Method for monitoring surfactant hydrolysis in biopharmaceutical formulations

Abstract

The present disclosure relates to methods for detecting, monitoring, and quantifying the free fatty acid content of pharmaceutical formulations. The method includes subjecting a sample of the formulation containing a surfactant to an extraction process to separate the free fatty acids in the sample from the surfactant. The method further includes derivatizing the separated free fatty acids to fatty acid esters for detection and quantification via gas chromatography. The field of the disclosure relates to methods for detecting, monitoring, and quantifying the hydrolysis of surfactants in biopharmaceutical formulations.

Description

[Technical Field]

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

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

[0003] Background of Various Embodiments Polysorbate (PS) is the most common nonionic surfactant used in biopharmaceutical formulations due to its excellent stabilizing properties for drug product (DP) formulations and its ability to protect proteins and other biologicals from aggregation and denaturation. However, residual lipase present in DP formulations can cause PS hydrolysis, resulting in the accumulation of free fatty acids (FFA), which drive undesirable particle formation in DP formulations. While methods have been developed to directly quantify FFA released from PS using liquid chromatography-mass spectrometry (LC-MS), many researchers have experienced significant interference from FFA in LC systems, leading to inaccurate quantification of this type of analyte. Therefore, there is a need for novel methods to accurately quantify FFA accumulation in biopharmaceutical formulations. The present disclosure overcomes these and other deficiencies in the art. Summary of the Invention [Means for solving the problem]

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

[0005] During the manufacture, distribution, storage, and administration of biopharmaceutical formulations, the therapeutic biological components of the formulation are exposed to several interfaces, such as glass, plastic polymers, stainless steel, air, ice crystals, and silicone oil, which can result in adsorption, denaturation, or aggregation of the therapeutic components. All of these processes reduce the effective concentration of the therapeutic biological agent in the formulation. Nonionic surfactants such as polysorbates are commonly used in biopharmaceuticals to protect the therapeutic biological components of the formulation, such as peptides, proteins, antibodies, and vaccines, from adsorption, aggregation, and related instability. Polysorbates are often the preferred surfactant choice due to their low toxicity and good stabilization properties. However, polysorbates can undergo enzyme-mediated degradation, resulting in the formation of free fatty acid degradation products, which can affect the functional properties of polysorbates and thereby induce instability of the biological components of the formulation. For this reason, it is important to have analytical approaches that can accurately characterize and quantify the presence of FFA degradants in pharmaceutical formulations, particularly during the development process, to ensure the production of formulations that will maintain product integrity during the subsequent manufacturing, storage, and administration processes.

[0006] Methods for detecting polysorbates and their degradation products, such as FFAs, in biopharmaceutical formulations have been described (see, for example, the 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 the detection of FFAs by LC-MS. However, these methods are often compromised by the presence of exogenous FFA contaminants in the LC-MS system, which prevents accurate FFA detection of test samples. Therefore, as described herein, we established a novel analytical approach using mixed-mode anion-exchange SPE coupled with gas chromatography-mass spectrometry (GC-MS) to enable simultaneous quantification of the major FFA degradation products formed from polysorbate degradation in pharmaceutical formulations with superior limits of quantitation (LLOQ = 0.5-2.8 ng / μL) 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 polysorbates in the sample to avoid simultaneous polysorbate derivatization and ensure accurate FFA measurement. The resulting GC-MS quantification method was validated for slope parallelism assessment, precision, accuracy, matrix effect, and extraction recovery in alternative versus authentic matrices, demonstrating its suitability for accurate absolute quantification of the major fatty acid degradation products of polysorbates.

[0007] Thus, described herein is a novel GC-MS-based approach for FFA determination in polysorbate-containing biopharmaceutical formulations that serves as an excellent method for the quality control of drug formulations in the pharmaceutical industry. [Brief explanation of the drawings]

[0008] [Figure 1]An example fatty acid extracted ion chromatograph from a blank solvent injection on a widely used LCMS system is shown. Chromatographic traces were collected from blank solvent injections in negative mode on a Thermo Orbitrap HFX high-resolution mass spectrometer. The total ion chromatogram (TIC) is shown as the top trace in Figure 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), all of which were detected in the blank solvent sample, are shown. [Figure 2] Representative extracted ion chromatograms of a solvent blank sample (blank; dashed trace) and FAME analytes in an alternative blank matrix (S9; solid trace) using a GCMS system are shown. [Figure 3] GC-MS analysis of internal standard fatty acid methyl ester (FAME) levels in the formic acid (FA)-methanol (MeOH) eluate samples (MB-C8 samples on the left of each graph) and the MeOH wash fractions (MB-C8 samples on the right of each graph) shows excellent retention of the internal deuterated standards (i.e., lauric acid shown in the left graph and stearic acid shown in the right graph) during the methanol wash, with negligible FAMES detected in the MeOH wash fractions. [Figure 4] Figure 1 shows a GC-MS analysis of a blank drug formulation matrix sample spiked with the indicated concentrations of FFA (lauric acid) standard. Measured FAMES levels are shown in both the FA MeOH eluate sample (bar on the left side of the graph) and the MeOH wash fraction (bar on the right side of the graph). Note the constant level of FAMES present in the methanol wash, which is generated by hydrolysis and derivatization of intact polysorbate from the formulation matrix. [Figure 5]Standard curves for each fatty acid of interest are shown, generated by spiking the indicated purified fatty acid standards into an alternative blank matrix containing PS20 as a surfactant at the concentrations indicated. Linear regression was performed with GraphPad Prism using 1 / x2 weighting. The lower limit of quantitation (LLOQ) was determined as the lowest calibration standard spiked with an acceptable % precision (<±20%). [Figure 6] Standard curves for each fatty acid of interest are shown, generated by spiking the indicated purified fatty acid standards into the authentic antibody drug formulation matrix at the concentrations shown. The lower limit of quantitation (LLOQ) was determined as the lowest calibration standard spiked with an acceptable % precision (<±20%). [Figure 7A] Figures 7A-7B show quantitative results (measured as FAMES) for several fatty acids using the anion-exchange SPE and GC-MS methods described herein. Figure 7A shows the results for lauric acid (left) and myristic acid (right), and Figure 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 shown in Figures 5 and 6. Fatty acid concentrations varied across drug product (DP) samples (i.e., DP1, DP2, DP3, DP4, and DP5) containing the indicated PS20 or PS80 surfactant and subjected to the indicated storage conditions. [Figure 7B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure relates to a method for quantifying free fatty acid content in a pharmaceutical formulation. The method includes providing a sample of a pharmaceutical formulation containing 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 includes derivatizing the separated free fatty acids into fatty acid esters. The derivatized fatty acid esters are separated and detected by gas chromatography. The method further includes quantifying the free fatty acid (FFA) content of the pharmaceutical formulation based on the separation and detection of the fatty acid esters.

[0010] According to this aspect of the disclosure, a pharmaceutical formulation is a composition comprising a pharmaceutical agent, a surfactant, and, optionally, one or more pharmaceutically acceptable excipients and / or vehicles. A pharmaceutical agent can be any biologically active drug or substance, including, but not limited to, a chemical compound, a nucleic acid molecule, a toxin, or a protein. In all embodiments, the drug is a protein, e.g., a peptide, polypeptide, protein, fusion protein, antibody (e.g., monoclonal antibody, multispecific antibody), antibody fragment (e.g., Fab, Fv, Fc, etc.), antibody derivative (e.g., scFv, diabody, minibody), antibody-drug conjugate, vaccine antigen, or any other protein product or protein product derivative. A protein drug 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.

[0011] The concentration of the drug in the pharmaceutical formulation is about 0.1% (w / v) to about 30% (w / v) of the formulation. For example, the drug in the pharmaceutical formulation can 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 all embodiments, the drug 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).

[0012] According to the method 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 buffers, bulking agents, osmolality adjusters, solubilizers, and preservatives. Additional acceptable excipients that can be included based on function and compatibility with the formulation are known in the art. For example, see Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated by reference in its entirety); Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated by reference in its entirety).

[0013] In all embodiments, the surfactant of the pharmaceutical formulation is a polysorbate surfactant. Polysorbates are a class of amphiphilic nonionic surfactants derived from ethoxylated sorbitan or isosorbide (a derivative of sorbitol) esterified with fatty acids. Polysorbates are commonly used in pharmaceutical formulations to prevent protein aggregation, denaturation, and surface adsorption.

[0014] Polysorbates share a common sorbitan head group in which each of the four hydroxyl groups is attached to a polyethylene glycol (PEG) chain (also known as a polyethylene oxide (POE) chain). Polysorbate types differ in the fatty acid side chain that is esterified by one of the PEG side chains. Polysorbates typically found in pharmaceutical formulations and therefore relevant to the methods described herein include, but are not limited to, 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).

[0015] Commercially available and pharmaceutical-grade polysorbates contain mixtures of structurally related compounds and contain a mixture of different fatty acid chains. For example, PS20 contains primarily lauric acid (40.0-60.0%), but also 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%). Similarly, PS80 contains primarily oleic acid (≧58.0%), but also 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%).

[0016] In one embodiment, the methods disclosed herein are utilized to quantify the 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 the free fatty acid content in pharmaceutical formulations comprising polysorbate 20. In another embodiment, the methods disclosed herein are utilized to quantify the free fatty acid content in pharmaceutical formulations comprising polysorbate 80. In yet another embodiment, the methods disclosed herein are utilized to quantify the free fatty acid content in pharmaceutical formulations comprising a mixture of polysorbate 20 and polysorbate 80.

[0017] The concentration of polysorbate in the pharmaceutical formulation is a concentration essential for stabilizing the components of the formulation, particularly the protein components of the formulation. In all embodiments, the concentration of polysorbate in the pharmaceutical formulation is about 0.001% to about 1% (w / v). In all embodiments, the concentration of polysorbate in the pharmaceutical formulation is about 0.01% to about 1% (w / v), about 0.1% to about 1% (w / v), about 0.001% to about 0.1% (w / v), about 0.01% to about 0.1% (w / v), or about 0.001% to about 0.01% (w / v). In all embodiments, 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 0.04 w / v%, about 0.05 w / v%, about 0.06 w / v%, about 0.07 w / v%, about 0.08 w / v%, about 0. ...6 w / v%, about 0 %, 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%.

[0018] Polysorbates are susceptible to various oxidation and hydrolysis pathways, with free fatty acids being the primary degradation products. Polysorbate degradation can reduce the functional properties of polysorbates, and the resulting fatty acid degradation products themselves can induce protein instability. As used herein, the term "free fatty acid" refers to the long aliphatic chain portion of fatty acid esters. FFAs are produced, for example, by the degradation of fatty acid esters. Free fatty acids can be in a solubilized form or can aggregate to form aggregates or particles. The methods described herein are suitable for detecting and quantifying the free fatty acid content of polysorbate-containing compositions. Free fatty acids that can be detected and quantified using the methods described herein include, but are not limited to, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, stearic acid, linoleic acid, palmitoleic acid, linolenic acid, and any combination thereof.

[0019] Caproic acid, also known as hexanoic acid, is a carboxylic acid derived from hexane with the chemical formula CH3(CH2)4COOH. Caproic acid has a carbon chain length of 6 (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 with the formula CH3(CH2)6COOH. Caprylic acid has a carbon chain length of 8 (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 with the formula CH3(CH2)8COOH. Capric acid has a carbon chain length of 10 (C10). Salts and esters of decanoic acid are called "decanoates" or "caprates." Lauric acid, also known as dodecanoic acid, has the formula CH3(CH2) 10 It is a saturated fatty acid with COOH. Lauric acid has a carbon chain length of 12 (C12). Salts and esters of lauric acid are known as "laurates." Myristic acid has the formula CH3(CH2) 12It is a saturated fatty acid with COOH. Myristic acid has a carbon chain length of 14 (C14). Its salts and esters are commonly called "myristate" or "tetradecanoate." Palmitic acid, also known as hexadecanoic acid, has the formula CH3(CH2) 14 It is a saturated fatty acid with COOH. Palmitic acid has a carbon chain length of 16 (C16). Salts and esters of decanoic acid are called "palmitates." Stearic acid, also known as octadecanoic acid, has the formula CH3(CH2) 16 Stearic acid is a saturated fatty acid with a carbon chain length of 18 carbon atoms (C18). Salts and esters of stearic acid are called "stearates." Oleic acid is a fatty acid with the chemical formula CH3(CH2)7CH=CH(CH2)7COOH. Oleic acid has a carbon chain length of 18 carbon atoms and one point of unsaturation (C18:1). Salts and esters of oleic acid are called "oleates." Linoleic acid is a saturated fatty acid with the formula COOH(CH2)7CH=CHCH2CH=CH(CH2)4CH3. Linoleic acid has a carbon chain length of 18 carbon atoms and two points of unsaturation (C18:2). Salts and esters of linoleic acid are called "linoleates."

[0020] The concentrations of free fatty acids that can be detected in pharmaceutical formulations using the methods described herein are from about 0.010 μg / mL to about 1000 μg / mL. In all embodiments, the methods described herein are suitable for detecting free fatty acids present in a formulation at concentrations of <500 μg / mL, <100 μg / mL, <50 μg / mL, <10 μg / mL, <5 μg / mL, <1 μg / mL, <0.9 μg / mL, <0.8 μg / mL, <0.7 μg / mL, <0.6 μg / mL, <0.5 μg / mL, <0.4 μg / mL, <0.3 μg / mL, <0.2 μg / mL, <0.1 μg / mL, <0.09 μg / mL, <0.08 μg / mL, <0.07 μg / mL, <0.06 μg / mL, <0.05 μg / mL, <0.04 μg / mL, <0.03 μg / mL, <0.02 μg / mL, or <0.01 μg / mL. In all embodiments, the methods described herein are suitable for detecting free fatty acids present in a formulation at a concentration of ≦0.1 μg / mL.

[0021] As described herein, the disclosed method involves subjecting a sample of a pharmaceutical formulation containing a surfactant, such as polysorbate, to a separation process to separate the surfactant (e.g., polysorbate) from free fatty acid degradation products present in the sample. Furthermore, because polysorbate undergoes derivatization, isolation of the fatty acid degradation products prior to derivatization into fatty acid esters is required to avoid false-positive signal generation. Fatty acids and polysorbates in a sample can be separated using separation techniques based on their distinctive physical or chemical properties, including, but not limited to, solid-phase extraction (SPE), liquid-liquid extraction (LLE), or electroextraction (EE). For example, because fatty acids contain carboxylic acid moieties that are not present in polysorbates, separation using anionic sorbent materials can adequately separate fatty acids from polysorbates present in a sample.

[0022] Thus, in one embodiment of the methods described herein, a pharmaceutical sample is subjected to a solid-phase extraction process. As used herein, the term "solid-phase extraction" refers to a process that partitions or separates compounds present in a liquid sample based on the compounds' differential affinity for a solid phase, i.e., an adsorbent. The solid phase is selected based on the type of interaction (e.g., ionic, hydrophobic, or polar) that will attract the analytes of interest (i.e., free fatty acids in the sample) so that the analytes of interest are retained on the solid phase and then eluted. Alternatively, the solid phase can be selected based on the type of interaction that will attract impurities in the sample, such that the analytes of interest pass through the stationary phase and are collected (while undesirable components are retained on the solid phase).

[0023] A solid-phase extraction process typically involves providing a sorbent with suitable properties for binding the compound or analyte of interest. Once the sorbent is equilibrated or conditioned, a sample containing the compound or analyte of interest is loaded or permeated through the sorbent under conditions that allow the analyte 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 it from the sorbent with an appropriate elution solution.

[0024] Suitable solid phase extraction phases for use in the methods described herein are those that have a preferential affinity for carboxylic acid moieties that are present on fatty acid digests but not on polysorbates, such as citrate, HSO, - , NO3 - , HSO3 - , NO2 - , Cl - , HCO3 - , HPO4 - , formate, acetate, propionate, F - , or OH - Suitable anionic stationary phases are commercially available, see for example anion exchange columns available from Waters™.

[0025] In one embodiment, the solid-phase extraction phase suitable for use in the methods described herein is a mixed-mode sorbent, e.g., a mixed-mode sorbent containing an anion exchange group. A mixed-mode sorbent provides at least two or more retention mechanisms for simultaneously extracting one or more compounds from a pharmaceutical sample. In one embodiment, the mixed-mode sorbent comprises an anion exchange and a reversed-phase sorbent. A reversed-phase sorbent is a hydrophobic stationary phase with a strong affinity for hydrophobic compounds. Mixed-mode sorbents containing anion exchange groups suitable for use in the methods disclosed herein are also commercially available. See, for example, Oasis MAX mixed-mode polymeric sorbent columns and plates available from Waters™.

[0026] Solid-phase extraction of a pharmaceutical sample containing polysorbates and fatty acids using an anion-exchange-based stationary phase, such as a mixed-mode anion-exchange and reversed-phase sorbent, retains both the polysorbates and the fatty acids on the stationary phase. The free fatty acid fraction is selectively recovered by elution with an acid-organic solution. For example, a suitable elution buffer includes methanol, acetonitrile, or ethyl acetate containing about 2% acid. In one embodiment, the elution buffer is methanol containing 2% acid. In one embodiment, the elution buffer is methanol containing 2% formic acid.

[0027] Once the fatty acids of a formulation sample have been separated from the polysorbate according to the methods described herein, the fatty acids are derivatized to form fatty acid esters, such as fatty acid methyl esters, which are more volatile and less polar than free fatty acids and therefore more amenable to gas chromatography separation and analysis. The derivatization of fatty acids to fatty acid esters can be carried out using acid- or base-catalyzed esterification or transesterification processes. Suitable short-chain alcohols for such reactions include, but are not limited to, methanol (to form methyl esters) and ethanol, 2-propanol, and butanol (to form ethyl esters). Suitable acid catalysts include, but are not limited to, boron trifluoride (BF), 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) (incorporated herein by reference in its entirety)). Suitable base catalysts include, but are not limited to, 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," Microbiol. 58:83-95 (2022) (incorporated herein by reference in its entirety)).Other suitable fatty acid derivatization methods include the use of potassium methanolate (KOCH3) / hydrochloric acid (HCl) or sodium methoxide (NaOCH3) 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 incorporated herein by reference in its entirety.

[0028] Exemplary methods for 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. Specifically, the derivatization reaction can involve incubating fatty acids isolated from a formulation sample with a 10% BF3-methanol solution at approximately 80°C for 1-3 hours with shaking. The derivatization reaction can be quenched by the addition of NaCl, and the fatty acid methyl esters (FAMES) can be extracted by the addition of hexane.

[0029] Alternative methods known in the art for preparing fatty acid esters suitable for GC analysis are also suitable for use in the methods of the present disclosure. For example, 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 Fatty Acids and Trans Fatty Acids in Bakery Products Using Gas Chromatography,”Sci.World J.906407(2014);Zotov et See the methods disclosed by W. et al., “Methodological Aspects of the Analysis of Fatty Acids in Biological Samples,” Applied Biochem. Microbiol. 58:83-95 (2022) (incorporated herein by reference in their entirety).

[0030] The derivative fatty acid esters, e.g., fatty acid methyl esters, are then separated and detected by gas chromatography. Gas chromatography (GC), also known as gas-phase chromatography and gas-liquid partition chromatography, is a common type of chromatography used to analyze compounds that can be vaporized without decomposition. GC functions by injecting a sample (e.g., a liquid sample) containing compounds, e.g., fatty methyl esters derivatized from fatty acids, into a mobile phase, which separates the compounds in a mixture in a pharmaceutical formulation sample as the mobile phase passes through a stationary phase. The mobile phase in GC is usually an inert gas such as helium, argon, nitrogen, or hydrogen, and the stationary phase is suitable for separating fatty acid esters according to carbon number (the number of carbon atoms in the fatty acid chain, excluding the methyl ester carbon) and degree of saturation. High resolution of fatty acid methyl esters can be achieved with polar and ultrapolar (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), incorporated herein by reference in its entirety). However, separation of fatty acid methyl esters can also be achieved with nonpolar stationary phases, as reviewed in K. Eder, "Gas Chromatography Analysis of Fatty Acid Methyl Esters," J. Chromatogr. B 671:113-131 (1995), incorporated herein by reference in its entirety.Thus, GC separation of fatty acid esters according to the methods of the present disclosure can be carried out using a nonpolar stationary phase (e.g., phases based on methylsilicone, dimethyl / diphenylpolysiloxane, and dimethylpolysiloxane), a polar stationary phase (e.g., phases based on polyethylene glycol, acidic polyethylene glycol, dimethyl / cyanopropylphenylpolysiloxane, and methylsilicone polymers), or a hyperpolar stationary phase (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) (incorporated herein by reference in its entirety)).

[0031] Detection and quantification of fatty acid esters after separation on a 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 the ester elutes is converted into an electronic signal, and the magnitude of the signal is plotted against time (time from injection) to generate a chromatogram. The time at which an ester elutes from the column and is detected is its retention time, i.e., the time it takes 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 the signal (area under the peak) indicates the fatty acid ester concentration when compared to a calibration curve generated from appropriate standards of known amounts.

[0032] Any suitable GC detector can be utilized in accordance with the methods described herein to detect the fatty acid esters after separation, for example, but not limited to, a flame ionization detector, a thermal conductivity detector, or a mass spectrometer.

[0033] In all embodiments of the methods described herein, the GC is coupled to a mass spectrometer for detection and quantification of separated fatty acid esters. A mass spectrometer is a device that can identify specific molecular species and quantify their mass. According to the methods described herein, a mass spectrometer is coupled to a gas chromatograph to achieve accurate detection and quantification of fatty acid esters, which serve as indicators of the identity and concentration of free fatty acids in a pharmaceutical formulation sample of interest. In one 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, but are not limited to, time-of-flight detection and tandem quadrupole (MS-MS).

[0034] The identity and quantification of fatty acids present in a formulation sample are determined by comparing the retention time of the fatty acid esters in the sample with the retention time of fatty acid esters similarly derived from individual purified fatty acid standards. The concentration of fatty acids in the sample is calculated by fitting the chromatographic peak area of ​​the derived fatty acid esters to a calibration curve generated using a dilution series of the purified fatty acid standards. The dilution series of the purified fatty acid standards can be made in a solvent blank or a surrogate matrix blank (each containing polysorbate), as described in the Examples herein. The surrogate matrix blank does not contain the residual lipase of the solvent blank, which may cause polysorbate hydrolysis and thereby interfere with standard curve generation.

[0035] The following examples, in which the invention is illustrated, are offered by way of illustration and not by way of limitation. [Example]

[0036] Materials and Methods To prepare for solid-phase extraction, samples of various 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, the specified concentrations of free fatty acids were added to an alternative blank matrix (20 mM histidine, 270 mM sucrose, 0.01% polysorbate 20, pH 6) before dilution as above.

[0037] Free fatty acids were extracted from drug formulation matrices using 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 1 mL of methanol followed by 1 mL of 5% NH4OH and another 1 mL of 5% NH4OH. After equilibration, the sample was slowly loaded onto the cartridge, ensuring that the vacuum force was sufficient to create a slow, dropwise flow rate through the cartridge. After sample loading, the SPE cartridge was washed twice with 1 mL of 5% NH4OH. After washing, a vacuum was applied to the cartridge to dry the sorbent for 5 minutes. After drying, the cartridge was washed with 1 mL of methanol. After the methanol wash, free fatty acids were eluted from the SPE cartridge by adding 1 mL of a solution of methanol and 2% formic acid. The SPE eluate was then dried under a stream of nitrogen gas.

[0038] To derivatize free fatty acids to fatty acid methyl esters (FAMES), 1 mL of 10% BF3 - Methanol was added to the dried sample. The solution was then incubated at 80°C for 2 hours with shaking at 400 rpm in a thermomixer. The derivatization reaction was then quenched by adding 100 μL of 5 M NaCl. FAMES was extracted from the solution by adding 500 μL of hexane and vortexing. For analysis, 200 μL of the resulting upper layer was removed and placed in an autosampler vial for GCMS analysis.

[0039] GCMS analysis was performed using an Agilent 8890 gas chromatograph (GC) coupled to a 5977B single quadrupole mass selective detector (MS). The column used to separate FAMES by gas chromatography was DB - The column used was a FastFAME 30 m × 250 μm × 0.25 μm column (Agilent). The sample injection volume was 1 μL, and the sample split ratio was 5:1. The carrier gas used was ultrapure 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 to 50 °C and held for 0.5 minutes. The temperature was then increased to 194 °C at a rate of 30 °C / min and held at this temperature for 3.5 minutes. The temperature was then increased 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 before injecting the next sample. The gas pressure was maintained at a constant 8 psi and a flow rate of 1.03 mL / min throughout the method. To detect FAMES, the following mass spectrometer settings were used: solvent delay = 3 min, mass range = 50–550 m / z, cycle time = 342.63 ms, step size = 0.1, and ionization mode was electron ionization (EI).

[0040] FAMES chromatographic peaks were analyzed by Quantitative Analysis. - My - Integrated into the Way software (Agilent), the FAMES concentration of the sample was calculated by comparing the peak area with a linear regression analysis obtained from the calibration curve.

[0041] Example 1: Quantification of Free Fatty Acids (FFA) Using Liquid Chromatography Mass Spectrometry Methods Susceptible to Unacceptable Levels of FFA Contaminant Interference Free fatty acids are commonly used in plastics manufacturing and are also present in many biological matrices routinely analyzed by scientists in biopharmaceutical laboratories. Additionally, free fatty acids can be difficult to efficiently remove from liquid chromatography autosamplers (where they can accumulate on seals, sampling needles, and sheets, as well as 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 the quantification of fatty acids in experimental samples, especially at lower concentrations.

[0042] Typical levels of fatty acid contamination in an LCMS system are illustrated in the chromatograph in Figure 1. Figure 1 shows an exemplary fatty acid extracted ion chromatograph from a blank solvent injection on a widely used LCMS system. Chromatographic traces collected from a blank solvent injection in negative mode on a Thermo Orbitrap HFX high-resolution mass spectrometer show high levels of contaminating signals 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 artificially high FFA levels in any test sample.

[0043] Example 2: Solid-phase extraction coupled with gas chromatography-mass spectrometry accurately quantifies FFA levels in pharmaceutical formulation samples To avoid the interference of fatty acid contaminants commonly present in LCMS systems, gas chromatography-mass spectrometry (GCMS) was evaluated as an alternative analytical platform for the quantification of fatty acids in pharmaceutical formulation samples. Fatty acids are not sufficiently volatile for direct analysis by GCMS and must therefore first be converted to fatty acid methyl esters by derivatization. As shown in Figure 2, no significant FAME interference was found in solvent blank samples using GCMS compared to the level of FAME interference in blank solvent injections with LCMS. Additionally, Figure 2 demonstrates that FAME analytes in the alternative blank matrix were well separated by the GCMS method described herein.

[0044] To analyze FFA concentrations in drug formulations containing polysorbates such as PS20 and PS80, it is necessary to separate free fatty acid degradants from intact polysorbates prior to derivatization, because any intact polysorbates in solution will be hydrolyzed to FAMES during derivatization. If left in the sample, intact polysorbates would result in artificially high free fatty acid measurements. To extract FFAs from solutions containing intact polysorbates, an anion exchange solid-phase extraction (SPE) sorbent was utilized as described in this method. SPE allows FFAs to be retained on the anion exchange resin, while neutral hydrophobic compounds are eluted during the methanol wash. The retained FFAs are then eluted with an acidic methanol elution step. Figure 3 shows excellent retention of two deuterated FFA standards using this approach, with negligible internal standard observed in the methanol wash fraction.

[0045] The methanol wash step contains appreciable levels of polysorbate (see Figure 3, bars in the right portion of each graph), which results in a substantial FAMES signal that significantly reduces the sensitivity and accuracy of FFA measurement unless anion exchange SPE is used to separate the FFA degradants from polysorbate prior to derivatization (see Figure 4).

[0046] The presence of residual lipase in drug formulations may induce polysorbate hydrolysis to release FFA analytes, which may interfere with standard curve generation in authentic drug formulations. Therefore, an alternative blank matrix containing no drug or residual lipase activity was selected for standard curve generation. To verify that the selected alternative matrix was equivalent to using the authentic drug formulation, a slope parallelism assessment of the FFA standard curves generated in the two matrices was used (see Figures 5 and 6). The slope parallelism test confirmed that the selected alternative matrix could be used for standard curve generation instead of the authentic matrix with comparable results (slope difference criterion: <±15%; Table 1).

[0047] [Table 1]

[0048] To obtain accurate quantitative measurements of the free fatty acid content in the drug formulation matrix, standard curves were generated by spiking 10 purified fatty acid standards ranging from 0.13 to 166.7 ng / μl into a surrogate blank matrix containing polysorbate (see Methods section for detailed matrix recipes). The calibration standards were processed according to the method described above, and the resulting curves are shown in Figure 5. The standard curves for all analytes were linear, with correlation coefficients (R 2 ) were found to be good at 0.99 for lauric, myristic, and palmitic acids, and 0.98 for stearic and oleic acids. The LLOQ for each analyte was determined as the lowest spiked calibration standard sample with an acceptable % precision (<±20%) and is shown in Figure 5.

[0049] Additionally, several other important assay performance characteristics, including precision, accuracy, matrix effect, and extraction recovery, were verified for the developed GCMS method, as discussed below.

[0050] Precision and accuracy. The precision and accuracy of the method were determined by relative error (RE%) and coefficient of variation (CV%), respectively. As shown in Table 2, the resulting variations did not exceed 15% for intra- and inter-assay CV% and 20% for RE% for both concentrations investigated, indicating that the established method is accurate and reliable.

[0051] [Table 2]

[0052] Matrix effect. Matrix effect was measured by comparing the peak area of ​​the FFA analyte in the authentic antibody drug formulation matrix with the peak area in a surrogate blank matrix at three different concentrations (Table 3). The matrix effect was found to be within the acceptance criteria of <±15, suggesting that no significant matrix effect was observed with the developed method.

[0053] [Table 3]

[0054] Extraction Recovery. Mixed-mode anion exchange solid-phase extraction (SPE) recovery was determined by comparing the MS responses of deuterated medium-chain (d23-lauric) and long-chain (d35-stearic) FFAs from pre-SPE and post-SPE spiked samples using the average results of four replicates. As presented in Table 4, extraction recoveries for both medium-chain and long-chain FFAs showed excellent recoveries (98.8% and 95.2%, respectively), with CV% less than 8% for all replicates. We concluded that the selected mixed-mode anion exchange solid-phase extraction can be used to extract medium- to long-chain FFAs (C12-C18) from antibody drug formulations.

[0055] [Table 4]

[0056] Several antibody drug product (DP) lots, i.e., DP1-DP5, that had previously been aged by incubation at various temperatures (5°C or 25°C) for specific periods of time (0, 6, or 12 weeks) were subjected to this analysis to evaluate whether prolonged incubation times at different temperatures affected fatty acid hydrolysis from polysorbates in the drug product formulations. The results demonstrated that prolonged incubation times, up to 12 weeks, resulted in increased FFA concentrations in the drug product, as shown by the quantitative graphs in Figures 7A and 7B. In some cases, incubation of the drug product at 25°C also appeared to increase the levels of FFAs present in the samples compared to the same drug product lots incubated at 5°C. This indicates that incubation temperature and storage time can affect fatty acid hydrolysis from polysorbates in drug products.

[0057] The analytical approach described herein, using mixed-mode anion-exchange SPE coupled with gas chromatography-mass spectrometry (GC-MS), allows for simultaneous quantification of the major FFA degradants formed from polysorbate degradation in drug formulations with superior limits of quantitation (LLOQ = 0.5-2.8 ng / μL) compared to previously reported GC-MS FFA detection methods. There are two known mechanistic pathways for polysorbate degradation: hydrolysis and autoxidation. The presence of FFA in drug formulation samples points to the occurrence of ester hydrolysis as one of the degradation mechanisms. By utilizing a mixed-mode ion-exchange SPE separation method, we demonstrate the ability to isolate free fatty acids from intact polysorbates, which allows for further investigation of the percentage of polysorbate degradation that occurs via the hydrolysis pathway.

[0058] In summary, the GC-MS quantification method described herein was validated for slope parallelism assessment, precision, accuracy, matrix effect, and extraction recovery in alternative versus authentic matrices and was proven suitable for accurate absolute quantification of the major fatty acid degradation products of polysorbates.

[0059] Each reference cited herein is incorporated by reference in its entirety for all purposes for which it teaches.

[0060] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual embodiments of the invention and functionally equivalent methods and components of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and the accompanying figures. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for quantifying free fatty acid content in a pharmaceutical formulation, 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; quantitating the free fatty acid content of the pharmaceutical formulation based on the separation and the detection of the fatty acid esters; A method comprising:

2. 10. 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 acid, linolenic acid, or combinations thereof.

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

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

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

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

7. The method of claim 6 , wherein the mixed-mode adsorbent comprises an anion exchange and reversed-phase adsorbent.

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

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

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

11. The method of any one of claims 1 to 10, wherein the free fatty ester is a free fatty methyl ester.

12. The derivatizing step comprises: The separated free fatty acids were treated with BF ３ - incubating with methanol to form fatty acid methyl esters.

13. The method of any one of claims 1 to 12, wherein the detecting is performed using a mass spectrometer.

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

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

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

17. 17. The method of claim 16, wherein the protein drug 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.

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