Accelerate Your Cell & Gene Therapy Workflows with Advanced Western Blotting Solutions
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Monitor AAV Capsid Identity, Purity, and Capsid Ratios During Product Purification
Simple Western immuno- and total protein assays can be used to monitor AAV capsid proteins throughout the purification process. From measuring the expression of novel AAV capsids to downstream manufacturing and QC, Simple Western systems can accurately measure product identity and purity where it is imperative. In this Application Note, learn how the Cell and Gene Therapy Consortium Catapult uses highly specific PROGEN antibodies in fully automated Simple Western assays to monitor and characterize AAV capsids during product purification.
Immunodetection of VP1, VP2 and VP3 proteins during purification from whole-cell lysate
Total Protein detection of VP1, VP2 and VP3 proteins during purification from whole-cell lysate.
The Next Generation of AAV Characterization Tools
As the gene therapy field continues to expand and companies increase the number of product batches manufactured per year, quality control requirements for product release have increased significantly. In this webinar, Tony Bou Kheir, PhD, from the Cell and Gene Therapy Catapult in London, showcases an advanced analytical platform for AAV vector characterization, addressing industry challenges in 1) assay precision, 2) standardization and throughput, and 3) enabling in-line product characterization in closed processes.
Limited in AAV sample availability? In this virtual poster, we describe a method to monitor the purification of AAV2 using automated capillary electrophoresis followed by immunoassay and total protein detection directly in the capillary, eliminating the need for SDS-PAGE. In this collaborative poster between the Cell & Gene Therapy Catapult, PROGEN and ProteinSimple, we describe a highly sensitive, fully automated assay which effectively reduces sample size requirements down to 3 µL of AAV sample, corresponding to approximately 400 pg or 1x108 genomic copies loaded per well.Download AAVAnalysis Poster
Identify Process-Related Impurities
Current methods for assessing the concentration of residuals like enzyme-linked immunosorbent assays (ELISAs), flow cytometry and traditional Western blotting are labor-intensive, prone to error and can also be misleading. Simple Western systems automate the process start to finish, and capture critical information on sizing and oligomerization, giving you more flexibility to build a comprehensive contaminant profile. The methods outlined in this application note for the evaluation and characterization of HCP, Protein A, GFP and BSA offer proof-of-concept methods for adaptation as quantitative and reliable protocols throughout the development process of a biotherapeutic.
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.
Ultra-High Sensitivity AAV Purity Measurements to Conserve Your Precious AAV samples
Many manufacturers of AAVs still rely on traditional SDS-PAGE with SYPRO Ruby staining to monitor purification. Unlike Simple Western, SYPRO Ruby staining is labor-intensive with many manual washing steps, generates large volumes of liquid waste, and requires special imaging equipment. Furthermore, SYPRO Ruby requires at least 1 ng of protein for reliable detection. In contrast Simple Western can reliably detect as little as 150 pg. In this application note, we demonstrate a Simple Western total protein assay workflow for AAV purity measurements that is more sensitive than SYPRO Ruby. Get the purity information you need while conserving your precious AAV samples!
Side-by-side comparison of high sensitivity AAV total protein assay on Simple Western (left) and SYPRO Ruby (right) shows Simple Western outperforms SYPRO Ruby in sensitivity in addition to improved automation and time to results.
SINGLE-CELL WESTERNS MEASURE GENE EDITING EFFICIENCY FOR SICKLE-CELL RESEARCH
Single-Cell Westerns measure protein expression in thousands of single cells per run. Their high sensitivity combined with the ability to use conventional Western blotting antibodies makes them particularly well-suited for quantifying gene therapy efficacy and tracking differentiation. For example, Milo can resolve hemoglobin subunits in single red blood cells (RBCs), something that is nearly impossible to do with flow cytometry, enabling researchers to track efficacy of sickle cell gene therapies. In addition, Milo offers the ability to quantify editing efficiency of hematopoietic stem cells (HSCs), quantify differentiation efficiency of HSCs into RBCs, and differentiate between hemoglobin subunits, measuring their expression with existing commercial reagents. Read this publication spotlight to learn how Milo's Single-Cell Westerns support clinical trials aimed at advancing gene therapies for Sickle Cell Disease and other hemoglobinopathies.
Single-Cell Western analysis of red blood cells from patients treated with LentiGlobin BB305 Drug Product. Figure adapted with permission from Bonner et al.
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