Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered<i>Komagataella phaffii</i>

Dalvie, Neil C.; Biedermann, Andrew M.; Rodriguez-Aponte, Sergio A.; Naranjo, Christopher A; Rao, Harish; Rajurkar, Meghraj P; Lothe, Rakesh R; Shaligram, Umesh; Johnston, Ryan S.; Crowell, Laura E.; Castelino, Seraphin; Tracey, Mary Kate; Whittaker, Charles A.; Love, J. Christopher

doi:10.1101/2021.04.15.440035

Cited by 7 publications

(2 citation statements)

References 30 publications

(23 reference statements)

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“…High-molecular weight species or aggregated recombinant proteins can impact final yields and may be hard to remove since they may have similar biophysical features as properly folded protein [ 30 ]. We previously reported manufacturing of the SARS-CoV-2 receptor binding domain (RBD), a promising candidate subunit vaccine antigen for COVID-19, in K. phaffii [ 31 , 32 ]. We observed a high-molecular weight species (RBD-HMW) in purified RBD samples purified by ion exchange chromatography.…”

Section: Resultsmentioning

confidence: 99%

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

et al. 2022

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Background Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. Results We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. Conclusions Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.

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Section: Resultsmentioning

confidence: 99%

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

et al. 2022

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“…Despite numerous immunological advantages of using the spike protein compared to RBD alone, the spike protein is more difficult to manufacture than RBD, which can be easily expressed and produced on a large scale in microbial hosts such as yeast. Therefore, RBD-based vaccines may help facilitate COVID-19 vaccine production and distribution in lower-income countries ( 11 , 60 – 62 ).…”

Section: Discussionmentioning

confidence: 99%

RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge

Wong

Russ

Miller

et al. 2022

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Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

et al. 2021

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Vaccination of the global population against COVID-19 is a great scientific, logistical, and moral challenge. Despite the rapid development and authorization of several full-length Spike (S) protein vaccines, the global demand outweighs the current supply and there is a need for safe, potent, high-volume, affordable vaccines that can fill this gap, especially in low- and middle-income countries. Whether SARS-CoV-2 S-protein receptor-binding domain (RBD)-based vaccines could fill this gap has been debated, especially with regards to its suitability to protect against emerging viral variants of concern. Given a predominance for elicitation of neutralizing antibodies (nAbs) that target RBD following natural infection or vaccination, a key biomarker of protection, there is merit for selection of RBD as a sole vaccine immunogen. With its high-yielding production and manufacturing potential, RBD-based vaccines offer an abundance of temperature-stable doses at an affordable cost. In addition, as the RBD preferentially focuses the immune response to potent and recently recognized cross-protective determinants, this domain may be central to the development of future pan-sarbecovirus vaccines. In this study, we review the data supporting the non-inferiority of RBD as a vaccine immunogen compared to full-length S-protein vaccines with respect to humoral and cellular immune responses against both the prototype pandemic SARS-CoV-2 isolate and emerging variants of concern.

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Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineeredKomagataella phaffii

Cited by 7 publications

References 30 publications

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge

Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

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