LA JOLLA, CA, United States

Nanoimaging Services, Inc.
LA JOLLA, CA, United States
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Short J.R.,Scripps Research Institute | Short J.R.,Illumina | Speir J.A.,Scripps Research Institute | Speir J.A.,Nanoimaging Services, Inc. | And 4 more authors.
Journal of Virology | Year: 2016

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are~50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. © 2016, American Society for Microbiology.

Zhao Q.,Xiamen University | Zhao Q.,Merck And Co. | Potter C.S.,Nanoimaging Services, Inc. | Potter C.S.,Scripps Research Institute | And 10 more authors.
Human Vaccines and Immunotherapeutics | Year: 2014

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant. In addition, we determined the three-dimensional structure of the VLPs, both alone and when interacting with neutralizing antibodies. Two modes of binding of two different neutralizing antibodies were apparent; for HPV type 11 saturated with H11.B2, 72 potential Fab binding sites were observed at the center of each capsomer, whereas for HPV 16 interacting with H16.V5, it appears that 60 pentamers (each neighboring 6 other pentamers) bind five Fabs per pentamer, for the total of 300 potential Fab binding sites per VLP. © 2014 Landes Bioscience.

Correia I.,AbbVie Bioresearch Center | Sung J.,Nanoimaging Services, Inc. | Burton R.,AbbVie Bioresearch Center | Jakob C.G.,Abbott Laboratories | And 3 more authors.
mAbs | Year: 2013

A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVDIg ™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVDIg ™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig ™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance. © 2013 Landes Bioscience.

Mulder A.M.,Nanoimaging Services, Inc. | Carragher B.,Nanoimaging Services, Inc. | Towne V.,Vaccine Manufacturing Science and Commercialization | Meng Y.,Merck And Co. | And 9 more authors.
PLoS ONE | Year: 2012

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. Methodology: The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). Principal Findings: SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images - confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing. Significance: Together, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines. © 2012 Mulder et al.

It is an object of the invention to provide methods and compositions for characterizing particles in a size range from about 1 m to about 10 nm, and preferably 5 m to about 5 nm. Using transmission electron microscopy and digital image processing techniques, the methods of the present invention can provide detailed information on the aggregation state of, for example, proteinaceous samples such as antibody-based pharmaceutical compositions. The methods can further permit assessment of the effect of storage, use, processing, and shipping conditions in such proteinaceous samples.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 193.83K | Year: 2010

DESCRIPTION (provided by applicant): Characterization of nanoparticles and biologics is a critical step in the development of important new pharmaceutical therapeutics as, in many cases, preclinical characterization is the rate-limiting phase of the commercialization process. Nanoparticle therapeutics poses unique challenges for characterization that require an interdisciplinary approach in which several orthogonal methods are required to provide a complete picture. Cryo-electron microscopy, with its unique capability for providing exact visual information of size, shape, morphology, and aggregation of a sample in its natural hydrated state, is a powerful additional tool in the armamentarium of characterization techniques applied to nanoparticles. NanoImaging Services, Inc. was established in 2007 in order to provide cryoEM imaging services specifically focused on the characterization of biologics and nanoparticles. Over the past 18 months it has become clear that there is a critical need for more quantitative analysis of multiple cryoEM images. The images must be processed, analyzed and evaluated with the goal of providing numerical data that can be used for assessing the characteristics of the samples and comparing these characteristics to the results obtained from other orthogonal methods. Quantitation is critical for assessing lot-to-lot comparisons, quality assurance and control, monitoring samples over time, and when providing data to the regulatory agencies. Here we request support to undertake a 6 month study to determine the feasibility of developing several quantitative characterization protocols that are designed to address specific needs in the research and development of new biologics and nanoparticles as therapeutics. Specifically we will develop quantitative tools to characterize particle morphology and aggregation state and test and validate the methods using a number of samples of high interest as pharmaceutical therapeutics. PUBLIC HEALTH RELEVANCE: Arguably two of the fastest growing sectors of the biopharmaceutical industry are the production of biologics as therapeutic agents and nanoparticles as drug delivery vehicles. There is considerable evidence that the physical properties of biologics and nanoparticles is tightly linked to their functional behavior and may thus be a determining factor in the biodistribution, safety, and efficacy of the pharmaceutical product. Cryo- electron microscopy, with its unique potential for providing quantitative information of size, shape, morphology, and aggregation of a sample in its natural hydrated state, is a powerful additional tool in the armamentarium of characterization techniques applied to nanoparticles.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.21M | Year: 2012

DESCRIPTION (provided by applicant): Aggregation in antibody therapeutics is a topic of increasing concern in the biopharmaceutical industry, as these aggregates are believed to play an undesirable role resulting in adverse health effects to patients by inducing immunogenicity, altering therapeutic pharmacokinetics, or decreasing efficacy of the drug product. While methods are available to count (and in some cases, image) visible, and to a lesser extent sub-visible, particles, there is currently no reliableway to count and identify aggregates with sizes smaller than 1 micron. Studies on the mechanism of antibody aggregation indicate a process of formation that begins with very small sub-micron sized seed aggregates, which suggests the value of sub-micron characterization as a predictive indicator of therapeutic stability. It is hypothesized that humanized antibody therapeutics break B-cell tolerance by presenting the self-antigen in a foreign array through ordered packing in aggregates; not unlike the mechanism by which viruses elicit a vigorous immune response. This poses the question-what are the cutting-edge technologies utilized by scientists to study the size, shape, and morphology of viruses, and are these technologies applicable to the problem of sub-micron aggregate characterization? Molecular microscopy is a non-invasive molecular imaging technology that uses advanced specimen preparation and imaging methods designed specifically to visualize complex biological samples, under conditions close to their native state. For well-ordered samples such as viruses, and virus-antibody complexes, the achievable resolution can be lt 0.4 nm. In order to address the problem of quantitative sub- micron aggregate characterization of monoclonal antibody therapeutics, our technology applies high- throughput molecular microscopy to directly visualize antibody monomers and aggregates at multiple scales of magnification to provide quantitative measures of antibody aggregate count, size, shape, and morphology as well asantibody monomer loss in a single experiment. This technology utilizes a complex and expensive TEM instrument and automated software that requires a significant level of expertise to achieve reliable and high- throughput results, and is thus not readily accessible as an in-house method. We propose to make this technology available by incorporating a robust Antibody Therapeutic Submicron Aggregate Characterization service into the armamentarium of fee-for-service TEM capabilities that we already offer tothe biopharmaceutical industry. To this end, our Phase II proposal has the following milestones: 1) Defining technology specifications and limitations in quantitative terms that are meaningful to antibody therapeutic formulations and characterization scientists, in consultation with protein therapeutic characterization expert, Dr. Kogan Bao (Allergan Pharmaceuticals, CA); 2) Validating the technology through comparison to accepted methodologies, in partnership with protein aggregation expert, Dr. John Carpenter (UC Denver, CO); and 3) Developing and implementing software algorithms to enhance technology throughput in a cost-effective manner. This will prepare us for Phase III, when we will provide trial service offerings to a select number of our existing pharmaceutical and biotechnology clients. PUBLIC HEALTH RELEVANCE: Aggregation in antibody therapeutics is a topic of increasing concern in the biopharmaceutical industry, because these aggregates are believed to play an undesirable role resulting inadverse health effects to patients; as a result, there is increasing pressure from the U.S. Food and Drug Administration to develop quantitative characterization tools for submicron aggregate particles in biopharmaceutical drug products. Our technology applies high-throughput molecular microscopy to identify and quantify sub-micron protein aggregates in antibody therapeutics. These new analytical methods will contribute to the armamentarium of analytical techniques aimed at helping reduce costs and improvesafety and efficacy of antibody therapeutics.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 223.24K | Year: 2014

DESCRIPTION (provided by applicant): Transmission electron microscopy is a valuable technique for characterizing biological therapeutics, vaccines, drug delivery vehicles, and nanoparticles. There is considerable evidence that links the physical propertiesof these samples to their bio-distribution, safety, and efficacy. TEM imaging allows direct observation and quantification of these parameters, which include size distributions, shape distributions, concentrations, aggregation states, and 2D and 3D structure determinations. This wealth of information is especially useful when researchers must troubleshoot problems of unknown origin. As bio-therapeutics and nanoparticles increase in size and complexity, TEM is becoming increasingly important as an orthogonal complement to other biophysical methods. Despite the obvious utility of TEM as a direct technique for describing the physicochemical properties of samples in a variety of relevant biological environments, the cost per sample is a significant barrier

PubMed | Nanoimaging Services, Inc.
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2015

Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety because of potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size-exclusion chromatography, dynamic light scattering, microflow imaging, and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three-dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semiquantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis.

PubMed | Nanoimaging Services, Inc. and Infectious Disease Research Institute
Type: | Journal: International journal of nanomedicine | Year: 2014

Development of lipid-based adjuvant formulations to enhance the immunogenicity of recombinant vaccine antigens is a focus of modern vaccine research. Characterizing interactions between vaccine antigens and formulation excipients is important for establishing compatibility between the different components and optimizing vaccine stability and potency. Cryogenic transmission electron microscopy (TEM) is a highly informative analytical technique that may elucidate various aspects of protein- and lipid-based structures, including morphology, size, shape, and phase structure, while avoiding artifacts associated with staining-based TEM. In this work, cryogenic TEM is employed to characterize a recombinant tuberculosis vaccine antigen, an anionic liposome formulation, and antigen-liposome interactions. By performing three-dimensional tomographic reconstruction analysis, the formation of a population of protein-containing flattened liposomes, not present in the control samples, was detected. It is shown that cryogenic TEM provides unique information regarding antigen-liposome interactions not detectable by light-scattering-based methods. Employing a suite of complementary analytical techniques is important to fully characterize interactions between vaccine components.

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