The overall goal of this procedure is to develop manufacturing cell lines for biologics with clonality and high productivity. This is accomplished by first transecting, the host cell with DNA, encoding the gene of interest. The next step of the procedure is to isolate high producing clones by facts, followed by documentation of colony formation by the clone select imager.
The final step is to screen and select highly productive clones. Ultimately, results can be obtained that show antibody production in continuous and fed batch cultures through reverse phase HPLC.Video. Demonstration of this method is critical as the fact sorting and pcan 80 dgl steps are difficult to learn because a complex setup is involved in these procedures, the following scientists from a laboratory will demonstrate these procedures for transfection of the host cell effect, sorting Jason Sanders for documentation of colony formation and Russell Kan for tecan sampling.
And Eliza To begin this procedure one day before transfection seed the host cells in a cell culture flask in 15 milliliters of growth.Medium. Incubate the cells in a 37 degree Celsius, 7.5%carbon dioxide incubator on the following day, prepare the transfection complexes first dilute eight micrograms, DNA in 800 microliters of growth medium without serum in a sterile micro centrifuge tube mixed gently. Next, dilute 40 microliters lip in 800 microliters of growth medium without serum in a polystyrene tube.
Add the diluted DNA to the diluted Lipofectamine. Mix gently and incubate for 30 minutes at room temperature to prepare the host cells for transfection. Remove the growth medium from cells and replace with 6.4 milliliters of growth medium without serum.
Add 1.6 milliliters of the transfection complexes to the flask. Mix gently by rocking the flask. Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator for six hours.
After six hours, add eight milliliters of growth medium to the flask and incubate at 37 degrees Celsius in a carbon dioxide incubator overnight on the following day. Remove medium by aspiration and add fresh growth medium to the flask. Incubate the cells overnight again after the overnight incubation.
Replace the growth medium with selection.Medium. Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator for two to three weeks. Two to three weeks after being transfected, the cells are ready for cloning by flow cytometry activated cell sorting dislodged cells from the flask and centrifuge at 800 RPM for five minutes at four degrees Celsius.
Resuspend the cells in selection medium, supplemented with 2%bovine serum albumin. Then add fluorescently labeled anti-human IgG antibody. Add a dilution of one to 20 incubate on ice for 15 to 30 minutes, and then wash twice with cold PBS resuspend cells in one XPBS in a polypropylene tube.
An example of stained cells is shown in this picture for aseptic sort on a BD fax aria prepare a 96 well cell culture plate that contains 200 microliters selection medium in each well. Aseptically load the tube with cells and analyze on the BD facts. Aria, record the results for stained and unstained cells respectively.
Set the gates and settings to sort single cells of the desired fluorescent staining profile into the 96 Well plate incubate the plate in a 37 degrees Celsius incubator for two weeks at day zero, day three, day seven, and day 13 after fact sorting document colony formation in the 96 well plate on a clone select imager to ensure selection of clones derived from a single cell to screen and select for highly productive clones. First document sample position in the receiving 96 well assay plate. Then use a robotic liquid handling system to transfer aliquots of 20 microliters supernatant into each well of the 96 well assay plate antibody production in the samples is analyzed by a sandwich.
Human IgG detecting ELI SA on the liquid handling system in the following way. First, the samples and a standard are loaded on pre-coded bio coat anti-human IgG assay plates. The plates are incubated at room temperature for two hours, followed by three PBS washes.
Next immuno pure goat anti-human IgG. HRP is added to the plates at a one to 2000 dilution and the plates are incubated for one hour at room temperature. After one hour, the plates are washed three times with PBS and then the A BTS substrate is added.
Finally, the plates are red on a plate reader using absorbance at 405 nanometers for detection. Using this automated platform, it was found that surface antibody level reflected by staining with fluorescent labeled anti-human IgG correlates with antibody productivity of the cell. As shown here for the top two clones generated from the cell population with high surface antibody expression, the specific productivity ranged from 50 to 70 picograms per cell per day.
In contrast, the antibody productivity was roughly 20 picograms per cell per day for the top two clones generated from the cell population with low surface antibody expression. Furthermore, cell lines developed by fact sorting are more stable with respect to antibody production than those that were cloned through manual limiting dilution. The specific productivity of the top clone clone one generated by manual limiting dilution dropped from roughly 30 PCD to roughly 10 PCD after culturing for 80 cell doublings.
On the other hand, the top sub clone generated by fax sorting clone two was able to maintain the specific productivity around 20 PCD for at least 100 cell doublings. Identification of the transgene integration site in the cell chromosome by fluorescent in situ hybridization demonstrated that clone one is a mixed population of phenotype A and phenotype B.In contrast, the fact sorted clone appeared to be a more homogeneous population with 99.5%of the cells of phenotype A.After watching this video, you should have a good understanding of how to develop manufacturing cell lines for the protection of therapeutic proteins.
