High-producing Cell Lines Research Articles

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.

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).

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.

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 cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Enhancement of anti-TNFα monoclonal antibody production in CHO cells through the use of UCOE and DHFR elements in vector construction and the optimization of cell culture media

Recently, there has been a high demand for anti-tumor necrosis factor-α monoclonal antibodies (mAbTNFα) in the treatment of rheumatoid arthritis and other autoimmune diseases. Thus, efficient strategies and stable high-producing cell lines need to be established to increase antibody production. In this study, we describe an efficient approach to establish a mAbTNFα high-producing clone through the optimization of expression vectors and cell culture media. The ubiquitous chromatin opening element (UCOE) and dihydrofolate reductase (DHFR)-based vectors encoding mAbTNFα were introduced into the CHO-DG44 cells using lipofection. Clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate, and isolating them by limiting dilution. Different media formulated with commercial feeds and media were also screened to develop an improved medium. The antibody produced by the selected clone was purified, characterized, and compared to standard adalimumab. Using our established protocol, a cell clone obtained from stable mAbTNFα-expressing cell pools showed a 3.8-fold higher antibody titer compared to stable cell pools. Furthermore, the highest antibody yield of selected clones cultured in fed-batch mode using improved medium was 2450 ± 30 µg/mL, which was 13.2-fold higher than that of stable cell pool cultivated in batch mode using a basal medium. The purified antibody had primary chemical and biological characteristics similar to those of adalimumab. Therefore, the use of UCOE and DHFR vectors in combination with the optimization of cell culture media may help in establishing stable and high-producing CHO cell lines for therapeutic antibody production.

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

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 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.

Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Identifying metabolic features and engineering targets for productivity improvement in CHO cells by integrated transcriptomics and genome-scale metabolic model

In this study, we presented an integrated systems biology approach to elucidate the key characteristics of cellular metabolism in Chinese hamster ovary (CHO) cells producing monoclonal antibodies (mAb). The cellular metabolism in high and low producers under batch conditions was interrogated dynamically both within and among cells. First, transcriptomics analysis was used to study the time-course change in the metabolic pathway within cells that was correlated with mAb productivity increase. Second, differentially regulated pathways between high and low producers were sought at each growth phase. Several up-regulated pathways were identified in the high producer at the late growth phase, including citrate cycle, oxidative phosphorylation, and pentose phosphate pathway. These activities were further analyzed by intracellular flux distributions estimated through a genome-scale CHO model. Our results revealed that these key pathways are identified to be characteristics of high mAb production, not only for the high-producing cell line but also a dynamic phenomenon in mAb-producing cell cultures. This study showed that the approach of integrating transcriptomics and flux analysis leads to a better understanding of cellular metabolism related to mAb productivity. In turn, this allows for the identification of metabolic bottlenecks and potential engineering targets for cell line development and process optimization.

Создание стабильной высокопродуктивной платформы на основе клеток CHO, адаптированных к химическому и механическому стрессам

Возрастающий спрос на получение рекомбинантных молекул требует создания не только стабильных, но и высокопроизводительных клеточных линий, пригодных для масштабного производства. В настоящей работе предложен дизайн вектора для эффективной и стабильной экспрессии рекомбинатных моноклональных антител в клетках CHO и схема адаптации линии CHO-S (реципиента) к высокой осмоляльности среды в условиях повышенного содержания хлорида натрия (до 100 мМ), лактата натрия (до 170 мМ) и скорости перемешивания в биореакторе до 240 об/мин. Показана эффективность полученной клеточной платформы на примере экспрессии моноклонального антитела IgG2/4k, позволяющей получать до 2,5 г/л продукта без амплификации гена интереса и оптимизации условий культивирования в режиме fed-batch.

Increased mAb production in amplified CHO cell lines is associated with increased interaction of CREB1 with transgene promoter.

Most therapeutic monoclonal antibodies in biopharmaceutical processes are produced in Chinese hamster ovary (CHO) cells. Technological advances have rendered the selection procedure for higher producers a robust protocol. However, information on molecular mechanisms that impart the property of hyper-productivity in the final selected clones is currently lacking. In this study, an IgG-producing industrial cell line and its methotrexate (MTX)-amplified progeny cell line were analyzed using transcriptomic, proteomic, phosphoproteomic, and chromatin immunoprecipitation (ChIP) techniques. Computational prediction of transcription factor binding to the transgene cytomegalovirus (CMV) promoter by the Transcription Element Search System and upstream regulator analysis of the differential transcriptomic data suggested increased in vivo CMV promoter-cAMP response element binding protein (CREB1) interaction in the higher producing cell line. Differential nuclear proteomic analysis detected 1.3-fold less CREB1 in the nucleus of the high productivity cell line compared with the parental cell line. However, the differential abundance of multiple CREB1 phosphopeptides suggested an increase in CREB1 activity in the higher producing cell line, which was confirmed by increased association of the CMV promotor with CREB1 in the high producer cell line. Thus, we show here that the nuclear proteome and phosphoproteome have an important role in regulating final productivity of recombinant proteins from CHO cells, and that CREB1 may play a role in transcriptional enhancement. Moreover, CREB1 phosphosites may be potential targets for cell engineering for increased productivity.

Genetic and Epigenetic Variation across Genes Involved in Energy Metabolism and Mitochondria of Chinese Hamster Ovary Cell Lines.

The increasingdemandfor biopharmaceutical products drives the search for efficient cell factories that are able to sustainably support rapid growth, high productivity, and product quality. As these depend on energy generation, here the genomic variation in nuclear genes associated with mitochondria and energy metabolism and the mitochondrial genome of 14 cell lines is investigated. The variants called enable reliable tracing of lineages. Unique sequence variations are observed in cell lines adapted to grow in protein-free media, enriched in signaling pathways or mitogen-activated protein kinase 3. High-producing cell lines bear unique mutations in nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND2 and ND4) and in peroxisomal acyl-CoA synthetase (ACSL4), involved in lipid metabolism. As phenotypes are determined not only by functional mutations, but also by the exquisite regulation of expression patterns, it is not surprising that ≈50% of the genes investigated here are found to be differentially methylated and thus epigenetically controlled, enabling a clear distinction of high producers, and cells adapted to a minimal, glutamine (Gln)-free medium. Similar pathways are enriched as those identified by genome variation. This strengthens the hypothesis that these phenomena act together to define cell behavior.

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single-Cell Level.

Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high-producing clonal cell lines. During cell line development, cell cloning using fluorescence-activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single-cell level to identify cell attributes predictive of productivity. IFC assays are developed to quantify the organelle content and recombinant heavy-chain (HC) and light-chain (LC) polypeptide as well as messenger RNA (mRNA) amounts in single cells. The assays are then validated against orthogonal standard flow cytometry, western blot, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. The authors describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single-cell level, allowing the isolation of such cells during the cloning process. From the analysis, HC polypeptide and mRNA are found to be predictive of productivity early in the culture; however, specific organelle content did not show any correlation with productivity.