A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product‐related impurity removal

et al. 2020

Preprint

Self Cite

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.

Section: Reduction Of Product-related Variants Enables a Common Purifmentioning

confidence: 99%

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

Dalvie

Brady

et al. 2020

Preprint

Self Cite

“…If removal of a specific product‐related variant is deemed necessary, a third column could be added to the chromatography process proposed here. Combined screening and in silico methods can also be used to design integrated purification processes for products with significant product‐related impurity removal challenges (Vecchiarello et al, 2019 ). Alternatively, changes could be made to the strain or the molecule itself to reduce the formation of this variant in the first place.…”

Section: Discussionmentioning

confidence: 99%

Development of a platform process for the production and purification of single‐domain antibodies

Biotech & Bioengineering

Goodwine

Holt

et al. 2021

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Single‐domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two‐step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

“…We previously demonstrated an integrated, continuous manufacturing system for producing biopharmaceutical proteins (InSCyT) [35]. Two core elements-microbial perfusion-based fermentation and integrated chromatographic operations [36,37]made it possible to rapidly and simultaneously develop processes for production and recovery on this system. We rst developed a single process for perfusion-based fermentation based on our prior experience producing similar molecules, and executed it to produce each antigen individually (Fig.…”

Section: Reduction Of Product-related Variants Enables a Common Puri mentioning

confidence: 99%

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

Dalvie

Brady

et al. 2020

Preprint

Self Cite

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.