Mechanisms of gut epithelial barrier impairment caused by food emulsifiers polysorbate 20 and polysorbate 80.

BACKGROUND: The rising prevalence of many chronic diseases related to gut barrier dysfunction coincides with the increased global usage of dietary emulsifiers in recent decades. We therefore investigated the effect of the frequently used food emulsifiers on cytotoxicity, barrier function, transcriptome alterations, and protein expression in gastrointestinal epithelial cells. METHODS: Human intestinal organoids originating from induced pluripotent stem cells, colon organoid organ-on-a-chip, and liquid-liquid interface cells were cultured in the presence of two common emulsifiers: polysorbate 20 (P20) and polysorbate 80 (P80). The cytotoxicity, transepithelial electrical resistance (TEER), and paracellular-flux were measured. Immunofluorescence staining of epithelial tight-junctions (TJ), RNA-seq transcriptome, and targeted proteomics were performed. RESULTS: Cells showed lysis in response to P20 and P80 exposure starting at a 0.1% (v/v) concentration across all models. Epithelial barrier disruption correlated with decreased TEER, increased paracellular-flux and irregular TJ immunostaining. RNA-seq and targeted proteomics analyses demonstrated upregulation of cell development, signaling, proliferation, apoptosis, inflammatory response, and response to stress at 0.05%, a concentration lower than direct cell toxicity. A proinflammatory response was characterized by the secretion of several cytokines and chemokines, interaction with their receptors, and PI3K-Akt and MAPK signaling pathways. CXCL5, CXCL10, and VEGFA were upregulated in response to P20 and CXCL1, CXCL8 (IL-8), CXCL10, LIF in response to P80. CONCLUSIONS: The present study provides direct evidence on the detrimental effects of food emulsifiers P20 and P80 on intestinal epithelial integrity. The underlying mechanism of epithelial barrier disruption was cell death at concentrations between 1% and 0.1%. Even at concentrations lower than 0.1%, these polysorbates induced a proinflammatory response suggesting a detrimental effect on gastrointestinal health.

Development of invaethosomes and invaflexosomes for dermal delivery of clotrimazole: optimization, characterization and antifungal activity.

The objective of this study was to develop novel invaethosomes (I-ETS) and invaflexosomes (I-FXS) to enhance the dermal delivery of clotrimazole (CZ). Twenty model CZ-loaded I-ETS and I-FXS formulations were created according to a face-centered central composite experimental design. CZ-loaded vesicle formulations containing a constant concentration of 0.025% w/v CZ and various amounts of ethanol, d-limonene, and polysorbate 20 as penetration enhancers were prepared using the thin film hydration method. The physicochemical characteristics, skin permeability, and antifungal activity were characterized. The skin permeability of the experimental CZ-loaded I-ETS/I-FXS was significantly higher than that of conventional ethosomes, flexosomes, and the commercial product (1% w/w CZ cream). The mechanism of action was confirmed to be skin penetration of low ethanol base vesicles through the disruption of the skin microstructure. The optimal I-ETS in vitro antifungal activity against C. albicans differed significantly from that of ETS and the commercial cream (control). The response surface methodology predicted by Design Expert® was helpful in understanding the complicated relationship between the causal factors and the response variables of the 0.025% w/v CZ-loaded I-ETS/I-FXS formulation. Based on the available information, double vesicles seem to be promising versatile carriers for dermal drug delivery of CZ.

Evaluating a Modified High Purity Polysorbate 20 Designed to Reduce the Risk of Free Fatty Acid Particle Formation.

PURPOSE: To evaluate a modified high purity polysorbate 20 (RO HP PS20)-with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20-as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particle formation upon hydrolytic degradation relative to HP PS20. METHODS: Analytical characterization of RO HP PS20 raw material included fatty acid ester (FAE) distribution, higher order ester (HOE) fraction, FFA levels and trace metals. Functional assessments included 1) vial and intravenous bag agitation; 2) oxidation via a placebo and methionine surrogate study; and 3) hydrolytic PS20 degradation studies to evaluate FFA particle formation with and without metal nucleation. RESULTS: Interfacial protection and oxidation propensity were comparable between the two polysorbates. Upon hydrolytic degradation, FFA particle onset was delayed in RO HP PS20. The delay was more pronounced when HOEs of PS20 were preferentially degraded. Furthermore, the hydrolytic degradants of RO HP PS20 formed fewer particles in the presence of spiked aluminum. CONCLUSION: This work highlights the criticality of having tighter control on long chain FAE levels of PS20 to reduce the occurrence of FFA particle formation upon hydrolytic degradation and lower the variability in its onset. By simultaneously meeting compendial PS20 specifications while narrowing the allowable range for each FAE and shifting its composition towards the shorter carbon chain species, RO HP PS20 provides a promising alternative to HP PS20 for biopharmaceutical DPs.

Light-Induced Histidine Adducts to an IgG1 Molecule Via Oxidized Histidine Residue and the Potential Impact of Polysorbate-20 Concentration.

PURPOSE: Histidine (His) undergoes light-induced reactions such as oxidation, crosslinking and addition. These reactions are initiated by singlet oxygen (1O2) to generate His photo-oxidation products, which are subject to nucleophilic attack by a non-oxidized His residue from another protein or by nucleophilic buffer components such as Tris and His. This report aims to identify light-induced His-adducts to a monoclonal antibody (mAb-1) due to the reaction of His molecules in the buffer with the photooxidized His residues under ICH light conditions. Since polysorbate-20 (PS-20) is a commonly used excipient in biotherapeutics formulation, it is also important to study the impact of PS-20 concentration on protein photostability. RESULTS: We identified and characterized light-induced His-adducts of mAb-1 by LC-MS/MS. We showed that the levels of light-induced His-adducts generally correlate with the solvent accessibility of His residues in the protein. In addition, the presence of PS-20 at concentrations commonly used in protein drug formulations can significantly increase the levels of light-induced His-adducts. CONCLUSIONS: Since His residues are present in a conserved region in the Fc domain, and may be present in the complementarity-determining region (CDR), the impact on the biological functions of the His-adducts observed here should be further studied to evaluate the risk of their presence.

Shape and Structure Formation of Mixed Nonionic-Anionic Surfactant Micelles.

Aqueous solutions of a nonionic surfactant (either Tween20 or BrijL23) and an anionic surfactant (sodium dodecyl sulfate, SDS) are investigated, using small-angle neutron scattering (SANS). SANS spectra are analysed by using a core-shell model to describe the form factor of self-assembled surfactant micelles; the intermicellar interactions are modelled by using a hard-sphere Percus-Yevick (HS-PY) or a rescaled mean spherical approximation (RMSA) structure factor. Choosing these specific nonionic surfactants allows for comparison of the effect of branched (Tween20) and linear (BrijL23) surfactant headgroups, both constituted of poly-ethylene oxide (PEO) groups. The nonionic-anionic surfactant mixtures are studied at various concentrations up to highly concentrated samples (ϕ ≲ 0.45) and various mixing ratios, from pure nonionic to pure anionic surfactant solutions. The scattering data reveal the formation of mixed micelles already at concentrations below the critical micelle concentration of SDS. At higher volume fractions, excluded volume effects dominate the intermicellar structuring, even for charged micelles. In consequence, at high volume fractions, the intermicellar structuring is the same for charged and uncharged micelles. At all mixing ratios, almost spherical mixed micelles form. This offers the opportunity to create a system of colloidal particles with a variable surface charge. This excludes only roughly equimolar mixing ratios (X≈ 0.4-0.6) at which the micelles significantly increase in size and ellipticity due to specific sulfate-EO interactions.

The Nature of the Increased Efficiency of Sorption-Stimulating Preparations Containing Non-Ionic Surfactants for Pre-Sowing Seed Treatment.

The effect of the introduction of a non-ionogenic surfactant Polysorbate 20 into a sorption preparation (CB-H-BYA) on the structure of sorbent layers formed on the surface of spring wheat seeds during their pre-sowing treatment has been studied using electron microscopy. According to the results, an increase in the efficiency of sorption preparations containing Polysorbate 20 is based on an intensification of the bentonite aggregate disintegration into individual montmorillonite particles and a formation of more dense protective sorption layer providing a better protection of seeds against allelotoxins on the seed surface. The introduction of non-ionogenic surfactants into the preparation increases a sorption capacity of a bentonite-humus complex that results in a decreased gibberellin activity in a solution. Therefore, to achieve the maximum physiological activity of gibberellin in a preparation solution, it is necessary to increase its concentration from 100 to 300 mg/L. As a result, the stimulating effect increases from 36 to 55%.

Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies.

PURPOSE: L-Histidine (L-His) and polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. METHODS: The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. RESULTS: Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. CONCLUSION: L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.

Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry.

PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism.

Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18-36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2-8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C.

Discrimination of polysorbate 20 by high-performance liquid chromatography-charged aerosol detection and characterization for components by expanding compound database and library.

Analyzing polysorbate 20 (PS20) composition and the impact of each component on stability and safety is crucial due to formulation variations and individual tolerance. The similar structures and polarities of PS20 components make accurate separation, identification, and quantification challenging. In this work, a high-resolution quantitative method was developed using single-dimensional high-performance liquid chromatography (HPLC) with charged aerosol detection (CAD) to separate 18 key components with multiple esters. The separated components were characterized by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) with an identical gradient as the HPLC-CAD analysis. The polysorbate compound database and library were expanded over 7-time compared to the commercial database. The method investigated differences in PS20 samples from various origins and grades for different dosage forms to evaluate the composition-process relationship. UHPLC-Q-TOF-MS identified 1329 to 1511 compounds in 4 batches of PS20 from different sources. The method observed the impact of 4 degradation conditions on peak components, identifying stable components and their tendencies to change. HPLC-CAD and UHPLC-Q-TOF-MS results provided insights into fingerprint differences, distinguishing quasi products.

A comparison of Polysorbates and Alternative Surfactants for Interfacial Stress Protection and Mitigation of Fatty Acid Particle Formation in the Presence of an Esterase.

The hydrolysis of polysorbate surfactants in large molecule drug product formulations caused by residual host cell proteins presents numerous stability concerns for pharmaceuticals. The fatty acids (FA) released by polysorbate hydrolysis can nucleate into particulates or challenge the conformational stability of the proteinaceous active pharmaceutical ingredient (API). The loss of intact polysorbate may also leave the Drug Product (DP) vulnerable to interfacial stresses. Polysorbate 20 and 80 are available in several different quality grades (Multi-compendial, Super Refined, Pure Lauric Acid (PLA)/Pure Oleic Acid (POA)). All variations of polysorbate as well as three alternative surfactants: Brij L23, Brij O20 and Poloxamer 188 were compared for their ability to protect against air-water interfacial stresses as well as their risk for developing particulates when in the presence of lipoprotein lipase (LPL) (Pseudomonas). Results show a meaningful difference in the timing and morphology of FA particle formation depending on the type of polysorbate used. All grades of polysorbate, while susceptible to hydrolysis, still offered sufficient protection to interfacial stresses, even when hydrolyzed to concentrations as low as 0.005 % (w/v). Alternative surfactants that lack an ester bond were resistant to lipase degradation and showed good protection against shaking stress.

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.

The effect of polysorbate 20 and polysorbate 80 on the solubility of quercetin.

Background: Quercetin acts as an antioxidant, anti-inflammatory, wound healing, and anti-aging so quercetin can be used as a topical preparation. However, it has low solubility in water at 0.01 mg/ml at 25oC. Increasing the solubility of quercetin in water was done by the addition of surfactants. Objective: This study compared the solubility of quercetin in Polysorbate 20 (P20) and Polysorbate 80 (P80) in a citrate buffer medium pH 4.5±0.2. Methods: The surfactants Polysorbate 80 and Polysorbate 20 differ in their alkyl chain length. Polysorbate 80 has an alkyl chain length of 18, while Polysorbate 20 has an alkyl chain length of 12. The concentrations of surfactant are above, below, and at the critical micelle concentration (CMC) values. The concentrations of quercetin were determined at the maximum wavelength by spectrophotometric method. Results: The results of the quercetin solubility test without surfactant were 3.89±0.59 mg/L. The results of the quercetin solubility test by adding Polysorbate 20 at a concentration of 42.0 ppm; 57.5 ppm; and 73.0 ppm were 3.62±0.72, 4.04±0.23 and 8.35±1.97 mg/L, respectively. While the solubility of quercetin by adding Polysorbate 80 at a concentration of 4.0 ppm, 11.5 ppm, and 19.0 ppm was 11.15±0.72, 11.37±1.23 and 14.17±1.96 mg/L, respectively. The solubility of quercetin is greater after the addition of surfactant Polysorbate 20 only at the concentration above the CMC value and the solubility of quercetin is greater with the addition of surfactant Polysorbate 80 at all concentrations. Surfactant Polysorbate 20 increases the solubility of quercetin in citrate buffer pH 4.5±0.2 only at concentrations above the CMC value of 2.14 times. Polysorbate 80 can increase the solubility of quercetin in citrate buffer pH 4.5±0.2 at concentrations below, at, and above CMC by 2.87, 2.92, and 3.63 times, respectively. Conclusion: Polysorbate 80 can increase the solubility of quercetin in citrate buffer pH 4.5±0.2 higher than Polysorbate 20.

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.

Combinational Inhibition of P-Glycoprotein-Mediated Etoposide Transport by Zosuquidar and Polysorbate 20.

P-glycoprotein (P-gp) limits the oral absorption of drug substances. Potent small molecule P-gp inhibitors (e.g., zosuquidar) and nonionic surfactants (e.g., polysorbate 20) inhibit P-gp by proposedly different mechanisms. Therefore, it was hypothesised that a combination of zosuquidar and polysorbate 20 may potentiate inhibition of P-gp-mediated efflux. P-gp inhibition by zosuquidar and polysorbate 20 in combination was assessed in a calcein-AM assay and in a transcellular etoposide permeability study in MDCKII-MDR1 and Caco-2 cells. Furthermore, solutions of etoposide, zosuquidar, and polysorbate 20 were orally administered to Sprague Dawley rats. Zosuquidar elicited a high level of nonspecific adsorption to various labware, which significantly affected the outcomes of the in vitro studies. Still, at certain zosuquidar and polysorbate 20 concentrations, additive P-gp inhibition was observed in vitro. In vivo, however, oral etoposide bioavailability decreased by coadministration of both zosuquidar and polysorbate 20 when compared to coadministration of etoposide with zosuquidar alone. For future formulation development, the present study provided important and novel knowledge about nonspecific zosuquidar adsorption, as well as insights into combinational P-gp inhibition by a third-generation P-gp inhibitor and a P-gp-inhibiting nonionic surfactant.

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.

Fabrication of UV-Protective Polyester Fabric with Polysorbate 20 Incorporating Fluorescent Color.

Technological advancement leads researchers to develop multifunctional materials. Considering such trends, this study aimed to conjugate dual functionality in a single material to satisfy aesthetic and functional necessities. We investigated the potentiality of polysorbate 20 to perform as an effective ultraviolet absorber to develop UV-protective fabric. Coumarin derivative (Benzoxazolyl type) disperse dyes are well-known as fluorescent colors. On the other hand, luminescence materials are conspicuous and viable for fashion trends. Deliberate utilization of this inherent property of the dye and incorporation of polysorbate fulfilled the need for dual functionality. In addition, the knitted fabric structure enhanced wearing comfort as well. The effect of polysorbate consolidated the PET fabric as an excellent UV absorber, exhibiting an ultraviolet protection factor (UPF) of 53.71 and a blocking percentage of more than 95% for both UVA and UVB. Surface morphology was studied by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FTIR) with attenuated mode was used to investigate chemical modification. Moreover, X-ray diffraction (XRD) investigated the crystallography of the surface. Reflectance spectrophotometric analysis unveiled the color strength (K/S) of the dyed polyester fabrics. Finally, light fastness assessment revealed that the developed samples could resist a certain amount of photo fading under a controlled testing environment with the increment of ratings towards betterment.

Identification of Key Excipients for the Solubilization and Structural Characterization of Lipoprotein Lipase, An Enzyme for Hydrolysis of Triglyceride.

Lipoprotein lipase (LPL) is an essential enzyme that hydrolyzes triglycerides in chylomicrons and very low-density lipoprotein into glycerol and fatty acids. One major hurdle in using LPL as a therapeutic has been its poor solubility/stability after purification. Solutions used to preserve purified LPL commonly contain either heparin, or concentrated glycerol and sodium chloride, resulting in hypertonic solutions. These solutions are not acceptable as pharmaceutical formulations. This paper describes the identification of a key excipient, sodium laurate, which can solubilize LPL in an isotonic environment without heparin or concentrated glycerol. A follow-up multi-variant study was performed to identify the effect of sodium laurate and its interaction with sodium chloride on the solubility and processing conditions of LPL. The LPL concentration (up to 14 mg/mL) achievable in pharmaceutically relevant and salt-free conditions was identified to be closely correlated to the concentration of sodium laurate, which was co-concentrated with LPL. The result that sodium laurate increases stability of LPL characterized by differential scanning calorimetry and UV absorbance spectra suggests that the mechanism of solubilization of LPL by sodium laurate is related to LPL structural stabilization. The findings indicate that substrates and their enzymatic products can be strong stabilizers for other protein molecules.

Simultaneous quantification of polysorbate 20 and poloxamer 188 in biopharmaceutical formulations using evaporative light scattering detection.

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method's practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate.

Dissolution of Polysorbate 20 Degradation Related Free Fatty Acid Particles in Intravenous Bag Solutions.

Degradation of Polysorbate 20 (PS20), a commonly used surfactant in drug product (DP) formulations, is a phenomenon of increasing concern to the biopharmaceutical industry. One of the most prevalent modes of PS20 degradation is enzymatic hydrolysis resulting from co-purified hydrolases that make their way into biologic DP formulations at trace levels. Enzymatic PS20 degradation results in generation of free fatty acids (FFAs) that have limited solubility in aqueous formulations and can form visible and/or sub-visible particles which is undesirable for parenteral DP stability and administration. Many therapeutic monoclonal antibodies are administered intravenously after first diluting the DP into an infusion solution (e.g., 0.9% normal saline, 0.45% half normal saline or 5% dextrose). The purpose of this work is to understand if FFA particles in the DP dissolve in intravenous solutions prior to administration. Our assessment indicates that visible and/or sub-visible particles that contain high levels of lauric, myristic and palmitic acids dissolve immediately upon dilution (at or exceeding two fold) regardless of the intravenous bag or solution type. Therefore, the risk is low of visible and/or sub-visible particles, comprised of FFAs in biopharmaceutical DPs, being intravenously administered to a patient.