Step I: Cell line submission, recovery, adoption and expansion. 2-3 weeks

1. Two vials of frozen hybridoma cells or one flask of live cells (> 2×10⁶) in culture medium
2. Formula of original culture medium
3. Species the cell line derived and production history, if available
4. Upon receipt, the cell line will be recovered, adopted to production medium, and expanded to obtain enough cells for mouse inoculation.

Step II: Inoculation. 2-3 weeks

1. 500 ml of in vitro production medium is transferred into a two-liter size roller bottle.
2. The expanded cell culture is transferred into the roller bottle to final cell density of 1-2×10⁵ cells/ml.
3. The cells are immediately placed on the roller device in a 37°C incubator.
4. The cells are cultured under 2-3 rpm rotation for two weeks.
5. The culture is monitored daily for cell growth.
6. If necessary, more roller bottles are inoculated using the cells from the existing roller bottle culture.

Step III: Harvest. 1 week

1. The culture is stopped by removal of the roller bottle from the incubator. The culture is stored at 2-8°C until all roller bottle cultures are done.
2. The cells are removed from the culture medium by centrifuging at 3,000-4,000rpm for 15 minutes or by filtering through the 1.0 µm filters.
3. The clarified culture supernatant is concentrated to 1/10 of the original volume and, if not immediately processed for purification, the processed antibody containing the medium is stored at -20°C.