Fully automated high-throughput immuno-µPlaque assay for live-attenuated tetravalent dengue vaccine development.

Dengue fever has remained a continuing global medical threat that impacts half of the world's population. Developing a highly effective dengue vaccine, with live-attenuated tetravalent vaccines as leading candidates, remains essential in preventing this disease. For the development of live virus vaccines (LVVs), potency measurements play a vital role in quantifying the active components of vaccine drug substance as well as drug product during various stages of research, development, and post-licensure evaluations. Traditional plaque-based assays are one of the most common potency test methods, but they generally take up to weeks to complete. Less labor and time-intensive potency assays are thus called for to aid in the acceleration of vaccine development, especially for multivalent LVVs. Here, we introduce a fully automated, 96-well format µPlaque assay that has been optimized as a high-throughput tool to evaluate process and formulation development of a live-attenuated tetravalent dengue vaccine. To the best of our knowledge, this is the first report of a miniaturized viral plaque method for dengue with full automation via an integrated robotic system. Compared to the traditional manual plaque assay, this newly developed method substantially reduces testing time by approximately half and allows for the evaluation of over ten times more samples per run. The fully automated workflow, from cell culture to plaque counting, significantly minimizes analyst hands-on time and improves assay repeatability. The study presents a pioneering solution for the rapid measurement of LVV viral titers, offering promising prospects for advancing vaccine development through high-throughput analytics.

Correlating Stability-Indicating Biochemical and Biophysical Characteristics with In Vitro Cell Potency in mRNA LNP Vaccine.

The development of mRNA vaccines has increased rapidly since the COVID-19 pandemic. As one of the critical attributes, understanding mRNA lipid nanoparticle (LNP) stability is critical in the vaccine product development. However, the correlation between LNPs' physiochemical characteristics and their potency still remains unclear. The lack of regulatory guidance on the specifications for mRNA LNPs is also partially due to this underexplored relationship. In this study, we performed a three-month stability study of heat-stressed mRNA LNP samples. The mRNA LNP samples were analyzed for their mRNA degradation, LNP particle sizes, and mRNA encapsulation efficiency. In vitro cell potency was also evaluated and correlated with these above-mentioned physiochemical characterizations. The mRNA degradation-cell potency correlation data showed two distinct regions, indicating a critical cut-off size limit for mRNA degradation. The same temperature dependence was also observed in the LNP size-cell potency correlation.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Greening automation: Wash and Re-use of disposable 384-well liquid handling tips to enable sustainable high-throughput vaccine development.

Laboratory automation uses large amounts of plastic consumables, generating substantial single-use plastic waste. Automated ELISAs are an indispensable analytical tool in vaccine formulation and process development. Current workflows, however, rely on disposable liquid handling tips. In progress toward sustainability, we developed workflows for washing 384-well format liquid handling tips, using nontoxic reagents, for re-use during ELISA testing. We estimate that this workflow reduces plastic and cardboard waste in our facility by 989 kg/year and 202 kg/year, respectively, without introducing new chemicals into our waste steam.

Quantitation of host cell proteins in biopharmaceuticals from chinese hamster ovarian and vero cell lines using capillary electrophoresis western blots.

Quantitation of host cell proteins (HCPs) is essential in the process of preparation of many biological and vaccine products. Common methods of quantitation include the widely applied enzyme-linked immunosorbent assays (ELISAs), mass spectrometry (MS) and other orthogonal assays. Prior to using these techniques, critical reagents need to be evaluated, for example, antibodies need to be assessed for HCP coverage. Percent of HCP coverage is often established by denatured 2D Western blot. However, ELISAs measure the amount of HCP only in a native state. There are limited studies linking reagents validated by 2D-Western to ensure adequate coverage in the final ELISA. ProteinSimple's newly developed capillary Western blot technology allows for separation, blotting, and detection of proteins in a semi-automated and simplified format. Capillary Westerns are similar to slab Westerns, with the added benefit of being quantitative. Here we outline the capillary Western method that links the 2D Western coverage and ultimately ELISAs for more efficient HCP quantitation. This study describes the development of the capillary Western analytical method to quantitively evaluate HCPs in Vero and Chinese Hamster Ovarian (CHO) cell lines. The amount of CHO HCPs decreases as the sample is purified as expected. Using this approach, we determined that the detected Vero HCPs amount was similar irrespective of denatured (capillary Western) versus native assay format (ELISA). This new method can also be potentially employed to quantitatively assess the anti-HCP antibody reagent coverage used in commercial HCP ELISA kits.

Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western.

A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.

Overcoming Biopharmaceutical Interferents for Quantitation of Host Cell DNA Using an Automated, High-Throughput Methodology.

The rapid development of biologics and vaccines in response to the current pandemic has highlighted the need for robust platform assays to characterize diverse biopharmaceuticals. A critical aspect of biopharmaceutical development is achieving a highly pure product, especially with respect to residual host cell material. Specifically, two important host cell impurities of focus within biopharmaceuticals are residual DNA and protein. In this work, a novel high-throughput host cell DNA quantitation assay was developed for rapid screening of complex vaccine drug substance samples. The developed assay utilizes the commercially available, fluorescent-sensitive Picogreen dye within a 96-well plate configuration to allow for a cost effective and rapid analysis. The assay was applied to in-process biopharmaceutical samples with known interferences to the dye, including RNA and protein. An enzymatic digestion pre-treatment was found to overcome these interferences and thus allow this method to be applied to wide-ranging, diverse analyses. In addition, the use of deoxycholate in the digestion treatment allowed for disruption of interactions in a given sample matrix in order to more accurately and selectively quantitate DNA. Critical analytical figures of merit for assay performance, such as precision and spike recovery, were evaluated and successfully demonstrated. This new analytical method can thus be successfully applied to both upstream and downstream process analysis for biologics and vaccines using an innovative and automated high-throughput approach.

Reverse-Phase Ultra-Performance Chromatography Method for Oncolytic Coxsackievirus Viral Protein Separation and Empty to Full Capsid Quantification.

Oncolytic virus immunotherapy is emerging as a novel therapeutic approach for cancer treatment. Immunotherapy clinical drug candidate V937 is currently in phase I/II clinical trials and consists of a proprietary formulation of Coxsackievirus A21 (CVA21), which specifically infects and lyses cells with overexpressed ICAM-1 receptors in a range of tumors. Mature Coxsackievirus virions, consisting of four structural virion proteins, (VPs) VP1, VP2, VP3, and VP4, and the RNA genome, are the only viral particles capable of being infectious. In addition to mature virions, empty procapsids with VPs, VP0, VP1, and VP3, and other virus particles are produced in V937 production cell culture. Viral protein VP0 is cleaved into VP2 and VP4 after RNA genome encapsidation to form mature virions. Clearance of viral particles containing VP0, and quantification of viral protein distribution are important in V937 downstream processing. Existing analytical methods for the characterization of viral proteins and particles may lack sensitivity or are low throughput. We developed a sensitive and robust reverse-phase ultra-performance chromatography method to separate, identify, and quantify all five CVA21 VPs. Quantification of virus capsid concentration and empty/full capsid ratio was achieved with good linearity, accuracy, and precision. ClinicalTrials.gov ID: NCT04521621 and NCT04152863.

SEC coupled with in-line multiple detectors for the characterization of an oncolytic Coxsackievirus.

V937 is an oncolytic virus immunotherapy clinical drug candidate consisting of a proprietary formulation of Coxsackievirus A21 (CVA21). V937 specifically binds to and lyses cells with over-expressed ICAM-1 receptors in a range of tumor cell types and is currently in phase I and II clinical trials. Infectious V937 particles consist of a ∼30 nm icosahedral capsid assembled from four structural viral proteins that encapsidate a viral RNA genome. Rapid and robust analytical methods to quantify and characterize CVA21 virus particles are important to support the process development, regulatory requirements, and validation of new manufacturing platforms. Herein, we describe a size-exclusion chromatography (SEC) method that was developed to characterize the V937 drug substance and process intermediates. Using a 4-in-1 combination of multi-detectors (UV, refractive index, dynamic and static light scattering), we demonstrate the use of SEC for the quantification of the virus particle count, the determination of virus size (molecular weight and hydrodynamic diameter), and the characterization of virus purity by assessing empty-to-full capsid ratios. Through a SEC analysis of stressed V937 samples, we propose CVA21 thermal degradation pathways that result in genome release and particle aggregation.

Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector.

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG-lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed-phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines.

Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines.

Messenger RNA (mRNA)-based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self-adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA-based vaccines encapsulated in lipid nanoparticles using commercial microchip-based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Characterization of gH/gL/pUL128-131 pentameric complex, gH/gL/gO trimeric complex, gB and gM/gN glycoproteins in a human cytomegalovirus using automated capillary western blots.

Human cytomegalovirus (HCMV) is currently a major cause of congenital disease in newborns and organ failure in transplant recipients. Despite decades of efforts, an effective vaccine against HCMV has yet to be developed. However, the discovery of pentameric gH complex on viral surface which contains potent neutralizing epitopes may help enable development of an effective vaccine. In our company ongoing Phase II clinical trial of whole-live virus HCMV vaccine (V160), the pentameric gH complex has been restored on the surface of live attenuated AD169 virus strain. The reconstructed HCMV virus contains a variety of surface glycoproteins including pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, gB glycoprotein, and gM/gN heterodimer complex. To further characterize this virus and enable the monitoring of multiple viral antigens during vaccine process development an effective and efficient analytical strategy was required to detect and quantify several viral surface proteins. In this paper, we present an innovative approach based on capillary western blot technology that allows fast and accurate quantitation of pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, and gB glycoprotein. This method is suitable for analyzing target proteins in multiple sample types including supernatants from infected cell culture, purification intermediates, concentration bulk, and the final vaccine product. In addition, the capillary western blot-based technology identified a previously unknown biochemical profile present in some HCMV viruses: triplet gH peaks of viral surface proteins in non-reducing environment, which could potentially present a new strategy for specificity and identity testing.

Recent advances in isoelectric focusing of proteins and peptides.

This review article describes the significant recent advances in Isoelectric Focusing from the period 2015-2020. The review highlights the principles and common challenges faced in Isoelectric Focusing as well as its applications. This review also details the recent advances in various modes of Isoelectric Focusing in various platforms and future directions for the technique.

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.

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting.

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Identification of adipocyte plasma membrane-associated protein as a novel modulator of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.

Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP-based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability-indicating making it suitable for both process and formulation development.

Quantitation of CRM197 using imaged capillary isoelectric focusing with fluorescence detection and capillary Western.

Maurice is a new instrument that can perform imaged capillary isoelectric focusing (icIEF). The standard detection for icIEF is UV absorbance at 280 nm, which limits its application to high protein concentration samples and non-complex samples. Here we describe an icIEF instrument with fluorescence detection. We demonstrate the advantage of using either icIEF with fluorescence detection or quantitative Western Blot to measure diphtheria toxin mutant CRM197 protein titer in crude cell lysates and purified samples. These two techniques have great potentials to become standard methods to analyze protein titers in crude cell lysate or other complex samples types.