Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

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.

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

INTRODUCTION: Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. METHODS: An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. RESULTS: Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 µs, which is lower than 56 µs for BSA and 16 µs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 µs, which confirms the reliability of MRPS measurements. CONCLUSION: The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

Relative Effectiveness of the MF59®-Adjuvanted Influenza Vaccine Versus High-Dose and Non-Adjuvanted Influenza Vaccines in Preventing Cardiorespiratory Hospitalizations During the 2019-2020 US Influenza Season.

BACKGROUND: Adults ≥ 65 years of age have suboptimal influenza vaccination responses compared to younger adults due to age-related immunosenescence. Two vaccines were specifically developed to enhance protection: MF59-adjuvanted trivalent influenza vaccine (aIIV3) and high-dose egg-based trivalent influenza vaccine (HD-IIV3e). METHODS: In a retrospective cohort study conducted using US electronic medical records linked to claims data during the 2019-2020 influenza season, we compared the relative vaccine effectiveness (rVE) of aIIV3 with HD-IIV3e and a standard-dose non-adjuvanted egg-based quadrivalent inactivated influenza vaccine (IIV4e) for the prevention of cardiorespiratory hospitalizations, including influenza hospitalizations. We evaluated outcomes in the "any" diagnosis position and the "admitting" position on the claim. A doubly robust methodology using inverse probability of treatment weighting and logistic regression was used to adjust for covariate imbalance. rVE was calculated as 100 * (1 - ORadjusted). RESULTS: The study included 4,299,594 adults ≥ 65 years of age who received aIIV3, HD-IIV3e, or IIV4e. Overall, aIIV3 was associated with lower proportions of cardiorespiratory hospitalizations with diagnoses in any position compared to HD-IIV3e (rVE = 3.9% [95% CI, 2.7-5.0]) or IIV4e (9.0% [95% CI, 7.7-10.4]). Specifically, aIIV3 was more effective compared with HD-IIV3e and IIV4e in preventing influenza hospitalizations (HD-IIV3e: 9.7% [95% CI, 1.9-17.0]; IIV4e: 25.3% [95% CI, 17.7-32.2]). Consistent trends were observed for admitting diagnoses. CONCLUSION: Relative to both HD-IIV3e and IIV4e, aIIV3 provided improved protection from cardiorespiratory or influenza hospitalizations.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

O1/W/O2 double emulsion gels based on nanoemulsions and Pickering particles for co-encapsulating quercetin and cyanidin: A functional fat substitute.

An O1/W/O2 double emulsion gel, as a functional fat substitute and based on nanoemulsions and hydrophobic Pickering particles, is prepared by two-step emulsification to co-encapsulate hydrophilic cyanidin and hydrophobic quercetin. Nanoemulsions loading quercetin are fabricated by Tween-80 and combining high-speed and high-pressure emulsification. Phytosterol nanoparticles stabilize the W-O2 interface of the secondary emulsion to load cyanidin in the W phase. The concentration of Tween-80 is optimized as 0.3% by the droplet size and viscosity of nanoemulsions. The structural stability of double emulsion gels will be weakened along with the increase of nanoemulsions, showing lower modulus and encapsulation efficiency (EE) and bigger droplets. In double emulsion gels, the EE of quercetin and cyanidin reaches 93% and 85.6%, respectively. Analysis of molecular interaction indicates that Tween-80 would decrease the in-situ hydrophobicity of phytosterol nanoparticles by hydrogen bonding adsorption, thereby weakening the emulsification. The pH-chromic 3D printing of double emulsion gels is designed according to the pH sensitivity of cyanidin. Texture profile analysis is performed to test the textural properties of 3D-printed objects. The simulated digestion is conducted on double emulsion gels. The double emulsion gel with fewer nanoemulsions is beneficial for protecting quercetin and improving the delivery due to the higher structural stability, while that with more nanoemulsions is conducive to the digestion of cyanidin and camellia oil due to weakened semi-solid properties. This double emulsion gel further simulates fat tissues by co-encapsulating hydrophilic and hydrophobic substances, promoting the application of fat substitutes in the food industry.

Long-term exposure to dietary emulsifier Tween 80 promotes liver lipid accumulation and induces different-grade inflammation in young and aged mice.

With the advent of industrialization, there has been a substantial increase in the production and consumption of ultra-processed foods (UPFs). These processed foods often contain artificially synthesized additives, such as emulsifiers. Emulsifiers constitute approximately half of the total amount of food additives, with Tween 80 being a commonly used emulsifier in the food industry. Concurrently, China is undergoing significant demographic changes, transitioning into an aging society. Despite this demographic shift, there is insufficient research on the health implications of food emulsifiers, particularly on the elderly population. In this study, we present novel findings indicating that even at low concentrations, Tween 80 suppressed the viability of multiple cell types. Prolonged in vivo exposure to 1 % Tween 80 in drinking water induced liver lipid accumulation and insulin resistance in young adult mice under a regular chow diet. Intriguingly, in mice with high-fat diet (HFD) induced metabolic dysfunction-associated steatotic liver disease (MASLD), this inductive effect was masked. In aged mice, liver lipid accumulation was replicated under prolonged Tween 80 exposure. We further revealed that Tween 80 induced inflammation in both adult and aged mice, with a more pronounced inflammation in aged mice. In conclusion, our study provides compelling evidence that Tween 80 could contribute to a low-grade inflammation and liver lipid accumulation. These findings underscore the need for increasing attention regarding the consumption of UPFs with Tween 80 as the emulsifier, particularly in the elderly consumers.

Adsorption behavior and the potential risk of As(V) in soils: exploring the effects of representative surfactants.

Arsenic contamination in soils poses a critical global challenge, yet the influence of surfactants on arsenic adsorption behavior is often underestimated. This study aims to investigate the effects of three representative surfactants, namely cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and polyethylene glycol anhydrous sugar alcohol monooleate (Tween 80), on arsenic adsorption behavior in soils. The adsorption isotherm shifts from a single Temkin model without surfactants to both the Langmuir and Temkin models in the presence of surfactants, indicating the simultaneous occurrence of monolayer and multilayer adsorption for arsenic in soils. Moreover, the surfactants can inhibit the adsorption and hasten the attainment of adsorption equilibrium. SDS displayed the most inhibitory effect on arsenic adsorption, followed by Tween 80 and CTAB, due to the competitive adsorption, electrostatic interaction, and hydrophobic interaction. Variations in zeta potential with different surfactants further elucidate this inhibitory phenomenon. Through orthogonal experiment analyses, pH emerges as a primary factor influencing arsenic adsorption in soils, with surfactant concentration and type identified as secondary factors. Temperature notably affects CTAB, with the adsorption inhibition rate plummeting to a mere 0.88% at 50 °C. Sequential extraction analysis revealed that surfactants enhanced the bioavailability of arsenic. The FTIR, XRD, SEM, and CA analyses further support the mechanism underlying the effect of surfactants on arsenic adsorption in soil. These analyses indicate that surfactants modify the composition and abundance of functional groups, hinder the formation of arsenic-containing substances, and improve soil compactness, smoothness, and hydrophilicity. This study provides valuable insights into the effect of surfactants in arsenic-contaminated soils, which is often ignored in previous work.

Digestion of surfactants does not affect their ability to inhibit P-gp-mediated transport in vitro.

While various non-ionic surfactants at low concentrations have been shown to increase the transport of P-gp substrates in vitro, in vivo studies in rats have shown that a higher surfactant concentration is needed to increase the oral absorption of e.g. the P-gp substrates digoxin and etoposide. The aim of the present study was to investigate if intestinal digestion of surfactants could be the reason for this deviation between in vitro and in vivo data. Therefore, Kolliphor EL, Brij-L23, Labrasol and polysorbate 20 were investigated for their ability to inhibit P-gp and increase digoxin absorption in vitro. Transport studies were performed in Caco-2 cells, while P-gp inhibition and cell viability assays were performed in MDCKII-MDR1 cells. Polysorbate 20, Kolliphor EL and Brij-L23 increased absorptive transport and decreased secretory digoxin transport in Caco-2 cells, whereas only polysorbate 20 and Brij-L23 showed P-gp inhibiting properties in the MDCKII-MDR1 cells. Polysorbate 20 and Brij-L23 were chosen for in vitro digestion prior to transport- or P-gp inhibiting assays. Brij-L23 was not digestible, whereas polysorbate 20 reached a degree of digestion around 40%. Neither of the two surfactants showed any significant difference in their ability to affect absorptive or secretory transport of digoxin after pre-digestion. Furthermore, the P-gp inhibiting effects of polysorbate 20 were not decreased significantly. In conclusion, the mechanism behind the non-ionic surfactant mediated in vitro P-gp inhibition seemed independent of the intestinal digestion and the results presented here did not suggest it to be the cause of the observed discrepancy between in vitro and in vivo.

AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans.

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Synergistic stabilization of garlic essential oil nanoemulsions by carboxymethyl chitosan/Tween 80 and application for coating preservation of chilled fresh pork.

Garlic essential oil (GEO) is a potential natural antioxidant and antimicrobial agent for food preservation, but its intrinsic low water-solubility, high volatility and poor stability severely limit its application and promotion. In this work, we investigated the synergistic stabilization of the GEO-in-water nanoemulsion using carboxymethyl chitosan (CCS) and Tween 80 (TW 80). Additionally, the nanoemulsion was fabricated through high-pressure microfluidization and utilized for the coating-mediated preservation of chilled pork. The garlic essential oil nanoemulsion (GEON) with 3.0 % CCS and 3.0 % TW 80 exhibited more homogeneous droplet size (around 150 nm) and narrower size distribution, while maintained long-term stability with no significant change in size during 30 d storage. Compared with free GEO, the GEONs exhibited a higher scavenging capacity to DPPH and ABTS free radicals as well as higher inhibitory effects against Escherichia coli and Staphylococcus aureus, suggesting that the encapsulation of GEO in nanoemulsion considerably improved its antioxidant and antibacterial activities. Furthermore, the results of coating preservation experiments showed that the GEON coating effectively expanded the shelf-life of chilled fresh pork for approximately one week. Altogether, this study would guide the development of GEO-loaded nanoemulsions, and promote GEON as a promising alternative for coating preservation of chilled fresh meat.

Combination drug therapy by herbal nanomedicine prevent multidrug resistance protein 1: promote apoptosis in Lung Carcinoma.

Given the numerous adverse effects of lung cancer treatment, more research on non-toxic medications is urgently needed. Curcumin (CUR) and berberine (BBR) combat drug resistance by controlling the expression of multidrug resistant pump (MDR1). Fascinatingly, combining these medications increases the effectiveness of preventing lung cancer. Their low solubility and poor stability, however, restrict their therapeutic efficacy. Because of the improved bioavailability and increased encapsulation effectiveness of water-insoluble medicines, surfactant-based nanovesicles have recently received a great deal of attention. The current study sought to elucidate the Combination drug therapy by herbal nanomedicine prevent multidrug resistance protein 1: promote apoptosis in Lung Carcinoma. The impact of several tween (20, 60, and 80) types with varied hydrophobic tails on BBR/CUR-TNV was evaluated. Additionally, the MDR1 activity and apoptosis rate of the BBR/CUR-TNV combination therapy were assessed. The encapsulation effectiveness of TNV was affected by the type of tween. With the TNV made from tween 60, cholesterol, and PEG (47.5: 47.5:5), more encapsulation effectiveness was attained. By combining CUR with BBR, especially when given in TNV, apoptosis increased. Additionally, when CUR and BBR were administered in combination, they significantly reduced the risk of MDR1 development. The current work suggests that the delivery of berberine and curcumin as a combination medication therapy via tween-based nanovesicles may be a potential lung cancer treatment.

Food additives impair gut microbiota from healthy individuals and IBD patients in a colonic in vitro fermentation model.

Intestinal fibrosis is a long-term complication of inflammatory bowel diseases (IBD). Changes in microbial populations have been linked with the onset of fibrosis and some food additives are known to promote intestinal inflammation facilitating fibrosis induction. In this study, we investigated how polysorbate 80, sucralose, titanium dioxide, sodium nitrite and maltodextrin affect the gut microbiota and the metabolic activity in healthy and IBD donors (patients in remission and with a flare of IBD). The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) with a static (batch) configuration was used to evaluate the effects of food additives on the human intestinal microbiota. Polysorbate 80 and sucralose decreased butyrate-producing bacteria such as Roseburia and Faecalibacterium prausnitzii. Both compounds, also increased bacterial species positively correlated with intestinal inflammation and fibrosis (i.e.: Enterococcus, Veillonella and Mucispirillum schaedleri), especially in donors in remission of IBD. Additionally, polysorbate 80 induced a lower activity of the aryl hydrocarbon receptor (AhR) in the three groups of donors, which can affect the intestinal homeostasis. Maltodextrin, despite increasing short-chain fatty acids production, promoted the growth of Ruminococcus genus, correlated with higher risk of fibrosis, and decreased Oscillospira which is negatively associated with fibrosis. Our findings unveil crucial insights into the potential deleterious effects of polysorbate 80, sucralose and maltodextrin on human gut microbiota in healthy and, to a greater extent, in IBD patients.

Toxicity of organic solvents and surfactants to the sea urchin embryos.

A comparative toxicity of widely applied organic solvents (methanol, ethanol, n-propanol, i-propanol, n-butanol, 2-butanol, i-butanol, t-butanol, 3-methoxy-3-methylbutanol-1 (MMB), ethylene glycol, diethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, glycerol, ethyl acetate, acetonitrile, benzene, dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, 2-pyrrolidone, and N-methyl-2-pyrrolidone) and surfactants (PEG 300, PEG 6000, Tween 20, Tween 80, miramistin, and Cremophor EL) was studied using a sea urchin embryo model. Sea urchin embryo morphological alterations caused by the tested chemicals were described. The tested molecules affected P. lividus embryo development in a concentration-dependent manner. The observed phenotypic anomalies ranged from developmental delay and retardation of plutei growth to formation of aberrant blastules and gastrules, cleavage alteration/arrest, and embryo mortality. Discernible morphological defects were found after embryo exposure with common pharmaceutical ingredients, such as glycerol, Tween 80, and Cremophor EL. In general, solvents were less toxic than surfactants. PEG 6000 PEG 300, DMSO, ethanol, and methanol were identified as the most tolerable compounds with minimum effective concentration (MEC) values of 3.0-7.92 mg/mL. Previously reported MEC value of Pluronic F127 (4.0 mg/mL) fell within the same concentration range. Toxic effects of methanol, ethanol, DMSO, 2-methoxyethanol, 2-ethoxyethanol, Tween 20, and Tween 80 on P. lividus embryos correlated well with their toxicity obtained using other cell and animal models. The sea urchin embryos could be considered as an appropriate test system for toxicity assessment of solvents and surfactants for their further application as solubilizers of hydrophobic molecules in conventional in vitro cell-based assays and in vivo mammalian models. Nevertheless, to avoid adverse effect of a solubilizing agent in ecotoxicological and biological experiments, the preliminary assessment of its toxicity on a chosen test model would be beneficial.

PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.

Efficient Combination Chemo-Sonodynamic Cancer Therapy Using Mitochondria-Targeting Sonosensitizer-Loaded Polysorbate-Based Micelles.

Sonodynamic therapy (SDT), utilizing ultrasound (US) and sonosensitizers, holds immense potential as a noninvasive and targeted treatment for a variety of deep-seated tumors. However, the clinical translation of SDT is hampered by several key limitations in sonosensitizers, especially their low aqueous stability and poor cellular uptake. In this study, non-ionic polysorbate (Tween 80, T80) was adopted to formulate effective nanocarriers for the safe and efficient delivery of sonosensitizers to cancer cells. Mitochondria-targeting triphenylphosphonium (TPP)-conjugated chlorin e6 (Ce6) sonosensitizer was loaded into T80-based micelles for efficient SDT. Pro-oxidant piperlongumine (PL) was co-encapsulated with TPP-conjugated Ce6 (T-Ce6) in T80 micelles to enable combination chemo-SDT. T80 micelles substantially enhanced the cellular internalization of T-Ce6. As a result, T80 micelles loaded with T-Ce6 and PL [T80(T-Ce6/PL)] significantly elevated intracellular reactive oxygen species (ROS) generation in MCF-7 human breast cancer cells upon US exposure. Moreover, T-Ce6 exhibited selective accumulation within the mitochondria, leading to efficient cell death under US irradiation. Importantly, T80(T-Ce6/PL) micelles caused cancer-specific cell death by selectively triggering apoptosis in cancer cells through PL. This study demonstrated the feasibility of using T80(T-Ce6/PL) micelles for efficient and cancer-specific combination chemo-SDT.

High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations.

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 - 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines.

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Enhancing oral bioavailability of an antifungal thiazolylhydrazone derivative: Development and characterization of a self-emulsifying drug delivery system.

RN104 (2-[2-(cyclohexylmethylene)hydrazinyl)]-4-phenylthiazole) is a thiazolylhydrazone derivative with prominent antifungal activity. This work aimed to develop a self-emulsifying drug delivery system (SEDDS) loaded with RN104 to improve its biopharmaceutical properties and enhance its oral bioavailability. Medium chain triglycerides, sorbitan monooleate, and polysorbate 80 were selected as components for the SEDDS formulation based on solubility determination and a pseudo-ternary phase diagram. The formulation was optimized using the central composite design in response surface methodology. The optimized condition consisted of medium chain triglycerides, sorbitan monooleate, and polysorbate 80 in a mass ratio of 65.5:23.0:11.5, achieving maximum drug loading (10 mg/mL) and minimum particle size (118.4 ± 0.7 nm). The developed RN104-SEDDS was fully characterized using dynamic light scattering, in vitro release studies, stability assessments, polarized light microscopy, and transmission electron microscopy. In vivo pharmacokinetic studies in mice demonstrated that RN104-SEDDS significantly improved oral bioavailability compared to free RN104 (the relative bioavailability was 2133 %). These results clearly indicated the successful application of SEDDS to improve the pharmacokinetic profile and to enhance the oral bioavailability of RN104, substantiating its potential as a promising antifungal drug candidate.