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.

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.

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.

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.

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.

[Effect of Tween 20 on enhancing extracellular polysaccharide synthesis by Pantoea alhagi NX-11].

Pantoea alhagi NX-11 exopolysaccharide (PAPS) is a novel microbial biostimulant that enhances crop resistance to salt and drought stress. It is biodegradable and holds promising applications in improving agricultural yield and efficiency. However, the fermentation process of PAPS exhibits a high viscosity due to low oxygen transfer efficiency, which hinders yield improvement and downstream processing. This study aimed to investigate the effects of seven oxygen carriers (Span 80, Span 20, Tween 80, Tween 20, glycerin, olive oil, and soybean oil) on fermentation yield. The results showed that the addition of 0.5% (V/V) Tween 20 significantly enhanced the production of PAPS. Moreover, the introduction of 0.5% (V/V) Tween 20 in a 7.5 L fermenter resulted in a PAPS titer of (16.85±0.50) g/L, which was 17.70% higher than that of the control group. Furthermore, the rheological characterization and the microstructure analysis of the polysaccharide products revealed that the characteristic structure of polysaccharides remained unchanged in the oxygen carrier treated group, but their viscosity increased. These findings may facilitate enhancing the biosynthesis efficiency of other polymer products.

Development and Evaluation of a Stable Oil-in-Water Emulsion with High Ostrich Oil Concentration for Skincare Applications.

This study investigates the development of an oil-in-water (O/W) emulsion enriched with a high concentration of ostrich oil, recognized for its abundant content of oleic acid (34.60 ± 0.01%), tailored for skincare applications. Using Span and Tween emulsifiers, we formulated an optimized emulsion with 20% w/w ostrich oil and a 15% w/w blend of Span 20 and Tween 80. This formulation, achieved via homogenization at 3800 rpm for 5 min, yielded the smallest droplet size (5.01 ± 0.43 µm) alongside an appropriate zeta potential (-32.22 mV). Our investigation into the influence of Span and Tween concentrations, types, and ratios on the stability of 20% w/w ostrich oil emulsions, maintaining a hydrophile-lipophile balance (HLB) of 5.5, consistently demonstrated the superior stability of the optimized emulsion across various formulations. Cytotoxicity assessments on human dermal fibroblasts affirmed the safety of the emulsion. Notably, the emulsion exhibited a 52.20 ± 2.01% inhibition of linoleic acid oxidation, surpassing the 44.70 ± 1.94% inhibition observed for ostrich oil alone. Moreover, it demonstrated a superior inhibitory zone against Staphylococcus aureus (12.32 ± 0.19 mm), compared to the 6.12 ± 0.15 mm observed for ostrich oil alone, highlighting its enhanced antioxidant and antibacterial properties and strengthening its potential for skincare applications. The optimized emulsion also demonstrates the release of 78.16 ± 1.22% of oleic acid across the cellulose acetate membrane after 180 min of study time. This successful release of oleic acid further enhances the overall efficacy and versatility of the optimized emulsion. Stability assessments, conducted over 6 months at different temperatures (4 °C, 25 °C, 45 °C), confirmed the emulsion's sustained physicochemical and microbial stability, supporting its promise for topical applications. Despite minor fluctuations in acid values (AV) and peroxide values (PV), the results remained within the acceptable limits. This research elucidates the crucial role of emulsification in optimizing the efficacy and stability of ostrich oil in skincare formulations, providing valuable insights for practical applications where stability is paramount.

Destabilization of a model O/W/O double emulsion: From bulk to interface.

Oil-in-water-in-oil (O/W/O) double emulsions are considered an advanced oil-structuring technology that can accomplish multi-functions to improve food quality and nutrition. However, this special structure is thermodynamically unstable. This study formulated a model O/W/O double emulsion with standard surfactants, Tween 80 (4 %) and polyglycerol polyricinoleate (PGPR, 5 %), using a traditional two-step method with different homogenization parameters. Cryo-SEM and GC-FID results show that O/W/O emulsions were successfully formulated, and the release rate (RR) of medium-chain triglycerides (MCT) oil from the inner oil to the outer oil phase increased significantly with 2nd homogenization speed increasing, respectively. Interestingly, the RR of all samples reached about 75 % after 2 months of storage, suggesting that O/W/O emulsions were highly unstable. To explain the observed instability, dynamic interfacial tension and interfacial rheology were performed using a drop shape tensiometer. Results demonstrated that unadsorbed Tween 80 in the intermediate aqueous phase was a key factor in markedly decreasing the interfacial properties of the outer PGPR-assembled film by affecting the interfacial rearrangement. Additionally, it was found that the MCT release showed a positive correlation with the Tween 80 concentration, demonstrating that the formed Tween 80 micelles could transport oil molecules to strengthen the emulsion instability. Taken together, this study reveals the destabilization mechanism of model O/W/O surfactants-stabilized emulsions from bulk to interface, providing highly relevant insights for the design of stable O/W/O double emulsions.

Effect of cationic surfactant on the physicochemical and antibacterial properties of colloidal systems (emulsions and microemulsions).

Colloidal systems have been used to encapsulate, protect and release essential oils in mouthwashes. In this study, we investigated the effect of cetylpyridinium chloride (CPC) on the physicochemical properties and antimicrobial activity of oil-in-water colloidal systems containing tea tree oil (TTO) and the nonionic surfactant polysorbate 80. Our main aim was to evaluate whether CPC could improve the antimicrobial activity of TTO, since this activity is impaired when this essential oil is encapsulated with polysorbate 80. These systems were prepared with different amounts of TTO (0-0.5% w/w) and CPC (0-0.5% w/w), at a final concentration of 2% (w/w) polysorbate 80. Dynamic light scattering (DLS) results revealed the formation of oil-swollen micelles and oil droplets as a function of TTO concentration. Increases in CPC concentrations led to a reduction of around 88% in the mean diameter of oil-swollen micelles. Although this variation was of only 20% for the oil droplets, the samples appearance changed from turbid to transparent. The surface charge of colloidal structures was also markedly affected by the CPC as demonstrated by the transition in zeta potential from slightly negative to highly positive values. Electron paramagnetic resonance (EPR) studies showed that this transition is followed by significant increases in the fluidity of surfactant monolayer of both colloidal structures. The antimicrobial activity of colloidal systems was tested against a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureaus) bacteria. Our results revealed that the inhibition of bacterial growth is observed for the same CPC concentration (0.05% w/w for E. coli and 0.3% w/w for S. aureus) regardless of TTO content. These findings suggest that TTO may not act as an active ingredient in polysorbate 80 containing mouthwashes.

Taurine, a Naturally Occurring Amino Acid, as a Physical Stability Enhancer of Different Monoclonal Antibodies.

Degradation of therapeutic monoclonal antibodies (mAbs) is a major concern as it affects efficacy, shelf-life, and safety of the product. Taurine, a naturally occurring amino acid, is investigated in this study as a potential mAb stabilizer with an extensive analytical characterization to monitor product degradation. Forced degradation of trastuzumab biosimilar (mAb1)-containing samples by thermal stress for 30 min resulted in high-molecular-weight species by more than 65% in sample without taurine compared to the sample with taurine. Samples containing mAb1 without taurine also resulted in higher Z-average diameter, altered protein structure, higher hydrophobicity, and lower melting temperature compared to samples with taurine. The stabilizing effect of taurine was retained at different mAb and taurine concentrations, time, temperatures, and buffers, and at the presence of polysorbate 80 (PS80). Even the lowest taurine concentration (10 mM) considered in this study, which is in the range of taurine levels in amino acid injections, resulted in enhanced mAb stability. Taurine-containing samples resulted in 90% less hemolysis than samples containing PS80. Additionally, mAb in the presence of taurine showed enhanced stability upon subjecting to stress with light of 365 nm wavelength, combination of light and H2O2, and combination of Fe2+ and H2O2, as samples containing mAb without taurine resulted in increased degradation products by more than 50% compared to samples with taurine upon subjecting to these stresses for 60 min. In conclusion, the presence of taurine enhanced physical stability of mAb by preventing aggregate formation, and the industry can consider it as a new mAb stabilizer.

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.

Comparative assessment of emulsifiers for in vitro ruminal gas production and fermentation measurements: Tween 80 is a suitable emulsifier.

Emulsifiers are essential for achieving a homogenous distribution of lipophilic supplements in in vitro rumen fluid incubations. Since emulsifiers can alter rumen fermentation, it is crucial to select one that minimally impacts fermentation parameters to reduce potential biases. This study aimed to evaluate seven emulsifiers' impact on in vitro ruminal fermentation using the Hohenheim Gas Test in order to identify the most inert emulsifier. Rumen fluids were collected from three non-lactating Original Brown-Swiss cannulated cows before morning feeding and incubated for 24 h with a basal diet in triplicates. The emulsifiers tested were ethanol, ethyl acetate, propylene glycol, glycerol, ethylene glycol, soy lecithin, and Tween® 80, each in two dosages (0.5% or 1% v/v). The untreated basal diet served as control. Compared to control, in vitro organic matter digestibility was enhanced by ethyl acetate (by 36.9 and 48.2%), ethylene glycol (by 20.6 and 20.1%), glycerol (by 46.9 and 56.8%) and soy lecithin (by 19.7 and 26.8%) at 0.5 and 1% dosage, respectively. Additionally, the 24-h methane production increased for ethanol (by 41.9 and 46.2%), ethylene glycol (by 50.5 and 51.5%), and glycerol (by 63.1 and 65.4%) for the 0.5 and 1% dosage, respectively, and 0.5% dosage for ethyl acetate (by 31.6%). The acetate molar proportion was 17.2%pt higher for ethyl acetate, and 25.5%pt lower for glycerol at 1% dosage, compared to the control. The propionate concentration was 22.1%pt higher 1% glycerol, and 15.2%pt and 15.1%pt higher for 0.5 and 1% propylene glycol, respectively, compared to the control. In summary, Tween® 80 did not significantly affect in vitro rumen fermentation parameters, making it the most suitable choice for in vitro incubations involving lipophilic substances in rumen fluid. Ethanol may be considered as an alternative emulsifier if methane production is not the variable of interest.

Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling.

Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.

Modular and tunable alternative surfactants for biopharmaceuticals provide insights into Surfactant's Structure-Function relationship.

Surface-induced aggregation of protein therapeutics is opposed by employing surfactants, which are ubiquitously used in drug product development, with polysorbates being the gold standard. Since poloxamer 188 is currently the only generally accepted polysorbate alternative, but cannot be ubiquitously applied, there is a strong need to develop surfactant alternatives for protein biologics that would complement and possibly overcome known drawbacks of existing surfactants. Yet, a severe lack of structure-function relationship knowledge complicates the development of new surfactants. Herein, we perform a systematic analysis of the structure-function relationship of three classes of novel alternative surfactants. Firstly, the mode of action is thoroughly characterized through tensiometry, calorimetry and MD simulations. Secondly, the safety profiles are evaluated through cell-based in vitro assays. Ultimately, we could conclude that the alternative surfactants investigated possess a mode of action and safety profile comparable to polysorbates. Moreover, the biophysical patterns elucidated here can be exploited to precisely tune the features of future surfactant designs.

The Development of a Novel Aflibercept Formulation for Ocular Delivery.

Aflibercept is a recombinant fusion protein that is commercially available for several ocular diseases impacting millions of people worldwide. Here, we use a case study approach to examine alternative liquid formulations for aflibercept for ocular delivery, utilizing different stabilizers, buffering agents, and surfactants with the goal of improving the thermostability to allow for limited storage outside the cold chain. The formulations were developed by studying the effects of pH changes, substituting amino acids for sucrose and salt, and using polysorbate 80 or poloxamer 188 instead of polysorbate 20. A formulation containing acetate, proline, and poloxamer 188 had lower rates of aggregate formation at 4, 30, and 40°C when compared to the marketed commercial formulation containing phosphate, sucrose, sodium chloride, and polysorbate 20. Further studies examining subvisible particles after exposure to a transport stress and long-term stability at 4°C, post-translational modifications by multi-attribute method, purity by reduced and non-reduced capillary electrophoresis, and potency by cell proliferation also demonstrated a comparable or improved stability for the enhanced formulation of acetate, proline, and poloxamer 188. This enhanced stability could enable limited storage outside of the cold chain, allowing for easier distribution in low to middle income countries.

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

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

[Preparation and quality evaluation of total flavonoids microemulsion of "Pueraria lobata-Hovenia dulcis"].

The effective components of flavonoids in the "Pueraria lobata-Hovenia dulcis" drug pair have low bioavailability in vivo due to their unstable characteristics. This study used microemulsions with amphoteric carrier properties to solve this problem. The study drew pseudo-ternary phase diagrams through titration compatibility experiments of the oil phase with emulsifiers and co-emulsifiers and screened the prescription composition of blank microemulsions. The study used average particle size and PDI as evaluation indicators, and the central composite design-response surface method(CCD-RSM) was used to optimize the prescription; high-dosage drug-loaded microemulsions were obtained, and their physicochemical properties, appearance, and stability were evaluated. The results showed that when ethyl butyrate was used as the oil phase, polysorbate 80(tween 80) as the surfactant, and anhydrous ethanol as the cosurfactant, the maximum microemulsion area was obtained. When the difference in results was small, K_(m )of 1∶4 was chosen to ensure the safety of the prescription. The prescription composition optimized by the CCD-RSM was ethyl butyrate(16.28%), tween 80(9.59%), and anhydrous ethanol(38.34%). When the dosage reached 3% of the system mass, the total flavonoid microemulsion prepared had a clear and transparent appearance, with average particle size, PDI, and potential of(74.25±1.58)nm, 0.277±0.043, and(-0.08±0.07) mV, respectively. The microemulsion was spherical and evenly distributed under transmission electron microscopy. The centrifugal stability and temperature stability were good, and there was no layering or demulsification phenomenon, which significantly improved the in vitro dissolution of total flavonoids.