Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell

2013 ◽  
Vol 29 (4) ◽  
pp. 980-985 ◽  
Author(s):  
Zhilan Hu ◽  
Donglin Guo ◽  
Shirley S.M. Yip ◽  
Dejin Zhan ◽  
Shahram Misaghi ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Stable Cell Line ◽  
Cell Line Development

scholarly journals Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery

2019 ◽  
Vol 20 (7) ◽  
pp. 1729 ◽  
Author(s):  
R. Lavoie ◽  
Alice di Fazio ◽  
R. Blackburn ◽  
Michael Goshe ◽  
Ruben Carbonell ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Mixed Mode ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Peptide Ligand ◽  
Host Cell Proteins ◽  
Ligand Discovery ◽  
Exchange Resins

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.

Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line

1988 ◽  
Vol 8 (6) ◽  
pp. 2316-2327
Author(s):  
C Ma ◽  
J E Looney ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I ◽  
Type Ii ◽  
Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

scholarly journals Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2407
Author(s):  
Sung Wook Shin ◽  
Dongwoo Kim ◽  
Jae Seong Lee
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Cell Line Development ◽  
Cho Cell Line ◽  
Vector Systems ◽  
Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

scholarly journals Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

1988 ◽  
Vol 8 (6) ◽  
pp. 2316-2327 ◽  
Author(s):  
C Ma ◽  
J E Looney ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I ◽  
Type Ii ◽  
Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

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