Fully automated high-throughput process development for the novel purification of rotavirus vaccines

Downstream processing of biopharmaceuticals, such as immunoglobulins, recombinant proteins and protein-based vaccines, traditionally requires multiple purification steps that can introduce cost and time-related limitations. To avoid these, alternative purification strategies are sought involving novel and efficient processes. Mixed-mode chromatography based unit operations can achieve these by making use of their multimodal functionality. This adheres to the goal of the Ultra-low cost Transferable Automated Platform for Vaccine Manufacture (ULTRA) project and its ultimate aim to deliver vaccines with a cost of goods less than 15 cents per dose. Herein, we show the application of mixed mode chromatography for the purification of a P2-VP8 (PATH) rotavirus antigen vaccine expressed in Pichia pastoris (MIT, MA, USA; J. Christopher Love). A high-throughput workflow was adopted for the development of the purification step which employed microscale chromatography with miniature columns. This was implemented on an automated liquid handling station (Tecan Evo 200). The performed scouting studies focused on combinations of different binding and elution conditions along with multiple resins and selectively accessing the ion exchange and hydrophobic integration mechanisms of the resins. Therefore, a wide range of conditions were assessed with walk-away automation. Conditions leading to high yields and purities were then scaled-up for verification purposes. This provided further evidence for the good scalability properties of the miniature chromatography column technique. The results from this study demonstrate that mixed mode chromatography can potentially lead to the establishment of a highly desirable, one-step purification of rotavirus vaccine upon further optimisation. Hence, downstream processing in rotavirus vaccine production can be de-bottlenecked with systematic process development activities leading to significantly improved whole process efficiencies. The utility of this method is that it has generated reproducible and scalable data with reduced sample requirements to just a few millilitres very early on in the development process. More broadly, this high-throughput methodology will enable the early purification screening of multiple vaccine candidates and enable their selection for further development based on ease of processing

Development of analytical characterization tools for process monitoring of adenovirus-based vaccines (ChAdOx and Ad5)

Product quality understanding is a critical part of viral vector vaccine manufacturing and regulation. Mass spectrometry is a technique that has widely been applied to protein-based therapeutics and could be used as a characterisation tool to monitor viral vector vaccine product quality. The ultimate objective of this Bill and Melinda Gates Foundation funded project is to enable vaccine manufacturing in Low and Middle-income countries (LMIC) through increased scientific understanding of viral vector vaccine manufacturing bottlenecks and therefore de-risking of vaccine development and manufacturing. Please click Download on the upper right corner to see the full abstract

Measurement of Adenovirus-Based Vector Heterogeneity

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities

Measurement of Adenovirus-Based Vector Heterogeneity (Dataset)

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. Interestingly, these proportions correlated with the sample’s infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify and track key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ~5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are thus promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring

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

© 2020 The Authors A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries