(T1030-02-11) First-in-Class 3D Bioprinting Technology for the Scalable Production of Complex Vaccines Targeting Influenza and Emerging Pathogens

Purpose: In this project, we aim to manufacture bioengineered P. pastoris-loaded F127-BUM microcarriers using advanced SMART 3D printing technology, minimizing exposure to cell-damaging forces. The printed microparticles will be flash-frozen at -80°C or lower directly on the print bed, followed by lyophilization to ensure stability, long-term storage, and ease of transport. The resulting dry microcarriers can be utilized in a variety of fermenters for recombinant protein production, improving operational efficiency and simplifying the production process.

Methods: Fabrication and Characterization of Polymeric Microcarriers Using Batch SMART Technology: Sodium alginate microparticles were prepared using the SMART process to encapsulate both free and engineered P. pastoris strains. Particles were created by extruding 0.3% sodium alginate solutions into a 100mM calcium chloride solution at 200 kPa pneumatic pressure. Development and Verification of Polymeric Microcarriers Incorporating Bioengineered P. pastoris: Bioengineered P. pastoris, optimized for stable recombinant protein expression, was used to develop polymeric microcarriers. The wild-type PAOX1 promoter was used. The target gene was cloned into expression plasmids pPZ-alpha carrying the wild-type AOX1 promoter. The plasmids were linearized and transformed into host P. pastoris by electroporation. Protein expression was confirmed using microfluidic capillary electrophoresis (mCE), SDS-PAGE, and Western Blot. Work has begun on recombinant protein candidates, including hemagglutinin for influenza vaccine, EBNA1 for EBV vaccine, and H5N1 for Avian Influenza vaccine. Morphology and Particle Size Analysis: The morphology of hydrogel-encapsulated yeast was observed under an optical microscope to assess the uniformity and integrity of the microcarriers. Quantification of Protein from P. pastoris Encapsulated Microcarriers: Protein expression analysis was conducted in both free yeast cells and yeast cells encapsulated with or without microparticles using ELISA. Stability Test of Free and Encapsulated Yeast: To evaluate the stability of bioengineered Pichia pastoris, both free yeast and hydrogel-encapsulated yeast were subjected to lyophilization. The samples were placed in a lyophilizer to remove moisture and converted into dry powder. The lyophilized samples were stored at room temperature for up to one month. At 7, 14, and 21 days, a portion of each sample was reconstituted in BMMY medium and incubated under standard culture conditions. The viability and protein expression levels were assessed at 24, 48, and 72 hours post-reconstitution to determine the long-term stability and functionality of the encapsulated yeast compared to free yeast. Setting up the Continuous Process: A prototype for continuous operation of the SMART platform was built, focusing on two major sections: a continuous feeding system and a continuous freezing system.

Results: Quantification of Protein from P. pastoris Encapsulated Microcarriers by ELISA: In the HA bioengineered P. pastoris group, yeast encapsulated with alginate hydrogel produced higher concentrations of HA in both the supernatant (1448.27 ng/ml) and pellet (149.12 ng/ml) compared to other groups. In the EBNA1 bioengineered P. pastoris group, yeast encapsulated with alginate hydrogel produced more protein in the supernatant (637.91 ng/ml) than other groups, with no significant difference observed in the pellet. In the H5N1 group, there was no specific difference in the supernatant between groups; however, the H5N1 expression level was higher in the pellet (26.04 ng/ml) compared to other groups. These observations suggest that encapsulating yeast cells within alginate microparticles can significantly enhance protein production. Stability Test of Free and Encapsulated Yeast: To assess whether hydrogel-encapsulated bioengineered yeast could endure the stress of a flash-freezing system, a preliminary experiment was conducted using a lyophilizer. The encapsulated yeast was lyophilized to produce dry powder, simulating the conditions of flash-freezing and subsequent stabilization. The lyophilized samples were stored at room temperature for up to 21 days. At intervals of 7, 14, and 21 days, the samples were reconstituted in BMMY medium and cultured. Viability and protein expression levels were measured at 24, 48, and 72 hours post-reconstitution. The results demonstrated that the encapsulated yeast retained its structural integrity and effectively produced the target vaccine protein (HA). Additionally, the concentration of the hydrogel-encapsulated bioengineered yeast group exceeded the upper limit of the standard curve, measuring over 745.45 ng/ml.

Conclusion: Our findings demonstrate that hydrogel encapsulation, combined with the SMART 3D printing technique, enhances protein expression and stability in bioengineered Pichia pastoris. Encapsulated yeast maintained consistent protein production even after lyophilization and prolonged storage, unlike free yeast, which exhibited a decline in expression. Additionally, microscopic analysis confirmed the successful formation of hydrogel microcarriers with well-defined structures. Moving forward, further characterization of additional yeast strains and optimization of the continuous manufacturing process will be essential to validate the scalability and robustness of this system for vaccine production.

Acknowledgements: R01 grant from FDA, CBER on novel vaccine technologies


Protein expression test by ELISA


Stability Test of Free and Encapsulated Yeast


Image of Encapsulated Yeast