Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro.

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197's transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.

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.

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.

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.

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.

Assessing the polysorbate degradation fingerprints and kinetics of lipases - how the activity of polysorbate degrading hydrolases is influenced by the assay and assay conditions.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stresses are polysorbate 20 (PS20) and polysorbate 80 (PS80). In recent years, numerous cases of hydrolytic polysorbate (PS) degradation in liquid formulations of biopharmaceuticals have been observed. Concomitant with the degradation of PSs, formulated proteins become inherently instable and more susceptible to aggregation. Furthermore, poorly soluble fatty acids (FA) are released from the PSs, which might lead to FA precipitation and the formation of visible and subvisible particles. Therefore, possible particle inducing factors have to be monitored closely. The major root cause of hydrolytic PS degradation in biologicals is the presence of enzymatic active host cell proteins (HCP), like lipases and esterases, which are co-purified with the active pharmaceutical ingredient. Such contaminants can be detected via their hydrolytic activity, either using ester-based substrates or PS itself. However, each approach has its up- and downsides, which makes the comparison of the results from other publications difficult. It was therefore the aim of the present study to investigate the impact of lipase specificities on the assay readouts. This study evaluates three different surrogate (model) lipases with distinctively different degradation kinetics and substrate specificities using specific analytical methods. The analytical panel contains on one hand two lipase activity assays with ester-based substrates, either detecting the release of para-nitrophenol or 4-methylumbelliferone, and on the other hand two PS-based monitoring analyses (fluorescence micelle assay and reverse phase high performance liquid chromatography - charged aerosol detection), which detect hydrolytic "activity" directly in the target substrate. Thereby, strengths and weaknesses of each assay are discussed, and recommendations are made for the respective use cases. Our results show that the determined lipase activities vary not only from assay to assay, but also significantly for the lipase tested, thus showing a different degradation fingerprint in the RP-HPLC-CAD chromatogram. This demonstrates that a comprehensive monitoring approach is essential to assess potential HCP contaminations.

Hydrolytic polysorbate 20 degradation - Sensitive detection of free fatty acids in biopharmaceuticals via UPLC-QDa analytics with isolator column.

The enzymatic hydrolysis of polysorbates, e.g. induced by specific host cell proteins in biologics, is a known risk factor regarding the potential particle formation in the product over time. One of the root causes for this observation is an increase in free fatty acids (FA) within the formulation, which indicates the need for convenient monitoring of FA release. This study presents a novel UPLC-QDa based method to evaluate the content of the FAs esterified to polysorbate 20 (PS20) after hydrolysis. The presented method is label-free, i.e. independent of elaborate fluorophore-labeling and able to directly measure the ionized FAs. Furthermore, the method allows the determination of released FAs as percentage of ester bond hydrolysis and as absolute concentration expressed in ng/mL. Additionally, we describe for the first time in FA analytics the application of an isolator column, to remove trace levels of FAs present in the eluents to improve the sensitivity of the method. Lastly, the capabilities of the newly developed method are proven in case studies with three different monoclonal antibodies, which display characteristic FA release patterns in PS20-containing formulations. In summary, we developed a reliable, sensitive method for FA quantification in biologics, which could also be used as a predictive tool, considering FA solubility, regarding the formation of particles.

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.

Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.

The endo-lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo-lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high-throughput screening against the cytotoxicity of ricin toxin, exhibits a broad-spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo-lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad-spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo-lysosome system-related cellular physiology and mechanisms of pathogenesis.

Primary resistance of human patients to botulinum neurotoxins A and B.

Botulinum neurotoxin serotypes A and B are successfully used to treat a variety of human diseases characterized by hyperactive peripheral nerve terminals. However, a number of patients are primary resistant to these pharmaceuticals, without having antitoxin-neutralizing antibodies. A straightforward explanation of this phenomenon posits that mutations of the toxin sites of interaction with their receptors or protein substrates prevent their neuroparalytic action. After a careful investigation of available human genomic databases, we conclude that it is very unlikely that humans are resistant to these two therapeutic neurotoxins because of mutations that would affect their binding or intracellular proteolytic actions.

High Conservation of Tetanus and Botulinum Neurotoxins Cleavage Sites on Human SNARE Proteins Suggests That These Pathogens Exerted Little or No Evolutionary Pressure on Humans.

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same time, this approach was aimed at testing the possibility that TeNT and BoNT may have acted as selective agents in the development of resistance to tetanus or botulism. We found that mutations of the SNARE proteins are very rare and concentrated outside the SNARE motif required for the formation of the SNARE complex involved in neuroexocytosis. No changes were found at the BoNT cleavage sites of VAMP and syntaxins and only one very rare mutation was found in the essential C-terminus region of SNAP-25, where Arg198 was replaced with a Cys residue. This is the P1' cleavage site for BoNT/A and the P1 cleavage site for BoNT/C. We found that the Arg198Cys mutation renders SNAP-25 resistant to BoNT/A. Nonetheless, its low frequency (1.8 × 10-5) indicates that mutations of SNAP-25 at the BoNT/A cleavage site are unlikely to account for the existence of BoNT/A resistant patients. More in general, the present findings indicate that tetanus and botulinum neurotoxins have not acted as selective agents during human evolution as it appears to have been the case for tetanus in rats and chicken.

Auranofin Inhibits the Enzyme Activity of Pasteurella multocida Toxin PMT in Human Cells and Protects Cells from Intoxication.

The AB-type protein toxin from Pasteurella multocida (PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold.

The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells.

Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.