Expression of human α1-proteinase inhibitor in Aspergillus niger

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Expression of human α₁-proteinase inhibitor in <it>Aspergillus niger</it>

Abstract Background Human α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for α1-PI in A. niger was successfully achieved to produce the secreted mature human r-α1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.</p

SDS-PAGE and Western blot (right panel) comparison of different α-PI's: 1- protein ladder; 2 and 6 - pd-α-PI standard; 3 - deglycosylated pd-α-PI; 4 - α-PI from (eluted from TALON beads); 5 - r-α-PI in the supernatant from D1526

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p

SDS-PAGE and Western blot (right panel) analysis of α-PI expression in D1526

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p> 1-protein ladder, 2 and 10 - pd-α-PI standard, 3–5 - supernatant from growth of the transformant #1 (30, 20, and 10 μL respectively), 6 – supernatant from growth of PYRG-transformant, 7–9 - supernatant from growth of the transformatant #2 (the 30, 20, and 10 μL respectively)

Diagram of the fusion region between glucoamylase (GLA) and α-PI coding region (A1-PI) within the pAN56-1 expression vector showing the KEX2 cleavage sequence (see abbreviations in the text)

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p

Development and Validation of an Innovative Analytical Approach for the Quantitation of Tris(Hydroxymethyl)Aminomethane (TRIS) in Pharmaceutical Formulations by Liquid Chromatography Tandem Mass Spectrometry

A novel COVID-19 vaccine (BriLife&reg;) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC&ndash;mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2&ndash;4.8 mg/mL). Specifically, the R2 of the standard curve was &gt;0.999, the recovery was &gt;92%, and the coefficient of variance (%CV) was &lt;12% and &lt;6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products

Toxicity and Local Tolerance of a Novel Spike Protein RBD Vaccine Against SARS-CoV-2, Produced Using the C1 Thermothelomyces Heterothallica Protein Expression Platform

Coronavirus disease 2019 (COVID-19) has caused the ongoing COVID-19 pandemic and there is a growing demand for safe and effective vaccines. The thermophilic Thermothelomyces heterothallica filamentous fungal host, C1-cell, can be utilized as an expression platform for the rapid production of large quantities of antigens for developing vaccines. The aim of this study was to evaluate the local tolerance and the systemic toxicity of a C1-cell expressed receptor-binding domain (C1-RBD) vaccine, following repeated weekly intramuscular injections (total of 4 administrations), in New Zealand White rabbits. The animals were sacrificed either 3 days or 3 weeks following the last dose. No signs of toxicity were observed, including no injection site reactions. ELISA studies revealed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G antibodies in the sera of C1-RBD-treated animals starting from day 13 post injection, that were further elevated. Histopathology evaluation and immunohistochemical staining revealed follicular hyperplasia, consisting of B-cell type, in the spleen and inguinal lymph nodes of the treated animals that were sustained throughout the recovery phase. No local or systemic toxicity was observed. In conclusion, the SARS-CoV-2 C1-RBD vaccine candidate demonstrated an excellent safety profile and a lasting immunogenic response against receptor-binding domain, thus supporting its further development for use in humans

Thermophilic Filamentous Fungus C1-Cell-Cloned SARS-CoV-2-Spike-RBD-Subunit-Vaccine Adjuvanted with Aldydrogel&reg;85 Protects K18-hACE2 Mice against Lethal Virus Challenge

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel&reg;&lsquo;85&rsquo;-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans