Visible light triggers the formation of reactive oxygen species in monoclonal antibody formulations.

Most monoclonal antibody formulations require the presence of a surfactant, such as polysorbate, to ensure protein stability. The presence of high concentrations of polysorbate have been shown to enhance photooxidation of certain protein drug products when exposed to visible light. The current literature, however, suggest that photooxidation of polysorbate only occurs when exposed to visible light in combination with UVA light. This is probable as peroxides present in polysorbate solutions can be cleaved homolytically in the UVA region. In the visible region, photooxidation is not expected to occur as cleavage of peroxides is not expected at these wavelengths. This report presents findings suggesting that the presence of one or more photosensitiser(s) in polysorbate must be a cause and is required to catalyse the aerobic oxidation of polysorbate solutions upon exposure to visible light. Our investigation aimed to clarify the mechanism(s) of polysorbate photooxidation and explore the kinetics and the identity of the generated radicals and their impact on monoclonal antibody (mAb) degradation. Our study reveals that when polysorbate solutions are exposed to visible light between 400 - 800 nm in the absence of proteins, discolouration, radical formation, and oxygen depletion occur. We discuss the initial formation of reactive species, most likely occurring directly after reaction of molecular oxygen, with the presence of a triplet state photosensitiser, which is generated by intersystem crossing of the excited singlet state. When comparing the photooxidation of PS20 and PS80 in varying quality grades, we propose that singlet oxygen possesses potential for reacting with unsaturated fatty acids in PS80HP, however, PS20HP itself exhibited no measurable oxidation under the tested conditions. The study's final part delves into the photooxidation behaviour of different PS grades, examining its influence on the integrity of a mAb in the formulation. Finally, we examined the effect of photooxidation on the integrity of monoclonal antibodies. Our findings show that the exposure to visible light in polysorbate-containing mAb solutions at high PS concentrations of 4 mg·ml-1 results in increased monoclonal antibody degradation, highlighting the need for cautious evaluation of the correct PS concentration to stabilise protein therapeutics.

All-in-one stability indicating polysorbate 20 degradation root-cause analytics via UPLC-QDa.

Polysorbates (PS) are the most frequently used surfactants to stabilize biologicals. Ironically, these excellent stabilizing non-ionic surfactants have inherent structural properties, which lead to instabilities of their own. Such PS degradation can be triggered by multiple root-causes, like chemical and enzymatic hydrolysis or oxidative degradation. This can on the one hand reduce the concentration of surface-active PS and on the other hand lead to the formation of unfavorable degradants, like poorly soluble free fatty acids (FFA), which may phase separate and form visible FFA particles. Due to the potential criticality of PS degradation in biopharmaceutical formulations, various analytics have been established in recent years not only to monitor the PS content but also to evaluate specific PS markers and crucial degradants. However, in most cases sample preparations and several analytical assays have to be conducted to obtain a comprehensive picture of potential PS degradation root-causes. Here we show a novel approach for PS degradation UPLC-QDa based root-cause analytics, which utilizes previously established analytics for (i) most relevant polysorbate 20 (PS20) esters, (ii) PS20 free fatty acids and (iii) a newly developed method for the evaluation of PS20 specific oxidation markers. Thereby, this triad of analytical methods uses the same sample preparation and detector, which reduces the overall necessary effort, time investment and sample volume. Furthermore, the innovative PS20 oxidation marker method allows to quantify specific concentrations of the determined markers by external calibration and possible perception of oxidative degradation processes prior to relevant losses of PS20 esters, which could serve as an early indication during formulation development. The applicability of this method set was verified using several PS20 containing stress samples, which cover the most relevant root-causes, including acidic and alkaline hydrolysis, enzyme mediated hydrolysis, oxidative AAPH stress and Fe2+/H2O2 mediated degradation as well as autoxidation via long-term storage at elevated temperatures. Overall, this analytical setup has shown to deliver in-depth data about PS20 degradation, which can be used to narrow down the causative stress without the necessity of fundamentally different methods. Therefore, it can be seen as all-in-one solution during sometimes troublesome development of biopharmaceutical formulations, that supports the elucidation of the PS degradation mechanism(s) and thus establish mitigation strategies.

Comparative Stability Study of Polysorbate 20 and Polysorbate 80 Related to Oxidative Degradation.

The surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80) are utilized to stabilize protein drugs. However, concerns have been raised regarding the degradation of PSs in biologics and the potential impact on product quality. Oxidation has been identified as a prevalent degradation mechanism under pharmaceutically relevant conditions. So far, a systematic stability comparison of both PSs under pharmaceutically relevant conditions has not been conducted and little is known about the dependence of oxidation on PS concentration. Here, we conducted a comparative stability study to investigate (i) the different oxidative degradation propensities between PS20 and PS80 and (ii) the impact of PS concentration on oxidative degradation. PS20 and PS80 in concentrations ranging from 0.1 mg⋅mL-1 to raw material were stored at 5, 25, and 40 °C for 48 weeks in acetate buffer pH 5.5 and water, respectively. We observed a temperature-dependent oxidative degradation of the PSs with strong (40 °C), moderate (25 °C), and weak/no degradation (5 °C). Especially at elevated temperatures such as 40 °C, fast oxidative PS degradation processes were detected. In this case study, a stronger degradation and earlier onset of oxidation was observed for PS80 in comparison to PS20, detected via the fluorescence micelle assay. Additionally, degradation was found to be strongly dependent on PS concentration, with significantly less oxidative processes at higher PS concentrations. Iron impurities, oxygen in the vial headspaces, and the pH values of the formulations were identified as the main contributing factors to accelerate PS oxidation.

How enzymatic hydrolysis of polysorbate 20 influences colloidal protein stability.

Polysorbates (PS) are esters of ethoxylated sorbitol anhydrides of different composition and are widely used surfactants in biologics. PSs are applied to increase protein stability and concomitant shelf-life via shielding against e.g., interfacial stresses. Due to the presence of specific lipolytic host cell protein (HCP) contaminations in the drug substance, PSs can be degraded via enzymatic hydrolysis. Surfactant hydrolysis leads to the formation of degradants, such as free fatty acids that might form fatty acid particles. In addition, PS degradation may reduce surfactant functionality and thus reduce the protection of the active pharmaceutical ingredient (API). Although enzymatic degradation was observed and reported in the last years, less is known about the relationship between certain polysorbate degradation patterns and the increase of mechanical and interfacial stress towards the API. In this study, the impact of specifically hydrolyzed polysorbate 20 (PS20) towards the stabilization of two monoclonal antibodies (mAbs) during accelerated shaking stress conditions was investigated. The results show that a specific enzymatic degradation pattern of PS20 can influence the colloidal stability of biopharmaceutical formulations. Furthermore, the kinetics of the appearance of visual phenomena, opalescence, and particle formation depended on the polysorbate degradation fingerprint as induced via the presence of surrogate enzymes. The current case study shows the importance of focusing on specific polysorbate ester fractions to understand the overall colloidal protein stabilizing effect. The performed study gives first insight into the functional properties of PS and helps to evaluate the impact of PS degradation in the formulation development of biopharmaceuticals in general.

Oxidation of polysorbates - An underestimated degradation pathway?

To ensure the stability of biologicals over their entire shelf-life, non-ionic surface-active compounds (surfactants) are added to protect biologics from denaturation and particle formation. In this context, polysorbate 20 and 80 are the most used detergents. Despite their benefits of low toxicity and high biocompatibility, specific factors are influencing the intrinsic stability of polysorbates, leading to degradation, loss in efficacy, or even particle formation. Polysorbate degradation can be categorized into chemical or enzymatic hydrolysis and oxidation. Under pharmaceutical relevant conditions, hydrolysis is commonly originated from host cell proteins, whereas oxidative degradation may be caused by multiple factors such as light, presence of residual metal traces, peroxides, or temperature, which can be introduced upon manufacturing or could be already present in the raw materials. In this review, we provide an overview of the current knowledge on polysorbates with a focus on oxidative degradation. Subsequently, degradation products and key characteristics of oxidative-mediated polysorbate degradation in respect of different types and grades are summarized, followed by an extensive comparison between polysorbate 20 and 80. A better understanding of the radical-induced oxidative PS degradation pathway could support specific mitigation strategies. Finally, buffer conditions, various stressors, as well as appropriate mitigation strategies, reagents, and alternative stabilizers are discussed. Prior manufacturing, careful consideration and a meticulous risk-benefit analysis are highly recommended in terms of polysorbate qualities, buffers, storage conditions, as well as mitigation strategies.

Existence of a superior polysorbate fraction in respect to protein stabilization and particle formation?

Biologicals including monoclonal antibodies are the current flagships in pharmaceutical industry. However, they are exposed to a multitude of destabilization conditions like for instance hydrophobic interfaces, leading to reduced biological activity. Polysorbates are commonly applied to effectively stabilize these active pharmaceutical ingredients against colloidal stress. Nevertheless, chemical instability of polysorbate via hydrolysis or oxidation results in degradation products that might form particles via phase separation. Polysorbates are mixtures of hundreds of individual components, and recently purer quality grades with reduced variations in the fatty acid composition are available. As the protective function of polysorbate itself is not completely understood, even less is known about its individual components, raising the question of the existence of a superior polysorbate species in respect to protein stabilization or degradation susceptibility. Here, we evaluated the protective function of four main fractions of polysorbate 20 (PS20) in agitation studies with monoclonal antibodies, followed by particle analysis as well as protein and polysorbate content determination. The commercially-available inherent mixtures PS20 high purity and PS20 all-laurate, as well as the fraction isosorbide-POE-monolaurate showed superior protection against mechanical-induced stress (visual inspection and turbidity) at the air-water interface in comparison to sole sorbitan-POE-monolaurate, -dilaurate, and -trilaurate. Fractions composed mainly of higher-order esters like sorbitan-POE-dilaurate and sorbitan-POE-trilaurate indicated high turbidities as indication for subvisible and small particles accompanied by a reduced protein monomer content after agitation. For the isosorbide-POE-monolaurates as well as for the inherent polysorbate mixtures no obvious differences in protein content and protein aggregation (SEC) were observed, reflecting the observations from visual appearance. However, absolute polysorbate concentrations vary drastically between different species in the actual formulations. As there are still open questions in respect to protein specificity or regarding mixtures versus individual components of PS20, further studies must be performed, to gain a better understanding of a "generalized" stabilizing effect of polysorbates on monoclonal antibodies. The knowledge of the characteristics of individual polysorbate species can have the potential to pave the way to superior detergents in respect to protein stabilization and/or degradation susceptibility.

Protein photodegradation in the visible range? Insights into protein photooxidation with respect to protein concentration.

Visible light (400-800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.

Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates.

Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.

Characterization of radicals in polysorbate 80 using electron paramagnetic resonance (EPR) spectroscopy and spin trapping.

Polysorbates are an important class of nonionic surfactants that are widely used to stabilize biopharmaceuticals. The degradation of polysorbate 20 and 80 and the related particle formation in biologics are heavily discussed in the pharmaceutical community. Although a lot of experimental effort was spent in the detailed study of potential degradation pathways, the underlying mechanisms are only sparsely understood. Besides enzymatic hydrolysis, another proposed mechanism is associated with radical-induced (auto)oxidation of polysorbates. To characterize the types and the origin of the involved radicals and their propagation in bulk material as well as in diluted polysorbate 80 solutions, we applied electron paramagnetic resonance (EPR) spectroscopy using a spin trapping approach. The prerequisite for a meaningful experiment using spin traps is an understanding of the trapping rate, which is an interplay of (i) the presence of the spin trap at the scene of action, (ii) the specific reactivity of the selected spin trap with a certain radical as well as (iii) the stability of the formed spin adducts (a slow decay rate). We discuss whether and to which extent these criteria are fulfilled regarding the identification of different radical classes that might be involved in polysorbate oxidative degradation processes. The ratio of different radicals for different scenarios was determined for various polysorbate 80 quality grades in bulk material and in aqueous solution, showing differences in the ratio of present radicals. Possible correlations between the radical content and product parameters such as the quality grade, the manufacturing date, the manufacturer, the initial peroxide content according to the certificate of analysis of polysorbate 80 are discussed.

Poloxamer 188 as surfactant in biological formulations - An alternative for polysorbate 20/80?

Surfactants are used to stabilize biologics. Particularly, polysorbates (Tween® 20 and Tween® 80) dominate the group of surfactants in protein and especially antibody drug products. Since decades drug developers rely on the ethoxylated sorbitan fatty acid ester mixtures to stabilize sensitive molecules such as proteins. Reasons are (i) excellent stabilizing properties, and (ii) well recognized safety and tolerability profile of these polysorbates in humans, especially for parenteral applications. However, over the past decade concerns regarding the stability of these two polysorbates were raised. The search of alternatives with preferably less reservations concerning degradation and product quality reducing issues leads, among others, to poloxamer 188 (e.g. Kolliphor® P188), a nonionic triblock-copolymer surfactant. This review sums up our current knowledge related to the characterization and physico-chemical properties of poloxamer 188, its analytics and stability properties for biological formulations. Furthermore, the advantages and disadvantages as a suitable polysorbate-alternative for the stabilization of biologics are discussed.

Assessing the polysorbate degradation fingerprints and kinetics of lipases - how the activity of polysorbate degrading hydrolases is influenced by the assay and assay conditions.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stresses are polysorbate 20 (PS20) and polysorbate 80 (PS80). In recent years, numerous cases of hydrolytic polysorbate (PS) degradation in liquid formulations of biopharmaceuticals have been observed. Concomitant with the degradation of PSs, formulated proteins become inherently instable and more susceptible to aggregation. Furthermore, poorly soluble fatty acids (FA) are released from the PSs, which might lead to FA precipitation and the formation of visible and subvisible particles. Therefore, possible particle inducing factors have to be monitored closely. The major root cause of hydrolytic PS degradation in biologicals is the presence of enzymatic active host cell proteins (HCP), like lipases and esterases, which are co-purified with the active pharmaceutical ingredient. Such contaminants can be detected via their hydrolytic activity, either using ester-based substrates or PS itself. However, each approach has its up- and downsides, which makes the comparison of the results from other publications difficult. It was therefore the aim of the present study to investigate the impact of lipase specificities on the assay readouts. This study evaluates three different surrogate (model) lipases with distinctively different degradation kinetics and substrate specificities using specific analytical methods. The analytical panel contains on one hand two lipase activity assays with ester-based substrates, either detecting the release of para-nitrophenol or 4-methylumbelliferone, and on the other hand two PS-based monitoring analyses (fluorescence micelle assay and reverse phase high performance liquid chromatography - charged aerosol detection), which detect hydrolytic "activity" directly in the target substrate. Thereby, strengths and weaknesses of each assay are discussed, and recommendations are made for the respective use cases. Our results show that the determined lipase activities vary not only from assay to assay, but also significantly for the lipase tested, thus showing a different degradation fingerprint in the RP-HPLC-CAD chromatogram. This demonstrates that a comprehensive monitoring approach is essential to assess potential HCP contaminations.

Complex Micellization Behavior of the Polysorbates Tween 20 and Tween 80.

Polysorbates (PSs, Tweens) are widely used surfactant products consisting of a sorbitan ring connecting up to four ethylene oxide (EO) chains of variable lengths, one or more of which are esterified with fatty acids of variable lengths and saturation degrees. Pharmaceutical applications include the stabilization of biologicals in solutions and the solubilization of poorly water soluble, active ingredients. This study characterizes the complex association behavior of compendial PSs PS20 and PS80, which is fundamentally different from that of single-component surfactants. To this end, a series of demicellization experiments of isothermal titration calorimetry with different PS concentrations are evaluated. Their experiment-dependent heats of titration are converted into a common function of the state of a sample, the micellar enthalpy Qm(c). These functions demonstrate that initial micelles are already present at the lowest concentrations investigated, 2 µM for PS20 and 10 µM for PS80. Initial micelles consist primarily of the surfactant species with the lowest individual critical micelle concentration (cmc). With increasing concentration, the other PS species gradually enter these micelles in the sequence of increasing individual cmc's and hydrophilic-lipophilic balance. Concentration ranges with pronounced slopes of Qm(c) can be tentatively assigned to the uptake of the major components of the PS products. Micellization and the variation of the micelle properties progress up to at least 10 mM PS. That means the published cmc values or ranges of PS20 and PS80 may be related to certain, major components being incorporated into and forming specific micelles but must not be interpreted in terms of an absence of micelles below and constant properties, e.g., the surface activity, of the micelles above these ranges. The micellization enthalpy curves differ quite substantially between PS20 and PS80 and, in a subtler fashion, between individual quality grades such as high purity, pure lauric acid/pure oleic acid, super-refined, and China grade.

Hydrolytic polysorbate 20 degradation - Sensitive detection of free fatty acids in biopharmaceuticals via UPLC-QDa analytics with isolator column.

The enzymatic hydrolysis of polysorbates, e.g. induced by specific host cell proteins in biologics, is a known risk factor regarding the potential particle formation in the product over time. One of the root causes for this observation is an increase in free fatty acids (FA) within the formulation, which indicates the need for convenient monitoring of FA release. This study presents a novel UPLC-QDa based method to evaluate the content of the FAs esterified to polysorbate 20 (PS20) after hydrolysis. The presented method is label-free, i.e. independent of elaborate fluorophore-labeling and able to directly measure the ionized FAs. Furthermore, the method allows the determination of released FAs as percentage of ester bond hydrolysis and as absolute concentration expressed in ng/mL. Additionally, we describe for the first time in FA analytics the application of an isolator column, to remove trace levels of FAs present in the eluents to improve the sensitivity of the method. Lastly, the capabilities of the newly developed method are proven in case studies with three different monoclonal antibodies, which display characteristic FA release patterns in PS20-containing formulations. In summary, we developed a reliable, sensitive method for FA quantification in biologics, which could also be used as a predictive tool, considering FA solubility, regarding the formation of particles.

Investigating photodegradation of antibodies governed by the light dosage.

The present study investigated the photodegradation of three different monoclonal antibodies (mAb) by visible light. Several chromatographic techniques, such as size-exclusion and hydrophobic interaction chromatography as well as mass spectrometry were used to measure relative changes of various oxidation related monoclonal antibody species. The results show that visible light is indeed capable of inducing the formation of protein photo-oxidation products, such as acidic, basic, hydrophilic, and several other protein species with altered physicochemical properties. Although, the formation rate of degradants of these three protein species was dependent on the light source's intensity (I), their yield is clearly correlated to the applied light dosage (ld), which is defined as the product of light intensity I and irradiation time t (light dosage = I·t). Hence, our findings indicate that the degradation of monoclonal antibodies can be described according to the Bunsen-Roscoe reciprocity law. This correlation can be useful to assess the impact of photodegradation of biologics with regards to changes in light intensity and/or duration of light exposure of the protein, e.g. during the manufacturing of biologics.

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.

An in-depth examination of fatty acid solubility limits in biotherapeutic protein formulations containing polysorbate 20 and polysorbate 80.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are polysorbate20 (PS20) and polysorbate 80 (PS80). In recent years, polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation. Since the emergence of particles in liquid formulations has to be avoided, it is important to prevent their formation. This study evaluates the solubility limits of selected FAs, which are likely to be released during the degradation of PS20 and PS80 in the presence of defined PS concentrations. Our results show that the solubility is highly dependent on the pH, the temperature, the used PS concentration and the aliphatic chain of respective FAs. Solubility of FAs, such as palmitic and oleic acid under the conditions determined in this study, are in the range of 3-130 µg·ml-1 (12-460 µM). Furthermore, the results allow making an estimation to which extent PS may degrade before particle formation in the drug product may be expected.

Acidic and alkaline hydrolysis of polysorbates under aqueous conditions: Towards understanding polysorbate degradation in biopharmaceutical formulations.

Polysorbate is one of the most commonly employed non-ionic surfactant in protein containing biological formulations, whereby, it can stabilize these biomolecules under different stress conditions. Despite the fact that polysorbates are present in almost 70% of currently marketed parenteral biological drugs, polysorbate degradation in biopharmaceutical formulations has emerged as a specific quality concern. Different degradation pathways have been explored in the recent years with the aim of understanding the root cause for polysorbate degradation in biopharmaceutical formulations. In an attempt to explore hydrolytic degradation of polysorbates in accelerated degradation conditions, we studied extreme pH conditions. We investigated specific polysorbate degradation profiles depending on acidic or alkaline solution conditions. The acidic and alkaline hydrolysis of polysorbate is monitored for the total content using a fluorescence micelle assay (FMA). Additionally, the compositional changes in polysorbates were detected using reversed phase high performance liquid chromatography coupled to a charged aerosol detector (RP-HPLC-CAD). We show that the stability of polysorbate against chemical hydrolysis is dependent upon selected pH condition and differ for polysorbate 20 and polysorbate 80. Additionally, we were able to show that a degradation pathway dependent fingerprint may support the identification of the degradation root cause.