Suvajyoti Guha

Quantitative determination of competitive molecular adsorption on gold nanoparticles using attenuated total reflectance-Fourier transform infrared spectroscopy.

Surface-sensitive quantitative studies of competitive molecular adsorption on nanoparticles were conducted using a modified attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy method. Adsorption isotherms for thiolated poly(ethylene glycol) (SH-PEG) on gold nanoparticles (AuNPs) as a function of molecular mass (1, 5, and 20 kDa) were characterized. We find that surface density of SH-PEG on AuNPs is inversely proportional to the molecular mass (M(m)). Equilibrium binding constants for SH-PEG, obtained using the Langmuir adsorption model, show the binding affinity for SH-PEG is proportional to M(m). Simultaneous competitive adsorption between mercaptopropionic acid (MPA) and 5 kDa SH-PEG (SH-PEG5K) was investigated, and we find that MPA concentration is the dominant factor influencing the surface density of both SH-PEG5K and MPA, whereas the concentration of SH-PEG5K affects only SH-PEG5K surface density. Electrospray differential mobility analysis (ES-DMA) was employed as an orthogonal characterization technique. ES-DMA results are consistent with the results obtained by ATR-FTIR, confirming our conclusions about the adsorption process in this system. Ligand displacement competitive adsorption, where the displacing molecular species is added after completion of the ligand surface binding, was also interrogated by ATR-FTIR. Results indicate that for SH-PEG increasing M(m) yields greater stability on AuNPs when measured against displacement by bovine serum albumin (BSA) as a model serum protein. In addition, the binding affinity of BSA to AuNPs is inhibited for SH-PEG conjugated AuNPs, an effect that is enhanced at higher SH-PEG M(m) values.

Electrospray-differential mobility analysis of bionanoparticles

Electrospray-differential mobility analysis (ES-DMA) is a versatile technique used to aerosolize bionanoparticles and measure their electrical mobility at ambient conditions. ES-DMA is similar to electrospray-mass spectrometry (ES-MS), but measures the effective particle size, rather than mass. It has a wide range of applications and nominally can be used to characterize biomolecules and nanoparticles ranging in size from a few nanometers (~3 nm) to several hundred nanometers, to obtain multimodal size distributions in minutes. Although both the ES and the DMA are mature technologies, they are finding increased use in combination to characterize particles in liquids. In this paper, we review ES-DMA, and how it has recently been used to characterize bionanoparticles such as polymers, proteins, viruses, bacteriophages and nanoparticle-biomolecule conjugates.

Method for determining the absolute number concentration of nanoparticles from electrospray sources.

We have developed a simple, fast, and accurate method to measure the absolute number concentration of nanoparticles in solution. The method combines electrospray differential mobility analysis (ES-DMA) with a statistical analysis of droplet-induced oligomer formation. A key feature of the method is that it allows determination of the absolute number concentration of particles by knowing only the droplet size generated from a particular ES source, thereby eliminating the need for sample-specific calibration standards or detailed analysis of transport losses. The approach was validated by comparing the total number concentration of monodispersed Au nanoparticles determined by ES-DMA with UV/vis measurements. We also show that this approach is valid for protein molecules by quantifying the absolute number concentration of Rituxan monoclonal antibody in solution. The methodology is applicable for quantification of any electrospray process coupled to an analytical tool that can distinguish monomers from higher order oligomers. The only requirement is that the droplet size distribution be evaluated. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas. This method eliminates the need for sample-specific calibration standards or detailed analysis of transport losses.

Quantification and Compensation of Nonspecific Analyte Aggregation in Electrospray Sampling

Electrospray (ES) sources are commonly used to introduce nonvolatile materials (e.g., nanoparticles, proteins, etc.) to the gas phase for characterization by mass spectrometry or ion mobility. Recent studies in our group using ES ion mobility to characterize protein aggregation in solution have raised the question as to whether the ES itself induces aggregation and thus corrupts the results. In this article, we develop a statistical model to determine the extent to which the ES process induces the formation of dimers and higher-order aggregates. The model is validated through ES differential mobility experiments using gold nanoparticles. The results show that the extent of droplet-induced aggregation is quite severe and previously reported cutoff criterion is inadequate. We use the model in conjunction with experiment to show the true dimer concentration in a protein solution as a function of concentration. The model is extendable to any ES source analytical system and to higher aggregation states. For users only interested in implementation of the theory, we provide a section that summarizes the relevant formulas.

Online Nanoparticle Mass Measurement by Combined Aerosol Particle Mass Analyzer and Differential Mobility Analyzer: Comparison of Theory and Measurements

A combination of a differential mobility analyzer (DMA) and aerosol particle mass analyzer (APM) is used to measure the mass of NIST Standard Reference Materials (SRM®) PSL spheres with 60 and 100 nm nominal diameter, and NIST traceable 300 nm PSL spheres. The calibration PSL spheres were previously characterized by modal diameter and spread in particle size. We used the DMA to separate the particles with modal diameter in a narrow mobility diameter range. The mass of the separated particles is measured using the APM. The measured mass is converted to diameter using a specific density of 1.05. We found that there was good agreement between our measurements and calibration modal diameter. The measured average modal diameters are 59.23 and 101.2 nm for nominal diameters of 60 and 100 nm (calibration modal diameter: 60.39 and 100.7 nm) PSL spheres, respectively. The repeatability uncertainty of these measurements is reported. For 300 nm, the measured diameter was 305.5 nm, which is an agreement with calibration diameter within 1.8%. The effect of spread in particle size on the APM transfer function is investigated. Two sources of the spread in “mono-dispersed” particle size distributions are discussed: (a) spread due to the triangular DMA transfer function, and (b) spread in the calibration particle size. The APM response function is calculated numerically with parabolic flow through the APM and diffusion broadening. As expected from theory, the calculated APM response function and measured data followed a similar trend with respect to APM voltage. However, the theoretical APM transfer function is narrower than the measured APM response.

Quantifying Ligand Adsorption to Nanoparticles Using Tandem Differential Mobility Mass Analysis

Although electrospray-differential mobility analyzers (ES-DMA) have been previously employed to characterize ligand binding to nanoparticles, absolute quantification of surface coverage can be inaccurate at times because of ligand conformational effects. In this Letter, we report a quantitative technique by in-flight coupling of a particle mass analyzer (APM) with ES-DMA, thus enabling a direct quantitative analysis of mass independent of particle size, material, morphology and conformation. We demonstrate the utility of ES-DMA-APM by studying two model complex systems (gold nanoparticle-bovine serum albumin and polystyrene bead-antibody) as a function of concentration and pH. Our results obtained with ES-DMA-APM are in excellent agreement with prior work. We anticipate that this will enhance the capabilities of online quantitative characterization of ligand binding to nanoparticles.

Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing

Abstract The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈109 particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use.

Physical Characterization of Icosahedral Virus Ultra Structure, Stability, and Integrity Using Electrospray Differential Mobility Analysis

We present a rapid and quantitative method to physically characterize the structure and stability of viruses. Electrospray differential mobility analysis (ES-DMA) is used to determine the size of capsomers (i.e., hexons) and complete capsids. We demonstrate how to convert the measured mobility size into the icosahedral dimensions of a virus, which for PR772 become 68.4 nm for vertex-to-vertex, 54.4 nm for facet-to-facet, and 58.2 nm for edge-to-edge lengths, in reasonable agreement with dimensions from transmission electron microscopy for other members of the family Tectiviridae (e.g., PRD1). These results indicate ES-DMAs mobility diameter most closely approximates the edge-to-edge length. Using PR772s edge length (36.0 nm) and the size of the major capsid hexon (≈8.4 nm) from ES-DMA with icosahedral geometry, PR772s T = 25 symmetry is confirmed and the number of proteins in the capsid shell is determined. We also demonstrate the use of ES-DMA to monitor the temporal disintegration of PR772, the thermal degradation of PP7, and the appearance of degradation products, essential to viral stability assays. These results lay groundwork essential for the use of ES-DMA for a variety of applications including monitoring of vaccine and gene therapy vector products, confirmation of viral inactivation, and theoretical studies of self-assembling macromolecular structures.

Bionanoparticles as candidate reference materials for mobility analysis of nanoparticles.

We propose bionanoparticles as a candidate reference material for determining the mobility of nanoparticles over the range of 6 × 10(-8)-5 × 10(-6) m(2)V(-1)s(-1). Using an electrospray differential mobility analyzer (ES-DMA), we measured the empirical distribution of several bionanoparticles. All of them show monomodal distributions that are more than two times narrower than the currently used calibration particles for mobility larger than 6 × 10(-8) m(2)V(-1)s(-1) (diameters less than 60 nm). We also present a numerical method to calculate corrected distributions of bionanoparticles by separating the contribution of the diffusive transfer function. The corrected distribution is about 20% narrower than the empirical distributions. Even with the correction, the reduced width of the mobility distribution is about a factor of 2 larger than the diffusive transfer function. The additional broadening could result from the nonuniform conformation of bionanoparticles and from the presence of volatile impurities or solvent adducts. The mobilities of these investigated bionanoparticle are stable over a range of buffer concentration and molarity, with no evidence of temporal degradation over several weeks.

Electrospray–Differential Mobility Analysis as an Orthogonal Tool to Size-Exclusion Chromatography for Characterization of Protein Aggregates

The biopharmaceutical industry characterizes and quantifies aggregation of protein therapeutics using multiple analytical techniques to cross-validate results. Here, we demonstrate the use of electrospray-differential mobility analysis (ES-DMA), a gas-phase and atmospheric pressure ion-mobility method for characterizing protein aggregates. Two immunoglobulin Gs are systematically heat treated to induce aggregation and characterized using size-exclusion chromatography (SEC) and ES-DMA. Although ES-DMA is a gas-phase characterization method, we find that aggregation kinetic rate constants determined by ES-DMA is in good agreement with those determined by SEC. ES-DMA appears to have a higher resolution and lower limit of detection as compared with SEC. Thus, ES-DMA can potentially become an important orthogonal tool for characterization of nascent protein aggregates in the biopharmaceutical industry.