Shortening timelines to generate more productive, stable, and high-expressing cell lines is the goal of any cell line development program. Here, we explore the advantages of combining of a technology for stable gene expression (GPEx®, Catalent) and an instrument for clonal selection (Beacon®, Berkeley Lights).

GPEx technology is a versatile system designed to insert genes of interest into a wide variety of mammalian host cells. The GPEx method is based on the use of replication defective retroviral vectors (retrovectors) to actively insert the desired genes into the genome of dividing cells. The majority of components from GPEx retrovectors are derived from Moloney murine leukemia virus. The vesicular stomatitis virus G protein is used as an envelope for the retrovector particle. These particles stably insert single copies of the transgene at multiple sites in the chromatin of dividing cells.

These integrated genes are maintained through subsequent cell divisions as if they were endogenous cellular genes. By controlling the number of retrovector particles accessing the cell, multiple gene insertions can be achieved without any of the traditional amplification steps. The advantages of the GPEx system include outstanding cell line stability, high gene expression due to gene insertion at active sites, ability to forgo selectable markers, and efficiency across a broad range of host cell lines. Once stably transduced GPEx cell pools are produced, they can be cloned and screened using the Beacon system.

Berkeley Lights has developed the Beacon platform, a flexible instrument that combines structured visible light technology and microfluidic design to automate isolation, growth, screening, and manipulation of thousands of monoclonal cell populations in parallel. The Beacon optofluidic platform uses OptoSelect™ OptoElectroPositioning technology (light cages) to place single cells into 1-nL pens on chips. The instrument pens, cultures, assays, images, ranks, and exports high-expressing clonal cell lines. Advantages of the Beacon system include high-throughput automated workflows, dramatically shortened timelines, and scalability to identify rare clones.

Combining GPEx technology with Beacon instrumentation creates a cell line development platform resulting in stable, higher expressing clones generated in shortened time frames.

Materials and methods

GPEx pool development.

Retrovectors were produced as described previously.1,2 A master cell bank of the GPEx-CHO cell line was the parent cell line for all data shown. Cell lines expressing antibodies were produced as shown in Figure 1. Retrovector transductions were performed at a multiplicity of infection of at least 1000 retrovector particles per CHO cell. To generate antibody-producing cell lines, an initial transduction of GPEx-CHO cells was performed using a retrovector containing the light chain gene. The light chain containing pool of cells was then transduced with a retrovector containing the heavy chain gene.

Bioprocessing Tutorial Figure 1
Figure 1. GPEx® process for cell line development

Upon completion of both transductions, the resulting pool of cells was then transduced a second time with light chain retrovector and two additional times with heavy chain retrovector for a total of five transduction cycles resulting in the final pool used for clonal selection. Pools were generated expressing two different antibody products. Antibody 1 was an easy-to-express molecule, and Antibody 2 was more difficult to express based on initial observations for each of the pools.

Clonal isolation and fed-batch productivity.

Clones were isolated either by one round of Beacon clonal selection or by two rounds of ClonePix™ selection.2 Resulting clones were evaluated for protein expression in a fed-batch shake flask using unoptimized conditions for Antibody 2 and semioptimized conditions for Antibody 1.

The media and feed for the unoptimized conditions were shown to be generally supportive for fed-batch productivity over a wide range of clones but were not optimized for these specific clones. The media and feed for the semioptimized conditions were shown to improve productivity for most clones expressing Antibody 1. Fed-batch cultures were monitored for glucose consumption and supplemented as needed. When harvest criteria were met, cultures were clarified and assayed for antibody concentration via protein A HPLC.

Monoclonality confirmation via the Beacon platform.

Experiments were performed to verify monoclonality using the Beacon platform. Clonal cell lines expressing either mScarlet or GFP were mixed 1:1 and loaded onto the Beacon system simultaneously. Individual cells were loaded into pens on the Opto-Select 1750b chip and allowed to culture for three days. On day 3, the chip was imaged with bright-field and fluorescence (green and red) microscopy.

Pens that initially contained only one cell, demonstrated good growth, and were brightly fluorescent for either GFP alone or mScarlet alone were exported from the pens and deposited onto a 96-well export plate. A “blank export” (fluid from the channel, not a pen) was made into every other well on the export plate to ensure that no cells were present to cross-contaminate the cells designated for export.

Cells were deposited horizontally across the 96-well plate, meaning every odd numbered column should contain either red or green cells, but every even-numbered column should be “blank” (no cells present). Plates were examined after export and imaged periodically during culture post export with bright-field and fluorescence (green and red) microscopy.

Results

For both Antibody 1 and Antibody 2, clonal selection via the Beacon system resulted in significantly higher expressing clonal cell lines. The top nine clones for Antibody 1 and the top 16 clones for Antibody 2 were Beacon system derived.

Bioprocessing Tutorial Figure 2
Figure 2. Semi-optimized shaking fed-batch productivity of high-expressing Antibody 1 clones derived by Beacon® platform (dark blue) or traditional (light purple) cloning methods

The advantage of Beacon clonal selection was more pronounced for the lower expressing antibody product. The average of selected Beacon platform or traditionally derived clones from Antibody 1 (Figure 2) and Antibody 2 (Figure 3) pools demonstrated a 1.5- and 3-fold increase, respectively, in titer for Beacon system–generated clones (Figure 4).

Bioprocessing Tutorial Figure 3
Figure 3. Unoptimized shaking fed-batch productivity of low-expressing Antibody 2 clones derived by Beacon® platform (dark blue) or traditional (light purple) cloning methods
Bioprocessing Tutorial Figure 4
Figure 4. Average productivity of selected clones for a low-expressing Antibody 2 and high Antibody 1

The Beacon platform imaged pens throughout the culture period from loading to export using bright-field and fluorescence (green and red) microscopy (Figure 5).

Bioprocesing Tutorial Figure 5
Figure 5. Beacon® system monoclonality confirmation with green and red fluorescent cells, from cell loading through end of culture

Additional confirmation of monoclonality was examined by field of view utilizing green and red fluorescence (Figure 6). To verify that clones exported off the chip remained monoclonal, export plates were imaged after export and periodically throughout the 96-well culture period. Wells that received exports from green pens contained only green cells, these were the only wells that contained green cells. No cells were observed in the wells expected to be blank, confirming there was no crossover of cells from exported pens (Figure 7).

Figure 6. An example of bright field, green channel, and red channel imaging for monoclonality validation
Bioprocessing Tutorial Figure 7
Figure 7. Red and green fluorescence imaging of a 96-well plate on day 10 of culture after export

Conclusions

Combining GPEx technology and the Beacon platform shortened timelines by 2½ months for cell line selection and resulted in clones with 1.5–3-fold higher titer. A more significant advantage was observed for a lower expressing antibody. All Beacon platform–derived cell lines shown in this data set were tracked and imaged throughout the cloning and export process, and imaging data on the chip and after export confirmed a high probability of monoclonality.

 

References
1. Bleck GT. 2005 An alternative method for the rapid generation of stable, high-expressing mammalian cell lines. Bioprocess. J. 2007; 5: 36–43.
2. Bleck, GT. 2010. GPEx® A Flexible Method for the Rapid Generation of Stable, High Expressing, Antibody Producing Mammalian Cell Lines. In: Shire S, Gombotz W, Bechtold-Peters K, Andya J, eds. Current Trends in Monoclonal Antibody Development and Manufacturing. New York, NY: Springer; 2010: 51–62.

Rachel Kravitz, PhD, is an R&D scientist, Wendy Vredenburgh is a scientist in cell line development, Victoria Chrostowski is cell line development manager, and Greg Bleck, PhD, is global head of R&D, biologics at Catalent Pharma Solutions.

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