scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thailin Lao-Gonzalez ◽  
Alexi Bueno-Soler ◽  
Arnelys Duran-Hernandez ◽  
Katya Sosa-Aguiar ◽  
Luis Eduardo Hinojosa-Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

AbstractThe high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

2020 ◽  
Author(s):  
Thailín Lao-González ◽  
Alexi Bueno Soler ◽  
Arnelys Duran Hernandez ◽  
Katya Sosa Aguiar ◽  
Luis Eduardo Hinojosa Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

Abstract The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

2020 ◽  
Author(s):  
Thailín Lao-González ◽  
Alexi Bueno Soler ◽  
Arnelys Duran Hernandez ◽  
Katya Sosa Aguiar ◽  
Luis Eduardo Hinojosa Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Screening Strategy ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Expression Levels ◽  
Short Period ◽  
Free Media

Abstract The high prices of biopharmaceutics or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceutics. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines in a short period of time with higher levels of expression. Here, we obtain trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein free media and suspension culture and lentiviral vectors. The results shown that early screening strategy allowed to obtain recombinant CHO cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular HC polypeptides by flow cytometry was a useful tool to characterize the homogeneity of cell population and our results suggest that might be used to predict the expression levels of monoclonal antibodies in early stages of cell line development process. Furthermore, using T-flask approach the assessment of the expression levels was studied in a setting more similar to that of a final production process. Finally, trastuzumab-expressing CHO-K1 clones were generated, characterized by batch experiments and preliminary results related to HER2-recognition capacity were successful. Further improvements in up-stream and down-stream steps related to this strategy will improve specific productivities and volumetric productivities of these clones.

scholarly journals BCG Substrains Change Their Outermost Surface as a Function of Growth Media

Vaccines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 40
Author(s):  
Sandra Guallar-Garrido ◽  
Farners Almiñana-Rapún ◽  
Víctor Campo-Pérez ◽  
Eduard Torrents ◽  
Marina Luquin ◽  
...  
Keyword(s):  
Culture Media ◽  
Chemical Properties ◽  
Growth Conditions ◽  
Neutral Red ◽  
Culture Conditions ◽  
Host Cells ◽  
Growth Media ◽  
Phenotypic Characteristics ◽  
Outermost Surface ◽  
Red Dye

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

scholarly journals Effect of Host Cells on Low- and Medium-Pressure UV Inactivation of Adenoviruses

2010 ◽  
Vol 76 (21) ◽  
pp. 7068-7075 ◽  
Author(s):  
Huiling Guo ◽  
Xiaona Chu ◽  
Jiangyong Hu
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Host Cells ◽  
Uv Disinfection ◽  
Human Embryonic Kidney ◽  
Experimental Conditions ◽  
Medium Pressure ◽  
Serotype 5 ◽  
Uv Dose

ABSTRACT UV disinfection is highly effective against most pathogens, with the exception of the adenoviruses (AD). To date, many studies have focused on low-pressure (LP) UV inactivation of AD, but little is known about the effect of medium-pressure (MP) UV inactivation of AD. Despite numerous studies of LP UV inactivation of AD, extreme variabilities in the LP UV dose requirements of AD had been observed because of differing experimental conditions used, such as the types of cell lines used for AD enumeration. This study therefore investigates the effect of three different host cell lines (PLC/PRF/5, human embryonic kidney 293 [HEK293], and XP17BE) on the LP and MP UV dose requirements of AD serotype 5 (AD5), AD40, and AD41 under similar experimental settings. Results showed that for 4-log inactivation of AD, LP UV and MP UV doses needed to be in the ranges of 123 to 182 mJ/cm2 and 65 to 90 mJ/cm2, respectively, when HEK293 and PLC/PRF/5 cells were used for enumeration. The UV doses required for MP UV inactivation of AD were significantly lower than those required for LP UV inactivation (P value < 0.05). When different cell lines were used for enumeration, UV dose requirements for AD differed. AD were portrayed to be most susceptible to UV (LP UV doses of <57 mJ/cm2 and MP UV doses of <42 mJ/cm2 for 4-log AD inactivation) when the XP17BE cells were used as the host cell. The use of different cell lines for AD enumeration affected LP UV dose results more significantly than MP UV dose results (P value < 0.05). Cell line variability factors for LP UV disinfection (CLLP) and MP UV disinfection (CLMP) for AD5, AD40, and AD41 enumerated with HEK293, PLC/PRF/5, and XP17BE cells were in the ranges of 1.0 to 3.2 and 1.0 to 2.5, respectively.

scholarly journals Growth properties and alloantigenic expression of murine lymphoblastoid cell lines.

1975 ◽  
Vol 142 (2) ◽  
pp. 378-390 ◽  
Author(s):  
R K Zwerner ◽  
R T Acton
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fetal Bovine Serum ◽  
Horse Serum ◽  
Culture Conditions ◽  
Lymphoblastoid Cell Lines ◽  
Stationary Growth ◽  
Growth Properties ◽  
Fetal Bovine ◽  
Logarithmic Growth

Murine lymphoblastoid cell lines were evaluated for their expression of Thy-1 and thymus leukemia (TL) differentiation alloantigens. Two culture conditions were shown to affect this expression. Cells grown in fetal bovine serum (FBS)-enriched medium expressed up to 15 times the amount of TL as cells grown in horse serum (HS)-enriched medium. Thy-1 expression was less affected by the type of serum used for culture. The phase of growth when the cells were harvested, was demonstrated to affect the expression of Thy-1. The expression of Thy-1.2 for one cell line examined, L-251A, during logarithmic growth was threefold greater than cells collected during either lag or stationary growth. When culture conditions were standardized a ranking of the amount of Thy-1 and TL expressed by several cell lines was made. All cell lines, except one, L-1210, expressed Thy-1. There was a 450-fold difference in the expression of Thy-1 between the cell lines evaluated. Seven cell lines expressed TL-1,2,3 with a ninefold difference in the amount of expression. The L-251A cell line was cultured in a 14 liter fermentor for a 26 day period. During this time TL and Thy-1 expression did not vary significantly, demonstrating that lymphoblastoid cell lines can be cultured on a continuous basis and will continue to express their surface alloantigens.

scholarly journals Evolution from adherent to suspension: systems biology of HEK293 cell line development

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Magdalena Malm ◽  
Rasool Saghaleyni ◽  
Magnus Lundqvist ◽  
Marco Giudici ◽  
Veronique Chotteau ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Hek293 Cell ◽  
Cholesterol Biosynthesis ◽  
Human Cell Line ◽  
Gene Copy ◽  
Cell Line Development ◽  
Suspension Systems

Abstract The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

Identification of regulatory pathways across different cell lines (NHBE and PBMC) in COVID-19 patients using transcriptome analysis v1

2021 ◽  
Author(s):  
Lavanya not provided C ◽  
Vidya Niranjan ◽  
Aajnaa not provided Upadhyaya ◽  
Arpita not provided Guha Neogi
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Analysis ◽  
Pathway Analysis ◽  
Gene Expression Analysis ◽  
Reference Genome ◽  
Host Cells ◽  
Human Reference Genome ◽  
Regulatory Pathways

The Sars-CoV-2 virus is a previously uncharacterized coronavirus and causative agent of the COVID-19 pandemic. Gene expression analysis followed by pathway analysis helps researchers to find possible key targets present in biological pathways of host cells that are targeted by the SARS-CoV-2 virus. This review considers the peripheral blood mononuclear cell line (PBMC) and the normal human bronchial epithelial (NHBE) cell line, both of which support SARS-CoV-2 viral replication. Pathway analysis between the healthy and patient samples of the respective cell lines shall provide useful insights on the COVID-19 disease. Initially, the datasets from the respective cell lines were collected from the NCBI databank. These datasets underwent further analysis and were mapped and aligned to the human reference genome. This outputs the file in the BAM format. The BAM files along with the human reference genome in the GFF format were uploaded to an open-source software called OmicsBox 2.0 for differential gene expression analysis. This resulted in the generation of a table containing the differentially expressed genes which were upregulated and downregulated. These gene lists were uploaded to various pathway analyzers that map the significant genes to the most significant pathways. In this project, KOBAS 3.0 and Enrichr were used for pathway analysis. The pathways obtained from the above-mentioned pathway analyzers were further narrowed down by manual comparison. It was observed that many pathways were similar between the NHBE and PBMC cell lines. However, they were also different in terms of their overall nature. In this project, many patterns were seen through the pathways obtained, however, further optimization and functionality studies must be performed in order to establish conclusive results on the scope of the COVID-19 disease. Expanding research on the scope of the disease by going back to the basics will generate new and valuable information about the virus. This knowledge will help us combat the disease in a better and appropriate manner.

scholarly journals 676 PD-L1 is induced by the periodontal pathogen porphyromonas gingivalis and can be blocked by small molecule gingipain inhibitors, including atuzaginstat

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A715-A715
Author(s):  
Shirin Arastu-Kapur ◽  
Mai Nguyen ◽  
Sean Broce ◽  
Joseph Vacca ◽  
Kirk Ehmsen ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Porphyromonas Gingivalis ◽  
Wnt Pathway ◽  
Host Cells ◽  
Periodontal Pathogen ◽  
Cancer Tissue ◽  
Dependent Manner

BackgroundThe periodontal pathogen Porphyromonas gingivalis (Pg) has been linked to esophageal and other cancers through epidemiology studies. Pg’s protease virulence factors known as gingipains have been identified in esophageal cancer tissue and correlate with worse disease prognosis. Anti-PD-1 antibodies have shown some success in esophageal cancer treatment, but further understanding of the induction of PD-L1 in esophageal cells is needed to identify potential treatment modalities. Pg has been shown to induce PD-L1 on the surface of infected cells, suggesting that the presence of Pg in esophageal cancer cells may contribute to PD-L1 expression and immune escape. One of the pathways known to induce PD-L1 is wnt pathway activation resulting in b-catenin translocation to the nucleus. Prior studies have demonstrated that Pg activates the wnt pathway by a non-canonical mechanism, leading to b-catenin nuclear localization.MethodsAn immortalized non-transformed esophageal cell line, Het-1A, was used to investigate the level of PD-L1 induction by Pg infection using quantitative immunofluorescence. PD-L1 expression was measured using irreversible gingipain inhibitors against lysine-gingipain (Kgp) and arginine-gingipain (Rgp). Pg-induced PD-L1 expression pathways were investigated by Western blot and qPCR. PD-L1 induction by Pg was characterized in cancer cell lines that have an endogenous level of PD-L1 expression, including tongue squamous cell carcinoma (SCC25) and neuroblastoma (SH-SY5Y). PD-L1 induction by Pg was assessed in a murine derived RAW macrophage cell line that is critical for anti-PD-1 responses.ResultsPg infection increased PD-L1 expression on Het-1A cells within 24 hours of infection and increased PD-L1 mRNA within 4 hours of infection. PD-L1 expression level correlated with cellular bacterial burden on the cells in a dose-dependent manner. PD-L1 expression was decreased by the Kgp inhibitor, atuzaginstat, or an Rgp inhibitor, COR613, and PD-L1 expression was completely blocked when both gingipain inhibitors were used together (figure 1). Pg also induced expression of PD-L1 on the surface of infected SCC-25, SH-SY5Y, and RAW cell lines. Western blot analysis and qPCR revealed that Kgp inhibition, but not Rgp inhibition, was able to inhibit the non-canonical activation of b-catenin and down regulation of classical wnt pathway effectors at both the mRNA and protein level.Abstract 676 Figure 1Gingipain inhibitors block PD-L1 induced by PgPg grown with and labeled by red fluorescent membrane-incorporated dye was pre-treated with vehicle or the compounds listed for 30 min. Het-1A cells were infected (MOI = 20) for 24 hours, washed, fixed and stained for visualization of the nuclei (DAPI, blue), PD-L1 protein (anti-PDL1 primary and secondary antibodies, green), and Pg infection (red). Images were captured with immunofluorescent confocal microscopy.ConclusionsIn host cells infected with Pg, gingipains mediate the induction of PD-L1 as a mechanism of immune evasion through the non-canonical activation of the wnt pathway. Further studies to elucidate induction mechanisms are in progress. In esophageal cancer and other cancers infected with Pg, combining gingipain inhibitors with anti-PD-1 therapy may improve treatment outcomes.

scholarly journals Multi-omics profiling of CHO parental hosts reveals cell line-specific variations in bioprocessing traits

2019 ◽  
Author(s):  
Meiyappan Lakshmanan ◽  
Yee Jiun Kok ◽  
Alison P. Lee ◽  
Sarantos Kyriakopoulos ◽  
Hsueh Lee Lim ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cho Cells ◽  
Cell Cycle Progression ◽  
Chinese Hamster ◽  
Suspension Cultures ◽  
Host Cells ◽  
Phenotypic Traits ◽  
Cell Factories

AbstractChinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines have been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11 and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N- and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics analysis indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N- and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.

scholarly journals Rich production media as a platform for CHO cell line development

2020 ◽  
Author(s):  
Yong Jae Kim ◽  
Sang Kyul Han ◽  
Seongtae Yoon ◽  
Chan-Wha Kim
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Mammalian Cells ◽  
Culture Media ◽  
Chinese Hamster ◽  
Cell Cloning ◽  
Cell Line Development ◽  
Solid Media ◽  
Semi Solid ◽  
Set Up

Abstract Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process.

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