RESUMO
Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method. However, progress is needed to increase the bioavailability. Transport of low molecular weight (Mw) biotherapeutics such as insulin and small molecule contrast agents such as lipiodol has been studied using X-ray computed tomography (CT). This analysis, however, does not translate to the investigation of higher Mw therapeutics, such as monoclonal antibodies (mAbs), due to differences in molecular and formulation properties. In this study, an iodinated fluorescein analog rose bengal (RB) was used as a radiopaque and fluorescent label to track the distribution of bovine serum albumin (BSA) compared against unconjugated RB and sodium iodide (NaI) via CT and confocal microscopy following injection into ex vivo porcine SC tissue. Importantly, the high concentration BSA-RB exhibited viscosities more like that of viscous biologics than the small molecule contrast agents, suggesting that the labeled protein may serve as a more suitable formulation for the investigation of injection plumes. Three-dimensional (3D) renderings of the injection plumes showed that the BSA-RB distribution was markedly different from unconjugated RB and NaI, indicating the need for direct visualization of large protein therapeutics using conjugated tags rather than using small molecule tracers. Whereas this proof-of-concept study shows the novel use of RB as a label for tracking BSA distribution, our experimental approach may be applied to high Mw biologics, including mAbs. These studies could provide crucial information about diffusion in SC tissue and the influence of injection parameters on distribution, transport, and downstream bioavailability.
Assuntos
Rosa Bengala , Soroalbumina Bovina , Tomografia Computadorizada por Raios X , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Animais , Rosa Bengala/química , Bovinos , Tomografia Computadorizada por Raios X/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Tela Subcutânea/diagnóstico por imagem , Tela Subcutânea/metabolismo , Suínos , Corantes Fluorescentes/químicaRESUMO
The increasing presence of 1,1,1,2-tetrafluoroethane (CF3CH2F) in the atmosphere has prompted detailed studies into its complex photodissociation behavior. Experiments focusing on CF3CH2F irradiation have unveiled an array of ions, with the persistent observation of the rearrangement product CHF2+ not yet fully understood. In this work, we combine density functional theory, coupled-cluster calculations with a complete basis set formalism, and atom-centered density matrix propagation molecular dynamics to investigate the energetics and dynamics of different potential pathways leading to CHF2+. We found that the two-body dissociation pathway involving an HF rearrangement, which was previously considered complex for CHF2+ formation, is actually straightforward but not likely due to the facile loss of HF. In contrast, our calculations reveal that the H elimination pathway, once thought of as a potential route to CHF2+, is not only comparably disadvantageous from both thermodynamic and kinetic points of view but also does not align with experimental data, particularly the lack of a rebound peak at m/z 101-102. We establish that the formation of CHF2+ is predominantly via the HF elimination channel, a conclusion experimentally corroborated by studies involving the trifluoroethylene cation CF2CHF+, a key intermediate in this process.
RESUMO
Diffusion and movement of subcutaneously injected biologics and high-concentration immunoglobulin G (IgG) therapeutics away from the injection site and through the subcutaneous (SC) tissue may be concentration dependent. This possibility was confirmed by in situ measurement of diffusion coefficients of unlabeled bovine IgG in phosphate-buffered saline within an in vitro hyaluronic acid matrix that represents the SC electrostatic environment. Diffusion decreased from 2.67 to 0.05 × 10-7 cm2 /s when IgG concentration increased from 25 to 73 mg/mL. The results demonstrated that in situ detection of unlabeled proteins within an in vitro SC environment provides another useful tool for the preclinical characterization of injectable biologics.
Assuntos
Produtos Biológicos , Ácido Hialurônico , Animais , Bovinos , Difusão , Imunoglobulina GRESUMO
There are currently more than 560 therapeutic monoclonal antibodies (mAbs) at various stages of research and clinical testing, including candidates for administration by subcutaneous (SC) injection. Preclinical studies based on in vitro measurements of high molecular weight proteins within simulated SC matrices are assisting laboratory studies of interactions of injectable biotherapeutic proteins within the SC environment in relation to bioavailability. We report a new method for directly measuring diffusion of unlabeled, high molecular weight proteins injected into an in vitro matrix that simulates the negatively charged environment of the SC. The matrix consists of 10 mg/ml HA in a repurposed cell culture chamber. The measurement consists of pipetting triplicate 20 µl protein samples into the matrix, placing the chamber in a laboratory scanner, activating tryptophan residues in the protein at 280 nm, and imaging the resulting protein fluorescence at 384 nm over a 0.5-4 h time period thus tracking protein movement. This facile approach enables mapping of protein concentration as a function of time and distance within the matrix, and determination of diffusion coefficients, D, within ±10%. Bovine IgG and BSA gave D = 2.3 ± 0.2*10-7 and 4.6 ± 0.2*10-7 cm2 /s at 24°C, respectively, for initial protein concentrations of 21 mg/mL.
Assuntos
Anticorpos Monoclonais , Ácido Hialurônico , Animais , Bovinos , Injeções Subcutâneas , Disponibilidade Biológica , DifusãoRESUMO
We report on single- and double-charge photofragment formation by synchrotron radiation, following C 1s core excitation and ionization and Cl 2p inner excitation and ionization of chlorobenzene, C6H5Cl. From a comparison of experimental near-edge X-ray absorption fine structure spectra and theoretical ab initio calculations, the nature of various core and inner shell transitions of the molecule and pure atomic features were identified. To shed light on the normal Auger processes following excitation or ionization of the molecule at the Cl 2p or C 1s sites, we addressed the induced ionic species formation. With energy resolved electron spectra and ion time-of-flight spectra coincidence measurements, the ionic species were correlated with binding energy regions and initial states of vacancies. We explored the formation of the molecular dication C6H5Cl2+, the analogue benzene dication C6H42+, and the singly charged species produced by single loss of a carbon atom, C5HnCl+. The appearance and intensities of the spectral features associated with these ionic species are shown to be strongly site selective and dependent on the energy ranges of the Auger electron emission. Unexpected intensities for the analogue double charged benzene C6H42+ ion were observed with fast Auger electrons. The transitions leading to C6H5Cl2+ were identified from the binding energy representation of high resolution electron energy spectra. Most C6H5Cl2+ ions decay into two singly charged moieties, but intermediate channels are opened leading to other heavy dicationic species, C6H42+ and C6H4Cl2+, the channel leading to the first of these being much more favored than the other.
RESUMO
This work discusses the possible HF formation routes via recombination reactions from CF3CH2F (R-134a) and its cation. The molecular properties of the main reagents were first evaluated at the M06-2X/cc-pVTZ level. Then, changes in energy (ΔE) for all reactions comprising a possible HF formation from the studied systems were evaluated at the CCSD(T)/CBS//M06-2X/cc-pVTZ level. With the aid of Intrinsic Reaction Coordinate (IRC) calculations for each path, it is found that the HF formation reaction takes place majorly through the "1,2" elimination, resulting in an olefin as the secondary product. In turn, the IRC associated with "2,2" reactions allowed to find a post-barrier complex between the carbene :CHCF3 and HF in its exit channel, with dissociation energy of â¼4 kcal mol-1. Similarly, the cationic system exhibits favoritism towards the "1,2" elimination, and an ion-dipole post-barrier complex is found. The ΔE of such a complex production is negative in both directions, indicating this complex (25.5 kcal/mol more stable than CF3CH2F+) should be a minimum on the R-134a cation surface. However, unlike the neutral "2,2" path, there is no F atom migration transition state for the 2,2-HF elimination from CF3CH2F+. Hence, the F migration is expected to occur simultaneously with the rest of the structural changes towards the ion-dipole complex. The rate coefficients computed at the current level of theory, including corrections for anharmonicity and hindered rotations, showed a reasonable agreement with the available experimental data, inspiring confidence in our predictions for the cationic system.
RESUMO
The measurement of yield stress and shear thinning flow behavior of slurries formed from unpretreated corn stover at solids loadings of 100-300 g/L provides a key metric for the ability to move, pump, and mix this lignocellulosic slurry, particularly since corn stover slurries represent a major potential feedstock for biorefineries. This study compared static yield stress values and flow hysteresis of corn stover slurries of 100, 150, 200, 250, and 300 g/L, after these slurries were formed by adding pellets to a cellulase enzyme solution (Celluclast 1.5 L) in a fed-batch manner. A rotational rheometer was used to quantitate relative yield stress and its dependence on processing history at insoluble solids concentrations of 4%-21% (wt/vol). Key findings confirmed previous observations that yield stress increases with solids loadings and reaches ~3000 Pa at 25% (wt/vol) solids concentration compared to ~200 Pa after enzyme liquefaction. While optimization of slurry forming (i.e., liquefaction) conditions remains to be done, metrics for quantifying liquefaction extent are needed. The method for obtaining comparative metrics is demonstrated here and shows that the yield stress, shear thinning and shear thickening flow behaviors of enzyme liquefied corn stover slurries can be analyzed using a wide-gap rheometry setup with relative measuring geometries to mimic the conditions that may exist in a mixing vessel of a bioreactor while applying controlled and precise levels of strain.