Tocilizumab produced with Conamax binds IL6R with a similar affinity as a reference sample of tocilizumab.

Binding was tested using an ELISA plate coated with IL6R. As control, trastuzumab produced by Conamax was used. As expected, this negative control antibody did not show any binding.</p

Codon usage table for <i>Aurantiochytrium acetophilum</i>.

Codon usage table for Aurantiochytrium acetophilum.</p

Strains used to confirm production of monoclonal antibodies and proteins.

Strains used to confirm production of monoclonal antibodies and proteins.</p

Enriched culture supernatant samples show ACE2 activity.

Proteolytic activity was determined using a fluorescently labeled probe. Non-specific proteolytic activity was determined by adding an ACE2 inhibitor. As a control, ACE2 supplied with the activity assay was used.</p

Fig 3 -

a. Expression and secretion of ACE2. Strain sCX00037 produced full-length ACE2, while strains sCX00148 produced a truncated form of ACE2. After prolonged incubation, some clipping of the produced ACE2 is noticeable. The specificity of the detection antibody was confirmed by the absence of bands in the wildtype samples. b. Titers of produced ACE2. Titers were determined using a sandwich ELISA. A steady increase in titers was seen, but as the western blot shows, this came with an increase in clipped ACE2.</p

S2 Raw images -

The emergence of COVID-19 as a global pandemic had sharply illustrated the limitations of research and development pipelines and scaled manufacturing. Although existing vaccines were created in record time, global deployment remains limited by regional production scales. Similarly, the most effective treatments for infected COVID-19 patients are also constrained by production scales as well as by the cost of production and thus expense per treatment. The need to produce these interventions more cost-effectively, at larger scales, in less time while retaining high quality is paramount. The ConamaxTM platform is based on a Thraustochytrid–an order of microorganisms well established in industry for world-scale production of omega-3 fatty acids by fermentation. Thraustochytrids, and the species Aurantiochytrium acetophilum in particular, possess a number of innate qualities which make it ideal for production of monoclonal antibodies and other biotherapeutic proteins. In this study, the Conamax system was used to produce several targets which may be relevant as interventions in the fight against COVID-19; an anti-SARS-CoV-2 antibody (CR3022), tocilizumab, and the ACE2 receptor. Our system was capable of producing all of these targets and each was assayed in vitro for an activity which confirmed proper structural folding. Purified CR3022 antibody produced from Conamax was capable of reducing the cytopathic effect of SARS-CoV-2. Conamax-derived tocilizumab was shown to bind to its target IL6R. Both the full-length and soluble versions of ACE2 protein produced in the Conamax platform exhibited ACE2-specific proteolytic activity. These data indicate that the Conamax platform has great potential in the production of therapeutic agents.</div

S1 Raw images -

The emergence of COVID-19 as a global pandemic had sharply illustrated the limitations of research and development pipelines and scaled manufacturing. Although existing vaccines were created in record time, global deployment remains limited by regional production scales. Similarly, the most effective treatments for infected COVID-19 patients are also constrained by production scales as well as by the cost of production and thus expense per treatment. The need to produce these interventions more cost-effectively, at larger scales, in less time while retaining high quality is paramount. The ConamaxTM platform is based on a Thraustochytrid–an order of microorganisms well established in industry for world-scale production of omega-3 fatty acids by fermentation. Thraustochytrids, and the species Aurantiochytrium acetophilum in particular, possess a number of innate qualities which make it ideal for production of monoclonal antibodies and other biotherapeutic proteins. In this study, the Conamax system was used to produce several targets which may be relevant as interventions in the fight against COVID-19; an anti-SARS-CoV-2 antibody (CR3022), tocilizumab, and the ACE2 receptor. Our system was capable of producing all of these targets and each was assayed in vitro for an activity which confirmed proper structural folding. Purified CR3022 antibody produced from Conamax was capable of reducing the cytopathic effect of SARS-CoV-2. Conamax-derived tocilizumab was shown to bind to its target IL6R. Both the full-length and soluble versions of ACE2 protein produced in the Conamax platform exhibited ACE2-specific proteolytic activity. These data indicate that the Conamax platform has great potential in the production of therapeutic agents.</div

Fig 1 -

a. CE-PAGE profile of purified CR3022. The Conamax-produced antibody was purified to an apparent 100% purity by a single Protein A chromatography step. Red curve: non-reduced sample, blue curve: reduced, showing heavy and light chains. b. Antibody CR3022 produced by Conamax was able to inhibit induced cytopathy by SARS-CoV-2 in Vero 76 cells. The EC50 was 61 μg/ml in this assay. Data shown here are averages of three observations per concentration of CR3022.</p

Histological analysis of leukocyte infiltration

Files contains data used to generate Figures 4-6

Histological score of inflammaed tissue

File contains histological score data used in Figure 3E, F