High-producing Cell Research Articles

Exploiting phenotypic heterogeneity to improve production of glutathione by yeast

BackgroundGene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product.ResultsA counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers.ConclusionsThe results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody–producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation.Key points• Proteome profiling of recombinant CHO cells under mild TM treatment.• Identified protein clusters are associated with the unfolded protein response (UPR).• The compared cell lines revealed noticeable disparities in protein expression levels.

Adeno-associated virus perfusion enhanced expression: A commercially scalable, high titer, high quality producer cell line process

With safety and efficacy demonstrated over hundreds of clinical trials in the last 30 years, along with at least six recent global marketing authorizations achieved since 2017, recombinant adeno-associated viruses (rAAVs) have been established asthe leading therapeutic gene transfer vector for rare, monogenic diseases. Significant advances in manufacturing technology have been made in the last few decades to address challenges with GMP production of rAAV products, although yield, cost, scalability, and quality remain a challenge. With transient transfection processes established as a manufacturing platform for multiple commercial AAV products, there remains significant yield, cost, robustness, and scalability constraints that need to be resolved to enable a reliable supply of rAAV products for global patient access. The development of stable producer cell lines for rAAV products has enabled scalability and, in some cases, improvements in productivity. Herein we describe a novel AAV perfusion-enhanced expression (APEX) process, resulting in higher maximum cell densities in the production bioreactor with a 3- to 6-fold increase in volumetric productivity. This process has been successfully demonstrated across multiple serotypes in large scale cell culture with titers approaching 1×1012 GC/mL. The APEX production platform marks a significant leap forward in the efficient and effective manufacturing of rAAV vector products.

Enhanced high β-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals

This study assessed Rhodotorula paludigena CM33's growth and β-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and β-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a β-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Qx) of 0.91 g/L.h and remarkable β-carotene production rates (Qp) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale β-carotene production for aquatic animal nutrition.

From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies.

The increasing demand for biosimilar monoclonal antibodies (mAbs) has prompted the development of stable high-producing cell lines while simultaneously decreasing the time required for screening. Existing platforms have proven inefficient, resulting in inconsistencies in yields, growth characteristics, and quality features in the final mAb products. Selecting a suitable expression host, designing an effective gene expression system, developing a streamlined cell line generation approach, optimizing culture conditions, and defining scaling-up and purification strategies are all critical steps in the production of recombinant proteins, particularly monoclonal antibodies, in mammalian cells. As a result, an active area of study is dedicated to expression and optimizing recombinant protein production. This review explores recent breakthroughs and approaches targeted at accelerating cell line development to attain efficiency and consistency in the synthesis of therapeutic proteins, specifically monoclonal antibodies. The primary goal is to bridge the gap between rising demand and consistent, high-quality mAb production, thereby benefiting the healthcare and pharmaceutical industries.

Expression of Bispecific Antibodies Using Cellular Display.

The identification and selection of high-producing cell lines can be a resource- and time-consuming process. The screening effort can be simplified by assessing the potential for high expression (or a desired product quality attribute) of the individual cell directly in a mix of cells. Here, we describe protocols for the use of such a cellular display technology. Using alternate splicing, two mRNA constructs are generated at tunable ratios. The first mRNA codes for the secreted product, the second mRNA attaches a transmembrane domain to the antibody and directs it to the cellular membrane. The design of the basic construct as well as efficient ways to tune the strength of the cellular display is detailed in this chapter. Further, enrichment methods are provided enabling the flow cytometric sorting of a cell population based on the quantity of cellular display or on the product quality (heterodimerization level of a bispecific antibody).

Peptonics: A new family of cell-protecting surfactants for the recombinant expression of therapeutic proteins in mammalian cell cultures.

Polymer surfactants are key components of cell culture media as they prevent mechanical damage during fermentation in stirred bioreactors. Among cell-protecting surfactants, Pluronics are widely utilized in biomanufacturing to ensure high cell viability and productivity. Monodispersity of monomer sequence and length is critical for the effectiveness of Pluronics-since minor deviations can damage the cells-but is challenging to achieve due to the stochastic nature of polymerization. Responding to this challenge, this study introduces Peptonics, a novel family of peptide and peptoid surfactants whose monomer composition and sequence are designed to achieve high cell viability and productivity at a fraction of chain length and cost of Pluronics. A designed ensemble of Peptonics was initially characterized via light scattering and tensiometry to select sequences whose phase behavior and tensioactivity align with those of Pluronics. Selected sequences were evaluated as cell-protecting surfactants using Chinese hamster ovary (CHO) cells expressing therapeutic monoclonal antibodies (mAb). Peptonics IH-T1010, ih-T1010, and ih-T1020 afforded high cell density (up to 3×107 cellsmL-1 ) and viability (up to 95% within 10 days of culture), while reducing the accumulation of ammonia (a toxic metabolite) by ≈10% compared to Pluronic F-68. Improved cell viability afforded high mAb titer (up to 5.5mgmL-1 ) and extended the production window beyond 14 days; notably, Peptonic IH-T1020 decreased mAb fragmentation and aggregation ≈5%, and lowered the titer of host cell proteins by 16% compared to Pluronic F-68. These features can improve significantly the purification of mAbs, thus increasing their availability at a lower cost to patients.

Enhanced exopolysaccharide production of Cordyceps militaris via mycelial cell immobilization on plastic composite support in repeated-batch fermentation

Repeated-batch fermentation with fungal mycelia immobilized in plastic composite support (PCS) eliminates the lag phase during fermentation and improves metabolite productivity. The strategy is implemented herein, and a novel modified PCS is developed to enhance exopolysaccharide (EPS) production from the medicinal fungus Cordyceps militaris. A modified PCS (SYE + PCS) was made by compositing polypropylene (PP) with a nutrient mixture containing soybean hull, peptone, yeast extract, and minerals (SYE+). The use of SYE + PCS has consistent cell productivity throughout the multiple fermentation cycles, which resulted in a more higher cell productivity after second batch compared to unmodified PCS. The cell grown on SYE + PCS also generates a higher yield of EPS (3.36, 6.93, and 5.72 g/L in the first, second, and third fermentation cycles, respectively) up to three-fold higher than the cell immobilized on unmodified PCS. It is also worth noting that the EPS from mycelium grown on SYE + PCS contains up to 2.3-fold higher cordycepin than those on unmodified PCS. The presence of nutrients in SYE + PCS also affects the hydrophobicity and surface roughness of the PC, improving mycelial cell adhesion. This study also provides a preliminary antioxidant activity assessment of EPS from immobilized C. militaris grown with SYE + PCS.

Development of an Expression Vector Engineering Strategy Based on tDNA Insulator Element for the Stable Expression of Vascular Endothelial Growth Factor Receptor-Fc Fusion Protein.

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.

Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Overexpression of peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺) in Chinese hamster ovary cells increases oxidative metabolism and IgG productivity

Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.

Weak promoters to drive selection marker expression: Improvement of cell line development process for therapeutic protein production in CHO-K1 cells.

Chinese Hamster Ovary cells have been widely used as host cells for production of recombinant therapeutic molecules. Cell line development is a decisive step, which must be carried out with an efficient process. In particular, degree of selection stringency is an important parameter for identification of rare, high-producing cell lines. In the CHOZN® CHO K1 platform, selection of top-producing clones is based on puromycin resistance, whose expression is driven by Simian Virus 40 Early (SV40E) promoter. In this study, novel promoters have been identified to drive expression of selection marker. Decrease of transcriptional activity compared to SV40E promoter was confirmed by RT-qPCR. Selection stringency was increased, as seen by decreased surviving rate of transfected mini-pools and longer recovery duration of transfected bulk pools. Several promoters led to a 1.5-fold increase of maximum titer and a 1.3-fold increase of mean specific productivity of the monoclonal antibody over the clone generation. Expression level was maintained stable over long term cultivation. Finally, productivity increase was confirmed on several monoclonal antibodies and fusion proteins. Lowering the strength of promoter for expression of selective pressure resistance is an efficient strategy to increase selection stringency, which can be applied on industrial CHO-based cell line development platforms.

Understanding the effect of temperature downshift on CHO cell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state.

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (qp ) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA)cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD+ /NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD+ /NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.

COMPARISON OF THE RESULTS OF CULTURING CELLS AND VIRUSES ON THE NEW SEPLIFE® LX-MC-DEX1 MICROCARRIER WITH EXISTING MICROCARRIERS

microcarriers are small solid particles 90–350 µm in size, on the surface of which cells grow in the form of a monolayer. The two main properties of microcarriers are cell adhesion and high cell production. According to the results of a comparative study conducted on various parameters, it was determined that each microcarrier has its own advantages and disadvantages. In our study, the properties of SEPLIFE® LX-MC-dex1 and Cytodex 3 microcarriers were compared. According to the results of the studies, it was found that SEPLIFE® LX-MC-dex1, containing an artificial substance, is not inferior to Cytodex 3 microcarrier widely used in production in all properties and ease of use. The main advantage of the SEPLIFE® LX-MC-dex1 microcarrier is its cost effectiveness, i.e. it is 5 times cheaper than Cytodex 3. In this regard, the SEPLIFE® LX-MC-dex1 microcarrier was found to be cost-effective.

Shortening the biologics clinical timeline with a novel method for generating stable, high-producing cell pools and clones.

Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell linegenome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

Diversity investigation of cultivable yeasts associated with honeycombs and identification of a novel Rhodotorula toruloides strain with the robust concomitant production of lipid and carotenoid

Oleaginous yeasts-derived microbial lipids provide a promising alternative feedstock for the biodiesel industry. However, hyperosmotic stress caused by high sugar concentration during fermentation significantly prevents high cell density and productivity. Isolation of new robust osmophilic oleaginous species from specific environment possibly resolves this issue to some extent. In this study, the cultivable yeast composition of honeycombs was investigated. Totally, 11 species of honeycomb-associated cultivable yeast were identified and characterized. Among them, an osmophilic yeast strain, designated as Rhodotorula toruloides C23 was featured with excellent lipogenic and carotenogenic capacity and remarkable cell growth using glucose, xylose or glycerol as feedstock, with simultaneous production of 24.41 g/L of lipids and 15.50 mg/L of carotenoids from 120 g/L glucose in 6.7-L fermentation. Comparative transcriptomic analysis showed that C23 had evolved a dedicated molecular regulation mechanism to maintain their high simultaneous accumulation of intracellular lipids and carotenoids and cell growth under high sugar concentration.

Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1–11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

Novel application of anti-human Fc nanobody for screening high-producing CHO cells for monoclonal antibody.

Animal‐derived anti‐IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)‐producing cells, but using animal‐derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage‐displayed immune llama VHH library, we identified new anti‐Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti‐Fc VHH‐GFP generated dramatically stronger FACS signals than AF488 conjugated anti‐IgG antibodies when used for staining mAb‐producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti‐Fc VHH‐GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost‐efficient anti‐Fc VHH‐GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal‐derived fluorescent antibodies.

Effect of co-overexpression of the cargo receptor ERGIC-53/MCFD2 on antibody production and intracellular IgG secretion in recombinant Chinese hamster ovary cells

Therapeutic antibodies are attractive biopharmaceuticals because of their high therapeutic effects, fewer side effects, and prolonged half-life in the blood. Chinese hamster ovary (CHO) cells are the most widely used host cell lines to produce therapeutic antibodies in industries. High-producing recombinant CHO cells can be established via overexpression of endogenous proteins. In this study, we focused on the intracellular traffic of an antibody-producing CHO cell line, CHO-HcD6. Assembled antibodies were accumulated in the endoplasmic reticulum (ER) in the cell. We hypothesized that the accumulation was due to the insufficient number of cargo receptors in the cell and focused on a cargo receptor, the ERGIC-53-MCFD2 complex, which transports expressed proteins from the ER to the Golgi apparatus. Overexpression of the cargo receptor transport was expected to improve antibody production. Exogenous ERGIC-53 and MCFD2 were transfected into CHO-HcD6 cells, and overexpressing CHO-HcD6 cells were constructed. As a result of overexpression, antibody productivity increased in batch cultivation. However, the chase assay results and immunofluorescence microscopic observations revealed intracellular IgG accumulation in the overexpressing cells. These results suggest that overexpression of cargo receptors not only promoted extracellular secretion but also enhanced the retention of intracellular antibodies.