(W1230-11-63) A Novel First-in-Class 3D Printing Technology for Advanced Manufacturing of Complex Vaccine Formulations against Influenza and Emerging Infectious Diseases

Purpose: In this project, we propose manufacturing bioengineered P. pastoris incorporated F127-BUM microcarriers using advanced SMART 3D printing technology, avoiding cell-destructive forces. The printed microparticles will be flash frozen at -80°C or lower on the print bed and then lyophilized for stability, storage, and transport. The final product can be used for recombinant protein production in various fermenters, enhancing and simplifying their operation.

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%, 1%, and 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. 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. 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: Identification and Characterization of Recombinant Yeast Monoclonal Colonies. Hemagglutinin (HA): Recombinant yeast monoclonal colonies were identified using colony PCR with specific primers: 5AOXF2 and HA-R1 (expected size: 1479 bp) and HA-F1 and TEF1-R1 (expected size: 1132 bp). The PCR results showed products of the expected sizes. The HA protein, with a theoretical molecular weight of 64 kDa and a His tag at the C-terminus, was detected using dot-blot analysis, revealing its expression levels in both the supernatant and ground cells of the engineered yeast strains. Epstein-Barr Nuclear Antigen 1 (EBNA1): Recombinant yeast monoclonal colonies were identified by colony PCR using primers 5AOXF2 and TEF1-R1 (expected size: 1699 bp). The PCR product size matched expectations. EBNA1 protein expression was confirmed by SDS-PAGE, Western blot, and dot-blot analyses. EBNA1 was secreted into the supernatant, with higher expression levels detected in the cells.
Highly Pathogenic Avian Influenza Antigen (H5N1): Recombinant yeast monoclonal colonies were identified by colony PCR using primers 5AOXF2 and H5N1-R1 (expected size: 1459 bp). The PCR product size was as expected. The H5N1 protein was detected by SDS-PAGE and Western blot, but not by dot-blot, indicating low expression levels. H5N1 was not secreted into the supernatant but was detected in some monoclonal cell pellets.
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.

Conclusion: We utilized three different bioengineered Pichia pastoris strains, encapsulated them with alginate hydrogel, and demonstrated enhanced protein production. For future experiments, we plan to encapsulate the yeast with F127-BUM hydrogel and run it through our continuous process. This will help determine if the protein expression system remains consistent throughout the entire manufacturing process. Confirming this will further validate the robustness and efficiency of our method.

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

EBNA1 protein expression was tested by western blot in (A) the supernatant and (B) the pellet, and by (C) dot blot.

Testing Protein Expression of bioengineered Pichia pastoris Using ELISA

SEM images of A) free yeast cells, B) yeast encapsulated alginate microparticles.