EP4662487A1 - Methods of monitoring surfactant hydrolysis in biopharmaceutical formulations - Google Patents

Abstract

The present disclosure relates to methods for detecting, monitoring, and quantifying free fatty acid content of a pharmaceutical formulation. These methods involve subjecting a sample of the formulation comprising a surfactant to an extraction process to separate free fatty acids in the sample from the surfactant. The methods further involve derivatizing the separated free fatty acids into fatty acid esters for detection and quantitation via gas chromatography.

Description

METHODS OF MONITORING SURFACTANT HYDROLYSIS IN BIOPHARMACEUTICAL FORMULATIONS

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63/443,523, filed February 6, 2023, is hereby claimed, and the disclosure thereof is hereby incorporated by reference herein.

FIELD

[0002] The field of this disclosure relates to methods for detecting, monitoring, and quantifying surfactant hydrolysis in biopharmaceutical formulations.

BACKGROUND OF VARIOUS EMBODIMENTS

[0003] Polysorbate (PS) is the most prevalent non-ionic surfactant used in biopharmaceutical formulations due to its excellent stabilizing properties for drug product (DP) formulations and its ability to protect proteins and other biologies from aggregation and denaturation. However, residual lipases present in DP formulations can cause PS hydrolysis, resulting in the accumulation of free fatty’ acids (FFAs) which drive undesired particulate formation in DP formulations. Although methods of direct quantification of FFAs released from PS have been developed using liquid chromatography-mass spectrometry' (LC-MS), many researchers experience high FFAs interference in the LC system, resulting in inaccurate quantification of these type of analytes. Accordingly, new methods for accurate quantification of FFA accumulation in biopharmaceutical formulations are needed. The present disclosure overcomes this and other deficiencies in the art.

SUMMARY OF VARIOUS EMBODIMENTS

[0004] A first aspect of the present disclosure is directed to a method for quantifying free fatty acid (FFA) content in a pharmaceutical formulation. This method involves providing a sample of a pharmaceutical formulation comprising a surfactant, and subjecting the formulation sample to a solid phase extraction (SPE) process to separate FFAs in the formulation sample from the surfactant. The method further involves derivatizing the separated free fatty’ acids into fatty acid esters. The derivative fatty acid esters are separated by gas chromatography and detected. The method further involves quantifying the FFA content of the pharmaceutical formulation based on the separation and detection of the fatty' acid esters.

[0005] During the production, distribution, storage and administration of a biopharmaceutical formulation, the therapeutic biologic components of the formulation are exposed to several interfaces, e.g. glass, plastic polymers, stainless steel, air, ice crystals, silicon oil. etc., that can lead to adsorption, denaturation, or aggregation of the therapeutic components. All of these processes decrease the effective concentration of the therapeutic biologic in the formulation. Non-ionic surfactants, such as polysorbates, are commonly used in biopharmaceutical formulations to protect the therapeutic biologic components of the formulations, e.g., peptides, proteins, antibodies, vaccines, etc., against adsorption, aggregation, and related instabilities. Polysorbates are often a preferred surfactant choice due to their low toxicity and good stabilizing properties. However, polysorbates are subject to enzyme-mediated degradation, resulting in the formation of free fatty acids degradants, which can affect the functional properties of the polysorbate, thereby inducing instability’ of the biological components of the formulation. For this reason, it is critical to have an analytical approach capable of accurately characterizing and quantifying the presence of FFA degradants in pharmaceutical formulations, especially during the development process, to ensure the production of a formulation that will maintain product integrity during subsequent manufacture, storage, and administration processes.

[0006] Methods for detecting polysorbates and its degradants, like FFAs, in biopharmaceutical formulations have been described (see e.g., review by Martos et al., '‘Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations,’’ J. Pharm. Sci. 106: 1722-1735 (2017)). Many of these methods involve detection of FFA by LC-MS. However, these methods are often compromised by the presence of exogenous FFA contaminants in the LC-MS system, which interfere with accurate test sample FFA detection. Accordingly, a novel analytical approach using mixed-mode anion exchange SPE combined with gas chromatography-mass spectrometry' (GC-MS) was established as described herein to enable simultaneous quantification of the major FFA degradants formed from polysorbate degradation in drug product formulations with superior lower limits of quantitation (LLOQ = 0.5-2.8 ng/pl) as compared to previously reported GC-MS FFA detection methods. In this method, sample processing prior to GC-MS analysis involves derivatization of FFAs to more volatile fatty' acid methyl esters (FAMEs). This approach requires separation of FFAs from polysorbate in the sample to avoid simultaneous polysorbate derivatization and ensure accurate FFA measurement. The resulting GC-MS quantification method was validated with respect to slope parallelism assessment in surrogate vs. authentic matrices, accuracy, precision, matrix effect, and extraction recovery, proving to be suitable for accurate absolute quantification of the major fatty' acid degradants of polysorbate. [0007] Thus, herein is described a novel GC-MS based approach for FFA measurement in polysorbate containing biopharmaceutical formulations to serve as a superior method for drug product formulation quality control in pharmaceutical industries.

BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 shows example fatty acid extracted ion chromatographs from a blank solvent injection on a heavily used LCMS system. Chromatograph traces were collected from a blank solvent injection in negative mode on a Thermo Orbitrap HFX high resolution mass spectrometer. The total ion chromatogram (TIC) is shown as the top trace in FIG. 1. Contaminating amounts of lauric acid (227.1955-227.20445 m/z; second trace from the top), palmitic acid (255.2249- 255.2351 m/z, third trace from the top), and stearic acid (283.2615-283.2671 m/z. bottom trace), where all detected in the blank solvent sample.

[0009] FIG. 2 shows representative extracted ion chromatograms of a solvent blank sample (Blank; dashed line trace) and FAME analytes in a surrogate blank matrix (S9; solid line trace) using GCMS system.

[0010] FIG. 3 shows GC-MS analysis of internal standard fatty acid methyl ester (FAME) levels in formic acid (FA)-methanol (MeOH) eluate samples (MB-C8 samples on the left-side of each graph) and MeOH wash fractions (MB-C8 samples on the right-side of each graph). Results demonstrate excellent retention of internal deuterated standards (i.e., lauric acid show n in the left graph and stearic acid shown in the right graph) during methanol wash resulting in negligible FAMES detected in MeOH wash fraction.

[0011] FIG. 4 shows GC-MS analysis of blank drug formulation matrix sample spiked with the indicated concentrations of FFA (lauric acid) standard. The FAMES levels measured in both FA MeOH eluate samples (bars on the left-side of the graph) and MeOH wash fractions (bars on the right-side of the graph) are depicted. Note the constant level of FAMES present in the methanol wash, which is generated by hydrolysis and derivatization of intact polysorbate from the for ulation matrix.

[0012] FIG. 5 show s the standard curves for each fatty acid of interest generated by spiking the indicated purified fatty acid standards into surrogate blank matrix containing PS20 as the surfactant at the indicated concentrations. Linear regression was performed in GraphPad Prism using 1/x² weighting. Lowber limits of quantitation (LLOQ) w ere determined as the lowest spiked calibration standard sample with tolerable % accuracy (< ± 20%).

[0013] FIG. 6 show s the standard curves for each fatty acid of interest generated by spiking the indicated purified fatty acid standards into authentic antibody drug formulation matrix at the indicated concentrations. Lower limits of quantitation (LLOQ) were determined as the lowest spiked calibration standard sample with tolerable % accuracy (< ± 20%).

[0014] FIGs. 7A-7B shows the quantitation results for several fatty acids (measured as FAMES) using the anion-exchange SPE and GC-MS method as described herein. FIG. 7A show's the results for lauric acid (left) and mystic acid (right) and FIG. 7B shows the results for palmitic acid (left) and stearic acid (right). Fatty acid concentrations were calculated by fitting the observed peak areas for each fatty acid onto the calibration curves depicted in FIGs. 5 and 6. Fatty acid concentration varied across the drug product (DP) samples (i.e., DPI, DP2, DP3, DP4, and DP5) which contained PS20 or PS80 surfactant as indicated and were subject to the indicated storage conditions.

DETAILED DESCRIPTION OF THE VARIOUS EMBODIMENTS

[0015] The present disclosure is directed to methods for quantifying free fatty acid content in a pharmaceutical formulation. This method involves providing a sample of a pharmaceutical formulation comprising a surfactant, and subjecting the formulation sample to a solid phase extraction (SPE) process to separate free fatty acids in the formulation sample from the surfactant. The method further involves derivatizing the separated free fatty acids into fatty acid esters. The derivative fatty acid esters are separated by gas chromatography and detected. The method further involves quantify ing the free fatty acid (FFA) content of the pharmaceutical formulation based on the separation and detection of the fatty acid esters.

[0016] In accordance with this aspect of the present disclosure, the pharmaceutical formulation is a composition comprising a pharmaceutical drug product, a surfactant, and, optionally, one or more pharmaceutically acceptable excipients and/or vehicles. The pharmaceutical drug product can be any biologically active drug product or substance, including, without limitation, a chemical compound, a nucleic acid molecule, a toxin, or a protein. In any embodiment, the drug product is a protein, such as, for example, a peptide, a polypeptide, a protein, a fusion protein, an antibody (e.g., monoclonal antibody, multispecific antibody), an antibody fragment (e.g., Fab, Fv, Fc, etc.), an antibody derivative (e.g., scFv, diabody, minibody), an antibody-drug conjugate, a vaccine antigen, or any other protein product or protein product derivative. The protein drug product can be a naturally occurring protein, a non-naturally occurring protein, a synthetically produced protein, a recombinant protein, a protein variant, or a protein derivative.

[0017] The concentration of the pharmaceutical drug product in the pharmaceutical formulation is from about 0.1% to about 30% (w/v) of the formulation. For example, the drug product in the pharmaceutical formulation may be about 1% to about 30% (w/v), about 5% to about 30% (w/v). about 5% to about 25% (w/v). about 5% to about 20% (w/v), about 5% to about 10% (w/v), about 10% to about 30% (w/v), about 10% to about 25% (w/v), about 10% to about 20% (w/v), or about 15% to about 25% (w/v). In any embodiment, the drug product in the pharmaceutical formulation is about 0.1% (w/v), about 0.5% (w/v), about 1% (w/v), about 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%, 10%. 11%. 12%. 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or about 30% (w/v).

[0018] In accordance with the methods of the present disclosure, the pharmaceutical formulation comprises a surfactant. In some embodiments, the pharmaceutical formulation comprises one or more additional excipients including, but not limited to, buffering agents, bulking agents, tonicity modifiers, solubilizing agents, and preservatives. Additional acceptable excipients that may be included based on function and compatibility with the formulation are known in the art, See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington 's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0019] In any embodiment, the surfactant of the pharmaceutical formulation is a polysorbate surfactant. Polysorbates are a class of amphipathic, non-ionic surfactants that are derived from ethoxylated sorbitan or isosorbide (derivative of sorbitol) esterified with fatty acids. Polysorbates are commonly used in pharmaceutical formulations to prevent protein aggregation, denaturation, and surface adsorption.

[0020] Polysorbates share a common sorbitan head group where each of the four hydroxyl groups is bound to a polyethylene glycol (PEG) chain (also known as polyethylene oxide (POE) chain). The types of polysorbates differ in the fatty acid side chain esterified with one of the PEG side chains. Polysorbates that are typically found in pharmaceutical formulations and, thus, relevant to the methods described herein include, without limitation, polysorbate 20 (PS20; polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (PS40; polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (PS60; polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (PS80; polyoxyethylene (20) sorbitan monooleate).

[0021] Commercial and pharmaceutical grade polysorbates comprise a mixture of structurally related compounds and contain a mixture of various fatty acid chains. For example, PS20 contains primarily lauric acid (40.0-60.0%), but also contains myristic acid (14.0-25.0%), palmitic acid (7.0-15.0%). oleic acid (< 11.0%), caprylic acid (< 10.0%), capric acid (< 10.0%), stearic acid (< 7.0%), linoleic acid (< 3.0%), and caproic acid (< 1.0%). Likewise, PS80 primarily contains oleic acid (> 58.0%), but also contains linoleic acid (< 18.0%), palmitic acid (< 16.0%), palmitoleic acid (< 8.0%), stearic acid (< 6.0%), myristic acid (< 5.0%), and linolenic acid (< 4.0%).

[0022] In one embodiment, the methods disclosed herein are utilized to quantify free fatty acid content in pharmaceutical formulations comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or any mixture thereof. In one embodiment, the methods disclosed herein are utilized to quantify free fatty acid content in a pharmaceutical formulation comprising polysorbate 20. In another embodiment, the methods disclosed herein are utilized to quantify free fatty⁷ acid content in a pharmaceutical formulation comprising polysorbate 80. In yet another embodiment, the method disclosed here is utilized to quantify free fatty acid content in a pharmaceutical formulation comprising a mixture of polysorbate 20 and polysorbate 80.

[0023] The polysorbate concentration in the pharmaceutical formulation is the concentration necessary⁷ to stabilize components of the formulation, in particular, protein components of the formulation. In any embodiment, the polysorbate concentration in the pharmaceutical formulation is between about 0.001% and about 1% (w/v). In any embodiment, the polysorbate concentration in the pharmaceutical formulation is between about 0.01% and about 1% (w/v), between about 0.1% and about 1% (w/v), between about 0.001% and about 0.1% (w/v), between about 0.01% and about 0.1% (w/v), or between about 0.001% and about 0.01% (w/v). In any embodiment, the concentration of polysorbate in the pharmaceutical formulation is about 0.001% w/v, about 0.002% w/v, about 0.003% w/v, about 0.004% w/v, about 0.005% w/v, about 0.006% w/v, about 0.007% w/v, about 0.008% w/v, about 0.009% w/v, about 0.01% w/v, about 0.015% w/v, about 0.02% w/v, 0.025% w/v, about 0.03% w/v, about 0.035% w/v, about 0.04% w/v, about 0.045% w/v, about 0.05% w/v, about 0.06% w/v, about 0.07% w/v, about 0.08% w/v, about 0.09% w/v, about 0. 1 % w/v, about 0.2% w/v. about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9% w/v, or about 1% w/v.

[0024] Polysorbates are susceptible to a variety of oxidative and hydrolytic degradation pathways, with a primary⁷ degradant being free fatty acids. Polysorbate degradation can decrease the functional properties of the polysorbate, and the resulting fatty acid degradants can, themselves, induce protein instability. As used herein, the term ‘'free fatty acid” refers to the long aliphatic chain moiety of the fatty⁷ acid ester. FFAs are produced, for example, by the degradation of fatty acid esters. Free fatty acids can be in a solubilized form or may aggregate to form aggregates or particles. The methods described herein are suitable for detecting and quantifying free fatty acid content of polysorbate containing compositions. The free fatty acids that can be detected and quantified using the methods described herein include, without limitation, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, stearic acid, linoleic acid, palmitoleic, linolenic, and any combination thereof.

[0025] Caproic acid, also known as hexanoic acid, is the carboxylic acid derived from hexane having the chemical formula CH3(CH2)4COOH. Caproic acid has a carbon chain length of six (C6). Salts and esters of decanoic acid are called “caproates'’. Caprylic acid, also known as octanoic acid, is a saturated fatty acid, and a carboxylic acid, having the formula CH3(CH2)eCOOH. Caprylic acid has a carbon chain length of eight (C8). Salts and esters of decanoic acid are called “caprylates”. Capric acid, also known as decanoic acid or decylic acid, is a saturated fatty acid of the formula CH3(CH2)sCOOH. Capric acid has a carbon chain length of ten (CIO). Salts and esters of decanoic acid are called “decanoates” or “caprates”. Lauric acid, also known as dodecanoic acid, is a saturated fatty acid having the formula CH3(CH2)ioCOOH. Lauric acid has a carbon chain length of twelve (Cl 2). The salts and esters of lauric acid are known as “laurates”. Myristic acid is a saturated fatty⁷ acid having the formula CHs(CH2)i2COOH. Myristic acid has a carbon chain length of fourteen (Cl 4). Its salts and esters are commonly referred to as “myristates” or “tetradecanoates”. Palmitic acid, also known as hexadecanoic acid, is a saturated fatty acid having the formula CH3(CH2)i4COOH. Palmitic acid has a carbon chain length of sixteen (C16). Salts and esters of decanoic acid are called “palmitates”. Stearic acid, also know n as octadecanoic acid, is a saturated fatty acid of the formula CH3(CH2)ieCOOH. Stearic acid has a carbon chain length of eighteen (Cl 8). The salts and esters of stearic acid are called “stearates”. Oleic acid is a fatty acid having the chemical formula CH3(CH2)?CH=CH(CH2)7COOH. Oleic acid has a carbon chain length of eighteen carbon atoms with one point of unsaturation (C 18:1). The salts and esters of oleic acid are called “oleates”. Linoleic acid is a saturated fatty acid having the formula COOH(CH2)7CH=CHCH2CH=CH(CH2)4CH3. Linoleic acid has a carbon chain length of eighteen carbon atoms and two points of unsaturation (C 18:2). The salts and esters of linoleic acid are called “linoleates”.

[0026] The concentration of free fatty⁷ acids that can be detected in the pharmaceutical formulations using the methods described herein is about 0.010 pg/mL to about 1000 pg/mL. In any embodiment, the method described herein is suitable for detecting free fatty acids present in the formulation at a concentration of < 500 pg/mL, < 100 pg/mL, < 50 pg/mL, < 10 pg/mL, < 5 pg/mL, < 1 pg/mL, < 0.9 pg/mL, < 0.8 pg/mL, < 0.7 pg/mL, < 0.6 pg/mL, < 0.5 pg/mL, < 0.4 pg/mL, < 0.3 pg/mL, < 0.2 pg/mL, < 0.1 pg/mL, < 0.09 pg/mL, < 0.08 pg/mL, < 0.07 pg/mL, < 0.06 pg/mL, < 0.05 pg/mL, < 0.04 pg/mL, < 0.03 pg/mL. < 0.02 pg/mL, or < 0.01 pg/mL. In any embodiment, the method descnbed herein is suitable for detecting free fatty acids present in the formulation at a concentration of < 0.1 pg/mL. [0027] As described herein, the methods of the present disclosure involve subjecting a sample of a pharmaceutical formulation comprising a surfactant, such as a polysorbate, to a separation process to separate the surfactant (e.g., polysorbate) from the free fatty acid degradants present in the sample. Because polysorbate is also subject to derivatization, isolating the fatty acid degradants prior to derivatization to fatty acid esters is required to avoid false positive signal generation. Fatty acids and polysorbate in a sample can be separated based on their distinctive physical or chemical properties using a separation technique, including, without limitation, solidphase extraction (SPE), liquid-liquid extraction (LLE), or electroextraction (EE). For example, because fatty acids comprise a carboxylic acid moiety that is absent in polysorbate, separation using an anionic based sorbent material can sufficiently separate fatty acids from polysorbate present in a sample.

[0028] Accordingly, in one embodiment of the methods described herein, the formulation sample is subject to a solid-phase extraction process. As used herein, a “solid-phase extraction” process refers to a process that partitions or separates compounds present in a liquid sample based on the differential affinities of the compounds for a solid phase, z.e., the sorbent. The solid phase is selected based on which type of interaction (e.g, an ionic, hydrophobic, or polar interaction) will attract the analytes of interest (i.e., the free fatty acids in the sample), such that the analytes of interest are retained on the solid phase and subsequently eluted. Alternatively, the solid phase can be selected based on which type of interaction will attract impurities in the sample, where the analyte(s) of interest pass through the stationary phase and are collected (while the undesired components are retained on the solid phase).

[0029] The solid phase extraction process typically involves providing a sorbent having characteristics suitable to bind the compound or analyte of interest. Once the sorbent is equilibrated or conditioned, the sample containing the compound(s) or analyte(s) of interest is loaded or percolated through the sorbent under conditions that allow the analyte(s) of interest to be retained on the sorbent. The sorbent is then washed one or more times to selectively remove any impurities, and the analyte or compound of interest is collected by eluting from the sorbent with an appropriate elution solution.

[0030] A suitable solid-phase extraction phase for use in the methods described herein is one that has preferential affinity for the carboxylic acid moiety' present on the fatty acid degradants, but absent on polysorbate, e.g, a stationary phase comprising anion-exchange groups such as citrate, HSOF, NOs’, HSOs’, Nt ’, Cl’, HCCE". HPOT. formate, acetate, propionate, F', or OH‘. Suitable anionic stationary phases are commercially available, see e.g, anion exchange columns available from Waters™. [0031] In one embodiment, a suitable solid-phase extraction phase for use in the methods described herein is a mixed-mode sorbent, for example a mixed-mode sorbent containing anion- exchange groups. A mixed-mode sorbent provides at least two or more retention mechanisms to simultaneously extract one or more compounds from the formulation sample. In one embodiment, the mixed-mode sorbent comprises an anion exchange and reversed-phase sorbent. The reversed- phase sorbent is a hydrophobic stationary phase, having a strong affinity for hydrophobic compounds. Mixed-mode sorbents comprising anion exchange groups suitable for use in the methods disclosed herein are also commercially available, see e.g., the Oasis MAX mixed-mode polymeric sorbent columns and plates available from Waters™.

[0032] Solid-phase extraction of the formulation samples comprising polysorbate and fatty acids using anion-exchange based stationary phase, e.g, the mixed mode anion exchange and reversed-phase sorbent, results in the retention of both polysorbate and fatty acids on the stationary phase. The free fatty acid fraction is selectively recovered by elution with an acid-organic solution. For example, suitable elution buffers comprise methanol, acetonitrile, or ethyl acetate containing ~2% acid solution. In one embodiment, the elution buffer is methanol containing 2% acid. In one embodiment, the elution buffer is methanol containing 2% formic acid

[0033] In accordance with the methods described herein, once the fatty acids of the formulation sample have been separated from the polysorbate, the fatty acids are derivatized to form fatty acid esters, e.g. , fatty acid methyl esters, which are more volatile and less polar than free fatty acids, and thus more amenable to gas chromatography separation and analysis. Derivatization of fatty acids to fatty acid esters can be performed using an acid-catalyzed or base-catalyzed esterification or transesterification reaction process. Suitable short-chain alcohols suitable for such reactions include, for example and without limitation, methanol (to form methyl esters) and ethanol (to form ethyl esters), 2-propanol. and butanol. Suitable acid catalysts include, for example and without limitation, boron trifluoride (BF3), boron trichloride, sulfuric acid, hydrogen chloride, acetyl chloride, aluminum chloride, aluminum trichloride, and p-toluenesulfonic acid (see e.g., Zotov et al., “Methodological Aspects of the Analysis of Fatty Acids in Biological Samples,” Applied Biochem. Microbiol. 58:83-95 (2022), which is hereby incorporated by reference in its entirety). Suitable base catalysts include, without limitation, sodium methoxide, potassium hydroxide, and ammonium derivatives such as tetramethylammonium hydroxide (TMAH) and trimethylphenylammonium hydroxide (TMPAH) (see e.g., Zotov et al., “Methodological Aspects of the Analysis of Fatty Acids in Biological Samples,” Applied Biochem. Microbiol. 58:83-95 (2022). which is hereby incorporated by reference in its entirety). Other suitable fatty acid derivatization methods include the use of potassium methanolate (KOCFbyhydrochloric acid (HC1) or sodium methoxide (NaOCHs) followed by (trimethylsilyl)diazomethane (TMS-DM) as described in Salimon et al., “Comparison of Two Derivation Methods for the Analysis of Fatty Acids and Trans Fatty Acids in Bakery Products Using Gas Chromatography,” Sci. World J. 906407 (2014), which is hereby incorporated by reference in its entirety.

[0034] Exemplary methods of preparing fatty acid methyl esters suitable for GC analysis are described in detail in the Examples herein. Accordingly, in one embodiment, the derivatization reaction utilizes methanol and boron trifluoride as described herein. In particular, the derivatization reaction can involve incubating fatty acids separated from a formulation sample with a 10% BF3-methanol solution at about 80°C for l-3hrs with shaking. The derivatization reaction can be quenched with the addition of NaCl and fatty acid methyl esters (FAMES) can be extracted with the addition of hexanes.

[0035] Alternative methods of preparing fatty acid esters suitable for GC analysis that are known in the art are also suitable for use in the methods of the present disclosure. See for example, methods disclosed by Ichihara and Fukubayashi, “Preparation of Fatty Acid Methyl Esters for Gas- Liquid Chromatography.” J. Lipid Res. 51(3):635-640 (2010); Cruz-Hernandez. C. and Destaillats, F., in Encyclopedia of Lipidomics, Wenk, M., Ed., Dordrecht: Springer, 2016; Cruz-Hernandez et al., “Quantification of Fatty Acids in Erythrocytes and Plasma by Fast Gas Chromatography,” J. Separation Sci. 40(16): 3289-3300 (2017); Salimon et al., “Comparison of Two Derivation Methods for the Analysis of Fath’ Acids and Trans Fatty Acids in Bakery Products Using Gas Chromatography,” Sci. World J. 906407 (2014); Zotov et al., “Methodological Aspects of the Analysis of Fatty Acids in Biological Samples,” Applied Biochem. Microbiol. 58:83-95 (2022), which are hereby incorporated by reference in their entirety.

[0036] The derivative fatty acid esters, e.g., fatty' acid methyl esters, are then separated by gas chromatography and detected. Gas chromatography (GC), also referred to as vapor-phase chromatography and gas-liquid partition chromatography, is a common type of chromatography used to analyze compounds that can be vaporized without decomposition. GC works by separating compounds in a mixture, e.g, fatty methyl ester derivatized from fatty acids in a pharmaceutical formulation sample, when a sample (e.g., liquid sample) containing the compounds is injected into a mobile phase and the mobile phase is passed through a stationary phase. The mobile phase of GC is usually an inert gas, such as helium, argon, nitrogen, or hydrogen, and the stationary phase is suitable for separating the fatty acid esters according to the carbon number (number of carbon atoms in the fatty acid chain, excluding the methyl ester carbon) and the degree of saturation. High resolution of fatty acid methyl esters can be achieved on polar columns and very polar (cyanopropyl) columns (see e.g., Goding et al., “Comparison of GC Stationary Phases for the Separation of Fatty Acid Methyl Esters in Biodiesel Fuels,'’ Anal. Bioanal. Chem. 405(18):6087- 94 (2013), which is hereby incorporated by reference in its entirety). However, separation of fatty acid methyl esters can also be achieved on non-polar stationary phases as reviewed in K. Eder, “Gas Chromatography Analysis of Fatty Acid Methyl Esters,” J. Chromatogr. B 671: 113-131 (1995), which is hereby incorporated by reference in its entirety. Accordingly, GC separation of fatty acid esters in accordance with the methods of the present disclosure can be carried out using non-polar stationary phases (e.g, phases based on methylsilicone, dimethyl/diphenylpolysiloxane, and dimethylpoly siloxane), polar stati onary phases (e.g., phases based on polyethylene glycol, acidified polyethylene glycol, dimethyl/cyanopropylphenylpolysiloxane, and methylsilicone polymer), or very polar stationary phases (e.g, phases based on cyanoethylsilicone, cyanopropylsilicone, or biscyanopropyl/dimethylsiloxane) all of which are commercially available (see e.g., K. Eder, “Gas Chromatography Analysis of Fatty Acid Methyl Esters,” J. Chromatogr. B 671 : 113-131 (1995), which is hereby incorporated by reference in its entirety).

[0037] Detection and quantitation of the fatty acid esters following separation on the GC column can be achieved via a detector that detects the fatty acid esters as they elute from the GC column. The interaction between the ester and the detector as it elutes is converted to an electronic signal, where the magnitude of the signal is plotted versus time (time from injection) and a chromatogram is generated. The time at which the ester elutes from the column and is detected is its retention time, z.e., the time taken for the ester to pass through the column (time from injection to detection). The retention time can be used to identify the ester. The magnitude of signal (area under the peak) is indicative of fatty acid ester concentration, when compared to a calibration curve generated from appropriate standard samples of known quantities.

[0038] Any suitable GC detector can be utilized in accordance with the methods described herein to detect the fatty acid esters following separation, for example and without limitation, a flame ionization detector, a thermal conductivity detector, or a mass spectrometer.

[0039] In any embodiment of the methods described herein, the GC is coupled to a mass spectrometer for detection and quantitation of the separated fatty' acid esters. A mass spectrometer is a device capable of identifying specific molecular species and quantifying its mass. In accordance with the methods described herein the mass spectrometer is coupled to the gas chromatograph to achieve accurate detection and quantitation of the fatty acid esters which serve as an indicator of the identity and concentration of free fatty acids in a pharmaceutical formulation sample of interest. In an embodiment, the mass spectrometer is a single quadrupole mass spectrometer. In another embodiment, the mass spectrometer is an ion trap mass spectrometer or a magnetic sector mass spectrometer. Other suitable detectors that can be coupled to the GC for detection of fatty acid methyl esters include, without limitation time of flight detection and tandem quadrupoles (MS-MS).

[0040] The identity and quantitation of the fatty acids present in a formulation sample are determined by comparison of the retention times of fatty acid esters in the sample to retention times of fatty acid esters similarly derived from individual purified fatty acid standards. The concentration of fatty acids in a sample is calculated by fitting the chromatographic peak areas of the derived fatty acid esters to calibration curves also generated using a dilution series of purified fatty acid standards. The dilution series of purified fatty acid standards can be made in a solvent blank or surrogate matrix blank (each containing polysorbate) as described in the Examples herein. The surrogate matrix blank does not contain residual lipases of the solvent blank that may cause polysorbate hydrolysis, thereby interfering with standard curve preparation.

[0041] The invention having been described, the following examples are offered by way of illustration, and not limitation.

EXAMPLES

Materials and Methods

[0042] To prepare for solid phase extraction, samples of different drug formulation lots were diluted 1 :4 with water and methanol containing deuterated fatty acid internal standards to a final composition of 50:50 aqueous: methanol solution. For calibration curves, specified concentrations of free fatty' acids were added to a surrogate blank matrix (20mM histidine, 270mM sucrose, 0.01% polysorbate 20 at a pH = 6) prior to dilution as described above.

[0043] Free fatty acids were extracted from drug formulation matrix using a mixed mode anion-exchange solid phase extraction (SPE, Waters Oasis MAX 96-well plate) as described below. First the SPE cartridge was connected to a vacuum manifold and equilibrated with ImL methanol, followed by ImL 5% NH4OH and another ImL 5% NH4OH. Following equilibration, the samples were slowly loaded onto the cartridge, ensuring that the vacuum force was sufficient to produce a slow drop by drop flow rate through the cartridge. After sample loading, the SPE cartridge was washed twice with ImL 5% NH4OH. After washing, vacuum was applied to the cartridge to dry' the sorbent for 5 minutes. After drying, the cartridge was washed with ImL methanol. Following the methanol wash, free fatty acids were eluted from the SPE cartridge by applying a solution of ImL of methanol + 2% formic acid. The SPE eluent was then dried under a stream of nitrogen gas.

[0044] To derivatize the free fatty⁷ acids to fatty⁷ acid methyl esters (FAMES), ImL of 10% BF3-methanol was added to the dried samples. This solution was then incubated in a thermomixer at 80°C for 2hrs with shaking at 400rpm. Next the derivatization reaction is quenched by adding 100 pL of 5M NaCl. FAMES were extracted from this solution by addition of 500 pL of hexanes and vortexing. For analysis, 200pL of the resulting upper layer was removed and introduced to an autosampler vial for GCMS analysis.

[0045] GCMS analysis was performed using an Agilent 8890 gas chromatograph (GC) coupled to an 5977B single quadrupole mass selective detector (MS). The column used for separating FAMES by gas chromatography was a DB-FastFAME 30m x 250pm x 0.25pm column (Agilent). Sample injection volume was IpL and the sample split ratio was 5: 1. The carrier gas used was ultra-pure helium (Praxair). The sample inlet was maintained at a temperature of 250°C. The gas chromatography temperature gradient was as follows: The initial temperature was set at 50°C and held for 0.5 minutes. Next, the temperature was ramped to 194°C at a rate of 30°C/min and held at this temperature for 3.5 minutes. Next, the temperature was ramped to 240°C at a rate of 5°C/min and held for 1 minute. Finally, the column was returned to 50°C and re-equilibrated at this temperature for 2 minutes prior to the next sample injection. The gas pressure was maintained at 8psi and 1.03 mL/minute constant flow rate throughout the method. The following mass spectrometer settings were used to detect FAMES: solvent delay = 3 minutes, mass range = 50- 550 m/z, cycle time was 342.63 ms, step size = 0.1 and the ionization mode was electron ionization (El).

[0046] FAMES chromatography peaks were integrated in Quant-My-Way software (Agilent) and FAMES concentrations for samples were calculated by comparing peak areas to the linear regression analysis obtained from the calibration curves.

EXAMPLE 1: Quantitation of Free Fatty Acids (FFA) Using Liquid Chromatography

Mass Spectrometry Vulnerable to Unacceptable Levels of FFA Contaminant Interference

[0047] Free fatty acids are commonly used in plastic manufacturing and are also present in many biological matrices that are routinely analyzed by scientists in biopharmaceutical laboratories. Additionally, free fatty acids can be difficult to efficiently remove from liquid chromatography autosamplers, where they can build up on seals, sampling needles and seats, and other components within the sample flow path. As a result, fatty' acid contamination is common in Liquid Chromatography Mass Spectrometry (LCMS) systems and can interfere with quantitation of fatty acids in experimental samples, particularly at lower concentrations.

[0048] Typical levels of fatty⁷ acid contamination in LCMS systems are shown in the chromatograph of FIG. 1. FIG. 1 shows exemplary⁷ fatty⁷ acid extracted ion chromatographs from a blank solvent injection on a heavily used LCMS system. Chromatograph traces collected from the blank solvent injection in negative mode on a Thermo Orbitrap HFX high resolution mass spectrometer show high levels of contaminating signal corresponding to lauric acid (227.1955- 227.2045 m/z, second trace from the top), palmitic acid (255.2249-255.2351 m/z, third trace from the top), and stearic acid (283.2615-283.2671 m/z, bottom trace). This level of interference in the blank solvent injection sample would cause an artificially high FFA level in any test sample.

EXAMPLE 2: Solid Phase Extraction Coupled with Gas Chromatograph Mass

Spectrometry Accurately Quantifies FFA Levels in Pharmaceutical Formulation Samples

[0049] To avoid the interference of fatty⁷ acid contamination commonly present in LCMS systems, gas chromatography mass spectrometry⁷ (GCMS) was evaluated as an alternative analytical platform for fatty⁷ acid quantitation in pharmaceutical formulation samples. Because fatty acids are not sufficiently volatile to be directly analyzed by GCMS, they must first be converted to fatty acid methyl esters by derivatization. As shown in Figure 2, in comparison to the level of FAMEs interference in the blank solvent injection on the LCMS, there is no significant FAMEs interference found in a solvent blank sample using GCMS. In addition, FIG. 2 shows that FAME analytes in the surrogate blank matrix were well separated with the GCMS method described herein.

[0050] To analyze FFA concentrations in drug formulations containing polysorbates like PS20 and PS80, it is necessary to separate free fatty acid degradants from intact polysorbate prior to derivatization. because any intact polysorbate in solution will be hydrolyzed to FAMES during derivatization. If allowed to remain in the sample the intact polysorbates would generate an artifactually high free fatty acid measurement. To extract FFA from solutions containing intact polysorbate, anion exchange solid phase extraction (SPE) sorbent was utilized as described in the Methods. SPE allows retention of FFA on the anion exchange resin while neutral hydrophobic compounds are eluted during the methanol wash. Retained FFAs are then eluted with an acidic methanol elution step. FIG. 3 demonstrates the excellent retention of two deuterated FFA standards using this approach, with negligible internal standard observed in the methanol wash fractions.

[0051] The methanol wash step does contain appreciable levels of polysorbate (see FIG. 3, bars on the right portion of each graph), which would give rise to a substantial FAMES signal that would greatly reduce the sensitivity and accuracy of the FFA measurements if anion-exchange SPE were not used to separate the FFA degradants from polysorbate prior to derivatization (see FIG. 4).

[0052] The presence of residual lipases in drug formulations can induce polysorbate hydrolysis and release FFA analytes, and this could interfere with standard curve preparation in an authentic drug formulation. Therefore, a surrogate blank matrix that does not contain drug product or residual lipase activity was selected for standard curve preparation. To validate the selected surrogate matrix was equivalent to using an authentic drug formulation a slope parallelism assessment of the FFA standard curve generated in the two matrices was used (see FIGs. 5 and 6). The slope parallelism test confirmed that the selected surrogate matrix can be used for standard curve preparation instead of authentic matrix with equivalent results (Slope difference criteria: < ±15 % Table 1) [0053] To obtain accurate quantitative measurements of free fatty acid content in drug formulation matrices, standard curves were generated by spiking in ten purified fatty acid standards ranging from 0.13 to 166.7 ng/pl into a surrogate blank matrix containing polysorbate (for detailed matrix recipe please see the Methods section). The calibration standards were processed according to the Method described above and the resulting curves are displayed in FIG. 5. The standard curves for all analytes were found to be linear with good correlation coefficient (R²) of 0.99 for Lauric, Myristic and Palmitic acid and 0.98 for Stearic and Oleic acid. The LLOQ for each analyte was determined as the lowest spiked calibration standard sample with tolerable % accuracy (< ±20 %) and are shown in FIG. 5.

[0054] Several other key assay performance attributes including accuracy, precision, matrix effect, and extraction recoven’ were also validated for the developed GCMS method as discussed below.

[0055] Accuracy & Precision. The accuracy and precision of the method were determined through Relative Error (RE%) and Coefficient of Variation (CV%) respectively. As shown in Table 2, the resulting fluctuations did not exceed 15% for intra- and inter-assays for CV% and 20% for RE% with both concentrations examined, indicating that the established method was accurate and reliable.

[0056] Matrix Effect. Matrix effect was measured by comparing the peak area of FFA analytes in authentic antibody drug formulation matrix to the peak area in surrogate blank matrix at three different concentrations (Table 3). The matrix effect was found to be within the acceptance criteria of < ±15 %, suggesting no significant matrix effect was observed with the developed method.

[0057] Extraction Recovery. The mixed mode anion-exchange solid phase extraction (SPE) recovery was determined by comparing the MS response of deuterated medium-chain (d23- lauric) and longer-chain (d35-stearic) FFAs from pre-SPE and post-SPE spiked samples using the average result of four replicates. As present in Table 4, the extraction recovery for both medium- and longer-chain FFAs showed excellent recovery (98.8% and 95.2%, respectively) with less than 8% CV% for all replicates. It was concluded that the selected mixed mode anion-exchange solid phase extraction could be used to extract medium to long chain FFA (C12-C 18) from antibody drug formulation.

[0058] Several antibody drug product (DP) lots, i.e., DP1-DP5, that had previously been aged by incubation for various temperatures (5°C or 25°C) over specified times (0, 6, or 12 weeks) were subjected to this analysis to assess whether prolonged incubation time at different temperatures impacted fatty acid hydrolysis from polysorbates in the drug product formulation. The results demonstrated that prolonged incubation of up to 12 weeks resulted in increased FFA concentrations in drug product as shown by the quantitative graphs of FIGs. 7A and 7B. In some cases, incubation of the drug product at 25°C also appeared to increase the levels of FFA present in the samples compared to the same drug lot incubated at 5°C. This indicates that incubation temperature and storage time may have an impact on fatty acid hydrolysis from polysorbate in drug products.

[0059] The analytical approach using mixed-mode anion exchange SPE combined with gas chromatography-mass spectrometry (GC-MS) as described herein enables simultaneous quantification of the major FFA degradants formed from polysorbate degradation in drug product formulations with superior lower limits of quantitation (LLOQ = 0.5-2.8 ng/pl) as compared to previously reported GC-MS FFA detection methods. There are two known mechanistic pathways of polysorbate degradation, i.e., hydrolysis and auto-oxidation. The presence of FFF in drug formulation samples points to the occurrence of ester hydrolysis as one of the degradation mechanisms. Utilizing the mixed mode ion exchange SPE separation method demonstrated that free fatty acids could be isolated from intact polysorbate, which allows for further examination of the percentage of polysorbate degradation that occurs through the hydrolysis pathway.

[0060] In summary, the GC-MS quantification method described herein was validated with respect to slope parallelism assessment in surrogate vs. authentic matrices, accuracy, precision, matrix effect, and extraction recovery, proving to be suitable for accurate absolute quantification of the major fatty acid degradants of polysorbate.

[0061] Each reference cited herein is hereby incorporated by reference in its entirety for all that it teaches and for all purposes.

[0062] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual embodiments of the invention, and functionally equivalent methods and components are invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method for quantifying free fatty acid content in a pharmaceutical formulation, said method comprising: providing a sample of a pharmaceutical formulation comprising a surfactant; subjecting the formulation sample to a solid phase extraction (SPE) process to separate free fatty acids in the sample from the surfactant; derivatizing the separated free fatty acids into fatty acid esters; separating the derivative fatty acid esters by gas chromatography; detecting the separated fatty' acid esters; and quantifying the free fatty acid content of the pharmaceutical formulation based on said separating and said detecting of tire fatty acid esters.

2. The method of claim 1 , wherein the free fatty acid content comprises fatty acids selected from caprylic acid, capric acid, caproic acid, lauric acid, myristic acid, palmitic acid, oleic acid, stearic acid, linoleic acid, palmitoleic, linolenic, or a combination thereof.

3. The method of claim 1 or claim 2, wherein the surfactant is polysorbate.

4. The method of claim 3, wherein the polysorbate is polysorbate 20, polysorbate 80, or a combination thereof.

5. The method of claim 3 or claim 4, wherein the polysorbate concentration in the pharmaceutical formulation is between 0.001% and 0.1% (w/v).

6. The method of any one of claims 1-5, wherein the SPE process utilizes a mixed-mode sorbent.

7. The method of claim 6, wherein tire mixed-mode sorbent comprises an anion exchange and reverse-phase sorbent.

8. The method of claim 7, wherein the free fatty acids are collected during the SPE process by eluting the free fatty’ acids from the mix-mode sorbent using an organic acid solution.

9. The method of claim 8, wherein the organic acid solution is a methanol/formic acid solution.

10. The method of claim 8 or claim 9, wherein the organic solution comprises 2% formic acid.

11. The method of any one of claims 1-10, wherein tire free fatty esters are free fatty methyl esters.

12. The method of claim 11, wherein said derivatizing comprises: incubating the separated free fatty⁷ acids with BF -methanol to form fatty acid methyl esters.

13. The method of any one of claims 1-12. wherein said detecting is carried out using a mass spectrometer.

14. The method of claim 13, wherein the mass spectrometer is a single quadrupole mass spectrometer.

15. The method of any one of claims 1-14, wherein said quantifying comprises: comparing the detected fatty acid esters from the formulation sample to one or more fatty acid ester calibration curves generated from fatty acid standard samples comprising known concentrations of corresponding fatty acids.

16. The method of any one of claims 1-15, wherein the pharmaceutical formulation comprises a drug product selected from a chemical compound, a nucleic acid drug product, or a protein drug product.

17. The method of claim 16, wherein the protein drug product is selected from a peptide therapeutic, a recombinant protein therapeutic, a fusion protein, an antibody, an antibody fragment, an antibody derivative, or an antibody-drug conjugate.