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Figure 1 shows a Cello system and its configuration: a plate-handling robot, up to three incubators, a cold storage unit, two liquid handling units with pipetting robots, and a high-resolution MAIA Scientific microscope. The high-resolution microscope is used for clone detection by imaging cell cultures in multiwell plates (Photo 1). The system records all process steps carried out on each individual well along with other associated data. That way, each clone can be traced easily, and detailed records are kept for regulatory purposes (8).
Photo 1:
Cello software can schedule multiple cell-line development experiments in parallel. Following a predefined procedure, the robot carries out all process steps involved in screening and expansion of static cultures, then selects clones for further analysis using assay data loaded into the system. As an option, single-cell cloning can be performed with a limiting dilution process.
Figure 2 shows a schematic of the cell-line development process we used. The colonies are assessed just as in the traditional manual procedure except that the procedure is highly automated. Following transfection, 96-well culture plates are placed in Cello incubators. The system automatically detects single colonies growing in those plates over a period of two to four weeks. The colonies could be prescreened off-line to remove low- or nonproducers based on an enzyme-linked immunosorbent assay (ELISA), and the remaining colonies then expanded to 24-well plates.
From that stage on, an automated off-line protein A high-performance liquid chromatography (HPLC) analytical method is used to select 20–30% of the best-producing clones, which are then expanded into six-well plates. About 15–20% of the remaining clones are then selected for further evaluation in shake flasks outside the Cello system (8). For measuring protein expression without a protein A HPLC technique, a high-throughput immunoassay from Gyros AB (www.gyros.com) is available for screening (9).
Demonstrated SuccessBoth Lonza's GS expression system and the DHFR-based OptiCHO expression system from Invitrogen (www.invitrogen.com) have been successfully adapted to the automated platform. A recent study using the GS system compared the quality of cell lines produced using the automated platform with those produced using a traditional manual procedure. As Figure 3 shows, monoclonal antibody concentrations produced by selected clones were comparable for both procedures (8).
Similar numbers were screened in both the manual (800) and automated (1,000) processes. The manual method was performed as two experiments because it was difficult to handle the same numbers the robot could. The automated procedure for screening cell lines was as described above. In the manual procedure, colonies were visually detected in 96-well plates three to five weeks after transfection. Single colonies were selected for ELISA, then manually expanded from 96-well to 24- and six-well plates. Based on IgG titers in the HPLC primary screen, selected clones were transferred to shake flasks (8).
Cell lines selected by both procedures were expanded to shake flasks. When the cell cultures had entered their exponential growth phase, they were evaluated in a batch process at 50-mL cultivation volume in 250-mL shake flasks.
The best performers were further examined during a 14-day fed-batch process. After harvest, their IgG concentration was measured and the top cell line studied further using a generic fed-batch process in stirred-tank bioreactors. The top-performing cell line from the manual procedure was cultivated to 20-L volume in a stainless steel stirred-tank bioreactor. The best cell line from the automated procedure was cultured to 25 L in a single-use bioreactor.
The IgG titer distribution of the batch cultures in 24-well plates and shake-flask batch cultures were similar for both processes. In addition, IgG titers for the top cell lines in both processes was comparably high. The automated procedure maintained cell growth and survival during cell-line development (8), which is a general prerequisite for the routine application of automation in cell line development. Similar expression levels and growth curves were found during a further experiment using the automated procedure on clones expressing a human IgG4 antibody.
Challenges and Future WorkThe study described here shows that an automated procedure for cell-line development can generate high-producing cell lines while reducing costs and manpower requirements. The robotic system allows both screening of many cell lines and simultaneous performance of several projects (8). Two different expression systems have been successfully implemented, with the OptiCHO system being most recent. Our main focus for ongoing work is on optimizing parameters in the automated procedure to shorten time lines and further increase expression levels as well as the number of high-producing cell lines achievable with the OptiCHO system.
Future improvements will focus on further reducing the manual handling required and increasing the capacity of the robotic system. This may involve reducing the number of culture plates handled in each project by removing low-producing transfectants at an earlier stage. However, it is important to be cautious. We have found it hard to predict from early stage results which clones will turn into high-producing cell lines in bioreactor fed-batch cultures (8).
