An in-depth examination of fatty acid solubility limits in biotherapeutic protein formulations containing polysorbate 20 and polysorbate 80.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are polysorbate20 (PS20) and polysorbate 80 (PS80). In recent years, polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation. Since the emergence of particles in liquid formulations has to be avoided, it is important to prevent their formation. This study evaluates the solubility limits of selected FAs, which are likely to be released during the degradation of PS20 and PS80 in the presence of defined PS concentrations. Our results show that the solubility is highly dependent on the pH, the temperature, the used PS concentration and the aliphatic chain of respective FAs. Solubility of FAs, such as palmitic and oleic acid under the conditions determined in this study, are in the range of 3-130 µg·ml-1 (12-460 µM). Furthermore, the results allow making an estimation to which extent PS may degrade before particle formation in the drug product may be expected.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Acidic and alkaline hydrolysis of polysorbates under aqueous conditions: Towards understanding polysorbate degradation in biopharmaceutical formulations.

Polysorbate is one of the most commonly employed non-ionic surfactant in protein containing biological formulations, whereby, it can stabilize these biomolecules under different stress conditions. Despite the fact that polysorbates are present in almost 70% of currently marketed parenteral biological drugs, polysorbate degradation in biopharmaceutical formulations has emerged as a specific quality concern. Different degradation pathways have been explored in the recent years with the aim of understanding the root cause for polysorbate degradation in biopharmaceutical formulations. In an attempt to explore hydrolytic degradation of polysorbates in accelerated degradation conditions, we studied extreme pH conditions. We investigated specific polysorbate degradation profiles depending on acidic or alkaline solution conditions. The acidic and alkaline hydrolysis of polysorbate is monitored for the total content using a fluorescence micelle assay (FMA). Additionally, the compositional changes in polysorbates were detected using reversed phase high performance liquid chromatography coupled to a charged aerosol detector (RP-HPLC-CAD). We show that the stability of polysorbate against chemical hydrolysis is dependent upon selected pH condition and differ for polysorbate 20 and polysorbate 80. Additionally, we were able to show that a degradation pathway dependent fingerprint may support the identification of the degradation root cause.

A hydroxylamine probe for profiling S-acylated fatty acids on proteins.

Reversible S-palmitoylation is a key regulatory mechanism of protein function and localization. There is increasing evidence that S-acylation is not restricted to palmitate but it includes shorter, longer, and unsaturated fatty acids. However, the diversity of this protein modification has not been fully explored. Herein, we report a chemical probe that combined with MS-based analysis allows the rapid detection and quantification of fatty acids linked to proteins. We have used this approach to profile the S-acylome and to show that the oncogene N-Ras is heterogeneously acylated with palmitate and palmitoleate. Studies on protein distribution in membrane subdomains with semisynthetic proteins revealed that unsaturated N-Ras presents an increased tendency toward clustering and higher insertion kinetic rate constants.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Effect of ectoine, hydroxyectoine and ß-hydroxybutyrate on the temperature and pressure stability of phospholipid bilayer membranes of different complexity.

Previous research has shown that ectoines fluidize lipid monolayers by increasing the liquid expanded region in DPPC monolayers and also decreasing the line tension responsible for the phase morphology. Here, we explored possible effects of the compatible osmolytes ectoine, hydroxyectoine and ß-hydroxybutyrate on lipid bilayer membranes, including effects of temperature and pressure. The effect of the protective osmolytes on the phase transition of DPPC bilayers was investigated by fluorescence spectroscopy, differential scanning calorimetry and pressure perturbation calorimetry. A slight change of the phase behavior was observed, which resulted in a stabilization of the gel phase, which may be caused by an alteration of the hydration properties at the lipid interface and H-bond and electrostatic interactions in the headgroup region. We then explored the cosolvents' effects on giant unilamellar vesicles (GUVs) formed by lipid mixtures exhibiting phase separation into liquid-ordered (lo) and liquid-disordered (ld) domains using BODIPY-PC and the DiI18 dye as labels. The presence of both, ectoine and hydroxyectoine showed significant effects on the lateral organization increasing the fluid domains. Moreover, we observed a considerable increase in the adhesion behavior of small vesicles onto GUV surfaces. Diffusion studies by fluorescence recovery after photobleaching experiments on POPC giant vesicles quantitatively showed a hydroxyectoine-induced increase of the diffusion coefficient values, clearly demonstrating an increase in the lateral mobility of lipid within the bilayer membrane. This study provides clear evidence for the fluidizing effect of the compatible solutes on bilayer lipid membranes. A marked effect, however, was only detected if phase separated domains exist.

Probing Colocalization of N-Ras and K-Ras4B Lipoproteins in Model Biomembranes.

Signaling of N-Ras and K-Ras4B proteins depends strongly on their correct localization in the cell membrane. In vivo studies suggest that intermolecular interactions foster the self-association of both N-Ras and K-Ras4B and the formation of nanoclusters in the cell membrane. As sites for effector binding, nanocluster formation is thought to be essential for effective signal transmission of both N-Ras and K-Ras4B. To shed more light on the spatial arrangement and mechanism underlying the proposed cross-talk between spatially segregated Ras proteins, the simultaneous localization of N-Ras and K-Ras4B and their effect on the lateral organization of a heterogeneous model biomembrane has been studied by using AFM and FRET methodology. It is shown that, owing to the different natures of their membrane anchor systems, N-Ras and K-Ras4B not only avoid assembly in bulk solution and do not colocalize, but rather form individual nanoclusters that diffuse independently in the fluid membrane plane.

Polysorbate degradation in biotherapeutic formulations: Identification and discussion of current root causes.

Biotherapeutic protein formulations are often high concentration liquid protein solutions, which are required to be stable under pharmaceutically relevant storage conditions and presence of external stress. Non-ionic detergents like polysorbate have been the most commonly used detergent to maintain formulation stability. Recently, particle formation in polysorbate containing biotherapeutic formulations has arisen as a major quality concern and potential patient risk factor. In this review, we provide a general overview into (i) degradation of polysorbates, (ii) polysorbate analytics, (iii) particle formation induced by polysorbate degradation and root causes thereof, (iv) particle composition and (v) various influencing factors that might lead to particle formation. Consequently, we explore the role of polysorbate degradation in particle formation. Additionally, various degradation pathways and the current discussed root causes are reviewed.

3D Molecular ToF-SIMS Imaging of Artificial Lipid Membranes Using a Discriminant Analysis-Based Algorithm.

Artificial lipid membranes play a growing role in technical applications such as biosensors in pharmacological research and as model systems in the investigation of biological lipid films. In the standard procedure for displaying the distribution of membrane components, fluorescence microscopy, the fluorophores used can influence the distribution of the components and usually not all substances can be displayed at the same time. The discriminant analysis-based algorithm used in combination with scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables marker-free, quantitative, simultaneous recording of all membrane components. These data are used for reconstruction of distribution patterns. In the model system used for this survey, a tear fluid lipid layer, the distribution patterns of all lipids correlate well in calculated ToF-SIMS images and epi-fluorescence microscopic images. All epi-fluorescence microscopically viewable structures are visible when using both positive and negative secondary ions and can be reproduced with high lateral resolution in the submicrometer range despite the very low signal intensity and a very low signal-to-noise ratio. In addition, three-dimensional images can be obtained with a subnanometer depth resolution. Furthermore, structures and the distribution of substances that cannot be made visible by epi-fluorescence microscopy can be displayed. This enables new insights that cannot be gained by epi-fluorescence microscopy alone.

UNC119A Decreases the Membrane Binding of Myristoylated c-Src.

Plasma membrane localization of myristoylated c-Src, a proto-oncogene protein-tyrosine kinase, is required for its signaling activity. Recent studies proposed that UNC119 protein functions as a solubilizing factor for myristoylated proteins, thereby regulating their subcellular distribution and signaling. The underlying molecular mechanism by which UNC119 regulates the membrane binding of c-Src has remained elusive. By combining different biophysical techniques, we have found that binding of a myristoylated c-Src-derived N-terminal peptide (Myr-Src) by UNC119A results in a reduced membrane binding affinity of the peptide, due to the competition of binding to membranes. The dissociation of Myr-Src from membranes is facilitated in the presence of UNC119A, as a consequence of which the clustering propensity of this peptide on the membrane is partially impaired. By these means, UNC119A is able to regulate c-Src spatially in the cytoplasm and on cellular membranes, and this has important implications for its cellular signaling.

Interaction of KRas4B protein with C6-ceramide containing lipid model membranes.

Ras proteins are oncoproteins which play a pivotal role in cellular signaling pathways. All Ras proteins' signaling strongly depends on their correct localization in the cell membrane. Over 30% of cancers are driven by mutant Ras proteins, and KRas4B is the Ras isoform most frequently mutated. C6-ceramide has been shown to inhibit the growth activity of KRas4B mutated cells. However, the mechanism underlying this inhibition remains elusive. Here, we established a heterogeneous model biomembrane containing C6-ceramide. C6-ceramide incorporation does not disrupt the lipid membrane. Addition of KRas4B leads to drastic changes in the lateral membrane organization of the membrane, however. In contrast to the partitioning behavior in other membranes, KRas4B forms small, monodisperse nanoclusters dispersed in a fluid-like environment, in all likelihood induced by some kind of lipid sorting mechanism. Fluorescence cross-correlation data indicate no direct interaction between C6-ceramide and KRas4B, suggesting that KRas4B essentially recruits other lipids. A FRET-based binding assay reveals that the stability of KRas4B proteins inserted into the membrane containing C6-ceramide is reduced. Based on the combined results obtained, we postulate a molecular mechanism for the inhibition of KRas4B mutated cells' activity through C6-ceramide.

Lateral Organization of Host Heterogeneous Raft-like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c-Src.

Membrane-bound c-Src non-receptor tyrosine kinase, unlike other acyl-modified lipid-anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale-clusters upon membrane binding of the Myr-Src (2-9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr-Src (2-9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity.

The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.

Binding of Vinculin to Lipid Membranes in Its Inhibited and Activated States.

Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2 in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2 embedded in lipid membranes. Activated Vn harbored on PIP2 clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.

Biophysical investigations of the structure and function of the tear fluid lipid layers and the effect of ectoine. Part B: artificial lipid films.

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure-area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.

Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: natural meibomian lipid films.

The tear fluid lipid layer is the outermost part of the tear film on the ocular surface which protects the eye from inflammations and injuries. We investigated the influence of ectoine on the structural organization of natural meibomian lipid films using surface activity analysis and topographical studies. These films exhibit a continuous pressure-area isotherm without any phase transition. With the addition of ectoine, the isotherm is expanded towards higher area per molecule values suggesting an increased area occupied by the interfacial lipid molecules. The AFM topology scans of natural meibomian lipid films reveal a presence of fiber-like structures. The addition of ectoine causes an appearance of droplet-like structures which are hypothesized to be tri-acyl-glycerols and other hydrophobic components excluded from the lipid film. Further the material properties of the droplet-like structure with respect to the surrounding were determined by using the quantitative imaging mode of the AFM technique. The droplet-like structures were found to be comparatively softer than the surrounding. Based on the observations a preliminary hypothesis is proposed explaining the mechanism of action of ectoine leading to the fluidization of meibomian lipid films. This suggests the possibility of ectoine as a treatment for the dry eye syndrome.

Size influences the effect of hydrophobic nanoparticles on lung surfactant model systems.

The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ~12 nm and ~136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ~136 nm NPs compared to minor effects in the presence of ~12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.