Advanced Western Blotting Solutions for Vaccine Development
Simple Western™ systems can help you develop and characterize your vaccine candidates and are used widely to improve reproducibility, quantitation, throughput and time to market. With the ability to separate proteins by either Size or Charge, Simple Western offers unparalleled speed and unique insights that have been leveraged throughout vaccine development workflows.
The Simple Western Advantage
By offering automation, reproducibility, sensitivity, and quick time to results, Simple Western has proven to be an invaluable tool to replace traditional Western blots and other immunoassay technologies across the entire vaccine development pipeline. It is also used as a second, orthogonal immunoassay technique for method development and characterization of critical quality attributes.
- Quantitation: Simple Western systems provide quantitative protein measurements with a 3 log dynamic range
- Reproducibility: Simple Western eliminates the unavoidable variation from traditional western blotting methods, with intra-assay CV’s of <15%.
- Sensitivity: Boost your sensitivity for immunoassays and total protein assays using Simple Western which can detect as little as 150 pg of protein in 3 µL of sample.
- Throughput: Simple Western can generate up to 96 data points in a single overnight run, all in a fully automated fashion! You can generate quantitative data and screen vaccine candidates with triplicate datapoints overnight – something that would take days, if not weeks, with traditional methods Western blotting.
- Method transferability: Using Compass for Simple Western, all data and records are digital, allowing for easy analysis, transfer and archiving between sites or from sponsors to CROs.
- Compliance: Compass software is 21 CFR Part 11 compliant and is GMP ready for your organization’s needs.
Simple Western systems have been used in numerous publications by both academic and biopharma groups in studies directly related to vaccine development. In this Scientific Review, we highlight some key publications which utilize Simple Western to accelerate vaccine development workflows.
Accelerate All Stages of Your Vaccine Development Workflow
Simple Western can be used to measure protein in crude cell lysates in upstream samples as well as in samples further downstream to detect impurities and assess final product release, stability, and characterization. In this Publication Spotlight, we provide more detail on how Simple Western was used to characterize the Zaire Ebola vaccine development in each step of the process and accelerate development of a critically needed vaccine amid a global health crisis.
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Process Stop Electropherogram obtained by Simple Western.
Impurity and Bioprocess Contaminant Screening With Simple Western
Simple Western assays have already been optimized for characterization of several different critical quality attributes including detecting process-related contaminants like host cell proteins, Protein A, GFP, and BSA, making it easy for you to get Simple Western integrated into your cell therapy workflow. In this application note, learn more about how Simple Western platforms detect contaminants in denatured and reduced samples, with the sensitivity needed to confirm if their concentration falls below regulatory guidelines.
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E. coli HCP antigen detection on Simple Western. Compass for Simple Western lane and graph views (A). The concentration of HCP lysate plotted against the average background-subtracted total peak area (B). E. coli HCP antigen was diluted in 0.1X Sample Buffer and titrated from 33.3 μg/mL to 1.23 μg/mL following default Simple Western protocols. Goat anti-E. coli HCP (1:10) was diluted in Antibody Diluent 2 (PN 042-203), followed by the anti-goat secondary HRP conjugate.
Beat Traditional SDS-PAGE Precision and Sensitivity for Purity Measurements
Purity of the final vaccine product is often confirmed using detection methods like SYPRO Ruby or Coomassie staining methods which reportedly require lower nanogram levels for reliable detection. The precision, sensitivity and flexibility of Simple Western rivals traditional approaches for detecting process-related contaminants. This is supported by the achievable R², LOD, LOQ and CV values.
With Simple Western, you can achieve limits of detection in the upper picogram range. In this application note, we describe the use of 5X total protein labeling reagent in conjunction with RePlexTM to achieve SYPRO-Ruby-beating total protein assay sensitivity which equips researchers to detect low abundance impurities, get the most data out of their precious samples and normalize their protein expression data with confidence.
Comparison of AAV total protein detection with SYPRO Ruby and 5X labeling reagent with Simple Western (A) . Simple Western analysis graph view (left), lane view (middle), and linearity analysis (right); (B) Side-by-side comparison of the 1:40 dilution on Wes (left) and SYPRO Ruby (right).
Rich Multi-Analyte Characterization of the Humoral Response With Simple Western Serology Assays
Compared to a single antigen assay, Simple Western serology assays can be used to detect serum antibodies against multiple different antigens simultaneously to give a more complete view into the humoral response. The SARS-CoV-2 Multi-Antigen Serology Module for Simple Western can be utilized to detect and quantify the humoral response to five key SARS-CoV-2 antigens in serum or plasma simultaneously in a single capillary while also providing molecular weight information of each antigen, all without compromising sensitivity.
Learn more about the SARS-CoV-2 Multi-Antigen Serology Module for Simple Western
Detection of all five antigens in a single capillary. The electropherogram resulting from the analysis is shown on the left and the lane view is shown on the right. Each antigen was detected with a humanized anti-His primary antibody and anti-human IgG HRP conjugate supplied in the SARS-CoV-2 Multi-Antigen Serology Module
In this application note, we show how the SARS-CoV-2 Multi-Antigen Serology Module allows for simultaneous detection of human serum IgG reactivity with 5 viral antigens from SARS-CoV-2, including the nucleocapsid, the spike protein, as well the spike protein’s S2, S1, and S1 RBD subunits. As reactivity with different antigens may indicate different stages of infectivity and neutralization, this kit provides a richer view into the immune response to SARS-CoV-2 in a single, quick, and easy assay.
Simple Western Streamlines Serum Antibody Analysis
Simple Western assays can also help measure serum antibody levels to confirm immune responses against an antigen, to test for antibody production in response to vaccination. In this application note, we used Simple Western to detect autoantibodies in lupus patient serum as a model system to generate proof-of-concept data for the assay.
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Normal and SLE patient serum samples were diluted 1:10 in Antibody Diluent 2 and introduced in the primary incubation step to capillaries already immobilized with known lupus antigens. Neither patient had autoantibodies for PCNA (A) while both had equivalent amounts of U1-snRNP A (D). Only the SLE patient had Ro/SS-A autoantibodies (B), and the SLE patient also had 40X more U1-snRNP 68/70 compared to the normal patient (C).
Poster: Characterization of a biological active soluble Human Cytomegalovirus gH/gL/pUL128/130/131 pentameric complex
In this poster, scientists from Merck present results from an attempt to better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization using soluble gH/gL/pUL128-131 pentameric complex (PC) and gH/gL from Chinese hamster ovary cells. Here the charge-based separation of Simple Western was utilized to quantitatively to assess the charge heretrogeneities of gH/gL and PC, purity of gH/gL/pUL128–131 PC and confirm the immunospecificity. The results highlight the importance of the gH/gL/pUL128-131 PC in HCMV vaccine design and emphasize the necessity to monitor the integrity of the PC during vaccine manufacturing process.
Simple Western webinars in vaccine development
Webinar: Application of Next Generation Analytical Tools for Vaccine Development
In this webinar, learn how scientists from Merck and Medgene Labs use Simple Western and other ProteinSimple platforms to assess proteins used in vaccine development for critical quality attributes such as identity and purity, that support the entire vaccine development process, from discovery to QC and lot release.
Webinar: Tackling the Challenges of Human Disease and Vaccine Research with Simple Western
In this on-demand webinar, speakers describe a range of applications utilizing the Simple Western technology to overcome some of their research challenges and get the answers they need.
Dr. Chris Esapa from the Mammalian Genetics Unit in MRC Harwell presents some of the challenges he has faced in quantifying protein expression levels using traditional Western blot analysis and how Simple Western has contributed to the study of molecular mechanisms using models of human disease enabling them to answer crucial scientific questions in disease processes relating to pathway analysis, and the role of post-translational modification such as phosphorylation, which is central to several of their research programs. John Loughney from Merck follows with a presentation on his vaccine development project hindered by the limitations of ELISA assays. He describes how Simple Western has allowed his team to detect specific impurities in vaccine process purification with additional specificity by size separation and increased precision and reduced analyst bench work.
Webinar: The Simple Western approach to Vaccine and Clinical Protein Research
Watch this on-demand webinar, where Melissa Hamm from Vaccine Analytical Development, Merk & Co. talks about a novel approach to vaccine development and Alice Fan, M.D., from Stanford University School of Medicine will discuss the application of Simple Western systems in a study involving nanoscale proteomics for profiling tumors as well as the therapeutic response to cancer.