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

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

Model construction and theoretical evaluation of the performance improvement of acetone-butanol-ethanol extractive fermentation by adding surfactant.

Severe butanol toxicity to the metabolism of solventogenic clostridia significantly impede the application of fermentative butanol as a biofuel. Liquid-liquid extraction is an efficient method to reduce the butanol toxicity by in-situ removing it in the extractant phase. Butanol mass transfer into extractant phase in static acetone-butanol-ethanol (ABE) extractive fermentation with biodiesel as the extractant could be enhanced by adding a tiny amount of surfactant such as tween-80. In the case of corn-based ABE extractive fermentation by Clostridium acetobutylicum ATCC 824 using biodiesel originated from waste cooking oil as extractant, addition of 0.14% (w/v) tween-80 could increase butanol production in biodiesel and total solvents production by 21% and 17%, respectively, compared to those of control under non-surfactant existence. Furthermore, a mathematical model was developed to elucidate the mechanism of enhanced ABE extractive fermentation performance. The results indicated that the mass transfer improvement was obtained by effectively altering the physical properties of the self-generated bubbles during ABE extractive fermentation, such as reducing bubble size and extending its retention time in extractant phase, etc. Overall, this study provided an efficient approach for enhancing biobutanol production by integration of bioprocess optimization and model interpretation.

Evaluation of the In-Use Stability of Monoclonal Antibody IV Admixtures Prepared from Drug Products Containing Polysorbate 20 Degraded by Host-Cell Lipases.

Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.

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.

[Evaluation of Malassezia furfur Biofilm Formation on Polypropylene Membrane]. / Polipropilen Membran Üzerinde Malassezia furfur Biyofilm Olusumunun Degerlendirilmesi.

The Malassezia yeast species colonize on the skin immediately after birth and could be found on the healthy skin flora for life. Although they are more frequently involved in the etiology of common skin infections in the community, particularly Malassezia furfur could cause life-threatening infections such as fungemia. Detection of biofilm during the colonization of these yeasts on the skin is an important criterion for its virulence. Since they are lipophilic yeasts, commonly used biofilm detection methods are not applicable to the Malassezia strains. The aim of the study was to describe the growth and measurement of M.furfur isolates on a polypropylene membrane to demonstrate their biofilm-forming capacities. Twenty-seven M.furfur strains colonized in the newborns were included in the study. Basically, sterile polypropylene membranes were placed on different polysorbates (tween 20, 40, and 80) which were spread on Sabouraud dextrose agar. Ten µl saline suspension of M.furfur was dropped on the polypropylene membrane and incubated in standard growth conditions for three days. Later, the visible colony was removed gently by washing with running water and the biofilm structure formed on the membrane was stained with safranin. The stained biofilm was photographed. Performing image analysis, the values obtained against background activity were digitized according to the specified protocol. Moreover, XTT reduction test was performed and the measured metabolic activity results were compared with the safranin-stained biofilm data. The safranin hydrolysis of the strains was measured spectrometrically. Twenty-five (92.6%) of the strains included in the study were stained with safranin, which indicated the presence of biofilm on the polypropylene membrane. The strains grown with tween 20 and tween 80 formed a higher biofilm layer density than those supplied with tween 40. Isolates with low and high biofilm-forming capacity were clearly separated by tween 20 (p< 0.05). XTT activity was detected in 26 (96.3%) isolates. No correlation was found between biofilm density obtained by the described method and XTT reduction. It was observed that hydrolysis of safranin did not affect the biofilm evaluation method. In this study, it was shown that as a result of sufficient diffusion through hydrophobic membranes, polysorbate-based growth factors could maintain measurement of the biofilm layer formed by lipophilic M.furfur strains. The best grouping properties for M.furfur were obtained with tween 20 which could determine low and high level of biofilm formation. Image analysis was used with high performance for this method. As conclusion, the utilization of different hydrophobic membranes and dyes would lead to the development of new techniques for the application in other lipophilic yeasts.

Comparison of rumen and abomasal infusions of an exogenous emulsifier on fatty acid digestibility of lactating dairy cows.

We evaluated the effects of infusing an exogenous emulsifier (polysorbates-C18:1) either into the rumen or abomasum on fatty acid (FA) digestibility and production responses of lactating dairy cows. Nine ruminally cannulated multiparous Holstein cows (170 ± 13.6 d in milk) were assigned to a treatment sequence in replicated 3 × 3 Latin squares with 18-d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water carrier only into the rumen and abomasum (control, CON), 30 g/d polysorbate-C18:1 (T80) infused into the rumen (RUM), or 30 g/d T80 infused into the abomasum (ABO). Emulsifiers were dissolved in water and delivered at 6-h intervals (total daily infusion was divided into 4 equal infusions per day). Cows were fed the same diet that contained [% diet dry matter (DM)] 32.2% neutral detergent fiber (NDF), 16.1% crude protein, 26.5% starch, and 3.41% FA (including 1.96% FA from a saturated FA supplement containing 28.0% C16:0 and 54.6% C18:0). Two orthogonal contrasts were evaluated: (1) the overall effect of T80 {CON vs. average of the T80 infusions [1/2 (ABO + RUM)]}, and (2) the effect of ABO versus RUM infusion. Compared with CON, infusing T80 increased the digestibilities of NDF (2.85 percentage units), total (4.35 percentage units), 16-carbon (3.25 percentage units), and 18-carbon FA (4.60 percentage units), and tended to increase DM digestibility and total and 18-carbon FA absorption. Compared with RUM, ABO decreased the intakes of total (28 g/d), 16-carbon (7 g/d), and 18-carbon FA (19 g/d); tended to increase the digestibility of total and 18-carbon FA; and had no effect on the absorption of total, 16-carbon, or 18-carbon FA. Production responses did not change among our treatments. In conclusion, infusing 30 g/d polysorbates-C18:1 increased NDF and total, 16-carbon, and 18-carbon FA digestibility. Compared with RUM, ABO tended to increase the digestibilities of total and 18-carbon FA; however, this may be related to the fact that ABO reduced the intakes of total, 16-carbon, and 18-carbon FA, not necessarily due to better emulsifying action per se. In summary, ABO and RUM both improved FA absorption.

Prediction of long-term polysorbate degradation according to short-term degradation kinetics.

Polysorbates (PSs) are a class of surfactants commonly used in the formulation of protein therapeutic agents to provide protection against denaturation and aggregation. When the PS in these drug formulations degrades, loss of stabilization of the protein therapeutic and formulation may occur, resulting in particulate formation or other undesirable changes in product critical quality attributes. Here, we present a simplified platform to predict long-term PS20 and PS80 degradation for monoclonal antibody drugs containing the PS-degrading enzyme lysosomal acid lipase. The platform was based on a temperature-dependent equation derived from existing PS20 degradation stability data. Accurate prediction of both PS20 and PS80 hydrolysis for as long as 2 years was achieved through short-term kinetics studies performed within 2 weeks. This platform substantially shortens the time required to determine the long-term stability of PS degradation and therefore can be used to guide the purification process and optimization of antibody formulations.

Tween-80 enhanced biodegradation of naphthalene by Klebsiella quasipneumoniae.

Accidental spillage of petroleum products and industrial activities result in various hydrocarbons in the environment. While the n-hydrocarbons are readily degraded, the polycyclic aromatic hydrocarbons (PAHs) are recalcitrant to natural degradation, toxic to aquatic life and are responsible for diverse health challenges in terrestrial animals; suggesting the need for faster and more eco-friendly ways of removing PAHs from the environment. In this study, the surfactant tween-80 was used to enhance a bacterium's intrinsic naphthalene biodegradation activity. Eight bacteria isolated from oil-contaminated soils were characterised using morphological and biochemical methods. The most effective strain was identified as Klebsiella quasipneumoniae using 16S rRNA gene analysis. High-Performance Liquid Chromatography (HPLC) analyses showed that the detectable concentration of naphthalene was decreased from 500 to 157.18 µg/mL (67.4%) after 7 d in the absence of tween-80, while 99.4% removal was achieved in 3 d in the presence of tween-80 at 60 µg/mL concentration. The peaks observed in the Fourier Transform Infra-Red Spectroscopy (FTIR) spectrum of control (naphthalene), which were absent in that of the metabolites, further established naphthalene degradation. Furthermore, Gas Chromatography-Mass Spectrometer (GCMS) revealed metabolites of single aromatic ring, such as 3,4-dihydroxybenzoic acid 4-hydroxylmethylphenol, which confirmed that the removal of naphthalene is by biodegradation. Tyrosinase induction and laccase activities suggested the involvement of these enzymes in naphthalene biodegradation by the bacterium. Conclusively, a strain of K. quasipneumoniae that can effectively remove naphthalene from contaminated environments has been isolated, and its biodegradation rate was doubled in the presence of non-ionic surfactant, tween-80.

Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability.

Polysorbate 80 is a non-ionic detergent derived from polyethoxylated sorbitan and oleic acid. It is widely used in pharmaceuticals, foods, and cosmetics as an emulsifier. Nav1.7 is a peripheral sodium channel that is highly expressed in sympathetic and sensory neurons, and it plays a critical role in determining the threshold of action potentials (APs). We found that 10 µg/mL polysorbate 80 either abolished APs or increased the threshold of the APs of dorsal root ganglions. We thus investigated whether polysorbate 80 inhibits Nav1.7 sodium current using a whole-cell patch-clamp recording technique. Polysorbate 80 decreased the Nav1.7 current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 250.4 µg/mL at a holding potential of -120 mV. However, the IC50 was 1.1 µg/mL at a holding potential of -90 mV and was estimated to be 0.9 µg/mL at the resting potentials of neurons, where most channels are inactivated. The activation rate and the voltage dependency of activation of Nav1.7 were not changed by polysorbate 80. However, polysorbate 80 caused hyperpolarizing shifts in the voltage dependency of the steady-state fast inactivation curve. The blocking of Nav1.7 currents by polysorbate 80 was not reversible at a holding potential of -90 mV but was completely reversible at -120 mV, where the channels were mostly in the closed state. Polysorbate 80 also slowed recovery from inactivation and induced robust use-dependent inhibition, indicating that it is likely to bind to and stabilize the inactivated state. Our results indicate that polysorbate 80 inhibits Nav1.7 current in concentration-, state-, and use-dependent manners when used even below commercial concentrations. This suggests that polysorbate 80 may be helpful in pain medicine as an excipient. In addition, in vitro experiments using polysorbate 80 with neurons should be conducted with caution.

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction.

The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The ß-carotene 15,15'-dioxygenase (BCO) cleaves ß-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated ß-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous ß-carotene biosynthetic genes, GGS1F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the ß-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in ß-carotene level, but less than 5% of the consumed ß-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.

Clove Oil Endorsed Transdermal Flux of Dronedarone Hydrochloride Loaded Bilosomal Nanogel: Factorial Design, In vitro Evaluation and Ex vivo Permeation.

The goal of this study was to develop a bilosomal gel formulation to enhance transdermal permeability of dronedarone hyrdrochloride (DRN) which suffers from poor oral absorption and limited bioavailability. To overcome this obstacle, bilosomes were successfully prepared using 23 full-factorial design. Span®40, cholesterol, sodium deoxycholate (bile salt), clove oil (permeability enhancer), and either Tween® 60 or Tween® 80 (edge activator) were used in bilosome preparation by ethanol injection method. In this design, independent variables were X1, edge activator type; X2, edge activator amount (mg); and X3, permeability enhancer concentration (% w/v). Optimal formula (B2) of the highest desirability of (0.776) demonstrated minimum vesicle size (VS) of 312.4 ± 24.42 nm, maximum absolute value of zeta potential (ZP) - 36.17 ± 2.57 mV, maximum entrapment efficiency (EE %) of 80.95 ± 3.01%, maximum deformability Index (DI) of 8.24 ± 1.26 g and maximum drug flux after 12 h (J12) of 21.23 ± 1.54 µg/cm2 h upon ex vivo permeation study. After 12 h, 70.29 ± 6.46% of DRN was released from B2. TEM identification of B2 showed spherical shaped nanosized vesicles which were physically stable for 3 months at different temperatures. B2 was incorporated into carboxymethylcellulose gel base for easiness of dermal application. B2 gel demonstrated good physical properties, non-Newtonian psuedoplastic flow, and enhanced release (57.0 ± 8.68% of DRN compared to only 13.3 ± 1.2% released from drug suspension after 12 h) and enhanced skin permeation.

A Mechanistic Understanding of Monoclonal Antibody Interfacial Protection by Hydrolytically Degraded Polysorbate 20 and 80 under IV Bag Conditions.

PURPOSE: Polysorbates (PS) contain polyoxyethylene (POE) sorbitan/isosorbide fatty acid esters that can partially hydrolyze over time in liquid drug products to generate degradants and a remaining intact PS fraction with a modified ester distribution. The degradants are composed of free fatty acids (FFAs) --primarily lauric acid for PS20 and oleic acid for PS80-- and POE head groups. We previously demonstrated that under IV bag agitation conditions, mAb1 (a surface-active IgG4) aggregation increased with increasing amounts of degradants for PS20 but not for PS80. The purpose of this work is to understand the mechanism behind this observation. METHODS: The surface tension of the remaining intact PS fraction without degradants was modeled and compared with that of enzymatically degraded PS solutions. Next, mAb1 aggregation in saline was measured in the presence of laurate and oleate salts during static storage. Lastly, colloidal and conformational stability of mAb1 in the presence of these salts was investigated through differential scanning fluorimetry and dynamic light scattering under IV bag solution conditions. RESULTS: The surface tension was primarily influenced by FFAs rather than the modified ester distribution of the remaining intact PS. MAb1 bulk aggregation increased in the presence of laurate but not oleate salts. Both salt types increased the melting temperature of mAb1 indicating FFA-mAb1 interactions. However, only laurate salt increased mAb1 self-association potentially explaining the higher aggregation propensity in its presence. CONCLUSION: Our results help explain the observed differences between hydrolytically degraded PS20 and PS80 in affecting mAb1 aggregation under IV bag agitation conditions.

In vitro assembly of chimeric virus-like particles composed of a porcine circovirus 2b capsid protein and a B-cell epitope of infectious bursal disease virus.

OBJECTIVES: To develop a method for in vitro assembly of recombinant proteins expressed in E. coli into chimeric virus-like particles (cVLPs). RESULTS: A fusion protein (Bepi-Cap-A) between capsid protein (Cap) of PCV2b and B cell epitope (Bepi) of IBDV was expressed in E. Coli, and purified. For assembling them into cVLPs (Bepi-Cap-VLP), the Bepi-Cap-A was suspended in buffer C [0.03% ("%" stands for "v/v" unless otherwise indicated) polyethylene glycol, 0.4 M Tris, 10 mM ß-mercaptoethanol, 5% glycerol, 0.02% (w/v) gellan gum, 0.1 M glycine, 0.03% Tween 80, 500 mM NaCl], and incubated. After centrifugation, the pellet was resuspended in buffer D [50 mM Na2HPO4, 50 mM NaH2PO4, 0.01% (w/v) gellan gum, 0.05 mM EDTA, 500 mM NaCl, 0.03% Tween 80, pH 6.5], and then dialyzed against dialysis buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 500 mM NaCl, 0.03% Tween 80, pH 6.5). The procedure resulted in typical and immunogenic Bepi-Cap-VLP. CONCLUSIONS: The data provide a method which is feasible for in vitro assembly of recombinant proteins into chimeric virus-like particles.

Extracellular production of azurin from Pseudomonas aeruginosa in the presence of Triton X-100 or Tween 80.

Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.

When SWEETs Turn Tweens: Updates and Perspectives.

Sugar translocation between cells and between subcellular compartments in plants requires either plasmodesmata or a diverse array of sugar transporters. Interactions between plants and associated microorganisms also depend on sugar transporters. The sugars will eventually be exported transporter (SWEET) family is made up of conserved and essential transporters involved in many critical biological processes. The functional significance and small size of these proteins have motivated crystallographers to successfully capture several structures of SWEETs and their bacterial homologs in different conformations. These studies together with molecular dynamics simulations have provided unprecedented insights into sugar transport mechanisms in general and into substrate recognition of glucose and sucrose in particular. This review summarizes our current understanding of the SWEET family, from the atomic to the whole-plant level. We cover methods used for their characterization, theories about their evolutionary origins, biochemical properties, physiological functions, and regulation. We also include perspectives on the future work needed to translate basic research into higher crop yields.

Effect of surfactants on the production of polysaccharides from Schizophyllum commune through submerged fermentation.

Schizophyllum commune (S. commune) polysaccharides are biomacromolecules with multiple biological activities and wide applications. In this study, polysaccharide production through submerged fermentation of S. commune using different surfactants was investigated. The addition of 1 g/L of polyoxyethylene sorbitan monooleate (Tween 80) at the beginning of the fermentation showed the best promotional effects on collective exopolysaccharide (EPS) production (which increased by 37.17%) while shortening the production cycle by 2 days. The monosaccharide composition of the EPS produced when the added Tween 80 was similar to that of the control; however, the molecular weight (Mw) was lower. Notably, the addition of Tween 80 significantly increased the ATP levels and the transcription levels of phosphoglucomutase and ß-glucan synthase genes in the polysaccharide synthesis pathway. The addition of Tween 80 reduced the pellet size of the mycelium compared to that of the control, but did not significantly change the microstructure of the mycelial cells. This study proposes an efficient strategy for the production of polysaccharides through submerged fermentation of S. commune, and elucidates the detailed mechanism of using Tween 80 as a fermentation stimulatory reagent.

Degradation of Polysorbate 20 by Sialate O-Acetylesterase in Monoclonal Antibody Formulations.

Polysorbates (PS) are surfactants commonly added in biologics formulations that can protect proteins from denaturation and aggregation. However, decreases in polysorbate 20 (PS20) content have been observed in some monoclonal antibody formulations, causing the formation of visible and/or subvisible particles that ultimately compromise the quality and stability of the therapeutic protein products. It was determined that the particles are mainly composed of free fatty acid, suggesting enzymatic hydrolysis of PS is responsible for the degradation of PS. Enrichment of host cell proteins (HCPs) by immunoprecipitation followed by shotgun proteomics have been utilized to identify the HCPs that can hydrolyze PS20. One HCP, sialate O-acetylesterase (SIAE), demonstrated strong enzymatic activity for PS20 degradation even at low concentration (<5 ppm level). Incubation of recombinant SIAE with PS20 resulted in a unique degradation pattern where the hydrolysis of monoester with short fatty acid chain (C12, C14) was observed but not the monoester with long fatty acid chain (C16, C18) or higher-order esters. SIAE was detected and quantitated in several formulated mAbs, and the amount of SIAE was positively correlated to PS20 degradation in these mAbs during incubation. Additional experiments also showed that when SIAE was depleted, PS20 degradation was diminished, suggesting a causality between SIAE and PS20 degradation. The lipase activity of SIAE is specific to PS20, but not to PS 80 (PS80), which contains monoesters with long chain fatty acid (C18) and higher-order esters. The specific esterase activity of SIAE on PS20 suggests a possible solution of using PS80 over PS20 to eliminate surfactant degradation in mAb products.

Enhanced exopolysaccharide production in submerged fermentation of Ganoderma lucidum by Tween 80 supplementation.

Bioactive polysaccharides extracted from Ganoderma lucidum (G. lucidum) have been widely applied in food and medicine for their multiple functions. In this study, G. lucidum exopolysaccharide (EPS) production in submerged fermentation was stimulated by Tween 80. The addition of 0.25% Tween 80 on day 3 gave a maximum production of mycelial biomass and EPS, with an increase of 19.76 and 137.50%, respectively. Analysis of fermentation kinetics showed that glucose was consumed faster after adding Tween 80, while the expression of EPS biosynthesis-related genes and ATP generation were greatly improved. Moreover, Tween 80 resulted in the significant accumulation of reactive oxygen species and increased cell membrane and cell wall permeability. The EPS from Tween 80-containing medium had higher contents of carbohydrate and uronic acid, lower molecular weight, and higher antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those of EPS produced in the absence of Tween 80. This study provides further evidence to clarify the stimulatory effects of Tween 80 in fermentation and provides a guide for the production of bioactive G. lucidum EPS.

Biotin biosynthesis affected by the NADPH oxidase and lipid metabolism is required for growth, sporulation and infectivity in the citrus fungal pathogen Alternaria alternata.

The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of H2O2, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.

Multimodal cellular redox nanosensors based on self-doped polyaniline nanocomposites.

We have successfully fabricated a nanocomposite, which is composed of polyaniline (PAni) and pyrene butyric acid (Pyba) via a solvent shift method, which was self-doped at a neutral pH value. This PAni nanocomposite can act as a fine nanoagent expressing absorbance, fluorescence, and Raman properties according to the surrounding pH values.