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Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals manufactured by well-established processes using Chinese Hamster Ovary (CHO) cells. Next-generation biomanufacturing using alternative hosts like Komagataella phaffii could improve the accessibility of these medicines, address broad societal goals for sustainability, and offer financial advantages for accelerated development of new products. Antibodies produced by K. phaffii, however, may manifest unique molecular quality attributes, like host-dependent, product-related variants, that could raise potential concerns for clinical use. We demonstrate here conservative modifications to the amino acid sequence of aglycosylated antibodies based on the human IgG1 isotype that minimize product-related variations when secreted by K. phaffii. A combination of 2-3 changes of amino acids reduced variations across six different aglycosylated versions of commercial mAbs. Expression of a modified sequence of NIST mAb in both K. phaffii and CHO cells showed comparable biophysical properties and molecular variations. These results suggest a path toward the production of high-quality mAbs that could be expressed interchangeably by either yeast or mammalian cells. Improving molecular designs of proteins to enable a range of manufacturing strategies for well-characterized biopharmaceuticals could accelerate global accessibility and innovations., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Background: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality., Results: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody., Conclusions: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes., (© 2024. The Author(s).)

Viruses, bacteria, and parasites frequently cause infections in the gastrointestinal tract, but traditional vaccination strategies typically elicit little or no mucosal antibody responses. Here, we report a strategy to effectively concentrate immunogens and adjuvants in gut-draining lymph nodes (LNs) to induce gut-associated mucosal immunity. We prepared nanoemulsions (NEs) based on biodegradable oils commonly used as vaccine adjuvants, which encapsulated a potent Toll-like receptor agonist and displayed antigen conjugated to their surface. Following intraperitoneal administration, these NEs accumulated in gut-draining mesenteric LNs, priming strong germinal center responses and promoting B cell class switching to immunoglobulin A (IgA). Optimized NEs elicited 10- to 1000-fold higher antigen-specific IgG and IgA titers in the serum and feces, respectively, compared to free antigen mixed with NE, and strong neutralizing antibody titers against severe acute respiratory syndrome coronavirus 2. Thus, robust gut humoral immunity can be elicited by exploiting the unique lymphatic collection pathways of the gut with a lymph-targeting vaccine formulation.

One of the greatest threats facing the planet is the continued increase in excess greenhouse gasses, with CO 2 being the primary driver due to its rapid increase in only a century. Excess CO 2 is exacerbating known climate tipping points that will have cascading local and global effects including loss of biodiversity, global warming, and climate migration. However, global reduction of CO 2 emissions is not enough. Carbon dioxide removal (CDR) will also be needed to avoid the catastrophic effects of global warming. Although the drawdown and storage of CO 2 occur naturally via the coupling of the silicate and carbonate cycles, they operate over geological timescales (thousands of years). Here, we suggest that microbes can be used to accelerate this process, perhaps by orders of magnitude, while simultaneously producing potentially valuable by-products. This could provide both a sustainable pathway for global drawdown of CO 2 and an environmentally benign biosynthesis of materials. We discuss several different approaches, all of which involve enhancing the rate of silicate weathering. We use the silicate mineral olivine as a case study because of its favorable weathering properties, global abundance, and growing interest in CDR applications. Extensive research is needed to determine both the upper limit of the rate of silicate dissolution and its potential to economically scale to draw down significant amounts (Mt/Gt) of CO 2 Other industrial processes have successfully cultivated microbial consortia to provide valuable services at scale (e.g., wastewater treatment, anaerobic digestion, fermentation), and we argue that similar economies of scale could be achieved from this research., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sergio Andre Rodriguez-Aponte, Neil Chandra Dalvie, and J. Christopher Love have filed a patent related to the RBD-L452K-F490W (RBD-J) sequence. J. Christopher Love has interests in Sunflower Therapeutics PBC, Honeycomb Biotechnologies, OneCyte Biotechnologies, QuantumCyte, and Repligen. J.C.L.’s interests are reviewed and managed under MIT’s policies for potential conflicts of interest. Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Saurabh Batwal, Rahul Chandrasekaran, Gaurav Nagar are employees of Serum Institute of India Pvt. Ltd. Sumi Biswas is an employee of SpyBiotech Limited., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Low-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low- and middle-income countries. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum-salt (Alhydrogel®, AH; AdjuPhos®, AP) and CpG 1018 adjuvants. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37°C) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro , the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Interestingly, the loss of in vivo potency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivo performance to enable successful formulation development of stable and efficacious subunit vaccines.

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., (© 2022. The Author(s).)

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (β RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (β/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (β RBD HBsAg+SWE) or without HBsAg (β RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only β/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with β RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC. IMPORTANCE Global COVID-19 vaccine distribution to low-income countries has been a major challenge of the pandemic. To address supply chain issues, RBD virus-like particle (VLP) vaccines that are cost-effective and capable of large-scale production were developed and evaluated for efficacy in preclinical mouse studies. We demonstrated that RBD-VLP vaccines protected K18-hACE2 mice against Alpha or Beta challenge similarly to Pfizer mRNA vaccination. Our findings showed that the VLP platform can be utilized to formulate immunogenic and efficacious COVID-19 vaccines.

To combat the HIV epidemic and emerging threats such as SARS-CoV-2, immunization strategies are needed that elicit protection at mucosal portals of pathogen entry. Immunization directly through airway surfaces is effective in driving mucosal immunity, but poor vaccine uptake across the mucus and epithelial lining is a limitation. The major blood protein albumin is constitutively transcytosed bidirectionally across the airway epithelium through interactions with neonatal Fc receptors (FcRn). Exploiting this biology, here, we demonstrate a strategy of "albumin hitchhiking" to promote mucosal immunity using an intranasal vaccine consisting of protein immunogens modified with an amphiphilic albumin-binding polymer-lipid tail, forming amph-proteins. Amph-proteins persisted in the nasal mucosa of mice and nonhuman primates and exhibited increased uptake into the tissue in an FcRn-dependent manner, leading to enhanced germinal center responses in nasal-associated lymphoid tissue. Intranasal immunization with amph-conjugated HIV Env gp120 or SARS-CoV-2 receptor binding domain (RBD) proteins elicited 100- to 1000-fold higher antigen-specific IgG and IgA titers in the serum, upper and lower respiratory mucosa, and distal genitourinary mucosae of mice compared to unmodified protein. Amph-RBD immunization induced high titers of SARS-CoV-2-neutralizing antibodies in serum, nasal washes, and bronchoalveolar lavage. Furthermore, intranasal amph-protein immunization in rhesus macaques elicited 10-fold higher antigen-specific IgG and IgA responses in the serum and nasal mucosa compared to unmodified protein, supporting the translational potential of this approach. These results suggest that using amph-protein vaccines to deliver antigen across mucosal epithelia is a promising strategy to promote mucosal immunity against HIV, SARS-CoV-2, and other infectious diseases.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain-hepatitis B surface antigen virus-like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>10 4 ) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log 10 ) and nasal mucosa (~2.9 log 10 ) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)