Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

Abstract

Background Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. Results We describe a holistic approach for the molecular design of recombinant protein antigens—considering both their manufacturability and antigenicity—informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. Conclusions This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

“…Insoluble or misfolded protein inside the host cells could lead to an unfolded protein response and subsequent degradation of the recombinant product, reducing its production. To further evaluate this relationship, we performed a genome-scale analysis of the yeast by RNA-sequencing and compared the gene expression to another strain capable of producing a subunit vaccine candidate for rotavirus (P2-VP4-P [8]) of similar size and complexity at commercially relevant productivities (exceeding 0.5 g/L/d) (23). This reference strain has no additional engineered genomic modifications, so we hypothesized that the observed differences in gene expression between the two strains were due to the host's response to the two different recombinant proteins.…”

“…We created a two-stage chromatographic method to purify RBD based on its biophysical characteristics and prior experience purifying heterologous proteins with similar molecular weight, isoelectric point, and hydrophobicity ( 21 – 23 ). We produced RBD in a 200-mL shake flask culture and purified quantities to assess the quality attributes of the protein ( Fig.…”

“…Small, conservative changes to a protein sequence can address quality attributes of the protein such as aggregation and also reduce strain on cellular functions to improve titer ( 23 ). We undertook a similar approach to molecular engineering for SARS-CoV-2 RBD.…”

Modular development enables rapid design of media for alternative hosts

“…To develop a medium for improved production of a recombinant protein, we chose to use a strain engineered to secrete a rotavirus‐derived subunit vaccine component, VP4‐P[8], as a model protein. We have previously demonstrated that this viral antigen can be expressed at high titer under the control of the methanol‐inducible pAOX1 promoter in BMMY media (Dalvie et al, 2021 ). Similar to our initial approach to optimize a medium for growing biomass, we first determined and optimized the concentrations of the sources for carbon and nitrogen, along with the pH.…”

“…Media for evaluating enhanced production were developed using a strain expressing a rotavirus‐derived subunit vaccine candidate, P[8], under the control of the pAOX1 promoter described previously (Dalvie et al, 2021 ). Biomass accumulation proceeded for 24 h; cells reached an initial induction density of ~20 OD/ml.…”