Absolute quantification method for protein concentration.

A fast and accurate assay to determine the absolute concentration of proteins is described based on direct measurement of droplet entrapped oligomer formation in electrospray. Here we demonstrate the approach using electrospray differential mobility analysis (ES-DMA), which can distinguish monomers and dimers from higher order oligomers. A key feature of the method is that it allows determination of the absolute number concentration of proteins eliminating the need for protein-specific calibration. The method was demonstrated by measuring the concentration of a NIST Standard Reference Material 927e (bovine serum albumin), a high-purity immunoglobulin G 1κ, and a formulated Rituximab. The method may be applied to any electrospray source, regardless of diagnostic tool (e.g., MS or ion-mobility, etc.), provided the electrospray is operated in a droplet-fission mode.

Characterization of polydopamine thin films deposited at short times by autoxidation of dopamine.

Current interest in melanin films derived from the autoxidation of dopamine stems from their use as a universal adhesion layer. Here we report chemical and physical characterization of polydopamine films deposited on gold surfaces from stirred basic solutions at times ranging from 2 to 60 min, with a focus on times ≤10 min. Data from Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical methods suggest the presence of starting (dopamine) and intermediate (C=N-containing tautomers of quinone and indole) species in the polydopamine films at all deposition times. A uniform overlayer analysis of the XPS data indicates that film thickness increased linearly at short deposition times of ≤10 min. At deposition times ≥10 min, the films appeared largely continuous with surface roughness ≈ ≤ 2 nm, as determined by atomic force microscopy (AFM). Pinhole-free films, as determined by anionic redox probe measurements, required deposition times of 60 min or greater.

Competitive adsorption-desorption of IgM monomers-dimers on silica and modified silica surfaces.

Understanding competitive adsorption-desorption of proteins onto surfaces is an important area of research in food processing and biomedical engineering. Here, we demonstrate, how electrospray-differential mobility analysis that has been traditionally used for characterizing bionanoparticles, can be used for quantifying complex competitive adsorption-desorption of oligomeric proteins or multiprotein systems using monomers and dimers of IgM as a model example onto silica and modified silica surfaces. Using ES-DMA, we show that IgM dimers show a preference to stay adsorbed to different surfaces although monomers adsorb more easily and desorption rates of monomers and dimers of IgM are surface-type-dependent and are not significantly affected by shear. We anticipate that this demonstration will make ES-DMA a popular "label-free" method for studying multicomponent multi-oligomeric protein adsorption to different surfaces in the future.

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.

A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins.

We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.

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.

Protein adsorption-desorption on electrospray capillary walls--no influence on aggregate distribution.

Adsorbed proteins on walls of glass capillaries used for electrospray (ES) can desorb and potentially affect size distributions and, thus, quantification of aggregates of proteins. In this study we use differential mobility analysis (DMA) to investigate the size distribution of various proteins eluting from bare and passivated glass capillaries. We found no significant differences in aggregate distributions from unpassivated capillaries at 'steady state' when compared to aggregate distributions from passivated capillaries implying that desorbing proteins do not influence protein aggregate distribution. Surface passivation with gelatin was found to be considerably more effective in limiting adsorption of two antibodies (Rituxan and polyclonal human IgG) compared to passivation with BSA. Gelatin passivation was also found to be stable for a few days and from a pH range of 4.8-9.0.

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.

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

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 ≈10(9)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.

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.

Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis.

We quantify the adsorption and desorption of a monoclonal immunoglobulin-G antibody, rituxamab (RmAb), on silica capillary surfaces using electrospray-differential mobility analysis (ES-DMA). We first develop a theory to calculate coverages and desorption rate constants from the ES-DMA data for proteins adsorbing on glass capillaries used to electrospray protein solutions. This model is then used to study the adsorption of RmAb on a bare silica capillary surface. A concentration-independent coverage of ≈4.0 mg/m(2) is found for RmAb concentrations ranging from 0.01 to 0.1 mg/mL. A study of RmAb adsorption to bare silica as a function of pH shows maximum adsorption at its isoelectric point (pI of pH 8.5) consistent with literature. The desorption rate constants are determined to be ≈10(-5) s(-1), consistent with previously reported values, thus suggesting that shear forces in the capillary may not have a considerable effect on desorption. We anticipate that this study will allow ES-DMA to be used as a "label-free" tool to study adsorption of oligomeric and multicomponent protein systems onto fused silica as well as other surface modifications.

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-DMA's mobility diameter most closely approximates the edge-to-edge length. Using PR772's edge length (36.0 nm) and the size of the major capsid hexon (≈8.4 nm) from ES-DMA with icosahedral geometry, PR772's 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.

Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers-exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the ß-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a ß-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.

Characterization of gold nanoparticles modified with single-stranded DNA using analytical ultracentrifugation and dynamic light scattering.

We report the characterization of gold nanoparticles modified with thiol-terminated single stranded DNA (ssDNA) using analytical ultracentrifugation. Dynamic light scattering was used to measure the diameter of bare and ssDNA modified gold nanoparticles to corroborate the predictions of our models. Sedimentation coefficients of nominally 10 and 20 nm diameter gold nanoparticles modified with thiol-terminated thymidine homo-oligonucleotides, 5-30 bases in length, were determined with analytical ultracentrifugation. The sedimentation coefficients of gold nanoparticles modified with ssDNA were found to decrease with increasing coverage of ssDNA and increasing length of ssDNA. The sedimentation coefficients of ssDNA modified gold particles were most closely predicted when the strands were modeled as fully extended chains (FEC). Apparent particle densities of bare gold nanoparticles calculated from measured sedimentation coefficients decreased significantly below that of bulk gold with decreasing size of nanoparticles. This finding suggests that hydration layer effects are an important factor in the sedimentation behavior for both bare and short ssDNA chain modified gold particles.

Packing and size determination of colloidal nanoclusters.

Here we demonstrate a rapid and quantitative means to characterize the size and packing structure of small clusters of nanoparticles in colloidal suspension. Clustering and aggregation play important roles in a wide variety of phenomena of both scientific and technical importance, yet characterizing the packing of nanoparticles within small clusters and predicting their aerodynamic size remains challenging because available techniques can lack adequate resolution and sensitivity for clusters smaller than 100 nm (optical techniques), perturb the packing arrangement (electron microscopies), or provide only an ensemble average (light scattering techniques). In this article, we use electrospray-differential mobility analysis (ES-DMA), a technique that exerts electrical and drag forces on the clusters, to determine the size and packing of small clusters. We provide an analytical model to determine the mobility size of various packing geometries based on the projected area of the clusters. Data for clusters aggregated from nominally 10 nm gold particles and nonenveloped viruses of various sizes show good agreement between measured and predicted cluster sizes for close-packed spheres.

Length distribution of single-walled carbon nanotubes in aqueous suspension measured by electrospray differential mobility analysis.

The first characterization of the length distribution of single-walled carbon nanotubes (SWCNT) dispersed in a liquid by electrospray differential mobility analysis (ES-DMA) is presented. Although an understanding of geometric properties of SWCNTs, including length, diameter, aspect ratio, and chirality, is essential for commercial applications, rapid characterization of nanotube length distributions remains challenging. Here the use of ES-DMA to obtain length distributions of DNA-wrapped SWCNTs dispersed in aqueous solutions is demonstrated. Lengths measured by ES-DMA compare favorably with those obtained from multiangle light scattering, dynamic light scattering, field flow fractionation with UV/vis detection, and atomic force microscopy, validating ES-DMA as a technique to measure SWCNTs of <250 nm in length. The nanotubes are previously purified and dispersed by wrapping with oligomeric DNA in aqueous solution and centrifuging to remove bundles and amorphous carbon. These dispersions are particularly attractive due to their amenability to bulk processing, ease of storage, high concentration, compatibility with biological and high-throughput manufacturing environments, and for their potential applications ranging from electronics and hydrogen-storage vessels to anticancer agents.

Selective binding of RNase B glycoforms by polydopamine-immobilized concanavalin A.

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.

Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy.

Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped murine polyomavirus (MPV) following: (i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.

A consensus rating method for small virus-retentive filters. I. Method development.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. One goal of the task force was to develop a test method for small virus-retentive filters. Because small virus-retentive filters present unique technical challenges, the test method development process was guided by laboratory studies to determine critical variables such as choice of bacteriophage challenge, choice of model protein, filtration operating parameters, target log10 reduction value, and filtration endpoint definition. Based on filtration, DLS, electrospray differential mobility analysis, and polymerase chain reaction studies, a final rating based on retention of bacteriophage PP7 was chosen by the PDA Virus Filter Task Force. The detailed final consensus filter method was published in the 2008 update of PDA Technical Report 41. Virus Filtration.

Determination of protein aggregation with differential mobility analysis: application to IgG antibody.

Here we describe the use of electrospray differential mobility analysis (ES-DMA), also known as gas-phase electrophoretic mobility molecular analysis (GEMMA), as a method for measuring low-order soluble aggregates of proteins in solution. We demonstrate proof of concept with IgG antibodies. In ES-DMA, aqueous solutions of the antibody protein are electrosprayed and the various aerosolized species are separated according to their electrophoretic mobility using a differential mobility analyzer. In this way, complete size distributions of protein species present from 3 to 250 nm can be obtained with the current set up, including distinct peaks for IgG monomers to pentamers. The sizes of the IgG and IgG aggregates measured by DMA were found to be in good agreement with those calculated from simple models, which take the structural dimensions of IgG from protein crystallographic data. The dependence of IgG aggregation on the solution concentration and ionic strength was also examined, and the portion of aggregates containing chemically crosslinked antibodies was quantified. These results indicate that ES-DMA holds potential as a measurement tool to study protein aggregation phenomena such as those associated with antibody reagent manufacturing and protein therapeutics.