Factors affecting the sialylation of Fc- fusion protein in recombinant CHO cell culture

Over the past decade, product titers from recombinant CHO cells have increased significantly. However, not only the product titer but also the product quality is important for therapeutic efficacy. When sodium butyrate (NaBu) was added to a culture of CHO cells producing Fc-fusion protein, it increased Fc-fusion protein titer, but it decreased sialylation of the Fc-fusion protein. Analysis of N-glycosylation-related genes revealed that altered expression patterns in st3gal3, neu 1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the Fc-fusion protein by NaBu. St3gal3, sialic transferase, was down-regulated and, nue1 and nue3, sialidases, were up-regulated by NaBu. Addition of LiCl decreased sialylation of Fc-fusion protein as well. However, unlike NaBu, LiCl did not affect the sialidase expression. Stressful culture conditions such as high ammonia concentration and hyperosmolality, which are encountered during the culture, also decreased the sialylation of Fc-fusion protein. In the case of ammonia, ammonia was found to increase the neu 1 and neu3 mRNA expression levels. Down-regulation of sialidases successfully increased the sialylation of Fc-fusion protein in the presence of ammonia. Interestingly, unlike ammonia treatment, hyperosmolality did not increase the sialidase mRNA expression level. Hyperosmolality appeared to increase the lysosomal exocytosis of sialidase 1 located in the lysosomes. Taken together, a number of factors can be seen to affect the sialyation of Fc-fusion protein in rCHO cell cultures, but through different mechanisms

Genomic understanding of clonal variation in recombinant CHO cells

In the current manufacturing platform for large-scale production of therapeutic proteins, recombinant Chinese hamster ovary (rCHO) cell line generation is probably the most time-consuming step. The high degree of phenotypic heterogeneity in the absolute transgene expression level and variable responses to culture conditions among the selected clones, which has been referred to as “clonal variation”, makes the cell line generation process laborious and time-consuming. Upon transfection, a transgene is randomly integrated into the chromosomes of the host cells, and the integration site of the transgene is believed to be responsible for this clonal variation. However, the lack of understanding the molecular basis involved in clonal variation has hindered rapid cell line generation with a predicted culture performance. With the availability of CHO genome sequences and targeted genome sequencing/editing technologies, we investigated “clonal variation” with an emphasis on the transgene integration sites and configuration of the integrated transgenes. rCHO cell clones expressing GFP, which were constructed by random integration of the GFP expression vector into CHOK1 host cells, revealed a remarkable variability in GFP expression at 37°C. Furthermore, they showed different responses to culture temperature shifts from 37°C to 33°C confirming the existence of clonal variation. Application of targeted sequencing by the proximity ligation to the transgenes enabled the mapping of the integrated transgenes, and thereby the genomic integration sites of the transgene in the representative rCHO cell clones showing different responses to hypothermia were identified. To determine whether the different responses of the rCHO clones to hypothermia were due to the different integration sites of the transgenes, rCHO cell clones expressing GFP were also constructed by CRISPR/Cas9-mediated targeted integration of an intact transgene into CHOK1 host cells at the integration sites that were identified in the rCHO cells exhibiting different responses to hypothermia. Surprisingly, the rCHO clones constructed with targeted integration, regardless of the integration site of the gene, had similar expression patterns in terms of the absolute expression level and responses to hypothermia. They all exhibited enhanced GFP expression with hypothermia. Evidence of several rearrangements in the integrated transgene was detected in the rCHO clones constructed with random integration, which may interrupt the normal function of regulatory elements, particularly promoters. Promoter replacement and dissection results support the crucial role of promoter elements in the differential transgene expression patterns at the identical genomic site. Taken together, we demonstrate the complex nature of “clonal variation” in rCHO cells which encompasses the concept of vector elements and their rearrangement upon random integration besides just the genomic integration sites. Further characterization of the interactions between the integration sites and vector regulatory elements together with controlled integration of transgenes could lead to the tailored control of recombinant gene expression in rCHO cells while minimizing clonal variation

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

Abstract To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

<p>Abstract</p> <p>Background</p> <p>Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis.</p> <p>Results</p> <p>From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM.</p> <p>Conclusions</p> <p>The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.</p

Reduction of metabolic waste products, ammonia and lactate, through the coupling of GS selection and LDH-A down-regulation in CHO cells

The cultivation of Chinese hamster ovary (CHO) cells for the production of therapeutic proteins inevitably accompanies the production of metabolic wastes, mostly ammonia and lactate. Ammonia alters cell growth, productivity and the glycosylation patterns of proteins, and lactate acidifies culture media, having negative effects on cell culture. A stable CHO cell line should be established for the manufacturing process of therapeutic proteins, and the development of stable cell lines is usually based on two expression systems: the dihydrofolate reductase (DHFR) system and the glutamine synthetase (GS) system. Compared to the DHFR system, the GS system produces a reduced level of ammonia because the GS protein uses ammonia to produce glutamine. In order to overcome the lactate accumulation problem, down-regulation of the lactate-producing enzyme, lactate dehydrogenase-A (LDH-A), has been shown to be effective. Engineering of the LDH-A gene has been applied for several CHO cell lines with the DHFR system, but there has been no trial which couples the ammonia reduction from the GS system and lactate reduction through cell engineering. In the present study, the GS system was used for the expression of therapeutic antibody in CHO cells, thereby reducing ammonia in the culture media. In addition, the LDH-A gene was down-regulated with shRNA to reduce lactate production. The antibody-producing cell line produces a reduced level of ammonia compared to the host cell line due to the over-expression of the GS protein. The down–regulation of the LDH-A gene in the antibody-producing cell line not only reduces the level of lactate but also further reduces the level of ammonia, accomplishing complete waste reduction. LDH-A down-regulation was also applied to the host cell lines of the GS system – the CHO-K1 cell line and the GS deficient CHO-K1 cell line. However, LDH-A down-regulated host cells could not survive the pool-selection process. Given that the GS system uses a glutamine-depleted condition as a form of selection pressure, enhanced glycolysis is inevitable and the down-regulation of LDH-A appears to hinder metabolic changes. Taken together, the application of LDH-A down-regulation in the producing cell line of the GS system successfully reduced both ammonia and lactate levels. However, LDH-A engineering could not be applied to the host cell lines because it inhibits the selection process of the GS system

Improving bone morphogenetic protein production in CHO cells by understanding its maturation, signaling, and endocytosis

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Accelerated homology-directed targeted integration of transgenes in CHO cells via CRISPR/Cas9 and fluorescent enrichment

Development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. With draft genome of several CHO cell lines and targeted genome editing technologies, rCHO cell line development based on site-specific integration has the potential to overcome the limitations of clonal heterogeneity. In a previous study, we demonstrated efficient and precise targeted gene integration in CHO cells using CRISPR/Cas9 technology and homology-directed repair (HDR) pathway1). However, it requires a selection process, which limits targeted integration of multiple transgenes at multiple sites due to a limited number of selection markers and a lengthy selection process. Here, we improved the targeted integration platform by applying fluorescent enrichment of transfected cells. The improved system is based on a fluorescent protein A linked Cas9 together with sgRNA towards the integration site and donor DNA harboring a fluorescent gene B outside homology regions. Involvement of fluorescent markers in constructs confers FACS enrichment of cells transfected with both Cas9 and donor DNA. Simultaneous introduction of GFP 2A peptide-linked Cas9 and sgRNA expression vectors together with donor plasmid has enabled precise targeted integration of large transgenes encoding model proteins including antibody, following transient expression and FACS enrichment. Subsequent selection of non-fluorescent clonal cells allowed for excluding cells with randomly integrated donor DNA. In this way, we not only shortened the clone screening time, but also generated clonal CHO cell lines with site-specific, marker-free (no antibiotic selection needed), and clean (no unwanted DNA present) targeted integration of GOI. Further improvement in targeted integration efficiency was additionally assessed by chemical treatment toward cell cycle arrest or nonhomologous end joining inhibition combined with fluorescent enrichment. Taken together, the present platform has the huge potential to accelerate targeted generation of stable production CHO cell lines in a rational way

Simplified method of making alginate-polylysine microcapsules for hybridoma cell culture using RPMI 1640 medium

The method of making alginate-poly-L-lysine microcapsules for hybridoma cell culture can be simplified by cultivating the cells in RPMI 1640 medium. Phosphate concentration in RPMI 1640 medium is sufficiently high to dissolve the alginate gel and, thereby, can be used to eliminate the step of citrate buffer treatment required for reliquefying the interior alginate gel.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42490/1/10542_2004_Article_BF00157433.pd