enGenes participates in the study of large scale production strategies for microbial carbohydrate-binding toxins

Scientists from recombinant proteins specialist CDMO enGenes Biotech GmbH (enGenes) have collaborated with researchers from Germany and the UK in a study that focuses on scalable production of the carbohydrate-recognizing toxins exploited for their significant role in the etiology of many human diseases.

Their research review paper ‘Microbial carbohydrate-binding toxins – From etiology to biotechnological application’ is due for publication in the October 2022 issue of the journal Biotechnology Advances [1] and has just been made available online.

Efficient expression

The study seeks to promote a greater understanding of how to efficiently express and purify recombinant toxins and their carbohydrate-binding domains to enable opportunities for the formulation of innovative biopharmaceuticals that can improve human health.

Lead author Natalia Danielewicz, a doctoral candidate at enGenes and a fellow of the Department of Biotechnology at Vienna’s University of Natural Resources and Life Sciences, and enGenes CEO Dr. Jürgen Mairhofer collaborated on the study with fellow researchers Francesca Rosato and Winfried Römer from the Faculty of Biology and the  Signaling Research Centers BIOSS and CIBSS at Albert Ludwigs University, Freiburg, Wenyue Dai from the Freiburg Institute for Advanced Studies (FRIAS), at the University of Freiburg, and Dr W. Bruce Turnbull, of the School of Chemistry and Astbury Centre for Structural Molecular Biology, at University of Leeds, UK.

The study was funded by synBIOcarb European Training Network, which has received funding from the European Union’s Horizon 2020 research and innovation program.

Applied glycoscience

It was inspired by an increased need for efficient and scalable manufacturing processes for carbohydrate-binding toxins, to meet a growing number of applied glycoscience therapeutic approaches that rely on the interactions of carbohydrates with proteins to provide innovations in the domain of vaccines, vaccine adjuvants, drug-delivery, neuronal tracers or sensors and detectors for small molecules and glycans.

The carbohydrate recognition domains  play essential roles in delivery of many microbial toxins to target organisms using different routes, from promoting pore formation to facilitating the entry of toxic enzymatic subunits into the host cell. These toxins are often stable proteins that are amenable to protein engineering and thus present an ideal opportunity for exploitation in innovative applications such as novel diagnostic tools, recognition of glycobiomarkers, therapeutics and platforms for vaccine and adjuvant designs.

Glycoproteins often exhibit disease-associated changes in their glycosylation, for example, during cancerogenesis, and as a result, can act as biomarkers for medical diagnostics. Carbohydrate-binding domains with an affinity for specific carbohydrate-based receptors can also be exploited as drug delivery systems, and new generation biopharmaceutical products and devices (e.g. glycoselective capture of tumour-derived exosomes).

Therefore, a greater understanding of carbohydrate-binding toxins in the context of biotechnological innovation can make a valuable contribution to advances in medicine.

Optimum production regimes

The study provides a comprehensive review covering four key topics:

• Status of toxin-based biopharmaceuticals currently in clinical studies or available on the market
• Use of toxins in developing tumour- and vaccine-directed pharmacological agents
• Choosing the best production regime for making relevant quantities of the desired toxins
• Purification strategies that exploit the toxins’ affinity towards different carbohydrates and glycans.

The study notes that to enable the production of carbohydrate-binding toxins in E. coli, upstream and downstream modules that provide suitable expression and purification strategies need to be coherent, requiring the development of a scalable system for the production of toxins that allows scale up from shake flask, through production in lab-scale bioreactors, to beyond pilot-scale (>100 L). Complementary to this, downstream purification must facilitate straightforward batch to batch reproducibility. Issues with manufacturability of these toxins are often related to their inherent properties, thereby being partly or fully toxic to host organisms such as E. coli, causing partial or even full cell lysis, posing a challenge for the entire manufacturing line.

Conclusions

The study shows that microbial carbohydrate-binding toxins have been successfully implemented in many biopharmaceutical applications, from cancer-targeting to vaccine development. Glyco-selectivity of bacterial toxins has been widely studied in vitro and in animal models, showing high potential in targeting glyco-surfaces for drug delivery or vaccine technology.

“Given the circumstances that the dosage of vaccines or drugs per patient can be high (e.g., 1 mg of recombinant CTxB for the vaccine DUKORAL®), large-scale production is often required for it to become a practical therapeutic option,” the authors note.

“Selecting a well-defined and optimized fermentation regime can improve overall productivity. Production supported by optimized carbohydrate-based affinity chromatography further enables effective bacterial toxin purification. Finding the right production regime of microbial toxins is thus considered important to provide cost-effective manufacturing of vaccines and diagnostic or therapeutic applications,” the authors continue.

“With the future rise of glycobiology the use of these important multi-functional protein complexes will become more and more state-of-the-art and with the advent of in-silico protein design and engineering the carbohydrate-binding functionalities can be crafted on promising effector proteins for cancer cell killing or drug delivery,” the study concludes.

Reference:

Danielewicz, N., Rosato, F., Dai, W., Römer, W., Turnbull, W.B. and Mairhofer, J. (2022). Microbial carbohydrate-binding toxins – From etiology to biotechnological application. Biotechnology Advances, [online] 59, p.107951. Available at: https://www.sciencedirect.com/science/article/pii/S0734975022000477

About enGenes Biotech

enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading-edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of the current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.

enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes, including its proprietary enGenes-X-press™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes-X-press™ has been successfully applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.

enGenes Biotech offers development and manufacturing services tailored the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.

For further information, visit: http://www.engenes.cc