Scalable niomanufacturing of recombinant enzymes for ex vivo conversion of group B red blood cells into ABO O-like cells

What do I want to biomanufacture, and why?

I propose the scalable biomanufacturing of a recombinant enzyme cocktail capable of removing group B antigens from donated red blood cells (RBCs), producing cells with an O-like phenotype within the ABO system. Blood group compatibility depends partly on carbohydrate antigens displayed on the RBC surface. In emergencies and blood shortages, group O RBCs are especially valuable because they lack A and B antigens. However, O-negative and other group O supplies are limited. In 2024, Jensen et al. identified exoglycosidases from the mucin-degrading gut bacterium Akkermansia muciniphila that can remove canonical and extended A and B blood group antigens. The study reported improved compatibility between enzyme-treated RBCs and group O plasma. This suggests that gut microorganisms can provide enzymes for expanding the supply of ABO-compatible blood. My proposed product is not a living bacterium administered to a patient. It is a purified recombinant enzyme cocktail used ex vivo in a controlled blood-processing system. The proposal focuses on group B RBCs because their enzymatic conversion currently appears more efficient and requires less enzyme than the conversion of group A cells. The resulting cells should be described as ABO O-like, rather than universally compatible blood, because other clinically important antigens, including Rh and Kell, would remain present.

What living system would manufacture it

Genes encoding selected A. muciniphila exoglycosidases would be synthesized, cloned and expressed separately in an established recombinant protein-production chassis such as Escherichia coli BL21(DE3). The engineered bacteria would be cultivated in stirred-tank bioreactors. After recombinant expression, the cells would be harvested and lysed. Each enzyme would be purified using chromatography and filtration, followed by endotoxin removal. The purified enzymes would then be combined at an optimized ratio and formulated as a standardized cocktail. The manufacturing workflow would therefore be: Enzyme-gene selection and plasmid construction. Transformation of the production strain. Fermentation and recombinant expression. Cell harvesting and lysis. Enzyme purification and endotoxin removal. Cocktail formulation and activity testing. Ex vivo treatment of group B RBCs. Washing and quality control of the treated cells.

How could automation improve the process?

Automation would be useful during both development and manufacturing. During development, a liquid-handling robot could prepare small expression cultures and test combinations of induction temperature, induction time and media composition. It could also create different enzyme ratios and concentrations in microplates. Automated assays could compare antigen removal at different treatment times and temperatures. Flow cytometry, antibody-based agglutination tests and compatibility tests with group O plasma could be used as readouts. Images and experimental metadata would be collected automatically so that the best conditions could be selected through a design-build-test-learn cycle. At manufacturing scale, sensors and control software would regulate temperature, pH, dissolved oxygen, agitation and feeding in the bioreactor. Automated chromatography, tangential-flow filtration and endotoxin testing would improve consistency between batches. A separate closed blood-processing device could control enzyme addition, incubation, washing and sampling of each RBC unit.

What would be a meaningful scale?

A meaningful initial target would be enough purified enzyme cocktail to treat 1,000 units of group B RBCs in a pilot blood-bank study. Jensen et al. estimated that approximately 8 mg of the limiting enzyme would be needed to treat one 200 mL unit of group B RBCs. Therefore: 1,000 RBC units × 8 mg per unit = 8,000 mg = 8 g of purified enzyme cocktail For an early process estimate, I will assume: Recombinant enzyme yield: 0.5 g/L of fermentation culture. Overall purification and formulation recovery: 50%. Required final product: 8 g. The required fermentation volume would be: 8 g ÷ (0.5 g/L × 0.50 recovery) = 32 L Because the enzymes would initially be produced separately, approximately two 20 L fermentations could provide enough material while allowing for process losses, quality-control samples and failed batches. A tentative manufacturing cycle could take: 1–2 days for fermentation and enzyme expression. 1 day for harvesting and purification. 1 day for formulation and quality control. This gives approximately 3–4 days per enzyme-production batch. Treatment of an individual RBC unit could require approximately 30 minutes of enzyme incubation, followed by washing and quality-control testing. The production yield, recovery and processing times are preliminary assumptions that would need experimental validation.

Important unknowns

Completeness and donor variability: Can the enzyme cocktail consistently remove canonical and extended group B antigens from RBCs obtained from genetically diverse donors? RBC safety and quality: Does enzyme treatment or subsequent washing affect membrane integrity, oxygen transport, deformability, storage life or transfusion survival? Manufacturing and clinical safety: Can the enzymes be produced economically with reproducible activity and acceptably low concentrations of endotoxin, host-cell proteins and residual enzymes? Before clinical use, the treated RBCs would require extensive preclinical validation, compatibility testing, storage studies and controlled clinical trials.

References

Jensen, M. et al. (2024). Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood. Nature Microbiology, 9, 1176–1188. https://doi.org/10.1038/s41564-024-01663-4 Rahfeld, P. et al. (2019). An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nature Microbiology, 4, 1475–1485. https://doi.org/10.1038/s41564-019-0469-7 Petzold, C. J. & Mukhopadhyay, A. (2025). From bench to biofactory: high-throughput technologies and automated workflows to accelerate biomanufacturing. Current Opinion in Biotechnology, 94, 103320. https://doi.org/10.1016/j.copbio.2025.103320

Use of AI

I used ChatGPT by OpenAI to facilitate the search for relevant scientific papers, help me identify information needed for the scale calculations, and improve the grammar and clarity of my writing because English is not my native language. I reviewed the cited sources and made the final decisions regarding the proposal, assumptions and calculations.