Production and Purification of Recombinant Antibodies from Mammalian Cells
Transcrição
Grow CHO cells in 200 milliliters of media into long Erlenmeyer baffled flasks. Combine 5 milliliters of CHO freestyle media with 50 micrograms of variable heavy clone DNA and 75 micrograms of variable light clone DNA in a 15-milliliter tube. Then, vortex the tube. Leave the mixture at room temperature for 5 minutes. Add 750 microliters of 1 milligram per milliliter polyethylene amine stock to the DNA solution, and aggressively vortex it for 30 seconds.
Leave the mixture at room temperature for an additional 5 minutes. Add the entire mixture to the CHO cells while manually shaking the flask. Immediately incubate the cells at 37 degrees Celsius while shaking at 130 RPM.
On the next day, add 2 milliliters of 100x anti-clumping agent and 2 milliliters of 100x antibacterial antimycotic solution to the cells. Incubate the flasks at 32 to 34 degrees Celsius with shaking at 130 RPM. Continue culturing the cells for 10 to 11 days, adding 10 milliliters of tryptone N1 feed and 2 milliliters of 100x glutamine supplement on every fifth day. Count the cells on every third day to ensure that cell viability remains above 80%. On day 10 or 11, harvest the medium for antibody purification by centrifuging the culture at 3,000 times g and 4 degrees Celsius for 40 to 60 minutes. Then, filter the clear media with a 0.22-micrometer bottle filter.
To purify the antibodies, equilibrate the protein A column with two column volumes of binding buffer. Pass the filtered media through the column at the flow rate of 1 milliliter per minute. Then wash the column with two column volumes of binding buffer. Use 5 milliliters of elution buffer to elute the antibody into 500-microliter fractions.
