Impact of raw materials on sialyation for a therapeutic protein

Total sialic acid content (TSAC) is a critical product quality attribute (CQA) for a therapeutic protein. Previous studies and results have suggested lot-to-lot variability of raw material, media powder and feed, have potential effects on total sialyation acid content (TSAC) at cell culture fluid (CCF) and harvested cell culture fluid (HCCF). Through searching literature, we found that potential components in media powder and feed such trace metal and vitamins can greatly modulate the sialyation acid level in vivo or in vitro. For instance, the sialic acid content was simultaneously increased in glycopeptides as well as gangliosides under lithium treatment1. Moreover, supplementation of vitamin A, B, C and E were found significant correlation with sialic acid levels in variety of organisms2, 3, & 4. Therefore, an investigation was performed to further examine the relationship between TSAC level and suggested component Multivariate analysis was performed to identify metal and vitamin candidates that potentially have positive or negative effect on sialyation acid level. Top candidates will be selected and experiments will then be performed to carefully evaluate their impacts. We thereby aim to establish the efficient platform to screen for critical regulators of raw materials on TSAC level. This study will provide the insight of understanding better control of product quality and establishes a methodology for identifying the rooting causes in cell culture media contributing to the variability of cell performance. References Edelfors et al. Acta Pharmacol Toxicol. 1981 Chitra et al. Indian Journal of Clinical Biochemistry 2008 Qiao et al. J Nutr Vitaminol 2013. Tarthan et al. process Biochemistry, 201

Cells lacking Hat1 show reduced virulence but persist in mouse kidneys.

<p>(A) Reduced growth rate of the <i>hat1</i>Δ/Δ strain was determined by measuring the OD₆₀₀ of cells growing in YPD at 30°C. (B) Cells lacking Hat1 are not cleared efficiently from kidneys. At the indicated time points, fungal burdens in kidneys of mice infected with <i>C</i>. <i>albicans</i> strains were determined and expressed as CFUs per gram kidney. Groups of 5–10 mice were analyzed at each time point and statistical significance was determined using the non-parametric Mann-Whitney-test. n.s.: not significant, *P<0.05 and **P<0.01 relative to the corresponding wild-type. (C) <i>hat1</i>Δ/Δ cells are defective in killing the host. Survival of mice infected with the indicated strains was monitored over 32 days post infection (p.i.). The data are presented as Kaplan-Meier survival curves. Groups of 6 mice were infected per <i>C</i>. <i>albicans</i> strain. Statistical significance was determined using the Log-rank test. ns: not significant; (D) Fungal burdens in kidneys of surviving mice from panel C were determined and expressed as CFUs per gram organ. One mouse infected with the <i>hat1</i>Δ/Δ strain was able to clear <i>Candida</i>. (E) The <i>cac2</i>Δ/Δ strain is not cleared efficiently from kidneys. Experiment was performed as described in (B). Groups of 4–5 mice were analyzed at each time point. (F) Infection with <i>hat1</i>Δ/Δ cells causes reduced kidney damage. Urea levels were determined in sera of infected mice at day 3 and 7 post infection. n.s.: not significant, *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p

Deletion of <i>HAT1</i> primarily leads to upregulation of genes.

<p>(A) Lack of Hat1 causes mainly induction of genes in logarithmically growing cells. Each dot corresponds to one protein-coding gene. The fold change in RNA expression between untreated wild-type and <i>hat1</i>Δ/Δ cells (y-axis) is plotted against the expression level of each gene in this dataset (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logCPM: log2 counts per million reads; logFC: log2 fold change; (B+C) Loss of Cac2 or Rtt109 causes almost exclusively upregulation of genes in logarithmically growing cells. Plots were created as described in (A). (D) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants in the absence of H₂O₂. (E) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants upon treatment with H₂O₂. (F) H₂O₂ repressed genes are upregulated in the <i>hat1</i>Δ/Δ mutant upon peroxide treatment. Each dot corresponds to one protein-coding gene. The -fold change in RNA expression between H₂O₂ treated wild-type and <i>hat1</i>Δ/Δ strains (y-axis) is plotted against the fold change between the wild-type without and with treatment (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logFC: log2 fold change; (A-F) Differentially regulated genes were defined by a fold change > = 2 and p-value <0.05.</p

Lack of histone chaperones mimics deletion of <i>HAT1</i>.

<p>(A) Loss of Cac2 increases H₂O₂ resistance. Deletion of <i>RTT106</i> or <i>HIR1</i> does not affect susceptibility to hydrogen peroxide. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days. (B) Deletion of <i>HAT1</i> or <i>CAC2</i> increases survival to transient hydrogen peroxide treatment. Exponentially growing cells were treated with the indicated concentrations of H₂O₂ for 2 hours. Cells were plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (C) Deletion of <i>HIR1</i> reduces voriconazole (Voric.) susceptibility. The <i>hat1hir1</i>Δ/Δ double deletion strain mimics lack of Hat1. Loss of Cac2 has only a minor effect and deletion of <i>RTT106</i> does not alter azole susceptibility. Experiment was performed as described in (A). (D) Increased azole tolerance of <i>hat1</i>Δ/Δ, <i>hir1</i>Δ/Δ and <i>hat1hir1</i>Δ/Δ was confirmed using a liquid growth inhibition assay. Logarithmically growing cells were diluted into medium containing the indicated concentrations of voriconazole (Voric.) and incubated at 30°C for 18 hours. OD₆₀₀ was determined and growth inhibition relative to untreated samples was calculated. Data are shown as mean + SD from three independent experiments. (E) Lack of Spt6 reduces H₂O₂ susceptibility. Experiment was performed as described in (B). Cells were treated with 10 mM H₂O₂. Data are shown as mean + SD from two independent experiments. (F) Deletion of <i>SPT6</i> increases H₂O₂ resistance and azole tolerance. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 5 days. (G) Reduction of histone gene dosage decreases H₂O₂ and azole susceptibility. Experiment was performed as described in (A). (B, D, E) *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding wild-type (Student's t-test).</p

Loss of Hat1 raises antioxidant enzyme activity and glutathione-mediated H₂O₂ resistance.

<p>(A) Faster <i>CAT1</i> induction increases catalase activity in <i>hat1</i>Δ/Δ cells. Catalase activity was determined in whole cell extracts isolated from cells before and after H₂O₂ treatment. Data are shown as mean + SD from three independent experiments. (B) Loss of Hat1 leads to increased glutathione peroxidase activity. GPx activity was determined in whole cell extracts isolated from cells before and after H₂O₂ treatment. Data are shown as mean + SD from two independent experiments. (C) Lack of <i>CAT1</i> does not abolish Hat1-mediated H₂O₂ resistance. Cells of the indicated strains were treated with 1 mM H₂O₂ for 2 hours, plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (D) Depletion of glutathione biosynthesis abolishes Hat1-mediated H₂O₂ resistance. Cells of the indicated strains were treated with H₂O₂ for 2 hours and plated on YPD plates containing glutathione. Colonies were counted to determine viability after growth for 3 days at 30°C. Data are shown as mean + SD from three independent experiments. (A-D) n.s.: not significant, *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding control (Student's t-test).</p

Specific functional gene groups are upregulated in cells lacking Hat1.

<p>(A) GO terms enriched among 2-fold significantly upregulated genes in logarithmically growing <i>hat1</i>Δ/Δ cells are shown. (B) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>rtt109</i>Δ/Δ strains only. (C) GO terms enriched within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>cac2</i>Δ/Δ strains only. (D) The panel shows GO terms found among genes significantly upregulated in the <i>hat1</i>Δ/Δ mutant only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains. (E) GO terms enriched among significantly upregulated genes in <i>hat1</i>Δ/Δ cells after treatment with H₂O₂ are shown. (F) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ strain only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains upon H₂O₂ treatment. (A-F) The corresponding p-values for the enrichment (empty bars) and the percentage of genes changed within the GO group (filled bars) are presented. The absolute number of regulated genes within a GO group is presented in brackets. Groups containing identical genes are depicted in the same color. Significantly regulated genes were defined by a p-value <0.05.</p

Deletion of <i>HAT1</i> and <i>HAT2</i> increases oxidative stress resistance and azole tolerance.

<p>(A) Cells lacking Hat1 or Hat2 show increased resistance to H₂O₂. Lack of both genes mimics the corresponding single deletion strains. (B) Deletion of <i>HAT1</i> increases resistance to <i>tert</i>-butyl hydroperoxide (tBOOH). Lack of Rtt109 does not affect tBOOH sensitivity. (C) Loss of Hat1 causes reduced susceptibility to voriconazole (Voric.) and itraconazole (Itrac.). Deletion of <i>HAT2</i> or <i>HAT1</i> and <i>HAT2</i> mimics loss of Hat1. (D) Deletion of <i>RTT109</i> or <i>RAD52</i> does not increase voriconazole tolerance. (A-D) Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days.</p

Higher ROS detoxification capacity of <i>hat1</i>Δ/Δ cells causes resistance to neutrophil killing.

<p>(A) Superoxide dismutases Sod4 and Sod5 are induced in <i>hat1</i>Δ/Δ cells. Expression levels of <i>SOD4</i> and <i>SOD5</i> in logarithmically growing cells were detected by RT-qPCR. Transcript levels were normalized to the expression level of the reference gene (RG) <i>RIP1</i>. Data are shown as mean + SD from 3 independent experiments. (B) Infection of macrophages with <i>hat1</i>Δ/Δ cells causes reduced ROS accumulation. ROS levels were determined by measuring luminol-dependent chemiluminescence [relative luciferase units (RLU) min^-1 per 1000 immune cells] in 2.5 min intervals during interaction of the indicated <i>C</i>. <i>albicans</i> strains with bone marrow-derived murine macrophages (BMDMs). One representative experiment is shown. Data were reproduced in three independent experiments. (C) Quantification of total ROS release upon interaction with BMDMs. Experiment was performed as described in (B). The area under the curve within 90 min of interaction was calculated. Data are shown as mean + SD from three independent experiments. (D) Cells lacking Hat1 show increased survival to neutrophil killing. Survival of <i>C</i>. <i>albicans</i> cells upon one hour interaction with murine bone marrow neutrophils was determined by plating and CFU counting. Data are shown as mean + SD from three independent experiments. (A-D) *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p

Resistance phenotypes caused by loss of Hat1 are specific for <i>C</i>. <i>albicans</i>.

<p>(A) Deletion of <i>HAT1</i> in <i>S</i>. <i>cerevisiae</i> (a), <i>C</i>. <i>glabrata</i> (b) and <i>S</i>. <i>pombe</i> (c) has no effect on H₂O₂ resistance. Exponentially growing cells were treated with 5 mM (a), 50 mM (b) or 20 mM (c) H₂O₂ for 2 hours. Cells were plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (B) Lack of Hat1 in <i>S</i>. <i>cerevisiae</i> (a) and <i>C</i>. <i>glabrata</i> (b) does not increase azole tolerance. Deletion of Hat1 in <i>S</i>. <i>pombe</i> reduces susceptibility to voriconazole (c). Logarithmically growing cells were diluted into medium containing 150 ng/ml (a), 1000 ng/ml (b) or 800 ng/ml (c) voriconazole and incubated at 30°C for 24 hours. OD₆₀₀ was determined and growth inhibition relative to untreated samples was calculated. Data are shown as mean + SD from three independent experiments. (C) <i>C</i>. <i>parapsilosis</i> (a) and <i>C</i>. <i>tropicalis</i> (b) <i>hat1</i>Δ/Δ cells show increased resistance to H₂O₂. Experiment was performed as described in (A). H₂O₂ concentrations were 50 mM (a) and 20 mM (b). (D) Loss of Hat1 in <i>C</i>. <i>parapsilosis</i> (a) and <i>C</i>. <i>tropicalis</i> (b) reduces susceptibility to voriconazole. Experiment was performed as described in (B). For <i>C</i>. <i>parapsilosis</i> cells were incubated for 41 hours prior to OD₆₀₀ measurement. Voriconazole concentrations were 50 ng/ml (a) and 200 ng/ml (b). (A-D) *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding wild-type (Student's t-test).</p

Lack of Hat1 accelerates induction of oxidative stress genes.

<p>(A) Catalase induction rate is strongly increased in <i>hat1</i>Δ/Δ cells. <i>CAT1</i> expression levels were measured by RT-qPCR after induction with 1.6 mM H₂O₂ at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) <i>PAT1</i>. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the <i>CAT1</i> locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the <i>CAT1</i> promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the <i>CAT1</i> locus. RNAPII levels were determined by ChIP at the <i>CAT1</i> CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. <i>GPX1</i> (a) and <i>GST1</i> (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the <i>GPX1</i> and <i>GST1</i> loci. RNAPII levels were determined by ChIP at the <i>GPX1</i> (a) and <i>GST1</i> (b) genes. (F+G) Loss of Cac2 increases the induction rate of both <i>GPX1</i> and <i>GST1</i> following H₂O₂ treatment. Experimental conditions were used as described in (A).</p