Visible light triggers the formation of reactive oxygen species in monoclonal antibody formulations.

Most monoclonal antibody formulations require the presence of a surfactant, such as polysorbate, to ensure protein stability. The presence of high concentrations of polysorbate have been shown to enhance photooxidation of certain protein drug products when exposed to visible light. The current literature, however, suggest that photooxidation of polysorbate only occurs when exposed to visible light in combination with UVA light. This is probable as peroxides present in polysorbate solutions can be cleaved homolytically in the UVA region. In the visible region, photooxidation is not expected to occur as cleavage of peroxides is not expected at these wavelengths. This report presents findings suggesting that the presence of one or more photosensitiser(s) in polysorbate must be a cause and is required to catalyse the aerobic oxidation of polysorbate solutions upon exposure to visible light. Our investigation aimed to clarify the mechanism(s) of polysorbate photooxidation and explore the kinetics and the identity of the generated radicals and their impact on monoclonal antibody (mAb) degradation. Our study reveals that when polysorbate solutions are exposed to visible light between 400---800 nm in the absence of proteins, discoloration, radical formation, and oxygen depletion occur. We discuss the initial formation of reactive species, most likely occurring directly after reaction of molecular oxygen, with the presence of a triplet state photosensitizer, which is generated by intersystem crossing of the excited singlet state, leading predominantly to the formation of reactive species such as singlet oxygen species. When comparing the photooxidation of PS20 and PS80 in varying quality grades, we propose that singlet oxygen possesses potential for reacting with unsaturated fatty acids in PS80HP, however, PS20HP itself exhibited no measurable oxidation under the tested conditions. The study's final part delves into the photooxidation behaviour of different PS grades, examining its influence on the integrity of a mAb in the formulation. Finally, we examined the effect of photooxidation on the integrity of monoclonal antibodies. Our findings show that the exposure to visible light in polysorbate-containing mAb solutions at high PS concentrations of 4 mg∙ml-1 results in increased monoclonal antibody degradation, highlighting the need for cautious evaluation of the correct PS concentration to stabilise protein therapeutics.

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.

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.

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.

Principles of Small-Molecule Transport through Synthetic Nanopores.

Synthetic nanopores made from DNA replicate the key biological processes of transporting molecular cargo across lipid bilayers. Understanding transport across the confined lumen of the nanopores is of fundamental interest and of relevance to their rational design for biotechnological applications. Here we reveal the transport principles of organic molecules through DNA nanopores by synergistically combining experiments and computer simulations. Using a highly parallel nanostructured platform, we synchronously measure the kinetic flux across hundreds of individual pores to obtain rate constants. The single-channel transport kinetics are close to the theoretical maximum, while selectivity is determined by the interplay of cargo charge and size, the pores' sterics and electrostatics, and the composition of the surrounding lipid bilayer. The narrow distribution of transport rates implies a high structural homogeneity of DNA nanopores. The molecular passageway through the nanopore is elucidated via coarse-grained constant-velocity steered molecular dynamics simulations. The ensemble simulations pinpoint with high resolution and statistical validity the selectivity filter within the channel lumen and determine the energetic factors governing transport. Our findings on these synthetic pores' structure-function relationship will serve to guide their rational engineering to tailor transport selectivity for cell biological research, sensing, and drug delivery.

Single Cell-like Systems Reveal Active Unidirectional and Light-Controlled Transport by Nanomachineries.

Cellular life depends on transport and communication across membranes, which is emphasized by the fact that membrane proteins are prime drug targets. The cell-like environment of membrane proteins has gained increasing attention based on its important role in function and regulation. As a versatile scaffold for bottom-up synthetic biology and nanoscience, giant liposomes represent minimalistic models of living cells. Nevertheless, the incorporation of fragile multiprotein membrane complexes still remains a major challenge. Here, we report on an approach for the functional reconstitution of membrane assemblies exemplified by human and bacterial ATP-binding cassette (ABC) transporters. We reveal that these nanomachineries transport substrates unidirectionally against a steep concentration gradient. Active substrate transport can be spatiotemporally resolved in single cell-like compartments by light, enabling real-time tracking of substrate export and import in individual liposomes. This approach will help to construct delicate artificial cell-like systems.

Thermodynamic Basis for Conformational Coupling in an ATP-Binding Cassette Exporter.

ATP-binding cassette (ABC) transporters constitute one of the largest protein superfamilies, and they mediate the transport of diverse substrates across the membrane. The molecular mechanism for transducing the energy from ATP binding and hydrolysis into the conformational changes remains elusive. Here, we determined the thermodynamics underlying the ATP-induced global conformational switching for the ABC exporter TmrAB using temperature-resolved pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We show that a strong entropy-enthalpy compensation mechanism enables the closure of the nucleotide-binding domains (NBDs) over a wide temperature range. This is mechanically coupled with an outward opening of the transmembrane domains (TMDs) accompanied by an entropy gain. The conserved catalytic glutamate plays a key role in the overall energetics. Our results reveal the thermodynamic basis for the chemomechanical energy coupling in an ABC exporter and present a new strategy to explore the energetics of similar membrane protein complexes.

Synthetic protein-conductive membrane nanopores built with DNA.

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10-3 s-1. Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells.

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis.