This article details the process of producing therapeutic antibodies using stably transfected mammalian cells. The methodology includes cell culture, nutrient supplementation, and harvesting techniques to maximize antibody yield.
Begin with a flask containing stably transfected mammalian cells in a serum-free medium supplemented with a non-ionic surfactant to prevent cell aggregation.
These cells carry a modified genome containing integrated heavy-and-light-chain genes for therapeutic antibody production.
Introduce tryptone, a nutrient-rich source of peptides and amino acids essential for cellular metabolism.
The cell utilizes these amino acids and peptides, and multiplies, resulting in an overgrowth that supports a higher antibody yield.
The expression of genes in the cells drives the production of heavy and light chain polypeptides.
In the endoplasmic reticulum, these chains fold and dimerize via inter-chain disulfide bonds, forming therapeutic antibodies.
In the Golgi apparatus, antibodies undergo post-translational modifications. They are then packaged into vesicles and released into the medium.
Incubate the cells for a longer duration to increase antibody production.
As cell viability begins to decline, harvest the supernatant.
Filter the supernatant containing therapeutic antibodies and store it at a lower temperature for later use.
Seed cells at 2 x 105 cells per milliliter in 100 milliliters of serum-free medium in two flasks. Incubate the cells at 37 degrees Celsius and 5% carbon dioxide at 120 rotations per minute for 24 hours. To the nutrient-supplemented culture, add tryptone to a final concentration of 0.5%. Then, use serum-free medium to equalize the volume of the unsupplemented culture. Incubate the cells for an additional seven days.
From the eighth day of culturing forward, count the cells daily using a hemocytometer to monitor cell viability. Once the cell viability reaches less than 80%, harvest the culture by centrifugation at 3,000 times g for 15 minutes. Then, filter-sterilize the supernatant through a 0.22-micrometer filter. Store the supernatants at 4 degrees Celsius in the short term or freeze at degrees Celsius long-term.
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