Optimization and validation of analytical affinity chromatography for the in-process monitoring and quantification of peptides containing a C-tag.

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 min 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.

Process intensification for the production of a C-tagged antimicrobial peptide in Escherichia coli - First steps toward a platform technology.

The production of antimicrobial peptides/proteins (AMPs) in sufficient quantities for clinical evaluation is challenging because complex peptides are unsuitable for chemical synthesis, natural sources have low yields, and heterologous systems often have low expression levels or require product-specific process adaptations. Here we describe the production of a complex AMP, the insect metalloproteinase inhibitor (IMPI), by adding a C-terminal C-tag to increase the yield compared to the unmodified peptide. We used a design of experiments approach for process intensification in Escherichia coli Rosetta-gami 2(DE3)pLysS cells and achieved a yield of 260 mg L-1, which is up to 30-fold higher than previously reported. The C-tag also enhanced product purity but had no effect on IMPI activity, making tag removal unnecessary and therefore simplifying process analytics and downstream processing. We have confirmed that the C-tag is compatible with the peptide and could form the basis of a platform technology for the expression, purification and detection of diverse AMPs produced in E. coli.