Establishment and Maintenance of Cell Lines
The development of cell lines ensures that they are stable in continuous culture, can be routinely cultured to high cell concentrations and produce consistent amounts of active specific antibody.
All cell lines are tested for mycoplasma infection. Mycoplasma is a common contaminant of cultured cell lines, and, if present, can adversely affect the growth rate and antibody secretion rate of the cells. If a mycoplasma infected cell line is grown in a laboratory, it is very difficult to avoid cross contamination into previously uninfected cultures. If a cell line is mycoplasma positive, it undergoes a regime of treatment, developed in the laboratory, until the mycoplasma is eliminated from cell cultures.
Stability of cell lines is monitored by limiting dilution cloning analysis. Cloning continues until all clones isolated are secreting specific antibody. At the final cloning stage, the best clone is chosen according to its growth rate, growth capability of cells in larger culture vessels, secreted antibody potency (measured by serological titration, flow cytometry, cellular assay or intensity of immunohistochemistry staining), and antibody concentration.
The specificity and immunoglobulin class and subclass of the antibody secreted by the best clone are carefully confirmed. The clone is then expanded in culture to provide sufficient cells to freeze a master cell bank and a working cell bank which are stored indefinitely in liquid nitrogen. One vial from each freezing is thawed to check the viability of the frozen cells.
Growth and Harvesting of Cell Lines
All antibodies are grown in in vitro cell culture conditions. When antibody is required, one vial of frozen cells is taken from the working cell bank and rapidly expanded in culture to the required final volume. If only small volumes of supernatant are required, the cells are grown in static flasks. If greater than 500 ml is required, the cells are grown in suspension spinner vessels or other systems.
The growth and secretion rate varies for each cell line and the conditions for growth must be optimised for each cell line. Growth and production of antibody is monitored daily by cell counts and quantified by ELISA. Following the peak of antibody production, cultures are harvested and cells removed by centrifugation and/or ultrafiltration followed by sterile filtration of supernatant. Each batch of supernatant is assessed for specificity, potency and antibody concentration by various QC tests ranging from ELISA, serology, flow cytometry, SDS PAGE, immunoblotting and cell based assays. Supernatant is then aseptically distributed into aliquots at a range of different volumes, and stored between -20 and -30°C until required. The activity of the frozen material is monitored.