June 26, 2022

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Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line

Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line

 

 

Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line.  When studying the proteome of CHO cells, it is important to consider post-translational modifications (PTMs), such as phosphorylation.

Phosphorylation can be used as a molecular switch to phosphorylate and dephosphorylate proteins. An example of this is protein kinase B, which regulates cell survival by being activated after phosphorylation of its Ser and Thr residues. It is estimated that about 30% of proteins are phosphorylated at any time in the cell).

PTMs can change the phenotype of proteins by covalent modification of amino acids or cleavage of proteins. If we consider phosphorylation events when studying the proteome of CHO cells, we can more accurately map the pathways that affect the phenotype of our interest.

 

 


How to determine the potential of CHO cell engineering as the goal to improve the titer and Qp of the product?

Researchers from Dublin City University in Ireland and Eli Lilly and Company published an article Global phosphoproteomicstudy of high/low specific productivity industrially relevant mAb producingrecombinant CHO cell lines. The study explained these points.

Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line


First of all, from the perspective of materials and methods.

1. Perform batch culture of CHO cell lines.

Each CDCL was cultured in duplicate culture flasks at 150 rpm, 6% CO2, and 36°C. The temperature on the 4th day changed to 32°C. The cells were cultured in the Kuhner shaker ISF1-X (Kuhner) for 17 days. On the 4th, 7th and 10th day, the cells were fed the feed produced by Eli Lilly. If necessary, the cells were also fed with glucose on the 12th and 14th days.

An automatic VicellTM XR cell viability analyzer was used to determine cell density, viability, cell size and cell volume. The productivity of the cell size was measured using the calculation method described below and previously published methods.


Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line


2. Extraction of total protein and digestion of protein in solution.

CDCLs with high and low Qp were grown in a 17-day feeding batch shaker study, and cell pellets were extracted on the 6th and 10th day of culture. The cell pellets were harvested by centrifugation at 1000 g for 5 minutes. The cell pellets were washed with phosphate buffered saline. Use 1mL lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 30mM Tris, pH8.5) to lyse 2 X 106 cells, then centrifuge at 14,000g for 15 minutes, add 0.5M DDT to the lysis buffer, Incubate at 56 °C for 20 minutes. The Bradford assay (Bio-rad) was used to determine the protein concentration. The filter-assisted sample preparation (FASP) method and the C18 peptide purification method were used to prepare 100 μg of each sample for LC-MS/MS analysis. The samples were digested with 1:200 Lys-c and 1:100 sequence-level modified trypsin (Thermo Fisher Scientific).


3. Enrichment of phosphopeptides and digestion of proteins in solution.

Use lysis buffer (8M urea, 50mM Tris, 75mM NaCl (pH 8.2) buffer, supplemented with 1X Halt protease inhi-bitors (Thermo Fisher Scientific)) to lyse cell pellets, and digest each cell lysate as described previously And enriched phosphopeptides (Kaushik et al., 2018). The samples were digested with 1:200 Lys-c and 1:100 sequence-level modified trypsin (Thermo Fisher Scientific).


4. LC-MS/MS total.

An Orbitrap mass analyzer was used for scanning with a resolution of 120,000 (at m/z 200), a maximum injection time of 50 ms, and an automatic gain control (AGC) value of 4 X 105. The top velocity acquisition algorithm is used to determine the fragmentation amount of the selected precursor ion. The isolation width is 1.6 Da and is used to isolate selected precursor ions in the quadrupole. After 60 seconds, the analyzed peptides are dynamically excluded, and only peptides with a charge state between 2+ and 7 will be excluded. The normalized collision energy of 28% was used to fragment the front cursor ions, and the generated MS/MS ions were measured. MS/MS scanning conditions are usually as follows: Joule’s AGC value is 2 X 104, and the maximum file time is 35 ms.


5. Gene ontology.

The GO database DAVID (https://david.ncifcrf.gov), TopGene (https://toppgene.cchmc.org) and STRING (https://string-db.org) were used to analyze all the differences List of expressed original proteins. The special gene symbols of the differentially expressed proteins are input into the gene ontology database. These gene ontology databases are used to identify biological functions and molecular processes, which are enriched in our list of differentially expressed proteins.


6. Statistical analysis.

A two-tailed Student’s t test was performed on all phenotypic parameters measured between high and low Qp CHO cell CDCLs. F-tests were performed on all data to determine whether equal or unequal variables should be used in the Student’s t-test. The F statistic value lower than the critical F value indicates that the variances are equal, and the F statistic value higher than the critical F value indicates that the variances are not equal. Data with P value ≤0.05 is considered low significance, ≤0.005 is considered significant, and ≤0.001 is considered highly significant.



The results obtained

The high and low Qp CDCLs were selected from industrially relevant IgG4-expressing CDCLs, which were produced as part of a cell line development project. These CDCLs were cultured in a 17-day feeding batch shaker study, and samples were collected for differential LC-MS/MS analysis. If the CDCL reaches a peak Qp of >20pg/cell/day, it is regarded as having a high Qp, and if it reaches a peak Qp of <20pg/cell/day, it is regarded as having a low Qp. CDCLs with high Qp were selected based on their high Qp and high titer, and only included in the group if they had a higher survival rate and VCD (Figure 1). CDCLs with low Qp were selected based on their low Qp and titer, and were only included in the group if they had a high survival rate and VCD (Figure 1). Choose the 6th and 10th day for LC-MS/MS analysis, because the high and low Qp CDCL maintained high viability and VCD at these time points. The viability of all CDCL and VCD began to decline after the 10th day. Day 6 represents the early exponential phase of growth, and day 10 represents the fixed phase of growth.

In addition to titer, Qp, survival rate, and VCD, all CDCLs have many characteristics. CDCLs that are similar in other phenotypic characteristics can be selected in order to have a cleaner background when comparing Qp. Two other parameters that may be important and that are statistically significantly different are:

1) Total cell density (TCD) (Figure 1E). On day 10, the cell density of low-Qp CDCLs increased significantly.

2) The integrated viable cell density (IVCD) (Figure 1F) was also found to be significantly higher in low-Qp CDCLs on day 10 and 14, indicating that the accumulation of viable cells in low-Qp CDCLs was higher at these time points. No statistical significance of cell volume 9 (Supplementary Figure 1A), cell size (Supplementary Figure 1B), transcript copy number (Supplementary Figure 1C) or gene copy number (Supplementary Figure 1D) was observed among the selected cell lines This makes the difference we observe in the proteome and phosphoprotein group more likely to be involved in the productivity phenotype.

Although the gene copy number tends to increase in high-Qp CDCLs, this is not statistically significant due to the high degree of difference in gene copy number among high-Qp CDCLs. Throughout the culture process, the waste and metabolites of the cells were also measured. There was no observation between high and low Qp CDCLs in lactate (Supplementary Figure 2A), ammonia (Supplementary Figure 2B), glutamine (Supplementary Figure 2C), glucose (Supplementary Figure 2D) or glutamate (Supplementary Figure 2E) The significant difference.

Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line


On the 6th and 10th day of culture, samples were taken from high and low Qp CDCLs for quantitative differential LC-MS/MS analysis. Using total protein LC-MS/MS analysis, more than 4000 total proteins were identified in each sample. An unlabeled LC-MS/MS analysis was then used to determine the total protein differentially expressed between high and low Qp CDCLs. The differentially expressed total protein list was determined to include only proteins recognized by 2 or more unique peptides, with a folding change of >1.5 and an analysis of variance of <0.05.

On the 6th day, 137 differently expressed total proteins that met these criteria were identified, of which 75 proteins had increased expression levels, and 61 proteins had decreased expression levels in high QpCDCLs. On the 10th day, 189 differently expressed total proteins were identified, of which 87 were increased in expression and 101 proteins were decreased in high QpCDCLs. We identified 30 differently expressed proteins on day 6 and day 10. At these two time points, it was also found that proteins related to tRNA aminoacylation promoted protein translation had different expressions (Table 1). All differentially expressed total proteins and phosphoproteins identified in this study are listed in Table 2.


Global Phosphorylated Proteomics Study on Recombinant CHO Cell Line

 

In this study, they also identified several other translation-related phosphoproteins and total proteins, which have different expressions between high and low Qp CDCL (Table 3). Many of these proteins have a special relationship with cell proliferation and the cell cycle of mitosis, and their expression is reduced in high-Qp CDCLs.

Compared with high-Qp CDCLs, these CDCLs may have a lower Qp because they direct their translation machinery to translate cell cycle-related mRNA, although in terms of its VCD, the cell does not seem to reap this reward. However, the low-Qp CDCL did show significantly higher TCD and higher on day 10 and higher IVCD on day 10 and 14.

These proteins and phosphoproteins can be further studied as potential targets for cell engineering of high-Qp CHO cell lines. Reducing the expression of these proteins in the CHO cell line can release the translation machinery, allowing it to focus on translating recombinant protein products. For example, the phosphorylation of EIF4G1 on Ser-1224 can use genome engineering tools, such as CRISPR/Cas9, to determine the specific function of the phosphate site.

Phosphoproteins such as RPL12 and NCL can also be targeted for knockout, trying to release translation capabilities and increase the translation of recombinant proteins. The results presented here also show that reducing the expression of these proteins has almost no negative impact on cell growth, which is ideal for recombinant protein production.



This study conducted a comprehensive phosphoproteomic investigation on high/low Qp industrial-related CHO cell lines, and these cell lines have been fully described at the phenotypic level. These results identify the differentially expressed proteins between high and low Qp CHO CDCLs, which can be further studied as a potential target for rationally engineering CHO cell lines to achieve high-standard production.

Proteins related to amino acid availability have been shown to have increased expression in high Qp CHO CDCLs. It was found that the phosphorylation of GCN2 at Thr-727 was increased in high-Qp CDCLs. The phosphorylation of GCN2 has been attributed to many cellular functions, including the response to stress; however, the role of Thr-727 phosphorylation detected here is unknown.

The results of this study also show that a major feature of low-Qp CDCLs is high levels of mRNA transcription, which promotes the progression of the cell cycle, although this benefit is not reflected in VCDs with low-Qp CDCLs.

 

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