Molecular Engineering Improves Antigen Quality and Enables Integrated Manufacturing of A Trivalent Subunit Vaccine Candidate for Rotavirus

Abstract

BackgroundVaccines 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. ResultsWe 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. ConclusionsThis 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.

Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost, Multi-Dose Vaccine Candidates: Case-Study With Non-Replicating Rotavirus (NRRV) Vaccine Antigens

“…The experimental outline begins with selection and production of each NRRV variant in K. phaffii followed by purification from the culture supernatant. 16 High-throughput analytical techniques were then used to evaluate and compare various key structural attributes of the proteins including primary structure/post-translational modifications, antibody binding, conformational stability (±the preservative thimerosal), and relative solubility. Stage 1 data were generated in 2e3 days using only~1 mg of each variant in solution (no aluminum adjuvant).…”

“…The design, preparation and characterization of these P [8] and P [4] variants expressed in K. phaffii are presented in detail elsewhere. 16 Fedbatch production and purification of the NRRV P [8] and P [4] proteins are described in brief in the Supplemental Methods section and in detail elsewhere. 17 Alhydrogel® adjuvant (2% aluminum hydroxide gel suspension, 10 mg/mL aluminum) was purchased from InvivoGen (San Diego, CA).…”

“…These results demonstrate the five P [8] samples are suitable for performing formulation developability experiments (see below), in which the parent protein is expressed with varying levels of truncated, glycosylated and non-native disulfide crosslinked species, and these species are eliminated by molecular engineering to facilitate line-of-sight towards low-cost production as reported elsewhere. 16 The compatibility of these five P [8] protein antigens with the vaccine preservative thimerosal was evaluated (Fig. 1).…”

“…9 The development of the series of NRRV antigen variants from K. phaffii, in terms of design rationale, experimental strategies for low-cost vaccine production, and immunological characterization, are presented in detail elsewhere. 16 We used these sequence variants of P [8] and P [4] NRRV as a case study to demonstrate proof-ofconcept for a two-stage formulation developability assessment workflow to rapidly assess formulation variables for recombinant vaccine antigens that could be applied to accelerate development of low-cost vaccine dosage forms targeted for use in low and middle income countries (LMICs). Stage 1 evaluates the physicochemical and immunochemical binding properties of each NRRV protein antigen, and Stage 2 examines the impact of various storage temperatures on conformational stability and antibody binding of downselected P [8] and P [4] NRRV variants, both in the presence and absence of an aluminum-adjuvant (Alhydrogel®, AH) and a vaccine preservative (thimerosal).…”

Crystallization of a nonreplicating rotavirus vaccine candidate

“…The P2‐VP8‐P[8] sequence was modified to improve product quality and expression titer (Dalvie et al, 2020). The modified P2‐VP8‐P[8] was expressed and secreted from Pichia pastoris ( Komagataella phaffii NRRL Y‐11430).…”