Analytical Performance Evaluation of Identity, Quality-Attribute Monitoring and new Peak Detection in a Platform Multi-Attribute Method Using Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies.

The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.

In silico method development for the reversed-phase liquid chromatography separation of proteins using chaotropic mobile phase modifiers.

Recent advances in biomedical and pharmaceutical processes has enabled a notable increase of protein- and peptide-based drug therapies and vaccines that often contain a higher-order structure critical to their efficacy. Hyphenation of chromatographic and spectrometric techniques is at the center of all facets of biopharmaceutical analysis, purification and chemical characterization. Although computer-assisted chromatographic modeling of small molecules has reached a mature stage across the pharmaceutical industry, software-based method optimization approaches for large molecules has yet to see the same revitalization. Conformational changes of biomolecules under chromatographic conditions have been identified as the major culprit in terms of sub-optimal modeling outcomes. In order to circumvent these challenges, we herein investigate the outcomes generated via computer-assisted modeling from using different chaotropic and denaturing mobile phases (trifluoroacetic acid, sodium perchlorate and guanidine hydrochloride in acetonitrile/water-based eluents). Linear and polynomial regression retention models using ACD/Labs software were built as a function of gradient slope, column temperature and mobile phase buffer for eight different model proteins ranging from 12 to 670 kDa (holo-transferrin, cytochrome C, apomyoglobin, ribonuclease A, ribonuclease A type I-A, albumin, y-globulin and thyroglobulin bovine). Correlation between experimental and modeled outputs was substantially improved by using strong chaotropic and denaturing modifiers in the mobile phase, even when using linear regression modeling as typically observed for small molecules. On the contrary, the use of conventional TFA buffer concentrations at low column temperatures required the used of polynomial regression modeling indicating potential conformational structure changes of proteins upon chromatographic conditions. In addition, we illustrate the power of modern computer-assisted chromatography modeling combined with chaotropic agents in the developing of new RPLC assays for protein-based therapeutics and vaccines.

Nanoparticle adsorption dynamics at fluid interfaces.

Understanding the dynamic adsorption of nanoparticles (NPs) at fluid interfaces is important for stabilizing emulsions and for the preparation of 2D NP-based materials. Here we show that the Ward-Tordai equations commonly employed to describe the dynamics of surfactant adsorption at a fluid interface combined with a Frumkin adsorption isotherm can be employed to model the diffusion-limited adsorption of NPs onto a fluid interface. In contrast to surfactants, an additional wetting equation of state (EOS) must be incorporated to characterize the dynamic interfacial tension during the adsorption of NPs to the oil-water interface. Our results show agreement between the model and experiments with NP area fractions <0.3. Slower dynamics are observed at larger area fractions, which are speculated to arise from polydispersity or re-organization at the interface. We show the model can be extended to the competitive adsorption between the NPs and a surface active species.

Competitive Adsorption between Nanoparticles and Surface Active Ions for the Oil-Water Interface.

Nanoparticles (NPs) can add functionality (e.g., catalytic, optical, rheological) to an oil-water interface. Adsorption of ∼10 nm NPs can be reversible; however, the mechanisms for adsorption and its effects on surface pressure remain poorly understood. Here we demonstrate how the competitive reversible adsorption of NPs and surfactants at fluid interfaces can lead to independent control of both the adsorbed amount and surface pressure. In contrast to prior work, both species investigated (NPs and surfactants) interact reversibly with the interface and without the surface active species binding to NPs. Independent measurements of the adsorption and surface pressure isotherms allow determination of the equation of state (EOS) of the interface under conditions where the NPs and surfactants are both in dynamic equilibrium with the bulk phase. The adsorption and surface pressure measurements are performed with gold NPs of two different sizes (5 and 10 nm), at two pH values, and across a wide concentration range of surfactant (tetrapentylammonium, TPeA+) and NPs. We show that free surface active ions compete with NPs for the interface and give rise to larger surface pressures upon the adsorption of NPs. Through a competitive adsorption model, we decouple the contributions of NPs wetting at the interface and their surface activity on the measured surface pressure. We also demonstrate reversible control of adsorbed amount via changes in the surfactant concentration or the aqueous phase pH.

Measurement of Anisotropic Particle Interactions with Nonuniform ac Electric Fields.

Optical microscopy measurements are reported for single anisotropic polymer particles interacting with nonuniform ac electric fields. The present study is limited to conditions where gravity confines particles with their long axis parallel to the substrate such that particles can be treated using quasi-2D analysis. Field parameters are investigated that result in particles residing at either electric field maxima or minima and with long axes oriented either parallel or perpendicular to the electric field direction. By nonintrusively observing thermally sampled positions and orientations at different field frequencies and amplitudes, a Boltzmann inversion of the time-averaged probability of states yields kT-scale energy landscapes (including dipole-field, particle-substrate, and gravitational potentials). The measured energy landscapes show agreement with theoretical potentials using particle conductivity as the sole adjustable material property. Understanding anisotropic particle-field energy landscapes vs field parameters enables quantitative control of local forces and torques on single anisotropic particles to manipulate their position and orientation within nonuniform fields.

Shape-controlled fabrication of cell-laden calcium alginate-PLL hydrogel microcapsules by electrodeposition on microelectrode.

In this study, we propose an electrodeposition method of fabricating shape-controlled calcium alginate-poly-L-lysine hydrogel microcapsules. The micro-patterned electrodes, which are produced by coating a patterned photoresist layer onto fluorine-doped tin oxide glass slide based on photolithography technique, are used for making different shapes of microcapsules. By the electrolysis of water in alginate gelation on micro-patterned anode electrode, the 2D alginate hydrogel structures embedded with yeast cells are formed on fluorine-doped tin oxide glass slide. Then, the 2D structures would be detached from the microelectrode surface and treated with given reagent to be transformed into 3D microcapsules while maintaining the ring and hexagon shapes. Finally, the yeast cells within the microcapsules are further promoted into compact tissues by cultivation. The experimental results indicate the method can successfully fabricate tissues which can maintain certain cells bioactivity after 24 h cultivation. The recommended method can lead to fabricating cell-laden scaffold for tissue engineering, biological studies and drug discovery with higher accuracy and efficiency.

Reversible Partitioning of Nanoparticles at an Oil-Water Interface.

The reversible adsorption of nanoparticles (NPs) to oil-water interfaces has been observed experimentally, however, models capable of interpreting and predicting the equilibrium partitioning of particles between bulk media and fluid interfaces are still lacking. Here we characterize the adsorption of 5 nm gold NPs functionalized with ion-pair ligands at the toluene-water interface. Partitioning of the NPs between the bulk aqueous phase and the interface is measured via absorbance spectroscopy for two different aqueous-phase pH values (11.0 and 11.7) and over several orders of magnitude of aqueous phase NP concentration. The surface pressure of the interfacial film in equilibrium with the bulk aqueous phase is measured using the pendant drop method. We determine the range of surface pressure where the adsorption is reversible as well as conditions under which the adsorbed NPs are irreversibly adsorbed at the oil-water interface. We analyze together the adsorption and surface pressure isotherms to obtain the two-dimensional equations of state (EOS) for the NPs in equilibrium with the bulk aqueous phase. The experimental data are then compared to the Frumkin models. We find that the adsorption isotherm and the equation of state show good agreement at low coverage with the Frumkin equations; however, both curves cannot be described with the same parameters. We also show that the low-coverage portion of the EOS can also be described by a wetting model. We hypothesize that deviations from models at higher coverage are likely due to nonequilibrium effects and possible coadsorption.

Cardioprotective activity of iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) are chemically inert materials and have been mainly used for imaging applications and drug deliveries. However, the possibility whether they can be used as therapeutic drugs themselves has not yet been explored. We reported here that Fe2O3 nanoparticles (NPs) can protect hearts from ischemic damage at the animal, tissue and cell level. The cardioprotective activity of Fe2O3 NPs requires the integrity of nanoparticles and is not dependent upon their surface charges and molecules that were integrated into nanoparticles. Also, Fe2O3 NPs showed no significant toxicity towards normal cardiomyocytes, indicative of their potential to treat cardiovascular diseases.

Preparation, characterization of 2-deoxy-D-glucose functionalized dimercaptosuccinic acid-coated maghemite nanoparticles for targeting tumor cells.

PURPOSE: To report a modified preparation and to systematically study the structure, magnetic and other properties of γ-Fe(2)O(3)-DMSA-DG NPs (2-deoxy-D-glucose (2-DG) conjugated meso-2,3-dimercaptosuccinic acid coated γ-Fe(2)O(3) nanoparticles) and test its ability to improve Hela tumor cells targeting in vitro compared to the γ-Fe(2)O(3)-DMSA NPs. METHODS: The conjugation of 2-DG on the surface of γ-Fe(2)O(3)-DMSA NPs was performed by esterification reaction and characterized. Acute toxicity was evaluated using MTT assay. Cellular uptake was investigated by Prussian blue staining and UV colorimetric assay. RESULTS: DG was successfully functionalized onto the surface of γ-Fe(2)O(3)-DMSA NPs; binding efficiency was ~60%. The mean diameter of single core of γ-Fe(2)O(3)-DMSA-DG NPs was 10 nm. Particle size and polydispersity index of its aggregates were 156.2 nm and 0.162, respectively. 2-DG-conjugated nanoparticles caused little cytotoxic effects on Hela cells at the concentration range of 0-600 µg/mL. When 2-DG-conjuated and non-conjugated nanoparticles were incubated with Hela cells for 4, 8 and 12 h, the 2-DG-conjugated nanoparticle showed significant amount of uptake in cells compared to their non-targeted counterparts. CONCLUSION: γ-Fe(2)O(3)-DMSA-DG NPs could be developed as a tumor-targeted probe for cervical cancer imaging and therapy.