenyl-l-naphthylamine (NPN). In some embodiments the fluorescent micelle assay uses a fluorescent detector, and the fluorescent detector is set to an excitation wavelength of 350 nm and an emission wavelength of 420 nm. [0094] The present disclosure provides a method of determining the concentration of a polysorbate, the method comprising providing a composition comprising a protein and a polysorbate, adding a poloxamer to the composition, wherein the concentration of the poloxamer is below the critical micelle concentration (CMC) of the poloxamer, and wherein the poloxamer has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate in the supernatant using an HPLC- fluorescent micelle assay.

[0095] The present disclosure provides a method of determining the concentration of polysorbate, the method comprising providing a composition comprising a protein and the polysorbate, adding a polyoxyethylene alkyl ether to the composition, wherein the concentration of the polyoxyethylene alkyl ether is below the critical micelle concentration (CMC) of the polyoxyethylene alkyl ether, and wherein the polyoxyethylene alkyl ether has a CMC mass value higher than the polysorbate CMC mass value, precipitating the protein in the composition to form a precipitate and a supernatant, and determining the concentration of the polysorbate in the supernatant using a fluorescent micelle assay.

[0096] In some embodiments, the precipitating comprises combining acetone with the composition.

[0097] All references cited herein, including patents, patent applications, papers, textbooks and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety.

EXAMPLES

EXAMPLE 1: Optimization of protein precipitation

[0098] Various methods were tested to determine the most efficient method of precipitating a monoclonal antibody from a drug product. Drug product containing the antibody (and free of polysorbate 80, i.e., PS80) was spiked to a target concentration of 0.01% (w/v) PS80. Various organic solvents (acetone or acetonitrile (ACN)) at various ratios and extraction numbers were used to precipitate the antibody, and then the antibody recovered was determined to measure the efficiency, as found in Table 1.

Table 1:

[0099] The results suggest acetone provided the best recovery and accuracy of the conditions tested.

[00100] The linearity, range, accuracy, precision, and carry over of the precipitated drug product (DP) were determined against a calibration curve in water (no precipitation, i.e., PP). WUP = Worked up. The results are presented in Table 2 and FIG. 1.

Table 2

[00101] Accuracy is not given when comparing the worked up DP against the calibration curve in water (no PP), standards are at 100% ± 22% of the nominal spiked value.

[00102] A full evaluation with the precipitation procedure using acetone was performed. The results are found in Table 3 and FIG. 2.

Table 3

[00103] Accuracy is not given when comparing the worked up DP against the calibration curve in water (PP), standards are at 100% ± 31% of the nominal spiked value.

EXAMPLE 2: Addition of second surfactant

[00104] To minimize the loss of PS80 that occurs during workup, experiments were performed where an additional surfactant (Brij and Poloxamer) was introduced to the DS before workup. For the controls, Poloxamer (FIG. 3) and Brij (FIG. 4) were added to the calibration curves in water. The HPLC-FMA signal is presented in FIG. 5.

[00105] Poloxamer does not have a significant impact on the HPLC-FMA signal. Brij enhances the signal of PS80. No significant signal was detected in STD 0 (standard not containing PC80 but either Brij or Poloxamer).

[00106] Linearity, range, accuracy (through the whole calibration range), carry-over, and specificity tests with DP samples containing one of the surfactants were determined. The samples were measured against a calibration curve in water (+additional surfactant), that was treated the same way as the standards in DS and against a calibration curve in water (+additional surfactant) that was not treated. The results are found in Table 4 and FIG. 6.

Table 4

[00107] The most promising values were obtained for the DP samples where poloxamer was introduced against a calibration curve in water (+ poloxamer) that was not treated. Linearity, range, carryover, precision, specificity showed acceptable results. Accuracy can be determined when comparing the worked up DP (+ poloxamer) against the calibration curve in water (+ poloxamer). Standards are at 100% ± 16% of the nominal spiked value. The most promising values were obtained for the DP when poloxamer was introduced against a calibration curve in water (+ poloxamer). [00108] Linearity, range, accuracy (through the whole calibration range), carry-over, and specificity tests with DP samples containing one of the surfactants were determined. See Table 5 and FIG. 7

Table 5

[00109] Linearity, range, carryover, precision, specificity showed acceptable results. Accuracy is given when comparing the worked up DP (+ poloxamer) against the calibration curve in water (+ poloxamer), standards are 100% ± 6% of the nominal spiked value.

[00110] Linearity, range, accuracy (Intermediate precision), carry-over, and specificity tests with DP samples containing one of the surfactants were determined. See Table 6 and FIG. 8.

Table 6

[00111] Linearity, range, carryover, precision, specificity showed acceptable results. Accuracy is given when comparing the worked up DP (+ poloxamer) against the calibration curve in water (+ poloxamer), standards are 100% ± 18% of the nominal spiked value.

[00112] This data suggests a work-up using acetone provides better recovery/accuracy compared to ACN. The loss of PS80 observed during workup could not be compensated by treating the standards in water the same way. However, the method including worked-up standards was near acceptable values for accuracy. Addition of poloxamer to the PS80 spiked standards in DP before precipitation was minimizing the coprecipitation of PS80.

[00113] The method in which acetone was used as the extraction solvent and poloxamer was added to the drug product to reduce coprecipitation was successfully evaluated for Linearity, range, carryover, precision, specificity. Intermediate precision was also successfully shown.

EXAMPLE 3: Preparation of reagents, mobile phases and solutions

[00114] Preparation of reagents

[00115] The preparation of reagents can be scaled up or down proportionally.

Table 7: Preparation of reagents

verify the correct preparation of the standards.

[00116] Preparation of mobile phase

[00117] The preparation of mobile phase can be scaled up and down proportionally. Table 8: Preparation of mobile phase

[00118] Preparation of conditioning solution

[00119] The conditioning solution is composed of the 0.0100% (w/v) PS80 in water. 700 pL are transferred into HPLC vials without insert:

Table 9: Preparation of conditioning solution

[00120] Preparation of blank

Table 10: Preparation of blank [00121] Preparation of blank [00122] Preparation of standards

[00123] The standard calibration curve is composed of four reference standards with 0.0025%, 0.0050%, 0.0100% and 0.0150% (w/v) PS80 in water, i.e. 25%, 50%, 100% and 150% of the target PS80 concentration of 0.0100% (w/v) PS80. Additionally, each standard contains 0.05 % (w/v) Poloxamer.

Table 11: Preparation of standards

Stability of the standards: 48 hours in the autosampler.

[00124] Preparation of samples Table 12: Preparation of samples

[00125] The work up procedure is applied to all API containing samples. - Aliquot 300 pL of sample into a 1.5 mL polypropylene tube.

- Add 600 pL of acetone with a positive displacement pipette (e.g. Gilson Microman, to avoid dripping) to the sample.

- Cap the tubes and vortex for 10 seconds directly after adding acetone. Make sure that the content of the tubes is mixed completely. If the batch of samples consists of more than 3 samples, vortex them again briefly directly before the centrifugation.

- Centrifuge at -12000 g for 10 minutes. The phases must be completely separated.

- Transfer 400 pL of supernatant into a clean 1.5 mL polypropylene tube without disturbing the pellet by using positive displacement pipette (e.g. Gilson Microman, to avoid dripping).

- Place the tubes in the vacuum centrifuge and evaporate to dryness at ambient temperature under vacuum (-overnight).

- Remove samples from the vacuum centrifuge and allow equilibrating to room temperature if required (-10 min).

- Reconstitute the sample by adding 133 pL of purified water.

- Vortex the reconstituted sample 4 times every 15 min for -5 s.

- Centrifuge the sample solution at -12000 g for 3 min before transferring 130 pL of sample to a fresh HPLC vial (make sure to use HPLC vials containing inlets to allow sufficient sample aspiration during HPLC injection when using low sample volume e.g. Infochroma #8002-SC-TPX3p). [00126] Preparation of control

[00127] Prepare one control sample at 100% of target - i.e. 0.0100% (w/v) - PS80 concentration and 0.05 % (w/v) Poloxamer. The control has to be prepared from a different stock solution than the standards (different weighing).

Table 13: Preparation of control

[00128] Procedure: Instrumental conditions

Table 14: Overview of instrumental conditions

[00129] Follow the following steps for the system preparation:

Table 15: System preparation

[00130] Sequence

[00131] Inject the conditioning solution until the area obtained is stable (at least 50 injections). The area of the last 5 injections should be within +/- 10% (see section [00133]). Inject the blank once, and then inject each of the four standard solutions once at the beginning and once after the sample solutions. Important: ensure that injections are performed from lowest to highest concentration. After each Standard 4 inject the blank and the control (the control is not injected after the last blank at the end of the sequence).

[00132] Inject each sample solution twice. If the end of the sequence is not reached after 10 injections of the sample solutions, the four standard solutions have to be re-injected before an additional set of ten sample solution injections can be performed. Evaluation of the PS80 content of the samples will be always done including both standard lines directly before and directly after the sample set.

Table 16: Example of analysis sequence

^l) This blank is not evaluated in the SST (see section 30); it serves the purpose of flushing the injection system

[00133] System suitability test (SST)

Table 17: SST requirements [00134] Evaluation of results

[00135] Integration

[00136] Using a suitable electronic integrator or computer system (e.g. Empower) identify the PS80 peak in the standards and sample chromatograms. In FIG. 10 an example chromatogram is shown. In this example an integration of the PS80 peak from around 0.80 min to 1.15 min would be done.

[00137] Standard curve

[00138] Generate a standard curve by plotting the PS80 peak area against the injected concentration (in % (w/v)) for each standard level. Use a linear-fit. Do not force the curve through zero.

[00139] Calculation

[00140] Determine the peak area of the main peak in the chromatograms of the standards and the samples by integration. Consider the peak area of each standard solution into the standard line as separate point.

[00141] A linear fit of the peak area against the concentration of PS80 in the standard solutions is performed. The standard calibration curve is comprised from 8 injections (4 standards injected before and 4 standards injected after the samples). The slope and intercept obtained for the standard regression line is used to calculate the PS80 concentration in the samples by the following equation:

CPS8O = (asampie - intercept) / slope

CPS8O: Concentration of PS80 in sample solution asampie: PS80 peak area in the sample

Intercept: obtained from linear fit of calibration curve

Slope: obtained from linear fit of calibration curve [00142] Sample acceptance criterion

[00143] The difference between the areas of the two injections per sample should be less than 10%.

[00144] Reporting

[00145] The content (% w/v) is reported as the mean result from the two sample injections with five decimal places.

EXAMPLE 4: Determination of Coprecipitation with Poylsorbate 20

[00146] Linearity, range, accuracy (through the whole calibration range of 0.0100% to 0.0600%) with DP samples containing Polysorbate 20 (PS20) was determined as described previously, using acetone as a precipitating solvent. The samples were measured against a calibration curve in water, that was treated the same way as the standards in DS. The results are presented in Table 18 and FIG. 11.

Table 18

[00147] Accuracy is given when comparing the calibration curve in DP against the calibration curve in water. All standards are 100% ±11% of the nominal spiked value. [00148] Linearity, range, accuracy (through the whole calibration range of 0.0100% to 0.0600%) with DP samples containing Polysorbate 20 (PS20) was determined as described previously, using acetone as a precipitating solvent but with poloxamer added as an additional surfactant. The samples were measured against a calibration curve in water, that was treated the same way as the standards in DS. The results are presented in Table 19 and FIG. 12.

Table 19

[00149] Accuracy is given when comparing the calibration curve in DP against the calibration curve in water. All standards are 100% ±4% of the nominal spiked value.

[00150] Similar to the results seen previously for Polysorbate 80, this data indicates that the addition of a second surfactant, Poloxamer, to a drug product comprising Polysorbate 20 minimized coprecipitation of the Polysorbate 20 during the protein precipitation with acetone. This allows for a more accurate determination of the Polysorbate 20 (or Polysorbate 80) concentrations in the drug product.