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.

Management of Metadata Types in Basic Cardiological Research.

INTRODUCTION: Ensuring scientific reproducibility and compliance with documentation guidelines of funding bodies and journals is a topic of greatly increasing importance in biomedical research. Failure to comply, or unawareness of documentation standards can have adverse effects on the translation of research into patient treatments, as well as economic implications. In the context of the German Research Foundation-funded collaborative research center (CRC) 1002, an IT-infrastructure sub-project was designed. Its goal has been to establish standardized metadata documentation and information exchange benefitting the participating research groups with minimal additional documentation efforts. METHODS: Implementation of the self-developed menoci-based research data platform (RDP) was driven by close communication and collaboration with researchers as early adopters and experts. Requirements analysis and concept development involved in person observation of experimental procedures, interviews and collaboration with researchers and experts, as well as the investigation of available and applicable metadata standards and tools. The Drupal-based RDP features distinct modules for the different documented data and workflow types, and both the development and the types of collected metadata were continuously reviewed and evaluated with the early adopters. RESULTS: The menoci-based RDP allows for standardized documentation, sharing and cross-referencing of different data types, workflows, and scientific publications. Different modules have been implemented for specific data types and workflows, allowing for the enrichment of entries with specific metadata and linking to further relevant entries in different modules. DISCUSSION: Taking the workflows and datasets of the frequently involved experimental service projects as a starting point for (meta-)data types to overcome irreproducibility of research data, results in increased benefits for researchers with minimized efforts. While the menoci-based RDP with its data models and metadata schema was originally developed in a cardiological context, it has been implemented and extended to other consortia at GÃuttingen Campus and beyond in different life science research areas.