RNAseq of two recombinant CHO cell lines differing only in the factor “produced monoclonal antibody\"
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ABSTRACT:
PROVIDER: PRJEB48931 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR7417870_1.fastq.gz | Fastqsanger.gz | |||
| ERR7417870_2.fastq.gz | Fastqsanger.gz | |||
| ERR7417863_1.fastq.gz | Fastqsanger.gz | |||
| ERR7417863_2.fastq.gz | Fastqsanger.gz | |||
| ERR7417864_1.fastq.gz | Fastqsanger.gz |
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Project description:We present the first comprehensive genome-wide view of CHO translational activity during recombinant expression by the application of ribosome profiling. The distribution of translational power in CHO cells was analyzed in the context of the recombinant mRNAs and the endogenous mRNA pool. In an antibody-producing CHO cell line, the recombinant mRNAs were found to be the most abundant transcripts and also sequestered a substantial amount of translating ribosomes (up to 15%). The recombinant mRNAs were translated as efficiently as the endogenous mRNAs, and changes in translation and transcription of the recombinant mRNAs were directly reflected in changes in specific productivity. Interestingly, improvements in bioprocess quality attributes were achieved by depleting the highly expressed and translated inert NeoR mRNA by siRNA-mediated knock-down. This resulted in improved cellular growth, which was accompanied by an 18% increase in antibody titers. This study is the first to carefully map the translatome of the CHO cell to the nucleotide level and to demonstrate how recombinant expression affects the cell on the translational level.
2016-12-21 | GSE79512 | GEO
Project description:Lysosomal storage diseases are rare life-threatening disorders caused by deficiency of a lysosomal enzyme, and delivery of a recombinant replacement enzyme is the primary therapy for several of these diseases. The structures of N-glycans on recombinant replacement enzymes are important for their therapeutic efficacy, biodistribution and circulation time. Here we use alpha-galactosidase A (GLA) and beta-glucoscerebrosidase (GBA) as representative replacement enzymes, and present a panel of genetically engineered CHO cells that enable production of lysosomal replacement enzymes with different N-glycan features. We also tested several GLA variants with distinct glycoforms in a Fabry mouse model and present evidence that GLA glycoforms capped with alpha 2-3 linked sialic acids may represent a new strategy to overcome the most critical problems of rapid clearance in liver and poor biodistribution found with current ERTs.
2019-05-31 | PXD013140 | Pride
Project description:Mature microRNA levels were analyzed in 4 producing CHO cell lines expressing 2 structurally different recombinant proteins at low and high level as well as in 1 non-producing CHO cell line. The goal was to identify microRNAs that are involved in heterologous protein formation and secretion. 5 recombinant CHO cell lines. 3 biological replicates each. All samples were hybridized against a common reference (pool of all total RNA samples).
2014-08-04 | E-GEOD-57023 | biostudies-arrayexpress
Project description:Mature microRNA levels were analyzed in 4 producing CHO cell lines expressing 2 structurally different recombinant proteins at low and high level as well as in 1 non-producing CHO cell line. The goal was to identify microRNAs that are involved in heterologous protein formation and secretion.
2014-08-04 | GSE57023 | GEO
Project description:Chinese Hamster ovary (CHO) cells are the main platform used to produce recombinant proteins in the biopharmaceutical industry and are commonly cultured in either fed-batch or perfusion mode. However, the optimization of the complex biological systems used in such processes is extremely challenging. Omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have applied proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The proteomics data show an increase of structural proteins as cell density increase, signs of oxidative stress and changes in glutathione metabolism at very high cell densities. Additionally, metabolomic profiling was carried out. See article “High cell density culture has a maintained exoproteome and metabolome” for more information.
2018-07-04 | PXD008760 | Pride
Project description:Background: Chinese hamster ovary (CHO) cells are the most widely used mammalian host for recombinant protein production. The primary production method is to construct stable, high-yielding cell lines to provide high-quality, low-cost products. The key to optimizing recombinant protein production is to modify engineered cell lines to obtain better growth, expression, or product quality characteristics. Site-specific integration technology can build monoclonal cell lines with ideal stability, yield, and epigenetics by integrating transgenes at stable transcriptional hotspots, becoming a potential tool for constructing genetically engineered cell lines. The previous study screened the C12orf35 site of CHO cells and built an engineered cell line expressing a mAb by CRISPR/Cas9. While the mechanism of site-specific integration of transgenes to achieve high-efficiency expression also needs to be further studied. In this study, we used the extracellular domains of CLL1 and CD33 as model proteins to construct engineered cell lines integrating at C12orf35. Then we studied the growth, expression, and passage stability of these cell lines and conducted transcriptomic studies. Method: In this study, the transgenes CLL1 and CD33 were site-specifically integrated into the C12orf35 site of the CHO-S cell by CRISPR/Cas9 technology. To obtain stable and high-yielding cell lines, we screened and identified monoclonal cell lines by 5’/3’ junction PCR, out-out PCR, and Western blot. Then we compared the growth characteristics of CLL1 and CD33 monoclonal cell lines with wild-type CHO-S cell lines to investigate the growth characteristics. We studied the protein yield and affinity of CLL1 and CD33 cell lines through protein purification, BCA quantification, and ELISA. In addition, we assessed the stability of monoclonal cell lines in long-term culture by studying the growth characteristics, protein yield, and protein quality of early (5th-10th passage) and late (about 20th-25th passage) cells. We performed transcriptomic studies on cell lines with different transgenes, including CLL1, CD33, and anti-PD-1 mAb, inserted at C12orf35. We analyzed the differential genes, GO enrichment, and KEGG enrichment of cell lines hoping to explore the reasons for the phenotypic changes of cell lines from the transcriptional level, as well as the high expression mechanism of C12orf35 site-specific integration cell lines. Results:Using the site-specific integration technology mediated by CRISPR/Cas9, we successfully built CLL1 and CD33 monoclonal cell lines integrated at the C12orf35 site. We evaluated the cell growth characteristics and protein expression properties. The results showed that the insertion of different transgenes at the C12orf35 site had no specific effect on the growth state of the cells. The proteins yields were about 120 mg/L and 160 mg/L, respectively. And the EC50 values of CLL1 and CD33 with the corresponding antibodies reached about 0.36 μg/mL and 0.1 μg/mL, respectively. In addition, the above indicators had similar properties between the 5th and 20th generations, indicating that the specific integration at C12orf35 can achieve both high expression of the target protein and production stability. Transcriptomic analysis showed that the integration of transgenes at the C12orf35 site of CHO cells mainly induced genes in the GO term of “Regulation of transcription, DNA-templated” differentially expressed. Further analysis screened that ATF4 and multiple ZFPs family members were significantly up-regulated. ATF4 and ZFPs are related to gene transcription and protein synthesis, which may be the mechanism of the stable and high expression of the cell lines. Besides, differential gene analysis showed that Car4 and CXCL12 were significantly up- and down-regulated in CD33 cells, respectively. And KEGG enrichment showed that Wnt, PI3K/AKT, and JAK/STAT pathways related to cell proliferation and apoptosis were significantly enriched in CD33 cells. These genes and pathways may be related to the growth state of CD33. In summary, this paper studied the growth characteristics, protein expression characteristics, and stability of the C12orf35 site-specific integration of recombinant CHO cell lines. Combined with transcriptomic analysis, this paper explored the stable and high expression mechanism of these cell lines, hoping to provide a theoretical reference for the subsequent construction and optimization of high-expression stable cell lines.
2023-05-31 | GSE203219 | GEO
Project description:Biologics have emerged as leading blockbuster therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here we identify bottlenecks limiting expression of challenging recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293. Surprisingly, one third of the challenging human proteins displayed an improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, a key set of genes with extreme expression variation was identified. Among these, ATF4 and SRP9 were validated as general productivity boosters in CHO. Further, we found that more heavily glycosylated products benefit more from the elevated activities of the N- and O-glycosyltransferases found in HEK293 cells. Collectively, our results demonstrate the utilization of HEK293 cells for expression rescue of human proteins and suggest a strategy for identification of secretory pathway components improving yield and quality in HEK293 and CHO.
2022-06-22 | GSE157729 | GEO
Project description:The aim of the study was to determine the epitope targeted by 31E10/E7 mouse monoclonal antibody (mAb). mAb 31E10/E7 was diluted at 1:2000 and incubated on a non-commercial Protein Microarray platform printed with NHBA specific recombinant protein fragments and full length NHBA of different variants.
2016-05-27 | GSE81378 | GEO
Project description:The mechanism of action behind the increased secretion capacity of the amplified progeny of CHO cells is investigated in this study through comparative transcriptomic analysis
2019-10-01 | GSE133511 | GEO
Project description:Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.
| S-EPMC4966638 | biostudies-literature