Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

IL‐17F co‐ ;expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL‐17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL‐17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL‐17F expression improves the efficiency of cell line subcloning processes. IL‐17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame. Biotechnol. Bioeng. 2013; 110: 1153–1163. © 2012 Wiley Periodicals, Inc.     

Avian embryos and related cell lines: A convenient platform for recombinant proteins and vaccine production

Chick embryos are a significant historical research model in basic and applied sciences. The embryonated eggs have been used for virus inoculation in order to vaccine production for nearly a century. Recently, avian eggs and cell lines derived from embryonated eggs have found wide application in biotechnology. This review will discuss about the unique characteristics of avian eggs in terms of safety, large scale and economical production of recombinant proteins. This system also provides the human‐like glycosylation on target proteins and therefore can be considered as a suitable host for biomanufacturing of humanized monoclonal antibodies and therapeutic proteins. Avian derived cell lines are an alternative for rapid vaccine manufacturing during a pandemic. Based on the latest knowledge in cell and animal transgenesis, the currently available germ cell‐mediated gene transfer system provides a more efficient strategy in gene targeting and creation of transgenic birds that lead to advancements in industrial, biotechnology, and biological research applications. This review covers the recent development of avian fertilized eggs and related cell lines in a variety of human biopharmaceuticals and viral vaccine manufacturing.     

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high‐producing clones among a large population of low‐ and non‐productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)‐based methotrexate (MTX) selection and glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS‐CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L‐MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS‐knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (～2%) of bi‐allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine‐dependent growth of all GS‐knockout cell lines. Full evaluation of the GS‐knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two‐ to three‐fold through the use of GS‐knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non‐producing and low‐producing cells after 25 µM L‐MSX selection, and resulted in a six‐fold efficiency improvement in identifying similar numbers of high‐productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS‐knockout cells on recombinant protein quality is also discussed. Biotechnol. Bioeng. 2012; 109:1007–1015. © 2011 Wiley Periodicals, Inc.     

Generation of stable, high-producing CHO cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture

Lentivirus‐derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence‐activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV‐transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co‐expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2‐day post‐infection, clonal cell lines with high eGFP‐specific fluorescence were recovered by FACS. These clones co‐expressed TNFR:Fc with yields of 50–250 mg/L in 4‐day cultures. The recovered cell lines maintained stable expression over 3 months in serum‐free suspension culture without selection. In conclusion, LV‐mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high‐producing recombinant cell lines. Biotechnol. Bioeng. 2011; 108:600–610. © 2010 Wiley Periodicals, Inc.     

Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed‐batch systems

The major challenge in the selection process of recombinant cell lines for the production of biologics is the choice, early in development, of a clonal cell line presenting a high productivity and optimal cell growth. Most importantly, the selected candidate needs to generate a product quality profile which is adequate with respect to safety and efficacy and which is preserved across cell culture scales. We developed a high‐throughput screening and selection strategy of recombinant cell lines, based on their productivity in shaking 96‐deepwell plates operated in fed‐batch mode, which enables the identification of cell lines maintaining their high productivity at larger scales. Twelve recombinant cell lines expressing the same antibody with different productivities were selected out of 470 clonal cell lines in 96‐deepwell plate fed‐batch culture. They were tested under the same conditions in 50 mL vented shake tubes, microscale and lab‐scale bioreactors in order to confirm the maintenance of their performance at larger scales. The use of a feeding protocol and culture conditions which are essentially the same across the different scales was essential to maintain productivity and product quality profiles across scales. Compared to currently used approaches, this strategy has the advantage of speeding up the selection process and increases the number of screened clones for getting high‐producing recombinant cell lines at manufacturing scale with the desired performance and quality. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:160–170, 2016     

Generation of genome-modified Drosophila cell lines using SwAP

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.     

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario.     

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells

Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over‐expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess‐relevant characteristics of Chinese hamster ovary (CHO) cell‐derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell‐cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub‐optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR‐transgenic CHO‐derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four‐fold increase compared to the parental production cell line. mTOR‐based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Biotechnol. Bioeng. 2011; 108:853–866. © 2010 Wiley Periodicals, Inc.     

Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in‐market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase‐mediated cassette exchange (RMCE) system to build a site‐specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT‐flanked mAb expression cassette, we generated a clonal cell line with good productivity, long‐term production stability, and low mAb gene‐copy number indicating the vector was located in a ‘hot‐spot.’ A SSI host cell line was made by removing the mAb genes from the ‘hot‐spot’ by RMCE, creating a ‘landing pad’ containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP‐based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened ‘time‐to‐clinic’ for therapeutic mAbs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1645–1656, 2015     

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

In Lonza Biologics' GS Gene Expression System?, recombinant protein‐producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine‐free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER‐resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb‐producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight‐fold and the concentration in harvest medium by two‐fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process‐driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017     

High‐throughput screening and selection of mammalian cells for enhanced protein production

The production of recombinant proteins for biotherapeutic use is a multibillion dollar industry, which has seen continual growth in recent years. In order to produce the best protein with minimal cost and time, selection methods are utilized during the cell line development process in order to select for the most desirable clonal cell line from a heterogeneous transfectant pool. Today, there is a vast array of potential selection methods available, which vary in cost, complexity and efficacy. This review aims to highlight cell line selection methods that exist for the isolation of high‐producing clones, and also reviews techniques that can be used to predict, at a small scale, the performance of clones at large, industrially‐relevant scales.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

High-throughput and automation advances for accelerating single-cell cloning,monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single-cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.     

B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the 'ADLib (Autonomously Diversifying Library) system', which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.     

Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production

The quality‐by‐design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design‐of‐experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus‐like particles from CHO‐cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009