Concentration Effects in the Interaction of Monoclonal Antibodies (mAbs) with their Immediate Environment Characterized by EPR Spectroscopy.
ABSTRACT: Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10-20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR "spin probing" assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions.
Project description:The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.
Project description:Polymer flooding is an enhanced oil recovery (EOR) process, which has received increasing interest in the industry. In this process, water-soluble polymers are used to increase injected water viscosity in order to improve mobility ratio and hence improve reservoir sweep. Polymer solutions are non-Newtonian fluids, i.e., their viscosities are shear dependent. Polymers may exhibit an increase in viscosity at high shear rates in porous media, which can cause injectivity loss. In contrast, at low shear rates they may observe viscosity loss and hence enhance the injectivity. Therefore, due to the complex non-Newtonian rheology of polymers, it is necessary to optimize the design of polymer injectivity tests in order to improve our understanding of the rheology behavior and enhance the design of polymer flood projects. This study has been addressing what information that can be gained from polymer injectivity tests, and how to design the test for maximizing information. The main source of information in the field is from the injection bottom-hole pressure (BHP). Simulation studies have analyzed the response of different non-Newtonian rheology on BHP with variations of rate and time. The results have shown that BHP from injectivity tests can be used to detect in-situ polymer rheology.
Project description:Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.
Project description:The N-nitroso-derivative of melatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NO* [oxidonitrogen*] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug, is its ability to release both melatonin and NO*, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-3H]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)2-Fe2+ (iron-N-methyl-D-glucamine dithiocarbamate) complex as a spin trap. The behaviour of NOM was compared with that of GSNO (S-nitrosoglutathione), a hydrophilic NO* donor. In the first minutes following [O-methyl-3H]NOM intraperitoneal injection, the radioactivity was found in organs (6% in the liver, 1% in the kidney and 0.6% in the brain), but not in the blood. In both liver and brain, the radioactivity content decreased over time with similar kinetics reflecting the diffusion and metabolism of NOM and of its metabolites. Based on the characterization and the quantification of the EPR signal in vitro with NOM or GSNO using (MGD)2-Fe2+ complex in phosphate-buffered solutions, the detection of these nitroso compounds was realized ex vivo in mouse tissue extracts. (MGD)2-Fe2+-NO was observed in the brain of NOM-treated mice in the first 10 min following injection, revealing that NOM was able to cross the blood-brain barrier, while GSNO was not.
Project description:Interspin distances between 0.8 nm and 2.0 nm can be measured through the dipolar broadening of the continuous wave (cw) EPR spectrum of nitroxide spin labels at X-band (9.4 GHz, 0.35 T). We introduce Gd(3+) as a promising alternative spin label for distance measurements by cw EPR above 7 Tesla, where the |-1/2? to |1/2? transition narrows below 1 mT and becomes extremely sensitive to dipolar broadening. To estimate the distance limits of cw EPR with Gd(3+), we have measured spectra of frozen solutions of GdCl3 at 8.6 T (240 GHz) and 10 K at concentrations ranging from 50 mM to 0.1 mM, covering a range of average interspin distances. These experiments show substantial dipolar broadening at distances where line broadening cannot be observed with nitroxides at X-band. This data, and its agreement with calculated dipolar-broadened lineshapes, show Gd(3+) to be sensitive to distances as long as ?3.8 nm. Further, the linewidth of a bis-Gd(3+) complex with a flexible ?1.6 nm bridge is strongly broadened as compared to the mono-Gd(3+) complex, demonstrating the potential for application to pairwise distances. Gd-DOTA-based chelates that can be functionalized to protein surfaces display linewidths narrower than aqueous GdCl3, implying they should be even more sensitive to dipolar broadening. Therefore, we suggest that the combination of tailored Gd(3+) labels and high magnetic fields can extend the longest interspin distances measurable by cw EPR from 2.0 nm to 3.8 nm. cw EPR data at 260 K demonstrate that the line broadening remains clear out to similar average interspin distances, offering Gd(3+) probes as promising distance rulers at temperatures higher than possible with conventional pulsed EPR distance measurements.
Project description:The microscopic structure of frozen aqueous sucrose solutions, over concentrations of 0-75% (w/v), is characterized by using multiple continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopic and relaxation techniques and the paramagnetic spin probe, TEMPOL. The temperature dependence of the TEMPOL EPR line-shape anisotropy reveals a mobility transition, specified at 205 K in pure water and 255 ± 5 K for >1% (w/v) added sucrose. The transition temperature is >>Tg, where Tg is the homogeneous water glass transition temperature, which shows that TEMPOL resides in the mesoscopic domain (mesodomain) at water-ice crystallite boundaries and that the mesodomain sucrose concentrations are comparable at >1% (w/v) added sucrose. Electron spin-echo envelope modulation (ESEEM) spectroscopy of TEMPOL-(2)H2-sucrose hyperfine interactions also indicates comparable sucrose concentrations in mesodomains at >1% (w/v) added sucrose. Electron spin-echo (ESE) detected longitudinal and phase memory relaxation times (T1 and TM, respectively) at 6 K indicate a general trend of increased mesodomain volume with added sucrose, in three stages: 1-15, 20-50, and >50% (w/v). The calibrated TEMPOL concentrations indicate that the mesodomain volume is less than the predicted maximally freeze-concentrated value [80 (w/w); 120% (w/v)], with transitions at 15-20% and 50% (w/v) starting sucrose. An ordered sucrose hydrate phase, which excludes TEMPOL, and a disordered, amorphous sucrose-water glass phase, in which TEMPOL resides, are proposed to compose a heterogeneous mesodomain. The results show that the ratio of ordered and disordered volume fractions in the mesodomain is exquisitely sensitive to the starting sucrose concentration.
Project description:The compression and shear viscoelasticities of egg-ceramide and its mixtures with sphingomyelin were investigated using oscillatory surface rheology performed on Langmuir monolayers. We found high values for the compression and shear moduli for ceramide, compatible with a solid-state membrane, and extremely high surface viscosities when compared to typical fluid lipids. A fluidlike rheological behavior was found for sphingomyelin. Lateral mobilities, measured from particle tracking experiments, were correlated with the monolayer viscosities through the usual hydrodynamic relationships. In conclusion, ceramide increases the solid character of sphingomyelin-based membranes and decreases their fluidity, thus drastically decreasing the lateral mobilities of embedded objects. This mechanical behavior may involve important physiological consequences in biological membranes containing ceramides.
Project description:For biotechnological drugs, it is desirable to formulate antibody solutions with low viscosities. We go beyond previous colloid theories in treating protein-protein self-association of molecules that are antibody-shaped and flexible and have spatially specific binding sites. We consider interactions either through fragment antigen (Fab-Fab) or fragment crystalizable (Fab-Fc) binding. Wertheim's theory is adapted to compute the cluster-size distributions, viscosities, second virial coefficients, and Huggins coefficients, as functions of antibody concentration. We find that the aggregation properties of concentrated solutions can be anticipated from simpler-to-measure dilute solutions. A principal finding is that aggregation is controllable, in principle, through modifying the antibody itself, and not just the solution it is dissolved in. In particular: (i) monospecific antibodies having two identical Fab arms can form linear chains with intermediate viscosities. (ii) Bispecific antibodies having different Fab arms can, in some cases, only dimerize, having low viscosities. (iii) Arm-to-Fc binding allows for three binding partners, leading to networks and high viscosities.
Project description:Dry-spinning method is extensively employed in fiber industry, comparing to the counter-part of wet-spinning process, it has advantages of environmentally friendly, high yield rate and no need for purification. Here, we report the synthesis of graphene oxide (GO) fibers via dry spinning GO inks with extremely high concentrations. The proper rheology properties of such GO inks allow us to dry spin GO fiber directly. Various dry spinning conditions are investigated, and the relationship between mechanical performance and micro-structure of the obtained GO fiber are established. We found that the existence of larger GO liquid crystal domains does not necessarily result to higher mechanical properties, and it is because those large GO liquid crystal domains evolve into thick GO films during drying process and thus prevent the intimate compaction of the whole GOF and leave behind gaps. This is detrimental for the mechanical properties, and thus the dry spin GOF are much weaker than that of wet spin ones. Importantly, Barus effects, that generally arise during the melt spinning of polymers, were not observed, indicating that caution must be taken when classical polymer rheology theories are applied to investigate the dynamic behaviors of GO solution.
Project description:Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 ?M nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.