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• W4387079717 abstract "Antimicrobial peptides and proteins (AMPs) are promising alternatives to conventional antibiotics for the treatment of infections caused by multidrug-resistant bacteria. The production of recombinant AMPs is facilitated by platform technologies such as the C-tag, a sequence of four C-terminal amino acids that allows immunoaffinity capture and purification. However, the detection and quantification of such products throughout the manufacturing process is a significant challenge. We therefore used a design of experiments approach to optimize a novel high-throughput analytical immunoaffinity chromatography method for the accurate quantification of AMPs containing a C-tag, resulting in minimal analyte carryover (98.8 ± 0.1% product elution). We then validated the method in accordance with International Conference on Harmonisation guideline Q2(R2). Validation confirmed that the method achieves high specificity, linearity, accuracy, and precision. We implemented in-process control and quantification throughout the manufacturing process, from cell lysis to the final purified product. We found that the lysate and acidic samples (pH < 2) can lead to deviations. However, following sample pretreatment, C-tag quantification reduced the error to ≤ 4%, which is potentially superior to current non-specific quantification methods such as UV absorbance and colorimetry. Implementing this method for in-process control and quantification throughout the manufacturing process achieves the reliable assessment of product quantity and quality. This method also offers improvements over the product-specific enzyme-linked immunosorbent assay currently used for C-tagged products because it has a higher precision, accuracy and throughput, with a measurement time of 2.5 minutes per sample. Our analytical affinity chromatography method is therefore a valuable tool for the quantification of AMPs as part of a novel platform technology approach for C-tagged products." @default.
• W4387079717 created "2023-09-28" @default.
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• W4387079717 date "2023-09-01" @default.
• W4387079717 modified "2023-10-09" @default.
• W4387079717 title "Optimization and validation of analytical affinity chromatography for the in-process monitoring and quantification of peptides containing a C-tag" @default.
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• W4387079717 doi "https://doi.org/10.1016/j.jchromb.2023.123899" @default.
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