Optimized quantification of fatty acids in complex media using advanced analytical techniques.

RATIONALE: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. METHODS: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. RESULTS: Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. CONCLUSIONS: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals.

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250 mg/mL).

Detailed profiling of polysorbate 80 oxidative degradation products and hydrolysates using liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant that is commonly used to stabilize proteins in biopharmaceutical formulations. PS80 undergoes oxidative and/or enzymatic degradation. However, because PS80 is a complex mixture consisting of many constituents, comprehensive evaluations of its oxidative degradation products are difficult and insufficient. METHODS: Our previously reported comprehensive liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based method for PS80 effectively provides an overall profile of PS80 components under simple LC conditions. In this study, we attempted to shorten the analysis time. Furthermore, PS80 was oxidatively degraded in a solution containing histidine and iron, and the oxidative degradation products were evaluated using a modified LC/MS/MS method. In addition, enzymatically hydrolyzed PS80 samples were analyzed. RESULTS: We succeeded in shortening the analysis time from 70 to 20 min while maintaining the resolution of the PS80 components of the same selected reaction monitoring transition. Both the previously reported oxidative degradation products and the newly discovered products were successfully detected, and their composition ratios and changes over time were observed. Changes in the hydrolysates over time are shown in the analysis of the hydrolyzed PS80 samples. CONCLUSIONS: This study clearly showed the presence of changes in PS80 oxidative and/or enzymatic degradation products, including those previously unreported. These results demonstrate that a detailed profiling of PS80 degradation products can be performed using LC/MS/MS, which is less expensive and more generally adopted than high-resolution MS.

Identification of nonvolatile organic compounds (NVOCs) in biopharmaceuticals through non-target analysis and quantification using complexation-precipitation extraction.

Single-use systems in biopharmaceutical manufacturing can potentially release chemical constituents (leachables) into drug products. Prior to conducting toxicological risk assessments, it is crucial to establish the qualitative and quantitative methods for these leachables. In this study, we conducted a comprehensive screening and structure elucidation of 23 leachables (nonvolatile organic compounds, NVOCs) in two antibody drugs using multiple (self-built and public) databases and mass spectral simulation. We identified 7 compounds that have not been previously reported in medical or medicinal extractables and leachables. The confidence levels for identified compounds were classified based on analytical standards, literature references, and fragment assignments. Most of the identified leachables were found to be plasticizers, antioxidants, slip agents or polymer degradants. Polysorbate (namely Tween) is commonly used as an excipient for protein stabilization in biopharmaceutical formulations, but its ionization in liquid chromatography-electrospray ionization mass spectrometry can interfere with compound quantification. To address this, we employed a complexation-precipitation extraction method to reduce polysorbate content and quantify the analytes. The developed quantitative method for target NVOCs demonstrated high sensitivity (limit of quantification: 20 or 50 µg/L), accuracy (recoveries: 77.2 to 109.5 %) and precision (RSD ≤ 8.2 %). Overall, this established method will facilitate the evaluation of NVOC safety in drug products.

High throughput FAMS - A fatty acid mass spectrometry method for monitoring polysorbate hydrolysis in QC.

Surfactant degradation in biopharmaceuticals has recently gained significant attention in the pharmaceutical industry. Specifically, hydrolytic degradation of polysorbates, leading to the release of free fatty acids potentially forming visible particles, is a key theme in technical development. To address this emerging topic, we present the development of a fully automated liquid-chromatography single quad mass detector method for the quantification of free fatty acids in biopharmaceuticals. For the first time, we have quantified the longer chain fatty acid degradation products of polysorbate, palmitic and stearic acid, allowing reliable detection and early critical insights for process improvements. This high-throughput method was validated underlining its robust performance in an interlaboratory trial as well as high flexibility allowing different robotic platforms and preparation techniques. The combination of automated sample preparation, separation by liquid chromatography and single quad mass detection makes the validated fatty acid mass spectrometry assay ready for routine use in a regulated environment.

Effects and Mode of Action of Oleic Acid and Tween 80 on Skin Permeation of Disulfiram.

Oral disulfiram (DSF) has been used clinically for alcohol dependence and recently has been found to have antitumor activity. A transdermal delivery system would be useful for maintaining drug concentration and reducing the frequency of administration of DSF for cancer treatment. Penetrating the stratum corneum (SC) barrier is a challenge to the transdermal delivery of DSF. Therefore, we investigated the promoting effects and mechanism of action of the combination of oleic acid (OA) and Tween 80 on the skin permeation of DSF. Hairless mouse skin was exposed to OA and Tween 80, combined in various ratios (1 : 0, 2 : 1, 1 : 1, 1 : 2, and 0 : 1). A permeation experiment was performed, and total internal reflection IR spectroscopic measurements, differential scanning calorimetry, and synchrotron radiation X-ray diffraction measurements were taken of the SC with each applied formulation. The combination of OA and Tween 80 further enhanced the absorption-promoting effect of DSF, compared with individual application. The peak of the CH2 inverse symmetric stretching vibration near the skin surface temperature was shifted by a high frequency due to the application of OA, and DSF solubility increased in response to Tween 80. We believe that the increased fluidity of the intercellular lipids due to OA and the increased solubility of DSF due to Tween 80 promoted the absorption of DSF. Our study clarifies the detailed mechanism of action of the skin permeation and promoting effect of DSF through the combined use of OA and Tween 80, contributing to the development of a transdermal preparation of DSF.

Profiling polysorbate 80 components using comprehensive liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant commonly used in pharmaceutical protein formulations and is composed of fatty acid (FA) esters of polyethoxylated sorbitan. However, commercial PS80 products contain substantial amounts of by-products. The development of simple and reliable methods for PS80 component analysis is challenging given the inherent heterogeneity. METHOD: We developed a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to profile the components of PS80. Semi-comprehensive LC-MS/MS analyses of 11 subspecies in three commercial PS80 products were performed to estimate the average degree of polymerization of the ethylene oxide units (Avg-n) in the molecules. Furthermore, three subspecies (polyoxyethylene sorbitan monoester, polyoxyethylene isosorbide monoester, and polyoxyethylene monoester) were analyzed to estimate the composition ratios of the seven ester-bonded FAs present in PS80. RESULTS: The Avg-n values of five polyoxyethylene sorbitan esters (none, mono, di, tri, and tetra), three polyoxyethylene isosorbide esters (none, mono, and di), and three polyoxyethylene esters (none, mono, and di) were 26.5-30.6, 12.1-14.6, and 11.4-15.8, respectively. These values were comparable regardless of the number of ester-bonded FAs. Each product had a similar FA composition ratio regardless of the differences in the subspecies. However, the obtained C18:2 values were higher than those reported in the product certificates. CONCLUSION: The proposed LC-MS/MS method evaluated the overall PS80 components, revealing the possibility of underestimation of ester-bonded linoleic acid using the conventional gas chromatography-mass spectrometry method. The similarity of Avg-n values and FA compositions among subspecies suggested the high reliability of these results, indicating that the presented approach may help in the quality control of PS80 formulations.

The Characterization of Polysorbate.

Polysorbates (PSs), including PS20 and PS80, are non-ionic surfactants widely used in the pharmaceutical industry to enhance drug solubility and stability. Despite their wide application, PSs are prone to degradation by either hydrolysis or oxidation in drug formulations during storage; therefore, a PS characterization method assessing protein products is needed for stability testing and for understanding the degradation pathway. In this article, we detail our protocol for sample preparation for forced degradation study and our instrumentation setup for PS profiling and quantitation in protein samples. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for forced degradation of polysorbate in protein samples Basic Protocol 2: Two-dimensional liquid chromatography coupled with charged aerosol detector or mass spectrometry to analyze polysorbate degradation.

Scaling Down Large-Scale Thawing of Monoclonal Antibody Solutions: 3D Temperature Profiles, Changes in Concentration, and Density Gradients.

PURPOSE: Scale-down devices (SDD) are designed to simulate large-scale thawing of protein drug substance, but require only a fraction of the material. To evaluate the performance of a new SDD that aims to predict thawing in large-scale 2 L bottles, we characterised 3D temperature profiles and changes in concentration and density in comparison to 125 mL and 2 L bottles. Differences in diffusion between a monoclonal antibody (mAb) and histidine buffer after thawing were examined. METHODS: Temperature profiles at six distinct positions were recorded with type T thermocouples. Size-exclusion chromatography allowed quantification of mAb and histidine. Polysorbate 80 was quantified using a fluorescent dye assay. In addition, the solution's density at different locations in bottles and the SDD was identified. RESULTS: The temperature profiles in the SDD and the large-scale 2 L bottle during thawing were similar. Significant concentration gradients were detected in the 2 L bottle leading to marked density gradients. The SDD slightly overestimated the dilution in the top region and the maximum concentrations at the bottom. Fast diffusion resulted in rapid equilibration of histidine. CONCLUSION: The innovative SDD allows a realistic characterisation and helps to understand thawing processes of mAb solutions in large-scale 2 L bottles. Only a fraction of material is needed to gain insights into the thawing behaviour that is associated with several possible detrimental limitations.

Stability Indicating Method for Polysorbate 80 in Protein Formulations.

Polysorbates (also known as "Tween") are common components of protein formulations used to minimize protein adsorption and stabilize the protein. These nonionic surfactants are heterogenous mixtures of fatty acids with a complex reversed-phase profile due to the inhomogeneity of the polymers present. Polysorbates can be oxidized, which can be hard to detect in the complex polymer profile. Further adding to the analytical challenge is the lack of a chromophore for the detection of these polymers. The routine analysis of polysorbates in protein formulations was greatly improved through the introduction of online solid-phase extraction (SPE) to simplify the polysorbate profile for quantification. However, this method combines many of the polysorbate polymers into a single peak for detection, thus limiting its effectiveness for detecting degradation. To address the need for a stability indicating method without the complexity of the reversed-phase profile, an optimized online SPE method was developed and investigated. Using polysorbate 80, this investigation shows that further expanding the step gradient can yield a profile that is stability indicating and available for routine testing of protein formulation.

Development and validation of a selective marker-based quantification of polysorbate 20 in biopharmaceutical formulations using UPLC QDa detection.

Polysorbates are widely used as non-ionic surfactant in biopharmaceutical formulations. Recently, the degradation of polysorbate moved into the focus of attention, because in several published studies it was described, that stability issues in polysorbate containing formulations were observed leading to the formation and appearance of sub-visible and visible particles. For this reason, monitoring of polysorbate and its degradation products is of importance throughout the development of parenterals. The aim of the study was to develop a method for the selective marker-based quantification of adequate polysorbate 20 components of interest without the need to apply derivatization or complex detection techniques. A single quadrupole mass (QDa) detector was used coupled to an ultra-high performance liquid chromatography (UPLC) system. Method development was based on a reversed phase-high performance liquid chromatography assay coupled to a charged aerosol detector (RP-HPLC CAD). Instead of a charged aerosol detector (CAD) a QDa detector was used in order to significantly improve the selectivity. The focus of this study is the development of the QDa based method for the analysis of polysorbate 20. Modifications of the mobile phase and the type of chromatography column allowed the separation of several components of polysorbate 20 from polar non-esterified to apolar higher order species. In addition, a multitude of components could be quantified by their individual m/z values. The peak assignment identified 676 compounds which originated from polysorbate 20. Some of these were selected and defined as marker components. It was shown that the developed method is capable to determine polysorbate 20 in different biopharmaceutical formulations. The proposed assay is based on a smart sample preparation as well as a unique calibration procedure that make the determination of several selected components achievable. Furthermore, it was successfully demonstrated that the analytical procedure is valid to reliably quantify several polysorbate 20 components at its 100% level (corresponds to 0.4 mg/mL intact polysorbate 20) and even at lower concentrations that occur e.g. in case of polysorbate 20 degradation. In conclusion, the method is beneficial to determine selected polysorbate 20 species during formulation development of biopharmaceuticals as well as during stability testing and trouble shooting.

Comparative analysis of docetaxel: physical and chemical characterisation of Taxotère® and generics.

Several cases of fatal enterocolitis have been described in association with the use of docetaxel (DTX), and this increase in adverse events has been concomitant with a change in formulation. Indeed in 2010, a new DTX-based presentation has been introduced in the form of a single ready-to-use vial by Sanofi-Aventis, presentation also used for generics. In this study, different available formulations were compared (Sanofi 2 vials, Sanofi 1 vial, Accord Healthcare, Kabi, Hospira) in terms of composition compliance with control specifications and simulated micelle behaviour to try to determine what could be the potential causes of this problem. This work had permitted to show that all the tested products complied with specifications in terms of dosage and purity. Variations in the composition of polysorbate 80 (PS80) have been observed but are probably too small to be responsible for the toxicity found in patients. However, we identified a difference in micelle size and release kinetics probably because of doubling concentration of ethanol in new formulation. As a result, we emphasised the importance in the case of DTX of conducting bioequivalence studies as expected in European Medicines Agency (EMA) guidance to ensure patient safety, even though these formulation changes might seem minor. Therefore, further studies are needed to explore the potential role of ethanol, PS80 and the unbound fraction of DTX in the development of enterocolitis in patients treated with DTX.

Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation.

Polysorbates are stabilizers typically required in therapeutic protein formulations. On account of their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects polysorbate 20 (PS20) and polysorbate 80 (PS80) against oxidative degradation. PS20 and PS80 solutions containing BHA, BHT-or as control without an antioxidant-were stressed by exposure to air at 40°C for 7 weeks. The following assays were performed: ferrous oxidation-xylenol orange assay, liquid chromatography coupled to UV and mass spectrometry (MS), pH measurement, liquid chromatography fluorescence micelle assay, headspace-gas chromatography coupled with MS. PS20 and PS80 solutions containing an antioxidant were found to be more stable, as demonstrated by lower peroxide levels, lower free fatty acid contents, stable pH, intact polysorbate micelle structure/composition, and less volatile degradants. PS20 and PS80 solutions containing BHT or BHA are more stable against oxidative degradation compared to nonstabilized solutions. It might be beneficial to formulate bulk polysorbate with the antioxidant(s) to ensure stabilization during all process steps.

Novel High-Throughput Assay for Polysorbate Quantification in Biopharmaceutical Products by Using the Fluorescent Dye DiI.

Polysorbates (PSs) are the most common surfactants in therapeutic protein formulations, and it is crucial to monitor their concentration along the life cycle of biopharmaceuticals. We developed a simple multi-well plate fluorescence-based assay for the rapid determination of PS20 and PS80 content in biopharmaceutical products. The method is based on the detection of the fluorescence emission intensity of the fluorescent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the presence of PSs at concentrations below their critical micelle concentration. This method can be applied for PS content determination in protein formulations (≤100 mg/mL) without the need of a previous protein removal step. The 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate assay implemented in multi-well plate format is suitable for high-throughput concentration screening. It has a linear range from 0.00020% to 0.0025% (w/v) PS20, and the limits of detection and quantification were 0.00020% and 0.00055% (w/v), respectively. This assay is markedly more selective and shows no or lower interferences due to hydrophobic components (e.g., silicone oil) potentially present in finished products than the fluorescence micelle assay based on N-phenyl-1-naphthylamine. It also provides comparable results for the PS content in liquid chromatography with charged aerosol detection analysis with protein removal, providing a fast alternative.

A development of high-throughput HPLC method for the polysorbate 80 quantitation in protein therapeutic products.

A quantitative analysis of polysorbate 80 is an essential part of a characterization of therapeutic protein products. There are many methods to perform such type of analysis, including spectrophotometry and HPLC. However, there is no high-throughput method with high sensitivity and accuracy. In our work, we optimized conditions of polysorbate 80 hydrolysis to reduce the sample preparation time and developed a new isocratic reversed phase HPLC method for the quantification of oleic acid, the principal product of the hydrolysis. The validation of developed method shows that it is characterized by the repeatability less than 2.7% with the LOD about 25 ng (125 ppb), LOQ about 100 ng (500 ppb) within the analytical range of 0.025 to 1.5 mg/ml polysorbate 80 concentrations, accuracy between 92and108% and high precision (coefficient of variation less than3.2%). The total time needed for the analysis was about 1 h. The method could be routinely used for the quality control during therapeutic proteins manufacturing.

Screening of Polysorbate-80 Composition by High Resolution Mass Spectrometry with Rapid H/D Exchange.

Polysorbate (PS) is a widely used polymeric excipient in biotherapeutic formulations to stabilize and protect protein drugs. Commercial PS is a highly heterogeneous mixture of structurally related components. PS composition can impact the stabilizer performance of PS in formulated protein drugs. Characterization of PS heterogeneity is, however, analytically challenging. In this work, a high-throughput screening protocol is presented for the profiling of the PS-80 polysorbate form using high resolution mass spectrometry (HRMS) coupled with a rapid hydrogen/deuterium (H/D) exchange in deuterated methanol. The protocol takes advantage of accurate mass measurements from HRMS analysis and utilizes H/D exchange-induced mass shifts that are characteristic to structures (particularly the number of terminal hydroxyl groups) of PS molecules to definitively identify species. In particular, mass shifts caused by deuterium uptake were used (1) to confirm molecular identities assigned by accurate mass measurements (which adds an extra level of identification confidence) and (2) to differentiate isomers that have an identical mass (thus, undistinguishable by high mass accuracy), but differ in the number of terminal hydroxyls. These data were input to an automated searching algorithm against a molecular mass database covering over 17000 potential PS-80 molecular species. The identified species were then visualized with Kendrick Mass Defect plots. The analysis protocol identified and profiled over 180 species from PS-80 samples in a high-throughput fashion without requiring chromatographic separation to reduce complexity of mixtures or tandem mass spectrometric analysis to conduct structural elucidation.

Insight into the Dissolution Molecular Mechanism of Ternary Solid Dispersions by Combined Experiments and Molecular Simulations.

With the increase concern of solubilization for insoluble drug, ternary solid dispersion (SD) formulations developed more rapidly than binary systems. However, rational formulation design of ternary systems and their dissolution molecular mechanism were still under development. Current research aimed to develop the effective ternary formulations and investigate their molecular mechanism by integrated experimental and modeling techniques. Glipizide (GLI) was selected as the model drug and PEG was used as the solubilizing polymer, while surfactants (e.g., SDS or Tween80) were the third components. SD samples were prepared at different weight ratio by melting method. In the dissolution tests, the solubilization effect of ternary system with very small amount of surfactant (drug/PEG/surfactant 1/1/0.02) was similar with that of binary systems with high polymer ratios (drug/PEG 1/3 and 1/9). The molecular structure of ternary systems was characterized by differential scanning calorimetry (DSC), infrared absorption spectroscopy (IR), X-ray diffraction (XRD), and scanning electron microscope (SEM). Moreover, molecular dynamic (MD) simulations mimicked the preparation process of SDs, and molecular motion in solvent revealed the dissolution mechanism of SD. As the Gordon-Taylor equation described, the experimental and calculated values of Tg were compared for ternary and binary systems, which confirmed good miscibility of GLI with other components. In summary, ternary SD systems could significantly decrease the usage of polymers than binary system. Molecular mechanism of dissolution for both binary and ternary solid dispersions was revealed by combined experiments and molecular modeling techniques. Our research provides a novel pathway for the further research of ternary solid dispersion formulations.

Prevalence of ethanol and other potentially harmful excipients in pediatric oral medicines: survey of community pharmacies in a Nigerian City.

OBJECTIVE: Excipients are needed in the formulation of oral liquid medicines intended for children; they have however been reported to trigger safety issues. This study evaluated the concentrations and prevalence of ethanol and other potentially harmful excipients in pediatric formulations marketed in South Eastern Nigeria in line with international labeling guidelines and allowable daily limits (ADL). The study sampled oral pediatric formulations offered for sale in registered pharmacies. Those with accessible information leaflets were assessed for the presence and quantity of previously flagged excipients with potential to harm the pediatric population. RESULT: Of the 380 oral pediatric medicines, 140 provided access to list/quantity of ingredients. 47.9% (67) of the formulations contain at least one of the flagged excipients while the remaining only listed the active ingredients. Ethanol had the highest occurrence (62.7%) and was more in cough/cold medicines. A homeopathic cough and cold remedy had concentration of 90% v/v. Ethanol and sucrose in some formulations exhibited concentrations with a potential of crossing their approved daily intake (ADI) (1-90% v/v and 1.7 g-3.7 g/5 ml respectively). Ethanol use in studied pediatric formulations was quite high, with ethanol-containing formulations being prescribed for children 0-6 years and older. Only 26 (38.8%) completely satisfied the labelling requirements for ethanol containing formulations.

In Situ Quantification of Polysorbate in Pharmaceutical Samples of Therapeutic Proteins by Hydrodynamic Profiling by NMR Spectroscopy.

Polysorbates are nonionic surfactants often used at variable levels in various formulations of protein therapeutics. Their quantification in pharmaceutical samples has posed an analytical challenge. Here we present an approach based on 1H NMR spectroscopy which can accurately estimate the concentration of polysorbate 80 (PS80) in intact pharmaceutical samples of an arbitrary formulation. The method, HAP-NMR (hydrodynamic profiling by NMR), is an extension of the protein fingerprint by line shape enhancement method (PROFILE) approach ( Poppe , L. ; Jordan , J. B. ; Lawson , K. ; Jerums , M. ; Apostol , I. ; Schnier , P. D. Anal. Chem. 2013 , 85 (20) , 9623 - 9629 ) and is based on the 1D 1H pulsed field gradient stimulated echo (PFGSTE) NMR experiment, which allows for the rectification of the 1D 1H NMR spectrum to a level suitable for a quantitative hydrodynamic analysis. Here we describe the methodology as applied to an antibody sample formulated in 9% (w/v) sucrose and with variable levels of PS80, ranging from 0.01% to 0.20% (w/v) sample concentrations. Equally important, we present evidence and propose a novel mechanism of how polysorbate stabilizes protein in pharmaceutical formulations.

A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics.

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.