Towards more tolerable subcutaneous administration: Review of contributing factors for improving combination product design.

Subcutaneous (SC) injections can be associated with local pain and discomfort that is subjective and may affect treatment adherence and overall patient experience. With innovations increasingly focused on finding ways to deliver higher doses and volumes (≥2 mL), there is a need to better understand the multiple intertwined factors that influence pain upon SC injection. As a priority for the SC Drug Development & Delivery Consortium, this manuscript provides a comprehensive review of known attributes from published literature that contribute to pain/discomfort upon SC injection from three perspectives: (1) device and delivery factors that cause physical pain, (2) formulation factors that trigger pain responses, and (3) human factors impacting pain perception. Leveraging the Consortium's collective expertise, we provide an assessment of the comparative and interdependent factors likely to impact SC injection pain. In addition, we offer expert insights and future perspectives to fill identified gaps in knowledge to help advance the development of patient-centric and well tolerated high-dose/high-volume SC drug delivery solutions.

Sub-Visible Particle Classification and Label Consistency Analysis for Flow-Imaging Microscopy Via Machine Learning Methods.

Sub-visible particles can be a quality concern in pharmaceutical products, especially parenteral preparations. To quantify and characterize these particles, liquid samples may be passed through a flow-imaging microscopy instrument that also generates images of each detected particle. Machine learning techniques have increasingly been applied to this kind of data to detect changes in experimental conditions or classify specific types of particles, primarily focusing on silicone oil. That technique generally requires manual labeling of particle images by subject matter experts, a time-consuming and complex task. In this study, we created artificial datasets of silicone oil, protein particles, and glass particles that mimicked complex datasets of particles found in biopharmaceutical products. We used unsupervised learning techniques to effectively describe particle composition by sample. We then trained independent one-class classifiers to detect specific particle populations: silicone oil and glass particles. We also studied the consistency of the particle labels used to evaluate these models. Our results show that one-class classifiers are a reasonable choice for handling heterogeneous flow-imaging microscopy data and that unsupervised learning can aid in the labeling process. However, we found agreement among experts to be rather low, especially for smaller particles (< 8 µm for our Micro-Flow Imaging data). Given the fact that particle label confidence is not usually reported in the literature, we recommend more careful assessment of this topic in the future.

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.

A Comparison between SARS-CoV-2 and Gram-Negative Bacteria-Induced Hyperinflammation and Sepsis.

Sepsis is a life-threatening condition caused by the body's overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients "mimics bacterial sepsis". Furthermore, the immune response to SARS-CoV-2 was described by others as "mirror image of sepsis". Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels.

Viscosity Prediction of High-Concentration Antibody Solutions with Atomistic Simulations.

The computational prediction of the viscosity of dense protein solutions is highly desirable, for example, in the early development phase of high-concentration biopharmaceutical formulations where the material needed for experimental determination is typically limited. Here, we use large-scale atomistic molecular dynamics (MD) simulations with explicit solvation to de novo predict the dynamic viscosities of solutions of a monoclonal IgG1 antibody (mAb) from the pressure fluctuations using a Green-Kubo approach. The viscosities at simulated mAb concentrations of 200 and 250 mg/mL are compared to the experimental values, which we measured with rotational rheometry. The computational viscosity of 24 mPa·s at the mAb concentration of 250 mg/mL matches the experimental value of 23 mPa·s obtained at a concentration of 213 mg/mL, indicating slightly different effective concentrations (or activities) in the MD simulations and in the experiments. This difference is assigned to a slight underestimation of the effective mAb-mAb interactions in the simulations, leading to a too loose dynamic mAb network that governs the viscosity. Taken together, this study demonstrates the feasibility of all-atom MD simulations for predicting the properties of dense mAb solutions and provides detailed microscopic insights into the underlying molecular interactions. At the same time, it also shows that there is room for further improvements and highlights challenges, such as the massive sampling required for computing collective properties of dense biomolecular solutions in the high-viscosity regime with reasonable statistical precision.

Oxidation of polysorbates - An underestimated degradation pathway?

To ensure the stability of biologicals over their entire shelf-life, non-ionic surface-active compounds (surfactants) are added to protect biologics from denaturation and particle formation. In this context, polysorbate 20 and 80 are the most used detergents. Despite their benefits of low toxicity and high biocompatibility, specific factors are influencing the intrinsic stability of polysorbates, leading to degradation, loss in efficacy, or even particle formation. Polysorbate degradation can be categorized into chemical or enzymatic hydrolysis and oxidation. Under pharmaceutical relevant conditions, hydrolysis is commonly originated from host cell proteins, whereas oxidative degradation may be caused by multiple factors such as light, presence of residual metal traces, peroxides, or temperature, which can be introduced upon manufacturing or could be already present in the raw materials. In this review, we provide an overview of the current knowledge on polysorbates with a focus on oxidative degradation. Subsequently, degradation products and key characteristics of oxidative-mediated polysorbate degradation in respect of different types and grades are summarized, followed by an extensive comparison between polysorbate 20 and 80. A better understanding of the radical-induced oxidative PS degradation pathway could support specific mitigation strategies. Finally, buffer conditions, various stressors, as well as appropriate mitigation strategies, reagents, and alternative stabilizers are discussed. Prior manufacturing, careful consideration and a meticulous risk-benefit analysis are highly recommended in terms of polysorbate qualities, buffers, storage conditions, as well as mitigation strategies.

Comparative Stability Study of Polysorbate 20 and Polysorbate 80 Related to Oxidative Degradation.

The surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80) are utilized to stabilize protein drugs. However, concerns have been raised regarding the degradation of PSs in biologics and the potential impact on product quality. Oxidation has been identified as a prevalent degradation mechanism under pharmaceutically relevant conditions. So far, a systematic stability comparison of both PSs under pharmaceutically relevant conditions has not been conducted and little is known about the dependence of oxidation on PS concentration. Here, we conducted a comparative stability study to investigate (i) the different oxidative degradation propensities between PS20 and PS80 and (ii) the impact of PS concentration on oxidative degradation. PS20 and PS80 in concentrations ranging from 0.1 mg⋅mL-1 to raw material were stored at 5, 25, and 40 °C for 48 weeks in acetate buffer pH 5.5 and water, respectively. We observed a temperature-dependent oxidative degradation of the PSs with strong (40 °C), moderate (25 °C), and weak/no degradation (5 °C). Especially at elevated temperatures such as 40 °C, fast oxidative PS degradation processes were detected. In this case study, a stronger degradation and earlier onset of oxidation was observed for PS80 in comparison to PS20, detected via the fluorescence micelle assay. Additionally, degradation was found to be strongly dependent on PS concentration, with significantly less oxidative processes at higher PS concentrations. Iron impurities, oxygen in the vial headspaces, and the pH values of the formulations were identified as the main contributing factors to accelerate PS oxidation.

How enzymatic hydrolysis of polysorbate 20 influences colloidal protein stability.

Polysorbates (PS) are esters of ethoxylated sorbitol anhydrides of different composition and are widely used surfactants in biologics. PSs are applied to increase protein stability and concomitant shelf-life via shielding against e.g., interfacial stresses. Due to the presence of specific lipolytic host cell protein (HCP) contaminations in the drug substance, PSs can be degraded via enzymatic hydrolysis. Surfactant hydrolysis leads to the formation of degradants, such as free fatty acids that might form fatty acid particles. In addition, PS degradation may reduce surfactant functionality and thus reduce the protection of the active pharmaceutical ingredient (API). Although enzymatic degradation was observed and reported in the last years, less is known about the relationship between certain polysorbate degradation patterns and the increase of mechanical and interfacial stress towards the API. In this study, the impact of specifically hydrolyzed polysorbate 20 (PS20) towards the stabilization of two monoclonal antibodies (mAbs) during accelerated shaking stress conditions was investigated. The results show that a specific enzymatic degradation pattern of PS20 can influence the colloidal stability of biopharmaceutical formulations. Furthermore, the kinetics of the appearance of visual phenomena, opalescence, and particle formation depended on the polysorbate degradation fingerprint as induced via the presence of surrogate enzymes. The current case study shows the importance of focusing on specific polysorbate ester fractions to understand the overall colloidal protein stabilizing effect. The performed study gives first insight into the functional properties of PS and helps to evaluate the impact of PS degradation in the formulation development of biopharmaceuticals in general.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

Protein-protein interactions in solutions of monoclonal antibodies probed by the dependence of the high-frequency viscosity on temperature and concentration.

Using a quartz crystal microbalance with dissipation monitoring (QCM-D), the complex high-frequency viscosity,  = η' - iη'', of concentrated solutions of a monoclonal antibody (mAb) was studied with respect to its dependence on temperature, T, and concentration, c. Lysozyme and bovine serum albumin (BSA) served as reference materials. Viscoelasticity was found for the mAb solution, while the reference materials behaved like Newtonian liquids. The QCM-D probes the solution's dynamics on the time scale of a few tens of nanoseconds. The processes of relaxation accessed with the QCM-D are not amenable to standard viscometry. The inverse loss tangent at 15 MHz (equal to η''/η' at 15 MHz, quantifying the elastic contribution to the oscillatory stress) was between 0.1 and 0.5 for the concentrated mAb solutions. It decreased with increasing temperature and decreasing pH. Activation energies of viscous flow, Ea,η, were derived from the functions η'(T). Ea,η was found to be higher for the mAb solutions than for water. No such increase was found for the reference materials. This difference evidences protein-protein interactions (PPIs) between the mAb molecules, which do not exist in the same way for lysozyme and BSA. The excipients citrate and arginine did not noticeably affect the mAb's high-frequency viscosity as determined with the QCM-D.

Antimicrobial Preservatives for Protein and Peptide Formulations: An Overview.

Biological drugs intended for multi-dose application require the presence of antimicrobial preservatives to avoid microbial growth. As the presence of certain preservatives has been reported to increase protein and peptide particle formation, it is essential to choose a preservative compatible with the active pharmaceutical ingredient in addition to its preservation function. Thus, this review describes the current status of the use of antimicrobial preservatives in biologic formulations considering (i) appropriate preservatives for protein and peptide formulations, (ii) their physico-chemical properties, (iii) their in-/compatibilities with other excipients or packaging material, and (iv) their interactions with the biological compound. Further, (v) we present an overview of licensed protein and peptide formulations.

Existence of a superior polysorbate fraction in respect to protein stabilization and particle formation?

Biologicals including monoclonal antibodies are the current flagships in pharmaceutical industry. However, they are exposed to a multitude of destabilization conditions like for instance hydrophobic interfaces, leading to reduced biological activity. Polysorbates are commonly applied to effectively stabilize these active pharmaceutical ingredients against colloidal stress. Nevertheless, chemical instability of polysorbate via hydrolysis or oxidation results in degradation products that might form particles via phase separation. Polysorbates are mixtures of hundreds of individual components, and recently purer quality grades with reduced variations in the fatty acid composition are available. As the protective function of polysorbate itself is not completely understood, even less is known about its individual components, raising the question of the existence of a superior polysorbate species in respect to protein stabilization or degradation susceptibility. Here, we evaluated the protective function of four main fractions of polysorbate 20 (PS20) in agitation studies with monoclonal antibodies, followed by particle analysis as well as protein and polysorbate content determination. The commercially-available inherent mixtures PS20 high purity and PS20 all-laurate, as well as the fraction isosorbide-POE-monolaurate showed superior protection against mechanical-induced stress (visual inspection and turbidity) at the air-water interface in comparison to sole sorbitan-POE-monolaurate, -dilaurate, and -trilaurate. Fractions composed mainly of higher-order esters like sorbitan-POE-dilaurate and sorbitan-POE-trilaurate indicated high turbidities as indication for subvisible and small particles accompanied by a reduced protein monomer content after agitation. For the isosorbide-POE-monolaurates as well as for the inherent polysorbate mixtures no obvious differences in protein content and protein aggregation (SEC) were observed, reflecting the observations from visual appearance. However, absolute polysorbate concentrations vary drastically between different species in the actual formulations. As there are still open questions in respect to protein specificity or regarding mixtures versus individual components of PS20, further studies must be performed, to gain a better understanding of a "generalized" stabilizing effect of polysorbates on monoclonal antibodies. The knowledge of the characteristics of individual polysorbate species can have the potential to pave the way to superior detergents in respect to protein stabilization and/or degradation susceptibility.

Protein photodegradation in the visible range? Insights into protein photooxidation with respect to protein concentration.

Visible light (400-800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.

Micro-flow imaging multi-instrument evaluation for sub-visible particle detection.

Sub-visible particles (SVPs) in pharmaceutical products are a critical quality attribute, and therefore should be monitored during development. Although light obscuration (LO) and microscopic particle count tests are the primary pharmacopeial methods used to quantify SVPs, flow imaging methods like Micro-Flow Imaging (MFI™) appear to overcome shortcomings of LO such as limited sensitivity concerning smaller translucent SVPs in the size range < 10 µm. Nowadays, MFI™ is routinely utilized during development of biologicals. Oftentimes multiple devices are distributed across several laboratories and departments. This poses challenges in data interpretation and consistency as well as in the use of multiple devices for one purpose. In this study, we systematically evaluated seven MFI™ instruments concerning their counting and size precision and accuracy, using an inter-comparable approach to mimic daily working routine. Therefore, we investigated three different types of particles (i) NIST certified counting standards, (ii) protein-coated particles, and (iii) stress-induced particles from a monoclonal antibody. We compared the results to alternative particle detection methods: LO and Backgrounded Membrane Imaging (BMI). Our results showed that the precision and accuracy of particle count and size, as well as the comparability of instruments, depended on the particle source and its material properties. The various MFI™ instruments investigated showed high precision (<15 %) and data generated on different instruments were of the same order of magnitude within pharmacopeial relevant size ranges for NIST certified counting standards. However, we found limitations in the upper and lower detection limits, contrary to the limits claimed by the manufacturer. In addition, proteinaceous and protein-containing particles showed statistically significant differences in particle counts, while the measured particle diameters of all sizes were quite consistent.

An Intercompany Perspective on Practical Experiences of Predicting, Optimizing and Analyzing High Concentration Biologic Therapeutic Formulations.

Developing high-dose biologic drugs for subcutaneous injection often requires high-concentration formulations and optimizing viscosity, solubility, and stability while overcoming analytical, manufacturing, and administration challenges. To understand industry approaches for developing high-concentration formulations, the Formulation Workstream of the BioPhorum Development Group, an industry-wide consortium, conducted an inter-company collaborative exercise which included several surveys. This collaboration provided an industry perspective, experience, and insight into the practicalities for developing high-concentration biologics. To understand solubility and viscosity, companies desire predictive tools, but experience indicates that these are not reliable and experimental strategies are best. Similarly, most companies prefer accelerated and stress stability studies to in-silico or biophysical-based prediction methods to assess aggregation. In addition, optimization of primary container-closure and devices are pursued to mitigate challenges associated with high viscosity of the formulation. Formulation strategies including excipient selection and application of studies at low concentration to high-concentration formulations are reported. Finally, analytical approaches to high concentration formulations are presented. The survey suggests that although prediction of viscosity, solubility, and long-term stability is desirable, the outcome can be inconsistent and molecule dependent. Significant experimental studies are required to confirm robust product definition as modeling at low protein concentrations will not necessarily extrapolate to high concentration formulations.

Toxicological and Safety Pharmacological Profiling of the Anti-Infective and Anti-Inflammatory Peptide Pep19-2.5.

Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.

Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates.

Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

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.

Characterization of radicals in polysorbate 80 using electron paramagnetic resonance (EPR) spectroscopy and spin trapping.

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.

Poloxamer 188 as surfactant in biological formulations - An alternative for polysorbate 20/80?

Surfactants are used to stabilize biologics. Particularly, polysorbates (Tween® 20 and Tween® 80) dominate the group of surfactants in protein and especially antibody drug products. Since decades drug developers rely on the ethoxylated sorbitan fatty acid ester mixtures to stabilize sensitive molecules such as proteins. Reasons are (i) excellent stabilizing properties, and (ii) well recognized safety and tolerability profile of these polysorbates in humans, especially for parenteral applications. However, over the past decade concerns regarding the stability of these two polysorbates were raised. The search of alternatives with preferably less reservations concerning degradation and product quality reducing issues leads, among others, to poloxamer 188 (e.g. Kolliphor® P188), a nonionic triblock-copolymer surfactant. This review sums up our current knowledge related to the characterization and physico-chemical properties of poloxamer 188, its analytics and stability properties for biological formulations. Furthermore, the advantages and disadvantages as a suitable polysorbate-alternative for the stabilization of biologics are discussed.