Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline for growth factor candidate selection and cell-based testing. (b) Western blot analysis of 11 high-value CFPS-derived growth factors. FGF-1, FGF-2, FGF-10, TNF-α, IL-1β, IFN-γ, and IL-15 were expressed in E. coli BL21 (DE3) in-house cell-free lysates. IL-6, EGF, IGF-1, and IL-3 were expressed in SHuffle-based in-house cell-free lysates. Detection was performed using an anti-His- HRP antibody. Data shown are from one representative biological replicate out of three independent experiments. (c) In vitro proliferation assay comparing on-demand, locally produced in Canada (blue) and commercial (green) FGF-1 growth factors in NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of growth factor. Luminescence was measured using a conventional plate reader. Data are shown as mean ± SD, n = 3. (d) In vitro proliferation assay comparing on-demand, locally produced (blue) and commercial (green) IL-3 growth factors in TF-1 cells. Cells were individually treated with varying concentrations (0.025, 0.05, 0.1, 0.5, 1, and 5 ng/mL). This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Growth factor expression and cell-based validation in a low-resource setting. In vitro proliferation assay using FGF-1 and NIH-3T3 cells. Cells were individually treated with varying concentrations (1, 5, 10, 50, 500, and 1000 ng/mL) of both in-house-produced (blue) and commercial (green) growth factors. This representative data was obtained using reagents produced on-site in Brazil. Relative fold proliferation was plotted as the fold-change compared to untreated, serum-starved cells under the same experimental conditions. Data are shown as mean ± SD, n = 3. Statistical differences were determined by two-way ANOVA with Šídák’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations are: ns, not significantly different; kDa, kilodaltons.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Selection, Western Blot, Derivative Assay, In Vitro, Proliferation Assay, Produced, Expressing, Biomarker Discovery

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic of the SARS-CoV-2 genome; the coding sequence for the SARS-CoV-2 nucleocapsid protein was optimized for translation in CFPS and ultimately used for Nuvax formulation. (b) Western blot analysis of CFPS-produced SARS-CoV-2 nucleocapsid protein exhibiting a distinct band at the expected molecular weight (49 kDa). The SARS-CoV-2 nucleocapsid protein from SinoBiologicals, a commercially available recombinant protein, was used as a positive control (Ctrl +). N (CFPS) refers to the antigen expressed using in-house CFPS reactions. The molecular weight ladder (in kilodaltons) is shown on the left. Data shown are from one representative biological replicate out of three independent experiments. (c) Endotoxin levels in CFPS-produced SARS-CoV-2 nucleocapsid protein. Three different strategies were used for antigen expression: 1) cell-free lysates made from E. coli BL21(DE3); 2) cell-free lysates from E. coli BL21(DE3) followed by endotoxin removal; 3) cell-free lysates prepared from ClearColi TM BL21(DE3), an engineered E. coli strain used to express endotoxin-free recombinant proteins, making it ideal for expression of therapeutic products. The dashed line indicates the standard guidelines for subunit-based vaccine formulations (<20 EU/mL). Data are shown as mean ± SD, n = 3. (d) Immunization schedule, indicating prime, boost and bleed timepoints, for the CFPS-derived Nuvax. Intramuscular (IM) vaccination was administered on days 0 (prime) and 15 (boost), and blood was drawn on days 7, 14, 21, and 28 to evaluate for induction of IgG production. (e) Robust neutralizing antibody response following Nuvax administration alongside group controls. Analysis was performed using ELISA and measured at OD 450 nm using a conventional plate reader. The data are presented as the mean ± SD for each group, with individual data points shown (control group that received only PBS, n = 5; control group that received only adjuvants, n = 5; Nuvax group, n = 5). Statistical differences were determined by two-way ANOVA with Tukey’s post hoc multiple comparisons test: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations are: ns, not significantly different; Ctrl, control; N, nucleocapsid; End, endotoxin; Ab, antibody; IM, intramuscular; CFPS, cell-free protein synthesis.

Article Snippet: E. coli BL21(DE3) (NEB, C2527I), ClearColi BL21(DE3) (Biosearch Technologies, 60810-1), E. coli SHuffle (NEB, C3028J), E. coli BL21 (DE3)-Gold-ΔLac (Addgene, 99247), and E. coli BL21(DE3) Star/CRISPRi+( ) strains were used for preparing cell-free lysates .

Techniques: Sequencing, Formulation, Western Blot, Produced, Molecular Weight, Recombinant, Positive Control, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control