Journal: Organic Process Research & Development

Article Title: Continuous Purification of a Conjugated Short Interfering RNA Therapeutic Using Anion Exchange Twin-Column Chromatography (MCSGP)

doi: 10.1021/acs.oprd.4c00513

Figure Lengend Snippet: Figure 1. Twin column chromatography system setup for MCSGP (Contichrom CUBE, YMC ChromaCon).

Article Snippet: Tables S2 and S3 detail specific MCSGP method parameters used to run “startup” and “main” methods on the Contichrom CUBE hardware.

Techniques: Column Chromatography

Journal: Organic Process Research & Development

Article Title: Continuous Purification of a Conjugated Short Interfering RNA Therapeutic Using Anion Exchange Twin-Column Chromatography (MCSGP)

doi: 10.1021/acs.oprd.4c00513

Figure Lengend Snippet: Figure 2. MCSGP process. (A) An MCSGP run with 4 cycles is shown as an example. A short startup step is needed to preload column 1 with feed before the main cyclic phase of MCSGP begins. Each cycle consists of an elution from column 1 (switch 1red) and an elution from column 2 (switch 2blue), respectively. Alternating elutions can continue indefinitely for “n” cycles until the feed is consumed. To finish the MCSGP run, a shutdown step consisting of a single elution without any refeeding is done. (B) A detailed schematic representation of a single MCSGP “switch” is shown. This consists of four phases: P1 = phase 1 (elution + regeneration); P2 = phase 2 (elution with weak recycling); P3 = phase 3 (product collection + refeeding); and P4 = phase 4 (elution with strong recycling). Columns are operated in parallel during P1 and P3; columns are interconnected for product recycling in P2 and P4. The position of the columns alternates switch to switch, allowing for continuous operation (yellow arrows indicate column interchange between switches). See Section 2.4 for a more complete description of the MCSGP process principle.

Article Snippet: Tables S2 and S3 detail specific MCSGP method parameters used to run “startup” and “main” methods on the Contichrom CUBE hardware.

Techniques:

Journal: Organic Process Research & Development

Article Title: Continuous Purification of a Conjugated Short Interfering RNA Therapeutic Using Anion Exchange Twin-Column Chromatography (MCSGP)

doi: 10.1021/acs.oprd.4c00513

Figure Lengend Snippet: Figure 3. Single-column AIEX chromatographic profiles and the MCSGP design. (A) Black line: representative chromatogram of pilot-scale single- column batch production run showing absorbance UV@290 nm vs column volumes (CV) overlaid with %B modifier concentration (purification using the AKTA pure 150 M). The pilot-scale method was downscaled 78.6× and run on the Contichrom CUBE. (B) MCSGP design chromatogram. Absorbance UV@280 nm is overlaid with product purity (area% HPLC-UV). Product purity was measured by HPLC analysis, and each dot represents an analyzed fraction. Phases P1 to P4, as described in Figure 2B, are highlighted showing where MCSGP recycling and product collection boundaries were set. (C) - The MCSGP Wizard v8.1 software simplifies method creation.

Article Snippet: Tables S2 and S3 detail specific MCSGP method parameters used to run “startup” and “main” methods on the Contichrom CUBE hardware.

Techniques: Concentration Assay, Purification, Software

Journal: Organic Process Research & Development

Article Title: Continuous Purification of a Conjugated Short Interfering RNA Therapeutic Using Anion Exchange Twin-Column Chromatography (MCSGP)

doi: 10.1021/acs.oprd.4c00513

Figure Lengend Snippet: Figure 4. (A, B) MCSGP chromatographic profiles showing absorbance UV@280 nm and %B (A) or UV@280 nm and conductivity (mS/cm) (B) plotted vs time (min). (A) Plot showing 13 consecutive MCSGP cycles including startup and shutdown (total of 27 product elutions). (B) Plot showing an overlay of 13 consecutive MCSGP cycles. (C, D) Plots showing the total area under the curve (AUC) in (mAU×mL) vs cycle number. Each dot is the sum of 2 elutions from 2 switches. (C) Black line: total AUC. Red line: AUC for the product collection window. (D) Blue line: AUC for the weak recycling phase. Green line: AUC for the strong recycling phase.

Article Snippet: Tables S2 and S3 detail specific MCSGP method parameters used to run “startup” and “main” methods on the Contichrom CUBE hardware.

Techniques:

Journal: Organic Process Research & Development

Article Title: Continuous Purification of a Conjugated Short Interfering RNA Therapeutic Using Anion Exchange Twin-Column Chromatography (MCSGP)

doi: 10.1021/acs.oprd.4c00513

Figure Lengend Snippet: Figure 5. MCSGP product quality evaluation. (A) Plot showing cycle to cycle MCSGP target product yield (%) and mass recovered (mg). (B) Plot showing cycle to cycle product purity HPLC-UV (area%) vs impurity N-1 content HPLC-UV (area%). (C) Analytical HPLC chromatograms (UV Abs at 260 nm, normalized and then zoomed in to show impurities). Blue line is the crude feed material (85 area% purity). Orange line is the 13 cycle MCSGP product pool (95.4 area% purity).

Article Snippet: Tables S2 and S3 detail specific MCSGP method parameters used to run “startup” and “main” methods on the Contichrom CUBE hardware.

Techniques:

Journal: bioRxiv

Article Title: Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response

doi: 10.1101/2023.06.27.546774

Figure Lengend Snippet: (A) Numbers of vitcylated proteins and sites identified in E0771 proteomic and E0771 proteomic incubated with vitC (1 mM for 12 hours) in indicated pH Tris-HCl buffer in vitro are summarized. E0771 proteomic was extracted by acetone. (B) Subcellular locations of lysine vitcylated proteins identified in E0771 (mouse) cells. The locations are classified as nuclear, cytosol, plasma membrane, extracellular, mitochondrial, cytosol_nuclear, and other compartments. (C) Top ten gene ontology molecular function enrichment of vitcylationo proteins identified in E0771 cells (mouse). (D) Top ten KEGG-based enrichment of lysine vitcylation proteins identified in E0771 cells (mouse). (E) Top ten gene ontology biological process enrichment of vitcylation proteins identified in E0771 cells (mouse). (F) Sequence probability logos of significantly enriched vitcylation site motifs for ±10 amino acids around the lysine vitcylation sites identified in Cal-51 cells (human) and E0771 cells (mouse). The size of each letter represents the frequency of the amino acid residue at that position. (G and H) Extracted ion chromatograms (left) and MS/MS spectra (right) from HPLC-MS/MS analysis of vitcylated peptides (mouse SMC1A, K129 (G) and mouse CKAP2L, K240 (H)) derived from E0771 (cellular peptide) respectively, their in vitro generated counterparts (synthetic peptide), and their mixture. (I and J) Extracted MS/MS spectra from HPLC-MS/MS analysis of 1- 13 C-vitcylated peptides and vitcylated peptides (mouse SMC1A, K129 (I) and mouse CKAP2L, K240 (J)) derived from E0771 cells (in cells, the lysine-containing 1- 13 C-vitcylated fragments and vitcylated fragments were marked by red and blue colors, respectively). (K) The pan anti-vitcylation antibody was tested for its reactivity with vitcylated peptide (pep. + vitC, peptide pre-incubated with 2 mM vitC at 37℃ for 3 hours) and cross-reactivity with unmodified (pep. and pep. (K/R) + vitC), DHA-derived modification peptide (pep. + DHA, peptide pre-incubated with 2 mM DHA at 37℃ for 3 hours), as well as synthetic acetylated, succinylated, leucylated and lactylated peptides (peptide sequence: Ac-VLSPKAVQRF).

Article Snippet: Cellular vitC assay kit (Cayman, 700420), ROS/superoxide detection assay kit (abcam, ab139476), and epigenase 5mC-hydroxylase TET activity/inhibition assay kit (Epigentek, P-3086-48) were used for cellular vitC level assay, ROS level assay, and TET activity assay according to the manufacturer’s instructions, respectively.

Techniques: Incubation, In Vitro, Clinical Proteomics, Membrane, Sequencing, Residue, Tandem Mass Spectroscopy, Derivative Assay, Generated, Modification

Journal: bioRxiv

Article Title: Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response

doi: 10.1101/2023.06.27.546774

Figure Lengend Snippet: (A) Intracellular vitC levels were measured in Cal-51, E0771, and PP cells cultured in different concentrations of vitC for 12 hours (n = 3). Data are represented as mean ± SEM. (B) Numbers of vitcylated proteins and sites identified in Cal-51 (human) and E0771 cells (mouse) are summarized. (C) Venn diagram of shared vitcylation proteins identified in Cal-51 cells (human) and E0771 cells (mouse). (D) Subcellular locations of lysine vitcylated proteins identified in Cal-51 cells. The locations are classified into nuclear, cytosol, plasma membrane, extracellular, mitochondrial, cytosol_nuclear, and other compartments. (E) Top ten gene ontology molecular function enrichment of vitcylated proteins identified in Cal- 51 cells. (F) Top ten KEGG-based enrichment of lysine vitcylated proteins identified in Cal-51 cells. (G) Top ten gene ontology biological process enrichment of vitcylated proteins identified in Cal- 51 cells. (H) Extracted ion chromatograms (left) and MS/MS spectra (right) from HPLC-MS/MS analysis of a vitcylated peptide (human GAPDH, K5) derived from Cal-51 cells (cellular peptide), its in vitro generated counterpart (synthetic peptide), and their mixture. The b ion refers to the N-terminal parts of the peptide, and the y ion refers to the C-terminal parts of the peptide (hereafter for HPLC- MS/MS analysis). (I) Extracted MS/MS spectra from HPLC-MS/MS analysis of 1- 13 C-vitcylated peptides and vitcylated peptides (human GAPDH, K5) derived from Cal-51 cells (in cells, the lysine-containing 1- 13 C-vitcylated fragments and vitcylated fragments were marked by red and blue colors, respectively). (J) Intracellular lysine vitcylation levels were measured from PP, E0771, Cal-51, and MCF7 cells cultured in medium containing a vehicle or vitC for 12 hours (2 mM vitC for PP and E0771 culture, 0.5 mM vitC for Cal-51 and MCF7 culture). Protein levels in each sample were normalized by coomassie staining, hereafter for global vitcylation detection. (K) Vitcylation signals of indicated cells were competed off by vitcylated peptide (Ac- VLSPKAVQRF peptide pre-incubated with 2 mM vitC at 37℃ for 3 hours). (L) Intracellular lysine vitcylation levels were measured from E0771 cells cultured in medium containing different concentrations of vitC for 12 hours. (M) Intracellular lysine vitcylation levels were measured in E0771 cells cultured in medium containing 2 mM vitC for the indicated times. (N) Intracellular lysine vitcylation levels were measured from E0771 cultured in medium containing a vehicle or 2 mM vitC for 12 hours under different pH conditions. See also and Tables S1-S6.

Article Snippet: Cellular vitC assay kit (Cayman, 700420), ROS/superoxide detection assay kit (abcam, ab139476), and epigenase 5mC-hydroxylase TET activity/inhibition assay kit (Epigentek, P-3086-48) were used for cellular vitC level assay, ROS level assay, and TET activity assay according to the manufacturer’s instructions, respectively.

Techniques: Cell Culture, Clinical Proteomics, Membrane, Tandem Mass Spectroscopy, Derivative Assay, In Vitro, Generated, Staining, Incubation

Journal: bioRxiv

Article Title: Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response

doi: 10.1101/2023.06.27.546774

Figure Lengend Snippet: (A and B) Top-ranked upregulated GO terms (A) and upregulated GSEA signatures (B) in E0771 cells treated with 1 mM vitC for 2 days (n = 2). (C) Extracted ion chromatograms (left) and MS/MS spectra (right) from HPLC-MS/MS analysis of a vitcylated peptide (human STAT1, K298) derived from Cal-51 cells (cellular peptide), its in vitro generated counterpart (synthetic peptide) and their mixture. (D) Extracted MS/MS spectra from HPLC-MS/MS analysis of vitcylated peptide (upper) and 1- 13 C-vitcylated peptide (lower) (human STAT1, K298) derived from Cal-51 cells. Lysine-containing vitcylated fragments and 1- 13 C-vitcylated fragments are marked by blue and red colors, respectively. (E) STAT1 vitcylation levels were measured from STAT1-GFP expressing cells (Cal-51 and PP cells) cultured in different pH mediums with or without vitC for 12 hours (2 mM vitC for PP cell culture, 0.5 mM vitC for Cal-51 cell culture). (F) pSTAT1 flow cytometric analysis of Cal-51 and PP cells cultured in different pH mediums with or without vitC for 2 days (2 mM vitC for PP cell culture, 0.5 mM vitC for Cal-51 cell culture, n = 3). Data are represented as mean ± SEM. ***p < 0.001, ****p < 0.0001. (G) STAT1 vitcylation levels were measured from STAT1-GFP expressing cells (Cal-51 and PP cells) cultured in different pH mediums with or without vitC for 12 hours (2 mM vitC for PP cell culture, 0.5 mM vitC for Cal-51 cell culture). (H) pSTAT1 flow cytometric analysis of Cal-51 and PP cells cultured in different pH mediums with or without vitC for 2 days (2 mM vitC for PP cell culture, 0.5 mM vitC for Cal-51 cell culture, n = 3). Data are represented as mean ± SEM. ***p < 0.001, ****p < 0.0001. (I) Measurement of STAT1-WT and STAT1-K298R vitcylation levels in PP-sgSTAT1_1 cell re- expressing STAT1-WT-GFP or STAT1-K298R-GFP cultured in 2 mM vitC-containing or control medium for 12 hours. (J) pSTAT1 flow cytometric analysis of PP-sgSTAT1_1 cell re-expressing STAT1-WT-GFP or STAT1-K298R-GFP cultured in different concentrations of vitC for 2 days (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (K) Nuclear translocation of STAT1 in PP-sgSTAT1_1 cell re-expressing STAT1-WT-GFP or re- expressing STAT1-K298R-GFP treated with 2 mM vitC for 2 days was assessed by immunofluorescence (scale bar, 50 μM). See also and Table S4.

Article Snippet: Cellular vitC assay kit (Cayman, 700420), ROS/superoxide detection assay kit (abcam, ab139476), and epigenase 5mC-hydroxylase TET activity/inhibition assay kit (Epigentek, P-3086-48) were used for cellular vitC level assay, ROS level assay, and TET activity assay according to the manufacturer’s instructions, respectively.

Techniques: Tandem Mass Spectroscopy, Derivative Assay, In Vitro, Generated, Expressing, Cell Culture, Control, Translocation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response

doi: 10.1101/2023.06.27.546774

Figure Lengend Snippet: (A and B) Upregulated (A) and downregulated (B) GSEA signatures were observed in E0771 cells treated with 2 mM vitC for 2 days (n = 3). (C) Levels of vitcylation of human STAT1 K298 were determined by HPLC-MS/MS analysis. Data are represented as mean ± SEM. (D) Sequence analysis of STAT1 K298 site from multiple vertebrate species. (E) Extracted ion chromatograms (left) and MS/MS spectra (right) from HPLC-MS/MS analysis of a vitcylated peptide (mouse STAT1, K298) derived from E0771 (cellular peptide), its in vitro generated counterparts (synthetic peptide), and their mixture. (F) Extracted MS/MS spectra from HPLC-MS/MS analysis of vitcylated peptides (upper) and 1- 13 C-vitcylated peptides (lower) (mouse STAT1, K298) derived from E0771 cells. The lysine- containing vitcylated fragments and 1- 13 C-vitcylated fragments were marked by blue and red colors, respectively). (G) Nuclear translocation of endogenous STAT1 in Cal-51 and PP cells cultured in vehicle- or vitC-containing medium for 2 days was assessed by immunofluorescence (0.5 mM vitC for Cal- 51 culture, 2 mM vitC for PP culture). Green represents anti-STAT1 and blue represents DAPI, with merged images allowing assessment of nuclear localization of STAT1, hereafter referred to as STAT1 immunofluorescence (scale bar, 50 μM). (H) Western blots for STAT1 and Actin in PP-sg_NC, PP-sgSTAT1_1 and PP-sgSTAT1_2 cells. (I) Western blots for STAT1 and Actin in PP-sgNC, PP-sgSTAT1_1 and PP-sgSTAT1 cells re- expressing STAT1-WT-GFP or re-expressing STAT1-K298R-GFP.

Article Snippet: Cellular vitC assay kit (Cayman, 700420), ROS/superoxide detection assay kit (abcam, ab139476), and epigenase 5mC-hydroxylase TET activity/inhibition assay kit (Epigentek, P-3086-48) were used for cellular vitC level assay, ROS level assay, and TET activity assay according to the manufacturer’s instructions, respectively.

Techniques: Tandem Mass Spectroscopy, Sequencing, Derivative Assay, In Vitro, Generated, Translocation Assay, Cell Culture, Immunofluorescence, Western Blot, Expressing

Journal: bioRxiv

Article Title: Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response

doi: 10.1101/2023.06.27.546774

Figure Lengend Snippet: (A) Quantitative PCR analysis of antigen processing and presentation genes expression in Cal-51 cells (n = 4) and PP cells (n = 3) treated with either vehicle or vitC for 2 days (0.5 mM vitC for Cal-51 cell treatment, 2 mM vitC for PP cell treatment). Data are represented as mean ± SEM. ****p < 0.0001. (B) Flow cytometry analysis of MHC class I expression in PP-sgControl, PP-sgSTAT1_1, and PP- sgSTAT1_2 cells cultured in different concentrations of vitC for 2 days (n = 3). Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) ROS levels were measured in Cal-51, E0771, and PP cells treated with various concentrations of vitC for 2 days (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Flow cytometric analysis of MHC/HLA class I expression on Cal-51, E0771, and PP cells treated with different concentrations of vitC for 2 days (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) TET activity was measured in Cal-51, E0771, and PP cells treated with vehicle or 10 μM Bobcat 339 for 6 hours (n = 3). Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) HIF1α level was measured in Cal-51, E0771, and PP cells treated with vehicle or 0.2 μM IOX2 for 6 hours. (G) Flow cytometric analysis of pSTAT1 and MHC/HLA class I expression in Cal-51, E0771, and PP cells cultured in medium supplemented with the vehicle, vitC (48 hours), 10 μM Bobcat 339 (52 hours), vitC + 10 μM Bobcat 339, 0.2 μM IOX2 (52 hours) or vitC + 0.2 μM IOX2 (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (H) Flow cytometry gating strategy for the DCs population. Hereafter for DCs gating. Representative plots are shown. (I) Flow cytometric analysis of H-2Kb and pSTAT1 expression on EL4-OVA cells treated with different doses of vitC for 3 days (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (J) Flow cytometry gating strategy for T cells population. Hereafter for T cells gating. Representative plots are shown. (K) Flow cytometric analysis of CD8 + T (OT-I) cells co-cultured with EL4-OVA cells pretreated with 2 mM vitC. T cells (CD45 + CD3 + CD8 + ) proliferation and activity were quantified as CFSE - and IFNγ + , TNFα + cells, respectively (n = 3). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Cellular vitC assay kit (Cayman, 700420), ROS/superoxide detection assay kit (abcam, ab139476), and epigenase 5mC-hydroxylase TET activity/inhibition assay kit (Epigentek, P-3086-48) were used for cellular vitC level assay, ROS level assay, and TET activity assay according to the manufacturer’s instructions, respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cell Culture, Activity Assay