When using CHO cells, the product (the mAb) is secreted from the cell and is found in the aqueous medium in the reactor.
mAking mAbs
We have covered several aspects of monoclonal antibodies (mAbs), but how do we produce these amazing molecules on a large scale so we can provide medicine for patients? It all starts with one small vial.
Cell Banks
Once development of a cell line is complete, a master cell bank (MCB) is produced by a GMP laboratory scale production of cells from the final cell line. Each vial of a master cell bank can be used to produce a working cell bank.
A working cell bank (WCB) is produced by growing a vial of master cell bank under specific, controlled conditions. The cells produced are genetically identical to the master cell bank. Both MCB and WCB are stored frozen at -80°C using liquid nitrogen (-196°C!).
Cell Growth
Starting from a small vial of working cell bank, we end up with a large bioreactor of cells and mAb. As the number of cells (cell mass) increases, the amount of mAb produced by the cell mass increases. Scientists work to maximize cell growth and the amount of mAb each cell can make. Changes in growth media and process parameters (pH, O2, temperature, feed) are optimized for each.
Once the processes of cell growth and mAb production are complete, the mAb must be separated from the cells and purified for use as a drug. See below for a diagram for mAb production and the next slide for purification steps.
Seed Expansion
Growth media are added at each expansion
step to allow cells to multiply.
Purification of mAbs
Purification of mAbs from the final bioreactor mixture varies from product to product, but an example is shown below. Purification uses multiple chromatography and filtration steps to separate the mAb from the cells/cell debris in the bioreactor.
(1) The first step to purification of mAbs is to remove the solution of mAbs from the cells/cell debris (pieces of cells). The mAb is much smaller than the cells and this is done by centrifugation and/or depth filtration.
Cells/cell debris are heavier and settle to the bottom of the centrifuge. The mAb remains suspended in the supernatant (the liquid portion).
(2) The next step is a series of chromatography steps where the mAbs are separated from smaller impurities. The mAb solution can be run through several different types of chromatography with each targeting removal of specific impurities.
Each column works on different principles. Columns are filled with resins (solid media) and the mAb mixture is run through them. As the mixture runs through the resin, different molecules move at different rates and the mAbs separated.
(3) After chromatography, the solution of mAb is nanofiltered any potential viruses present.
This filtration uses a cartridge with a fine pore size.
(4) The filtered solution is run through ultrafiltration/diafiltration (UF/DF = filtration through a membrane that adjusts the concentration and components of the aqueous media to provide stability of the drug product).
Larger particles (mAb) are retained behind the membrane smaller particles move through the membrane.
(5) After a final bioburden reduction filtration, the mAb solution can be used to fill syringes/vials or can be further processed prior to filling depending on the need of the final drug product.
