Commercial manufacturing of therapeutic monoclonal antibodies (MAbs) commonly uses mammalian cells to generate large quantities of a drug. Identifying cell lines that stably produce high protein titers is, therefore, a critical part of biopharmaceutical development. Unfortunately, identifying suitable cell lines is traditionally a time-consuming, labor-intensive process. That's because their productivity and stability can vary enormously, so large numbers of clones must be screened to find those with both the highest yield and a desired level of product quality (1).

Cell-line development is unsurprisingly seen as a major bottleneck in biopharmaceutical development (2,3). With companies facing pressure to reduce costs and shorten development times, techniques to improve the efficiency of this process are in demand. Several methods have been developed, including fluorescence-activated cell sorting (FACS) such as the BD FACS system from BD Biosciences (www.bdbiosciences.com) and the ClonePix FL system from Genetix, Ltd. (www.genetix.com) (4) to speed up cell-line selection and identify high-producing cell lines at an early stage in cell-line development (2,3,4,5). However, limited progress has been made in reducing the manpower costs involved in expanding several clones in cell-line development. We present an automated approach for cell-line development based on the Cello robotic system from The Automation Partnership (www.autoprt.co.uk). We believe it offers significant advantages over manual techniques.

PRODUCT FOCUS: BIOPHARMACEUTICALS

PROCESS FOCUS: PRODUCTION

WHO SHOULD READ: R&D AND PROCESS DEVELOPMENT

KEYWORDS: CELL-LINE DEVELOPMENT, DRUG DISCOVERY, AUTOMATION, CHO CELLS, ROBOTICS

LEVEL: INTERMEDIATE

Problems with Traditional Approaches

The most common mammalian cell lines used to manufacture commercial quantities of therapeutic antibodies are derived from Chinese hamster ovary (CHO) cells. Traditionally gene expression systems based on dehydrofolate reductase (DHFR) and the more recent glutamine synthetase (GS) from Lonza (www.lonza.com) have been used for development of cell lines suitable for commercial manufacturing.

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In traditional CHO-based expression systems, plasmid DNA is integrated into host-cell chromosomes during transfection. The plasmids contain the genes required for cells to manufacture a therapeutic antibody and selection markers required to identify those cells that do express the desired protein. However, productivity of cell lines generated using these expression systems varies enormously because plasmid DNA is randomly integrated, and drug yield depends on the integration site (6).

Generally, few transfected CHO cells will be high producers, and the number of high-producing clones detected depends on the number screened (2). Screening many clones and expansion of selected cell cultures to larger scales is time-consuming and labor-intensive. In addition, tracking documentation becomes a challenge with large numbers of clones. Colony screening is traditionally done manually using microscopy. It is followed by expansion of a large number (200–1,000) of clones into static cultures that are then screened for IgG titers in batch culture. Selected cell lines are expanded into a suspension culture for further evaluation using batch and fed-batch cultures grown in shake flasks and bioreactors.

Cell-line selection techniques using fluorescence technology were developed to be a less labor-intensive alternative to traditional screening for colonies and productivity. Commonly, such systems select high-producing cells by imaging fluorescently-tagged antibodies that bind to a drug protein. However, these techniques are applicable only during early selection. Expansion of static cultures — and subsequent evaluations in suspension cultures — still have to be performed manually or using additional equipment. We chose instead to study how that process could be automated to make cell-line development more efficient and less labor-intensive. To further cut down required man-hours, this automation approach can be combined with fluorescence techniques as well. Several other expression systems have been developed in recent decades for improvement of titer or shortened timelines (7).

Benefits of Automation

An automated concept for developing high-producing cell lines for biopharmaceutical production has now been successfully demonstrated (8). The approach uses a Cello robotic system, which can operate 24 hours a day and performs optical clone screening using automated microscopy and scale-up of static cultures. This system enables high-throughput, off-line screening of IgG titer in batch cultures at different stages during scale-up. Its software records all process steps and associated data in a relational database together with clone images to facilitate detailed documentation of the selection process.

We have shown that the automated approach generates cell lines of equal quality to those produced by traditional manual selection procedures. Throughput of cell-line generation is increased while maintaining or even reducing cell-line development costs. Our models show that up to three times as many cell lines can be screened by the same number of staff using this automated technique while continuing to screen large numbers of clones. Large screens also provide a higher chance of finding high-producing cell lines. Furthermore, automatic tracking of clones through the expansion process gives an added advantage of providing proper documentation for a strictly regulated environment.