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Ensuring the quality and safety of biopharmaceutical products requires the effective separation of monoclonal antibodies (mAbs) from host cell proteins (HCPs). A major challenge in this field is the enzymatic hydrolysis of polysorbates (PS) in drug products. This study addresses this issue by investigating the removal of polysorbate‐degrading HCPs during the polishing steps of downstream purification, an area where knowledge about individual HCP behavior is still limited. We investigated the separation of different mAb formats from four individual polysorbate degrading hydrolases (CES1F, CES2C, LPLA2, and PAF‐AH) using cation exchange (CEX) and mixed‐mode chromatography (MMC) polishing steps. Our research identified a key challenge: The similar elution behavior of mAbs and HCPs during chromatographic separation. To investigate this phenomenon, we performed high‐throughput binding screenings for recombinant polysorbate degrading hydrolases and representative mAb candidates on CEX and MMC chromatography resins. We then employed a three‐step strategy that also served as a scale‐up process, optimizing separation conditions and leading to the successful removal of specific HCPs while maintaining high mAb recovery rates (>96%). This strategy involved the use of surface response models and miniature columns for screening, followed by validation on larger columns using a chromatography system. Our results highlight the critical role of the inherent properties of mAbs for successful separation from HCPs. These results underscore the need to tailor the purification process to leverage the slight differences in binding behavior and elution profiles between mAbs and specific HCPs. This approach lays the foundation for developing more effective strategies for overcoming the challenge of enzymatic polysorbate degradation, paving the way for improved quality and safety in biopharmaceutical products. [ABSTRACT FROM AUTHOR]

This study reports the improvement and validation of a colorimetric method to quantify polysorbates (20, 60, 65, and 80) in food by measuring absorbance at 620 nm using ultraviolet–visible spectrophotometry. The method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and measurement uncertainty. The coefficient of determination was linear (r2 ≥ 0.9991) over the measured concentration range of 50–1000 mg/L. The LOD and LOQ were 2.3–4.9 and 7.0–15.0 mg/kg, respectively. Intra-day and inter-day accuracy and precision were 91.9–104.1% and 0.1–1.1% RSD, and 91.6–103.8% and 0.4–5.0% RSD, respectively. The result of inter-laboratory recovery was 90.9–99.8% and the measurement uncertainty was < 16% with the compliance of the CODEX recommendation. Sauce, bread, whipped cream, rice cake, ice cream, and various other polysorbate-labeled food products (n = 229, detection range; N.D.–16,442.3 mg/kg) distributed in Korea were analyzed to confirm the applicability of the analytical method. [ABSTRACT FROM AUTHOR]

Purpose: Polysorbates are the most commonly used surfactants in formulations to stabilize therapeutic proteins against interfacial stresses. Polysorbates can undergo oxidative or enzyme-mediated hydrolytic degradation to produce free fatty acids (FFAs) and subvisible particles in formulations. To determine which product related variables contribute to PS20 degradation, we investigated the effects of storage temperature, formulation, pH, presence of hydrolytic enzymes, and specific fatty acid composition on different grades of PS20 in relation to their PS20 degradation profile and consequently the quality of protein drug products. Methods: Bevacizumab and T-DM1 were reformulated in the freshly prepared therapeutic protein formulations containing either compendial PS20 or non-compendial PS20 with high % lauric acid and spiked with exogenous esterase or lipase. The release of FFAs and formation of particles were monitored at 4°C and 37°C. Protein quality was assessed for secondary structures, purity, and biological activity. Results: Hydrolytic release of FFAs and formation of subvisible particles were found to be dependent on grades of PS20, types of enzymes used, incubation temperature, and pH. Esterase- or lipase-mediated degradation of PS20 and formation of subvisible particles in drug formulation showed no significant impact on the biological activity and stability of therapeutic proteins against degradation or aggregation. Conclusions: Our study suggests that degradation of PS20 and formation of FFA particles depend on the fatty acid composition of PS20, types of hydrolytic enzymes, pH, and temperature. The presence of FFA subvisible particles showed no significant impact on the purity and biological activity of the therapeutic proteins under the tested conditions. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

The aim of this study was to assess the impact of physiological factors, namely tear fluid and lysozyme enzyme, as well as surfactant polysorbate, on the release profile from solid lipid microparticles (SLM), in the form of dispersion intended for ocular application. Indomethacin (Ind) was used as a model drug substance and a release study was performed by applying the dialysis bag method. Conducting release studies taking into account physiological factors is expected to improve development and screening studies, as well as support the regulatory assessment of this multi-compartment lipid dosage form. The effect of the lysozyme was directly related to its effect on lipid microparticles, as it occurred only in their presence (no effect on the solubility of Ind). Polysorbate also turned out to be an important factor interacting with the SLM surface, which determined the release of Ind from SLM. However, in study models without tear fluid or lysozyme, the release of Ind did not exceed 60% within 96 h. Ultimately, only the simultaneous application of artificial tear fluid, lysozyme, and polysorbate allowed for the release of 100% of Ind through the SLM dispersion. The examination of the residues after the release studies indicated the possibility of releasing 100% of Ind from SLM without complete degradation of the microparticles' matrix. The incubation of SLM with tear fluid confirmed a similar influence of physiological factors contained in tear fluid on the surface structure of SLM as that observed during the in vitro studies. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies require careful formulation due to their inherent stability limitations. Polysorbates are commonly used to stabilize mAbs, but they are prone to degradation, which results in unwanted impurities. KLEPTOSE® HPβCD (hydroxypropyl beta-cyclodextrin) has functioned as a stable stabilizer for protein formulations in our previous research. The current study investigates the collaborative impact of combining polysorbates and HPβCD as excipients in protein formulations. The introduction of HPβCD in formulations showed it considerably reduced aggregation in two model proteins, bevacizumab and ipilimumab, following exposure to various stress conditions. The diffusion interaction parameter revealed a reduction in protein–protein interactions by HPβCD. In bevacizumab formulations, the subvisible particle counts per 0.4 mL of samples in commercial formulations vs. formulations containing both HPβCD and polysorbates subjected to distinct stressors were as follows: agitation, 87,308 particles vs. 15,350 particles; light, 25,492 particles vs. 6765 particles; and heat, 1775 particles vs. 460 particles. Isothermal titration calorimetry (ITC) measurement indicated a weak interaction between PS 80 and HPβCD, with a KD value of 74.7 ± 7.5 µM and binding sites of 5 × 10–3. Surface tension measurements illustrated that HPβCD enhanced the surface activity of polysorbates. The study suggests that combining these excipients can improve mAb stability in formulations, offering an alternative for the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

Progesterone (P4), commonly administered in high doses for endometrial cancer palliative management, has limitations in current delivery systems. This preliminary in vitro drug release study introduces electrospun patches to offer a new perspective on P4 delivery. The study aimed to assess the influence of the surfactant polysorbate 80 (PS80) on the release of P4 from polycaprolactone (PCL) fibers. The PS80 effects are examined to inform the fine‐tuning of the fibre generation process. Patches developed, PCL wet (with PS80) and PCL dry (without PS80), showed encapsulation efficiencies of 76% and 42%, respectively. The dose levels studied are 6.1 mg for PCL wet and 4.4 mg for PCL dry samples. Molecular studies show that higher surfactant levels improved P4‐polymer mixing, enhancing dissolution and release rates. Patches with PS80 released 66% of the drug in 17 h, while those without released only 51%. Release data best fit the Weibull model, showcasing promise for these patches in transdermal P4 delivery. This study offers a non‐invasive option compared to traditional methods and underscores the need for further research to confirm the patches' clinical effectiveness for potential use in gynecological oncology. [ABSTRACT FROM AUTHOR]

The hydrophilic–lipophilic balance (HLB) is a valuable parameter used to determine the relative hydrophobicity of a compound based on its chemical structure. This semi-empirical parameter has been instrumental in formulating oil-in-water and water-in-oil emulsions using well-characterized ingredients with known HLB values. However, recent trends toward the use of minimally processed "virgin" oils of therapeutic or nutritional value may render the reported "required HLB" values inaccurate. Minimally processed oils can contain numerous compounds at varying or unknown concentrations, rendering the HLB value incalculable. Factors such as regional source, growing season, and processing method contribute to the variability in oil composition. Furthermore, the solubilization of lipophilic bioactives in oils can significantly alter the HLB of the oil phase in a concentration-dependent manner. This complicates the formulation of emulsions, as the HLB values of both the oil phase and emulsifiers must be closely matched to achieve stable formulations. This study presents a simple and efficient experimental method to determine an HLB value of a complex ingredient without resorting to lengthy Design-of-Experiment (DoE) matrices and trial-and-error approaches. The optimal HLB of a formulation can be determined from a series of experiments in which two well-characterized surfactants of known HLB values are mixed at varying proportions with an unknown oil phase, where the HLB of the oil is considered to match the HLB value of the surfactants combined at the proportion at which nanoemulsions with the smallest, most stable oil droplets are formed. Similarly, when the HLB values of the oil phase and other contributing components are precisely known, the unknown HLB of a complex natural surfactant can be calculated. These calculations assist in formulating emulsions efficiently and effectively by ensuring optimal compatibility among all the components. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Thermal stability of polysorbate-degradative enzymes was assessed with chemical proteomics and biophysical studies. • Brief heat treatment inactivated polysorbate-degradative enzymes and had minimal impact on the therapeutic antibody. • Heat inactivation of protein-A pool decelerated polysorbate degradation. Polysorbate degradation in biotherapeutics formulations is an industry-wide problem and mainly caused by residual host cell-derived enzymes. We present a proof-of-concept study of a control strategy which takes advantage of lower thermal stability of such enzymes relative to therapeutic proteins. We profiled heat sensitivity of host cell–derived enzyme activity with chemical proteomics and observed that PLA2G7 became inactive after brief heating. Further biophysical studies indicated that these enzymes were less thermally stable than a monoclonal antibody. Importantly, brief heat treatment had minimal impact on the stability of the antibody. Consequently, heat inactivation of polysorbate-spiked protein-A pool decelerated polysorbate degradation. This study suggests that heat inactivation of host cell–derived enzymes could be a control stragy for polysorbate degradation. [ABSTRACT FROM AUTHOR]

Nanoemulsions exhibit a wide range of practical applications owing to their smaller particle size, stability, and enhanced bioavailability. Conventional surfactants widely used in nanoemulsion formulations namely, polysorbate 80, macrogolglycerol ricinoleate, and 4‐octylphenol polyethoxylate have been associated with anaphylactoid responses, alterations in normal flora, cytotoxicity and tissue damage. The use of less toxic, environment‐friendly microbial biosurfactants in the formulation of nanoemulsions have been demonstrated to manifest stability, slow drug release, enhanced skin permeability, and bioactivity. The type and composition of mixed microbial biosurfactants used in nanoemulsions can decrease the droplet size and synergistically increase the stability. Moreover, a lower concentration of drug‐loaded surfactin‐based nanoemulsion synergistically inhibited the proliferation of cancer cells by 50% (IC50). The scope of the current review is to focus on the prospect of substituting conventional surfactants with biosurfactants obtained from microbial origin for nanoemulsion formulations. It also aims to draw attention to the paucity of research in the combinatorial study of biosurfactants and functional drugs used in nanoemulsions. The review emphasizes the bioactivity of microbial biosurfactant‐based nanoemulsions and their applications in diverse sectors. The differences in emulsion stability and size of microbial biosurfactant and conventional surfactant‐based nanoemulsions have been highlighted. The study intends to promote comparative research between nanoemulsions prepared using conventional surfactants and microbial biosurfactants. [ABSTRACT FROM AUTHOR]

Surfactants are commonly used in the therapeutic protein manufacturing process as stabilizer. Polysorbate‐20 (PS‐20) is one of the most commonly used surfactants to mitigate protein aggregation in the therapeutic protein formulation. It has been observed that polysorbate can be adsorbed by sterile filters during the final filtration process, which poses risk of uneven distribution and potentially reduced polysorbate concentration in final products. In this study, we evaluated the PS‐20 adsorption behavior using commonly used sterilizing‐grade microfiltration (MF) membranes via a customized filtration set‐up. The effect of membrane properties, including the membrane materials, modification, and layer configuration were studied. In addition, the effect of PS‐20 concentration was evaluated and the PS‐20 adsorption amount by each type of membranes was determined quantitatively and showed good agreement with the in‐process adsorption results. Finally, the selection of sterile filters and strategy of pre‐flush are discussed for ensuring accurate PS‐20 content in the final therapeutic protein drug product. [ABSTRACT FROM AUTHOR]

[Display omitted] Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air–water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Protein stability is fundamental to maintain pharmaceutical efficacy in the nascent field of biologics. One particular property that is essential for therapeutic effect is retention of the folded 3-dimensional conformation, i.e. once unfolding has occurred the biologic is often rendered inactive. In this work we propose a modified form of a recently published UV spectroscopic method that identifies protein unfolding. In this study we determine concentration limits to avoid protein unfolding of two model surfactants, namely polysorbate 20 and polysorbate 80, by correlating surfactant concentration with percentage ‘unfolded’ for three model proteins. For each scenario two distinct regions were observed, firstly surfactant concentrations at which no unfolding had occurred, followed by a second region whereby unfolding steadily increased with surfactant concentration. In general for the combinations analysed in this study, this second region began to appear around ten times below the critical micellar concentration of each surfactant, regardless of the protein or polysorbate chosen. It is therefore proposed that this adapted method could be used by researchers in the early stages of formulation development as a convenient and simple screening tool to confirm the ‘onset of unfolding’ concentration for protein-surfactant formulations, thus helping to optimise surfactant concentration selection in pharmaceutical formulations to maintain the benefits of surfactants yet avoid inadvertent unfolding.

Polysorbate 80 (PS80), a chemical substance composed of sorbitol, ethylene glycol, and fatty acids, is commonly used in pharmaceutical drug products to stabilize formulations. However, recent studies have demonstrated that PS80 may hydrolyze over time and the released free fatty acids (FFAs) may lead to particle formation. Naming conventions of fatty acids in current pharmacopeia and in products' certificates of analysis (CoA) of PS80 do not typically distinguish between isomeric species of fatty acids in PS80. Thus, methods to fully characterize the fatty acid species present in PS80 raw materials are needed to enhance quality control strategies of pharmaceuticals using PS80. Here, extended effort is taken to characterize fatty acids in hydrolyzed PS80 raw materials and elucidate the identities of isomeric fatty acid species. In this work, a method was developed and optimized for separation and detection of fatty acids in alkaline hydrolyzed PS80 raw materials using ultra performance liquid chromatography (UPLC) with ultra-violet (UV) detection and evaporative light scattering detection (ELSD). Fatty acids not specified in the current pharmacopeias were detected in PS80 raw material by the developed LC-UV-ELSD method including conjugated forms of linoleic and linolenic fatty acid species. Their identities were orthogonally confirmed by retention time agreement with analytical standards, accurate mass by high resolution mass spectrometry, UV absorbance, and proton nuclear magnetic resonance spectroscopy. The detected conjugated fatty acids are theoretically more hydrophobic and less soluble than their unconjugated counterparts and may increase the propensity of PS80 to form particles upon hydrolysis. This work highlights the need for better quality control of PS80 raw material, as it may eventually play a critical role in product quality of therapeutic proteins. [ABSTRACT FROM AUTHOR]

Purpose: Polysorbate oxidation can potentially lead to protein degradation and loss of potency, which has been a challenge for the pharmaceutical industry for decades. Many factors have been reported to impact polysorbate oxidation rate, including types of elemental impurities, peroxide content, pH, light exposure, grades of polysorbate, etc. Even though there are many publications in this field, the impact of primary container closure system on PS80 oxidation has not been systematically studied or reported. The purpose of the current study is to close this gap. Methods: Placebo PS80 formulations were prepared and filled into different container-closure systems (CCS), including different types of glass vials and polymer vials. Oleic acid content was monitored on stability as a surrogate value for PS80 content, which will decline upon oxidation. ICP-MS analysis and metal spiking studies were carried out to correlate the PS80 oxidation rate with metals leached from primary containers. Results: PS80 degrades via oxidation at the fastest rate in glass vials with high coefficient of expansion (COE), followed by glass vials with low coefficient of expansion, while polymer vials minimized the oxidation of PS80 in most formulation conditions explored in this paper. ICP-MS analysis demonstrated that 1) 51 COE glass has more metal leachables than 33 COE glass in this study; and 2) More metal leachables correlates with faster PS80 oxidation. Metal spiking studies confirmed the hypothesis that aluminum and iron have a synergistic catalysis effect on PS80 oxidation. Conclusions: Primary containers of drug products play a significant role in the rate of PS80 oxidation. This study revealed a new major contributor to PS80 oxidation and potential mitigation strategy for biological drug products. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies require careful formulation due to their inherent stability limitations. Polysorbates are commonly used to stabilize mAbs, but they are prone to degradation, which results in unwanted impurities. KLEPTOSE® HPβCD (hydroxypropyl beta-cyclodextrin) has functioned as a stable stabilizer for protein formulations in our previous research. The current study investigates the collaborative impact of combining polysorbates and HPβCD as excipients in protein formulations. The introduction of HPβCD in formulations showed it considerably reduced aggregation in two model proteins, bevacizumab and ipilimumab, following exposure to various stress conditions. The diffusion interaction parameter revealed a reduction in protein–protein interactions by HPβCD. In bevacizumab formulations, the subvisible particle counts per 0.4 mL of samples in commercial formulations vs. formulations containing both HPβCD and polysorbates subjected to distinct stressors were as follows: agitation, 87,308 particles vs. 15,350 particles; light, 25,492 particles vs. 6765 particles; and heat, 1775 particles vs. 460 particles. Isothermal titration calorimetry (ITC) measurement indicated a weak interaction between PS 80 and HPβCD, with a KD value of 74.7 ± 7.5 µM and binding sites of 5 × 10–3. Surface tension measurements illustrated that HPβCD enhanced the surface activity of polysorbates. The study suggests that combining these excipients can improve mAb stability in formulations, offering an alternative for the biopharmaceutical industry.

The aim of this study was to assess the impact of physiological factors, namely tear fluid and lysozyme enzyme, as well as surfactant polysorbate, on the release profile from solid lipid microparticles (SLM), in the form of dispersion intended for ocular application. Indomethacin (Ind) was used as a model drug substance and a release study was performed by applying the dialysis bag method. Conducting release studies taking into account physiological factors is expected to improve development and screening studies, as well as support the regulatory assessment of this multi-compartment lipid dosage form. The effect of the lysozyme was directly related to its effect on lipid microparticles, as it occurred only in their presence (no effect on the solubility of Ind). Polysorbate also turned out to be an important factor interacting with the SLM surface, which determined the release of Ind from SLM. However, in study models without tear fluid or lysozyme, the release of Ind did not exceed 60% within 96 h. Ultimately, only the simultaneous application of artificial tear fluid, lysozyme, and polysorbate allowed for the release of 100% of Ind through the SLM dispersion. The examination of the residues after the release studies indicated the possibility of releasing 100% of Ind from SLM without complete degradation of the microparticles’ matrix. The incubation of SLM with tear fluid confirmed a similar influence of physiological factors contained in tear fluid on the surface structure of SLM as that observed during the in vitro studies.

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.

Concern has arisen about hypersensitivity reactions in patients with allergic reactions to drugs containing polyethylene glycol (PEG) or polysorbate 80 (PS80), excipients of currently available anti-SARS-CoV-2 mRNA vaccines. However, the actual utility of PEG and PS80 skin allergy testing is currently still debated. We retrospectively analyzed all cases of patients on whom we performed allergometric skin tests for PEG and PS80 in the context of a pre-vaccination screening (for patients with multiple hypersensitivity reactions to drugs for which these excipients were among the suspected agents) or following suspected hypersensitivity reactions to anti-SARS-CoV-2 vaccines. A total of 134 tests were performed for PEG and PS80, eight of which produced uninterpretable results (due to dermographism or non-specific reactions). Of the remaining 126 cases (85 pre-vaccinal and 41 post-vaccine reactions), 16 (12.7%) were positive for PEG and/or PS80. Stratifying by clinical indication, there were no statistically significant differences in the proportion of positive tests between patients evaluated in the context of the pre-vaccination screening and those evaluated after a vaccine reaction (10.6% vs. 17.1%, respectively, p = 0.306). Allergometric skin tests for PEG and PS80 in our case series resulted positive in an unexpectedly high proportion of patients, suggesting that testing for allergy to these two excipients should not be ignored in case of reasonable clinical suspicion. [ABSTRACT FROM AUTHOR]

Background: The mechanism of allergic reactions to COVID-19 mRNA vaccines has not been clarified. Polyethylene glycol (PEG) is a potential antigen in the components of vaccines. However, there is little evidence that allergy after COVID-19 mRNA vaccination is related to PEG. Furthermore, the role of polysorbate (PS) as an antigen has also not been clarified. The objective of this study was to investigate whether PEG and PS allergies are reasonable causes of allergic symptoms after vaccination by detecting PEG-specific and PS-specific antibodies. Methods: Fourteen patients who developed immediate allergic reactions to BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccines and nineteen healthy controls who did not present allergic symptoms were recruited. Serum PEG-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) and PS-specific IgE and IgG were measured by enzyme-linked immunosorbent assay. Skin tests using PEG-2000 and PS-80 were applied to five patients and three controls. Results: Serum levels of PEG-specific IgE and IgG in patients with immediate allergic reactions to the COVID-19 mRNA vaccine were higher than those in the control group. Serum levels of PS-specific IgE in patients with allergy to the vaccine were higher than those in patients of the control group. Intradermal tests using PEG verified the results for PEG-specific IgE and IgG. Conclusions: The results suggest that PEG is one of the antigens in the allergy to COVID-19 mRNA vaccines. Cross-reactivity between PEG and PS might be crucial for allergy to the vaccines. PEG-specific IgE and IgG may be useful in diagnosing allergy to COVID-19 mRNA vaccines.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions. [ABSTRACT FROM AUTHOR]

In recent years, many fast drying techniques such as spray-drying are being explored as alternatives to biopharmaceutical freeze-drying. Thus, it is essential to understand how the processability of commonly used excipients will be affected when these new techniques are employed. This study reports a series of observations outlining how the thermally-induced oxidative degradation of polysorbates (PS) evolves in liquid to solid transitions, such as those expected in spray-drying. Firstly, the impact of different evaporation rates on the oxidative degradation of aqueous solutions of two different PS types namely, PS20 and PS80, were screened via evaporative solvent casting. The latter revealed that the evaporation rate could critically impact the rate-limiting steps of PS thermal oxidation. In addition, the potential of saccharides as excipients to mitigate the thermal oxidation of PS80 under slow and fast evaporation conditions was investigated. Five different saccharide excipients were screened, i.e., trehalose dihydrate, maltodextrin, hydroxypropyl-β-cyclodextrin, and Dextran 40. Under slow evaporation conditions, only trehalose dihydrate seemed to be beneficial in avoiding the thermal oxidation of PS80. For fast evaporation conditions, hydroxypropyl-β-cyclodextrin prevented the oxidative degradation of PS80. This implies that distinct strategies to mitigate PS oxidative degradation might be necessary depending on the drying process and rates. [ABSTRACT FROM AUTHOR]

While protein medications are promising for treatment of cancer and autoimmune diseases, challenges persist in terms of development and injection stability of high‐concentration formulations. Here, the extensional flow properties of protein‐excipient solutions are examined via dripping‐onto‐substrate extensional rheology, using a model ovalbumin (OVA) protein and biocompatible excipients polysorbate 20 (PS20) and 80 (PS80). Despite similar PS structures, differences in extensional flow are observed based on PS identity in two regimes: at moderate total concentrations where surface tension differences drive changes in extensional flow behavior, and at small PS:OVA ratios, which impact the onset of weakly elastic flow behavior. Undesirable elasticity is observed in ultra‐concentrated formulations, independent of PS identity; higher PS contents are required to observe these effects than in analogous polymeric excipient solutions. These studies reveal novel extensional flow behaviors in protein‐excipient solutions, and provide a straightforward methodology for assessing the extensional flow stability of new protein‐excipient formulations. [ABSTRACT FROM AUTHOR]

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. 1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. [ABSTRACT FROM AUTHOR]

Over seventy percent of marketed monoclonal antibody therapeutics contain between 0.001% and 0.1% (w/v) polysorbate, as it has a generally beneficial stabilizing effect that increases drug product shelf life. However, polysorbate has also been shown to contribute to particle formation due to auto-oxidation and hydrolysis, 1 which results in free fatty acids and subsequent fatty acid particle formation. Although the impact of fatty acid particles on the safety and efficacy of drug products has not been fully evaluated, it is advantageous to mitigate particle formation due to degradation of polysorbate, improving the consistency of a product's quality attributes (in this case particulate levels) throughout its lifecycle. In this report, we describe a simple experimental assay to rapidly generate fatty acid particles. Further, we show that the presence of human serum albumin (HSA) is sufficient to prevent the formation of fatty acid particles. Separately, we demonstrate that HSA can also rapidly and completely solubilize pre-formed particles. These results point to a highly plausible mechanistic explanation of previous observations and diminishes concern regarding low levels of particles in the final drug product. [ABSTRACT FROM AUTHOR]

The emulsification potential of plant-based emulsifiers, that is, pea (PPI) and lentil (LPI) proteins (4%), corn arabinoxylans (CAX, 1%), and legume protein-arabinoxylan mixtures (4% proteins + 0.15 or 0.9% CAX), was evaluated by assessing: the surface tension and potential of emulsifiers, emulsifier antinutritional contents, emulsion droplet size, emulsion physical stability, and vitamin E bioaccessibility from 10% oil-in-water emulsions. Tween 80 (2%) was used as a control. All emulsions presented small droplet sizes, both fresh and upon storage, except 4% LPI + 0.9% CAX emulsion that exhibited bigger droplet sizes (d(4,3) of approximately 18.76 μm vs 0.59 μm for the control) because of droplet bridging. Vitamin E bioaccessibility from emulsions stabilized with the combination of 4% PPI and either 0.15% or 0.9% CAX (28 ± 4.48% and 28.42 ± 3.87%, respectively) was not significantly different from that of emulsions stabilized with Tween 80 (43.56 ± 3.71%), whereas vitamin E bioaccessibility from emulsions stabilized with individual emulsifiers was significantly lower. • Legume proteins and/or arabinoxylans present good emulsifying capacities. • All tested emulsions present a very good stability over time (21 days). • Pea protein and arabinoxylans as emulsifiers promote vitamin E bioaccessibility. • Legume proteins and arabinoxylans can be an alternative to Tween 80 as emulsifiers. [ABSTRACT FROM AUTHOR]

[Display omitted] Adequate stabilization is essential for marketed protein-based biopharmaceutical formulations to withstand the various stresses that can be exerted during the pre- and post-manufacturing processes. Therefore, a suitable choice of excipient is a significant step in the manufacturing of such delicate products. Histidine, an essential amino acid, has been extensively used in protein-based biopharmaceutical formulations. The physicochemical properties of histidine are unique among amino acids and could afford multifaceted benefits to protein-based biopharmaceutical formulations. With a pKa of approximately 6.0 at the side chain, histidine has been primarily used as a buffering agent, especially for pH 5.5–6.5. Additionally, histidine exhibited several affirmative properties similar to those of carbohydrates (e.g., sucrose and trehalose) and could therefore be considered to be an alternative approach to established protein-based formulation strategies. The current review describes the general physicochemical properties of histidine, lists all commercial histidine-containing protein-based biopharmaceutical products, and discusses a brief outline of the existing research focused on the versatile applications of histidine, which can act as a buffering agent, stabilizer, cryo-/lyo-protectant, antioxidant, viscosity reducer, and solubilizing agent. The interaction between histidine and proteins in protein-based biopharmaceutical formulations, such as the Donnan effect during diafiltration of monoclonal antibody solutions and the degradation of polysorbates in histidine buffer, has also been discussed. As the first review of histidine in protein biopharmaceuticals, it helps to deepen our understanding of the opportunities and challenges associated with histidine as an excipient for protein-based biopharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Extracts of oleogum resins of Boswellia trees possess anti-inflammatory activities. Micellar formulations have been developed to increase the oral bioavailability of bioactive boswellic and lupeolic acids. The current single-dose crossover clinical trial compares for the first time pharmacokinetics/pharmacodynamics of two Boswellia serrata nutraceuticals, native Biotikon® BS-85 and micellar Boswellia-Loges®. After oral administration of the study preparations (800 mg) to 20 healthy volunteers, plasma concentrations of 8 boswellic and lupeolic acids were measured by using HPLC-MS/MS for up to 48 h Blood samples collected 2 and 5 h after drug administration were stimulated for 24 h with endotoxic lipopolysaccharide. The release of proinflammatory cytokines analyzed by flow cytometry was used as readout of the pharmacodynamic properties of the preparations. Registration: German Clinical Trials Register (DRKS) No. DRKS00027369. Administration of the micellar extract significantly increased C max, AUC 0–48 , and shortened T max for all boswellic and lupeolic acids compared to native extract. Accordingly, their relative bioavailability increased to 1,720-4,291 % with the highest difference for acetyl-11-keto-β-boswellic acid (AKBA). Both preparations reduced the release of TNF-α and the native formulation diminished also IL-1β and IL-6. However, no significant differences were observed between the preparations, except for a higher decrease in IL-1β by the native formulation Biotikon® BS-85. In a lymphocytic gene reporter cell line, both nutraceuticals similarly inhibited the NF-κB transcription factor activity as well as the TNF-α release, yet the native formulation Biotikon®BS-85 was more efficient in inhibiting TNF-α. Administration of the micellar Boswellia serrata nutraceutical increased the oral bioavailability of boswellic and lupeolic acids. Yet, the increase in plasma concentration did not enhance the anti-inflammatory efficacy of the micellar extract compared to the native extract in this ex vivo model. [Display omitted] [ABSTRACT FROM AUTHOR]

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase–mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold KD reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels.Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden–Fletcher–Goldfarb–Shanno; CHO, Chinese Hamster Ovary; KD, Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab’), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.

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

Polysorbate (also known as "Tween") 80 (PS80) is the most common surfactant in biopharmaceuticals. Here, we report a sensitive, and specific platform analytical method using reversed-phase high performance liquid chromatography employing a charged aerosol detector (HPLC-CAD), that offers the ability to quantitate intact PS80 and can distinguish between low recovery of PS80 and degraded PS80 by also monitoring several PS80 degradants which offers multiple improvements over various previously reported polysorbate analytical methods. The method employs protein/monoclonal antibody precipitation and is designed to be a quality control (QC) lab-friendly method. The method is distinguishable from other published methods because the quantification of the PS80 mono-esters is free from interference from known PS80 degradants. The method has demonstrated acceptable accuracy, precision, specificity, and sensitivity through qualification studies. The specificity afforded by the method allows for employment in forced degradation studies to discern kinetic information. Lastly, the method also has promising applications with other polysorbates (PS60, PS40, and all-laurate PS20) and shows acceptable performance with several types of biopharmaceutical proteins. [ABSTRACT FROM AUTHOR]

Polysorbates (PS 20 and PS 80) are the most widely used surfactants in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption. To date, around 70% of marketed therapeutic antibodies contain either PS 20 or PS 80 in their formulations. However, polysorbates are chemically diverse mixtures, which are prone to degradation by oxidation and hydrolysis to produce peroxides and fatty acids, which, in turn, induce protein oxidation, aggregation, and insoluble particle formation. These will negatively impact protein quality and stability. Thus, polysorbate degradation has emerged as one of the major challenges in the development and commercialization of therapeutic protein products. KLEPTOSE® HPβCD (hydroxypropyl beta-cyclodextrin), a new multifunctional excipient, has been shown to provide protein stabilization functions in biopharmaceutical downstream processes and in their final formulations. This study aims to evaluate HPβCD, a new molecule of its class, against polysorbates as a stabilizer in biologics formulations. In this study, the chemical stability of KLEPTOSE® HPβCDs is compared with polysorbates (20 and 80) under various stress conditions. When subjected to heat stress, HPβCDs show little change in product recovery (90.7–100.7% recovery for different HPβCDs), while polysorbates 20 and 80 show significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively. When subjected to other chemical stressors, namely, autoclave, light, and oxidative stresses, HPβCD remains almost stable, while polysorbates show more severe degradation, with 95.5% to 98.8% remaining for polysorbate 20 and 85.5% to 97.4% remaining for polysorbate 80. Further, profiling characterization and degradation analysis reveal that chemical structures of HPβCDs remain intact, while polysorbates undergo significant hydrolytic degradation and oxidation. Lastly, the physicochemical stability of monoclonal antibodies in formulations is investigated. When subjected to light stress, adalimumab, as a model mAb, formulated in the presence of HPβCD, shows a significant decrease in protein aggregation, and superior monomer and total protein recovery compared to PS 80-containing formulations. HPβCD also reduces both agitation and thermal stress-induced protein aggregation and prevents subvisible particle formation compared to PS 80. [ABSTRACT FROM AUTHOR]

The mechanism of allergic reactions to COVID-19 mRNA vaccines has not been clarified. Polyethylene glycol (PEG) is a potential antigen in the components of vaccines. However, there is little evidence that allergy after COVID-19 mRNA vaccination is related to PEG. Furthermore, the role of polysorbate (PS) as an antigen has also not been clarified. The objective of this study was to investigate whether PEG and PS allergies are reasonable causes of allergic symptoms after vaccination by detecting PEG-specific and PS-specific antibodies. Fourteen patients who developed immediate allergic reactions to BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccines and nineteen healthy controls who did not present allergic symptoms were recruited. Serum PEG-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) and PS-specific IgE and IgG were measured by enzyme-linked immunosorbent assay. Skin tests using PEG-2000 and PS-80 were applied to five patients and three controls. Serum levels of PEG-specific IgE and IgG in patients with immediate allergic reactions to the COVID-19 mRNA vaccine were higher than those in the control group. Serum levels of PS-specific IgE in patients with allergy to the vaccine were higher than those in patients of the control group. Intradermal tests using PEG verified the results for PEG-specific IgE and IgG. The results suggest that PEG is one of the antigens in the allergy to COVID-19 mRNA vaccines. Cross-reactivity between PEG and PS might be crucial for allergy to the vaccines. PEG-specific IgE and IgG may be useful in diagnosing allergy to COVID-19 mRNA vaccines. [Display omitted] [ABSTRACT FROM AUTHOR]

Background. Allergic patients may develop reactions following COVID-19 vaccination more frequently than non-allergic individuals. The aim of our study was to assess the risk of reactions in high-risk allergic patients vaccinated for COVID-19 at the University Health Agency Giuliano-Isontina (ASUGI) of Trieste (northeastern Italy). Methods. Patients were considered at high risk for allergic reactions in case of: prior anaphylactic reaction to any drug/vaccine; multiple drug allergy; intolerance to polyethylene glycol (PEG) or polysorbate 80 (PS80) containing drugs; and mast cell disorders. High-risk allergic patients were immunized in hospital by a dedicated allergy team supported by resuscitation staff. Patients were interviewed over the phone one month after vaccination to complete a structured questionnaire investigating signs and symptoms developed after immunization. Results. From March 2021 to February 2022, 269 patients with a history of severe allergic reactions were assessed, of whom 208 (77.3%) eventually received COVID-19 vaccination, 50 (18.6%) refused to be immunized, 10 (3.7%) were deferred for medical reasons and one was declared exempted due to testing positive for PS80. Mild reactions (urticaria, angioedema, rhinitis, erythema) to COVID-19 vaccines were reported by 30.3% of patients, 8.7% within 4 h and 21.6% > 4 h after immunization. No anaphylactic events were observed. Although they were 80 times (3.8%) more prevalent than in COVID-19 vaccinees from the general population (0.047%), vaccine allergic reactions in high-risk patients were mainly mild and late, more likely affecting women (OR = 3.05; 95% CI 1.22–7.65). Conclusions. High-risk allergic patients with urticaria and angioedema may experience mild flare-ups of mast cell activation-like symptoms following COVID-19 vaccination, supporting antihistamine premedication before vaccination and to be continued for one week afterwards. [ABSTRACT FROM AUTHOR]

Polysorbate is a key excipient included in formulations of therapeutic proteins to help prevent aggregation and surface adsorption. The stability of both polysorbate and therapeutic proteins can be compromised by oxidative degradation. In general, polysorbate is added to formulations at concentrations above the critical micelle concentration (cmc). To date, however, few experiments have quantitatively addressed the extent of extra- and intra-micellar oxidation of polysorbate in pharmaceutically relevant buffers. This study utilizes 2,2′-azobis(2-methylpropionamidine)dihydrochloride (AAPH), a peroxyl radical-generating initiator, C11-BODIPY(581/591), a lipid peroxidation probe, and fluorescence spectroscopy to reveal that both intra- and extra-micellar oxidation proceed in pharmaceutically relevant phosphate and histidine buffers. It is further demonstrated that the relative extent of oxidation observed in the intra- and extra-micellar compartments is similar irrespective of the buffer system. [ABSTRACT FROM AUTHOR]

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs. [ABSTRACT FROM AUTHOR]

[Display omitted] During the filling process of a biopharmaceutical drug product (DP), a liquid DP film might creep up the inner vial wall which is barely discernible, appears as milky-white haze after lyophilisation and is known as fogging. Creeping and fogging are mainly dependent on the primary packaging material surface and its hydration, vial preparation process as well as DP composition. The occurrence of both can impede visual inspection and might lead to DP rejection. Hence, our studies focused on the early detection of liquid solution and glass vial surface interaction directly after filling. For a fast and highly sensitive evaluation a novel video-based analysis was used. To our knowledge, this is the first time a MATLAB®-algorithm-based video analysis was applied to quantitatively determine creeping time-resolved. Furthermore, creeping in dependence of vial processing sites, surfactant type and concentration, filling temperature, and vial format were investigated. The results were verified using orthogonal conventional methods such as surface tension, wetting behaviour, and contact angle measurements, as well as ToF-SIMS, ICP-MS, and SEM. Additionally, the methods applied were assessed regarding their cross-validation capability. The observations indicate that the vial preparation process can have a pronounced impact on alteration of the glass vial surface and related creeping behaviour of the filled solution. [ABSTRACT FROM AUTHOR]

During the initial rollout of coronavirus disease 2019 (COVID-19) vaccination in Singapore, the Ministry of Health (MOH) issued a recommendation that patients with a history of any previous vaccine allergy be referred to an allergist for further review of their suitability to proceed with mRNA-based COVID-19 vaccines. Patients fulfilling the above criterion were divided into three groups: immediate reaction (Group A), delayed reaction (Group B) and no/irrelevant reaction (Group C). They were subjected to either a skin prick test (SPT) and intradermal test (IDT) with polyethylene glycol (PEG) or polysorbate-containing products; direct injection with the Pfizer BNT162b2 vaccine in the allergy clinic; or injection at community vaccination centres, respectively. Groups A and B were also invited to complete a questionnaire survey on post-vaccination reactions, and blood sampling pre-vaccination and 1 h after the first dose of the BNT162b2 vaccine to measure immunoglobulin (Ig) G, IgM and IgE antibodies to the Pfizer BNT162b2 vaccine via ELISA assays immobilised with the BNT162b2 vaccine, as well as levels of allergic cytokines interleukin (IL)-4 and IL-33, complement C5a and the endothelial activation marker intercellular adhesion molecule-1 (ICAM-1). Groups A and B comprised 62 (20.5%) patients each. In Group A, two subjects (3.2%) with equivocal IDT results tolerated both doses of the BNT162b2 vaccine without major allergic reactions. The remaining 60 (96.8%) in Group A and 62 (100%) in Group B completed both doses of BNT162b2 vaccination without major adverse reactions. Among the 99 who completed the questionnaire survey, 13 (13%) patients reported mild allergic reactions after the first dose of the vaccine. Immunoglobulin (Ig) G and M antibodies, but not IgE antibodies to the Pfizer BNT162b2 vaccine were detected in 67 subjects prior to vaccination. The presence of anti-Pfizer BNT162b2 IgG and IgM prior to vaccination did not result in major allergic reactions nor increases in Th2-related cytokines (IL-4, IL-33), complement activation products (C5a) or endothelial activation (ICAM-1). The majority of those with suspected reactions to non-COVID-19 polysorbate-containing vaccines tolerated the BNT162b2 vaccine. Excipient skin tests for PEG and polysorbate prior to vaccination are unnecessary. [ABSTRACT FROM AUTHOR]

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy. [ABSTRACT FROM AUTHOR]

Concern has arisen about hypersensitivity reactions in patients with allergic reactions to drugs containing polyethylene glycol (PEG) or polysorbate 80 (PS80), excipients of currently available anti-SARS-CoV-2 mRNA vaccines. However, the actual utility of PEG and PS80 skin allergy testing is currently still debated. We retrospectively analyzed all cases of patients on whom we performed allergometric skin tests for PEG and PS80 in the context of a pre-vaccination screening (for patients with multiple hypersensitivity reactions to drugs for which these excipients were among the suspected agents) or following suspected hypersensitivity reactions to anti-SARS-CoV-2 vaccines. A total of 134 tests were performed for PEG and PS80, eight of which produced uninterpretable results (due to dermographism or non-specific reactions). Of the remaining 126 cases (85 pre-vaccinal and 41 post-vaccine reactions), 16 (12.7%) were positive for PEG and/or PS80. Stratifying by clinical indication, there were no statistically significant differences in the proportion of positive tests between patients evaluated in the context of the pre-vaccination screening and those evaluated after a vaccine reaction (10.6% vs. 17.1%, respectively, p = 0.306). Allergometric skin tests for PEG and PS80 in our case series resulted positive in an unexpectedly high proportion of patients, suggesting that testing for allergy to these two excipients should not be ignored in case of reasonable clinical suspicion.

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17 , 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO 2 −), a strong reductant which reduces Fe3+, O 2 , and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O 2 by carbon dioxide radical anions and/or intermediary Fe2+. [ABSTRACT FROM AUTHOR]

The presence of subvisible or visible particles in mAb formulations can pose significant challenges to pharmaceutical development as it can lead to reduced shelf life, batch rejection, and recalls. Among all type of particles, proteinaceous particles are the most concerning due to their potential role in immunogenicity. Nevertheless, the underlying mechanism for protein particle formation remains poorly understood. Past research highlighted the importance of interfaces and mechanical agitation in causing protein particle formation. Current research suggests that fatty acids, as impurities present in excipients or as a result of polysorbate degradation, can also induce protein assembly and promote particle formation. In this work, we assessed oleic and lauric acid for their impact on particle formation as each represents the main hydrolysis product of PS80 or PS20, respectively. It was found that co-existence of either fatty acids with 10 mg/mL mAb A can cause protein particles, with a similar morphology to those observed previously in mAb formulations. FTIR spectra showed that the particles are proteinaceous, heterogeneous in its composition, but contain corresponding fatty acids. Interestingly, it was found that oleic acid is significantly more effective in causing protein particles than lauric acid in these experiments. This suggests that PS20 containing formulations might have a lower likelihood to have protein particles compared to PS80 containing mAb formulations if hydrolysis of polysorbate were to occur. Lastly, the presence of 0.01% polysorbate in the mAb A formulation was able to fully mitigate the effect of fatty acids and reduce the protein particles significantly, suggesting a potential mechanism where interfacial action is involved. The present study can help to understand the root cause for protein particles in a mAb formulation where fatty acids are introduced because of polysorbate hydrolysis. With further work, it will help to shed light into product control strategy as well as design approaches for robust mAb products. [ABSTRACT FROM AUTHOR]

The occurrence of visible particles over the shelf-life of biopharmaceuticals is considered a potential safety risk for parenteral administration. In many cases, particle formation resulted from the accumulation of fatty acids released by the enzymatic hydrolysis of the polysorbate surfactant by co-purified host cell proteins. However, particle formation can occur before the accumulated fatty acids exceed their expected solubility limit. This early onset of particle formation is driven by nucleation phenomena e.g. the presence of metal cations that promote the formation and growth of fatty acid particles. To further characterize and understand this phenomenon, we assessed the potential of different metal cations to induce fatty acid particle formation using a dynamic light scattering assay. We demonstrated that the presence of trace amounts of multivalent cations, in particular trivalent cations such as aluminum and iron, may act as nucleation seed in the process of particle formation. Finally, we developed a mitigation strategy for metal-induced fatty acid particles that deploys a chelator to reduce the risk of particle formation in biopharmaceutical formulations. [ABSTRACT FROM AUTHOR]

A liquid chromatography-mass spectrometry (LC–MS) method was developed to provide a fingerprint of polysorbate 80 (PS80) subspecies that enables identification of PS80 degradation pathway. The developed method demonstrates unique monoester peak profile of PS80 from different vendors, attributed by differences in relative abundance of the fatty acid monoesters. The LC–MS method was also applied to examine the susceptibility of PS80, at different grades, to auto-oxidation and hydrolysis. PS80 oxidative degradation induced by iron or occurred in open bottle without nitrogen overlay was found to follow the same pathway, but at a much faster rate in the former scenario. The oxidation preferentially occurs at the double bond of fatty acid chains, thus providing explanation on the faster degradation observed in PS80 at Chinese Pharmacopia (ChP) grade than at multi-compendial (MC) grade. In contrast, the difference in susceptibility of MC and ChP grade PS80 against esterase-induced hydrolysis in placebo was not pronounced. The method was also able to provide a fingerprint to identify both PS80 hydrolysis and oxidation in mAb drug product stability samples, but it required a solid phase extraction step to remove protein prior to the analysis. [ABSTRACT FROM AUTHOR]

[Display omitted] Monoclonal antibodies (mAbs) and excipients can degrade owing to different stress factors they encounter during their life cycle or after administration in human body. This can result in the formation of aggregates and particulates. As particles can evoke an immune response in patients, it becomes increasingly important to monitor their fate after administration. In this study, we used a protein-free serum model to assess the fate of mAb and polysorbate (PS) particles under physiologic conditions. Commonly encountered stress conditions such as pH, temperature, extrusion, and shaking were chosen to generate mAb particles. Alkaline hydrolysis was used to generate PS particles. The fate of aggregates and particles was evaluated in serum and histidine buffer. We observed that depending on the nature of stress and the environment particles are subjected to, the fate of particles can differ substantially. The mAb aggregates generated by pH stress, showed reduction in HMWS from 26% to 6% over 14 days in human serum filtrate. PS particles dissolved at 37 °C but remained unaltered in Histidine at 5 °C. Our results reinforce the need to track the fate of particles generated during drug product development upon exposure to physiologic conditions. [ABSTRACT FROM AUTHOR]

Background: During the COVID‐19 pandemic focus has been on polyethylene glycol (PEG) and polysorbate as these excipients are constituents in the first vaccines and possible elicitors of allergic reactions to the vaccines. We aimed to evaluate the possibility of vaccinating patients with PEG and/or polysorbate allergy against COVID‐19. Methods: Twenty‐five patients with a history of an allergic reaction to drugs, vaccines and mouth hygiene products containing PEG or polysorbate and sensitization (skin test or in vitro test) or a positive challenge were included. We re‐evaluated 19 of 21 patients diagnosed before 2021 and four new patients by skin prick tests (SPT) and Basophil Histamine Release (BaHR) for PEGs, polysorbates and approved COVID‐19 vaccines as well as measurement of specific IgE (PEG 2000, 10,000). Patients were offered vaccination based on decision points from the primary diagnosis and re‐evaluation. Results: Most common primary elicitors were depot‐steroids and laxatives. Most patients had experienced more than one reaction. SPT was superior to BaHR test although many SPTs became negative over time. After careful re‐evaluation three patients were successfully vaccinated with the Pfizer/BioNTech vaccine. Three were vaccinated before referral. Eleven were offered the Johnson‐Johnson vaccine; four were vaccinated successfully, seven abstained. Six patients could not be vaccinated with PEG or polysorbate containing vaccines. Conclusion: Hypersensitivity to excipients in COVID‐19 vaccines constitutes a risk to patients with allergy to PEG or polysorbates. After diagnostic evaluation, a safe COVID‐19 vaccine could be offered to most patients, the remainders will await new vaccines containing different excipients. [ABSTRACT FROM AUTHOR]