Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Standards efforts and landscape for rapid microbial testing methodologies in regenerative medicine.

The Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery (SCB) supports the development and commercialization of regenerative medicine products by identifying and addressing industry-wide challenges through standards. Through extensive stakeholder engagement, the implementation of rapid microbial testing methods (RMTMs) was identified as a high-priority need that must be addressed to facilitate more timely release of products. Since 2017, SCB has coordinated efforts to develop standards for this area through surveys, weekly meetings, workshops, leadership in working groups and participation in standards development organizations. This article describes the results of these efforts and discusses the current landscape of RMTMs for regenerative medicine products. Based on discussions with stakeholders across the field, an overview of traditional culture-based methods and limitations, alternative microbial testing technologies and current challenges, fit-for-purpose rapid microbial testing and case studies, risk-based strategies for selection of novel rapid microbial test methods and ongoing standards efforts for rapid microbial testing are captured here. To this end, SCB is facilitating several initiatives to address challenges associated with rapid microbial testing for regenerative medicine products. Two documentary standards are under development: an International Organization for Standardization standard to provide the framework for a risk-based approach to selecting fit-for-purpose assays primarily intended for cell and gene therapy products and an ASTM standard guide focused on sampling methods for microbial testing methods in tissue-engineered medical products. Working with the National Institute of Standards and Technology, SCB expects to facilitate the process of developing publicly available microbial materials for inter-laboratory testing. These studies will help collect the data necessary to facilitate validation of novel rapid methods. Finally, SCB has been working to increase awareness of, dialog about and participation in efforts to develop standards in the regenerative medicine field.

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Summary of the National Institute of Standards and Technology and US Food And Drug Administration cell counting workshop: Sharing practices in cell counting measurements.

The emergence of cell-based therapeutics has increased the need for high-quality, robust and validated measurements for cell characterization. Cell count, being one of the most fundamental measures for cell-based therapeutics, now requires increased levels of measurement confidence. The National Institute of Standards and Technology (NIST) and the US Food and Drug Administration (FDA) jointly hosted a workshop focused on cell counting in April 2017 entitled "NIST-FDA Cell Counting Workshop: Sharing Practices in Cell Counting Measurements." The focus of the workshop was on approaches for selecting, designing and validating cell counting methods and overcoming gaps in obtaining sufficient measurement assurance for cell counting. Key workshop discussion points, representing approximately 50 subject matter experts from industry, academia and government agencies, are summarized here. A key conclusion is the need to design the most appropriate cell counting method, including control/measurement assurance strategies, for a specific counting purposes. There remains a need for documentary standards for streamlining the process to develop, qualify and validate cell counting measurements as well as community-driven efforts to develop new or improved biological and non-biological reference materials.

FDA and NIST collaboration on standards development activities supporting innovation and translation of regenerative medicine products.

The development of standards for the field of regenerative medicine has been noted as a high priority by several road-mapping activities. Additionally, the U.S. Congress recognizes the importance of standards in the 21st Century Cure Act. Standards will help to accelerate and streamline cell and gene therapy product development, ensure the quality and consistency of processes and products, and facilitate their regulatory approval. Although there is general agreement for the need of additional standards for regenerative medicine products, a shared understanding of standards is required for real progress toward the development of standards to advance regenerative medicine. Here, we describe the roles of standards in regenerative medicine as well as the process for standards development and the interactions of different entities in the standards development process. Highlighted are recent coordinated efforts between the U.S. Food and Drug Administration and the National Institute of Standards and Technology to facilitate standards development and foster science that underpins standards development.

Evaluating the quality of a cell counting measurement process via a dilution series experimental design.

BACKGROUND AIMS: Cell counting measurements are critical in the research, development and manufacturing of cell-based products, yet determining cell quantity with accuracy and precision remains a challenge. Validating and evaluating a cell counting measurement process can be difficult because of the lack of appropriate reference material. Here we describe an experimental design and statistical analysis approach to evaluate the quality of a cell counting measurement process in the absence of appropriate reference materials or reference methods. METHODS: The experimental design is based on a dilution series study with replicate samples and observations as well as measurement process controls. The statistical analysis evaluates the precision and proportionality of the cell counting measurement process and can be used to compare the quality of two or more counting methods. As an illustration of this approach, cell counting measurement processes (automated and manual methods) were compared for a human mesenchymal stromal cell (hMSC) preparation. RESULTS: For the hMSC preparation investigated, results indicated that the automated method performed better than the manual counting methods in terms of precision and proportionality. DISCUSSION: By conducting well controlled dilution series experimental designs coupled with appropriate statistical analysis, quantitative indicators of repeatability and proportionality can be calculated to provide an assessment of cell counting measurement quality. This approach does not rely on the use of a reference material or comparison to "gold standard" methods known to have limited assurance of accuracy and precision. The approach presented here may help the selection, optimization, and/or validation of a cell counting measurement process.

Polyaspartic Acid Concentration Controls the Rate of Calcium Phosphate Nanorod Formation in High Concentration Systems.

Polyelectrolytes are known to greatly affect calcium phosphate (CaP) mineralization. The reaction kinetics as well as the CaP phase, morphology and aggregation state depend on the relative concentrations of the polyelectrolyte and the inorganic ions in a complex, nonlinear manner. This study examines the structural evolution and kinetics of polyaspartic acid (pAsp) directed CaP mineralization at high concentrations of polyelectrolytes, calcium, and total phosphate (19-30 mg/mL pAsp, 50-100 mM Ca2+, Ca/P = 2). Using a novel combination of characterization techniques including cryogenic transmission electron microscopy (cryo-TEM), spectrophotometry, X-ray total scattering pair distribution function analysis, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), it was determined that the CaP mineralization occurred over four transition steps. The steps include the formation of aggregates of pAsp stabilized CaP spherical nanoparticles (sNP), crystallization of sNP, oriented attachment of the sNP into nanorods, and further crystallization of the nanorods. The intermediate aggregate sizes and the reaction kinetics were found to be highly polymer concentration dependent while the sizes of the particles were not concentration dependent. This study demonstrates the complex role of pAsp in controlling the mechanism as well as the kinetics of CaP mineralization.

Quantifying the sensitivity of the network structure and properties from simultaneous measurements during photopolymerization.

We present a method that combines experimental and computational approaches to assess a comprehensive set of structural and functional evolution during a network formation process via photopolymerization. Our work uses the simultaneous measurement of the degree of conversion, polymerization stress, the change in reaction temperature, and shrinkage strain in situ. These measurements are combined with the theory of viscoelastic materials to deduce the relaxation time and frequency-dependent modulus of the polymerizing network. The relaxation time and degree of conversion are used to demonstrate the effect of processing parameters (e.g. curing protocol adjusted by the light intensity) in creating different network structures for the same initial resin. We describe experimental trends using effective medium calculations on a cross-linked polymer network model. In particular, we show that the effect of curing conditions on the spatial heterogeneity in crosslink density can be quantified using multiparametric measurements and modeling. Collectively, the present method is a way to examine holistically the complex structural and functional evolution of the network formation process.

Experimental and statistical methods to evaluate antibacterial activity of a quaternary pyridinium salt on planktonic, biofilm-forming, and biofilm states.

Robust evaluation and comparison of antimicrobial technologies are critical to improving biofilm prevention and treatment. Herein, a multi-pronged experimental framework and statistical models were applied to determine the effects of quaternary pyridinium salt, 4-acetyl-1-hexadecylpyridin-1-ium iodide (QPS-1), on Streptococcus mutans in the planktonic, biofilm-forming and biofilm cell states. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined via common methods with novel application of statistical approaches combining random effects models and interval censored data to estimate uncertainties. The MICs and MBCs for planktonic and biofilm-forming states ranged from 3.12 to 12.5 µg ml-1, with biofilm values only ≈ 8 times higher. Potent anti-biofilm activity and reactive structural features make QPS-1 a promising antibacterial additive for dental and potentially other biomedical devices. Together, the experimental framework and statistical models provide estimates and uncertainties for effective antimicrobial concentrations in multiple cell states, enabling statistical comparisons and improved characterization of antibacterial agents.

Kinetics of aggregation and crystallization of polyaspartic Acid stabilized calcium phosphate particles at high concentrations.

Bone is an important material to study due to its exceptional mechanical properties and relevance with respect to hard tissue regeneration and repair. A significant effort has been directed toward understanding the bone formation process and the production of synthetic bone mimicking materials. Here, the formation and structural evolution of calcium phosphate (CaP) was investigated in the presence of relatively high concentrations of calcium, phosphate, and polyaspartic acid (pAsp) using dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). The incipient CaP aggregates were comprised of spherical nanoparticles (diameter ≈ 3-4 nm); they became preferentially aligned over time and eventually transformed into nanorods. The nanorods remained stable in suspension with no signs of further aggregation for at least four months. Detailed cryo-TEM suggested that the CaP nanorods formed through an oriented attachment mechanism. These results show that the reaction concentration greatly influences the mechanism and final properties of CaP. Mechanistic insights gained from this study will facilitate better design and fabrication of bioinspired materials.

Toward a common standard for data and specimen provenance in life sciences.

Open and practical exchange, dissemination, and reuse of specimens and data have become a fundamental requirement for life sciences research. The quality of the data obtained and thus the findings and knowledge derived is thus significantly influenced by the quality of the samples, the experimental methods, and the data analysis. Therefore, a comprehensive and precise documentation of the pre-analytical conditions, the analytical procedures, and the data processing are essential to be able to assess the validity of the research results. With the increasing importance of the exchange, reuse, and sharing of data and samples, procedures are required that enable cross-organizational documentation, traceability, and non-repudiation. At present, this information on the provenance of samples and data is mostly either sparse, incomplete, or incoherent. Since there is no uniform framework, this information is usually only provided within the organization and not interoperably. At the same time, the collection and sharing of biological and environmental specimens increasingly require definition and documentation of benefit sharing and compliance to regulatory requirements rather than consideration of pure scientific needs. In this publication, we present an ongoing standardization effort to provide trustworthy machine-actionable documentation of the data lineage and specimens. We would like to invite experts from the biotechnology and biomedical fields to further contribute to the standard.

Different kinetic pathways of early stage calcium-phosphate cluster aggregation induced by carboxylate-containing polymers.

Acidic proteins are critical to biomineral formation, although their precise mechanistic function remains poorly understood. A number of recent studies have suggested a nonclassical mineralization model that emphasizes the importance of the formation of polymer-stabilized mineral clusters or particles; however, it has been difficult to characterize the precursors experimentally due to their transient nature. Here, we successfully captured stepwise evolution of transient CaP clusters in mineralizing solutions and studied the roles of functional polymers with laser light scattering (LLS) to determine how these polymers influence the stability of nanoclusters. We found that the polymer structure can alter CaP aggregation mechanisms, whereas the polymer concentration strongly influences the rate of CaP aggregation. Our results indicate that the ability of acidic biomolecules to control the formation of relatively stable nanoclusters in the early stages may be critical for intrafibrillar mineralization. More importantly, LLS provided information about the size and the structural evolution of CaP aggregates, which will help define the process of controlled biomineralization.

Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR).

Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.

Cooperative calcium phosphate nucleation within collagen fibrils.

Although "chaperone molecules" rich in negatively charged residues (i.e., glutamic and aspartic acid) are known to play important roles in the biomineralization process, the precise mechanism by which type I collagen acquires intrafibrillar mineral via these chaperone molecules remains unknown. This study demonstrates a mechanism of cooperative nucleation in which three key components (collagen, chaperone molecules, and Ca(2+) and PO(4)(3-)) interact simultaneously. The mineralization of collagen under conditions in which collagen was exposed to pAsp, Ca(2+), and PO(4)(3-) simultaneously or pretreated with the chaperone molecule (in this case, poly(aspartic acid)) before any exposure to the mineralizing solution was compared to deduce the mineralization mechanism. Depending on the exact conditions, intrafibrillar mineral formation could be reduced or even eliminated through pretreatment with the chaperone molecule. Through the use of a fluorescently tagged polymer, it was determined that the adsorption of the chaperone molecule to the collagen surface retarded further adsorption of subsequent molecules, explaining the reduced mineralization rate in pretreated samples. This finding is significant because it indicates that chaperone molecules must interact simultaneously with the ions in solution and collagen for biomimetic mineralization to occur and that the rate of mineralization is highly dependent upon the interaction of collagen with its environment.

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Effects of sample preparation on bacterial colonization of polymers.

Characterization of materials developed for medical usage frequently includes studies in which the materials are inoculated with bacteria in order to assess bacterial colonization and biofilm formation. Observed differences in bacterial growth are typically considered to be due to the material or the incubation conditions. To our knowledge, the method used to prepare the materials has generally not been considered with regard to its influence on bacterial colonization. The objective of this study was to determine the effects that various preparation methods exert on bacterial colonization of polymer disks. Polymer disks of the same dimethacrylate composition were photopolymerized: (1) between untreated glass slides, (2) between polyester release film, (3) between glass slides treated with an alkyl silane, (4) between glass slides treated with a perfluorinated silane, or (5) with one free surface in an argon-purged chamber. Surface chemistry was quantified using X-ray photoelectron spectroscopy, hydrophobicity was assessed by water contact angle, and topography was characterized using atomic force microscopy. The disks were inoculated with Streptococcus mutans for 4 h, fixed, and visualized using confocal laser scanning microscopy. Differences among all groups were found with regard to surface chemistry, hydrophobicity, topography, and bacteria morphology, density, and coverage, indicating that the method of sample preparation strongly affects both the surface properties and the initial bacterial colonization. Polymerization on untreated slides was selected as the preferred method of preparation due to minimal material transfer to the polymer and consistent, reproducible bacterial colonization.

Exploring cellular contact guidance using gradient nanogratings.

Nanoscale surface features that mimic extracellular matrix are critical environmental cues for cell contact guidance and are vital in advanced medical devices in order to manipulate cell behaviors. Among them, nanogratings (line-and-space gratings) are common platforms to study geometric effects on cell contact guidance, especially cell alignment, but generally are one pattern height per platform. In this study, we developed a strategy to fabricate controlled substrates with a wide range of pattern shapes and surface chemistries and to separate surface chemistry and topography effects. As a demonstration of this strategy, six nanograting platforms on three materials were fabricated and applied to examine and differentiate the effects of surface topography and surface chemistry on cell contact guidance of murine preosteoblasts. All of the six platforms contained the same gradient in pattern height (0 to ≈350 nm). They were prepared using nanoimprint lithography and annealing for thermoplastic materials (low molecular weight polystyrene (PS) and polymethylmethacrylate (PMMA)) and photoimprint for a thermoset material (a cross-linked dimethacrylate (DMA)). Each material contains two platforms that are only different in line-and-space pitch (420 or 800 nm). The DMA nanogratings had a reverse line-and-space profile to those of the PS and PMMA nanogratings. Using these platforms, a full range of cell alignment, from randomly orientated to completely parallel to the grating direction was achieved. Results from focal adhesion assays and scanning electronic microscopy indicated a change in cell-substrate contact from a noncomposite state (full contact) to a composite state (partial contact between cell and substrate) as pattern height increased. These gradient platforms allowed for the separation of surface chemistry and surface topography to provide insight into the mechanisms responsible for cell contact guidance on nanopatterned surfaces.

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation.

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (beta-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. beta-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of beta-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of beta-sheet content. The increased beta-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.