Establishment and Maintenance of Cell Lines
The work with the cell lines ensured that they are stable in continuous culture, can be cultured to high cell concentrations on a routine basis 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, making it difficult to expand cell cultures into large growth vessels. If a mycoplasma infected cell line is grown in a laboratory, it is very difficult to avoid cross contamination into previously uninfected cultures. All new cell lines are kept in "quarantine" (in a separate incubator, and not handled at the same time as other lines) until they have been tested and shown to be free of mycoplasma. Several methods have been used for mycoplasma detection including a culture method, a DNA hybridisation kit, PCR based method and most recently a biochemical test which detects the activity of mycoplasmal enzymes. If a cell line is mycoplasma positive, it undergoes a regime of treatment, developed in the laboratory, with the antibiotic ciprofloxacin. This is effective in eliminating mycoplasma from cell cultures. Treatment continues until the culture gives a negative result with the mycoplasma test. Once a treated line is mycoplasma test negative, normal work continues on the line, but, it is retested at later stages of growth.
Stability of cell lines is monitored by limiting dilution cloning analysis1. All lines are cloned at least three times after primary fusion, and cloning continues until all clones isolated are secreting specific antibody. At the final cloning stage, several clones are compared for
1. Growth rate
2. Growth capability of cells in larger culture vessels
3. Secreted antibody potency (measured by serological titration, flow cytometry or intensity of staining immunoblots)
4. Antibody concentration measured by double antibody sandwich class specific ELISA2.
The best clones are selected (optimal growth/ high level of secretion of specific antibody/highest potency for the lowest antibody concentration). Comparison of antibody concentration and potency is important if the avidity of the secreted antibody is to be maintained and/or improved. We have observed significant variation in antibody avidity between different sister clones3.
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. Because we have optimised conditions for producing antibodies at high levels there is no need for in vivo ascitic fluids to be produced. 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. Higher concentrations of antibody can be achieved using hollow fibre technology . The concentration obtained varies from cell line to cell line but concentrations 10 times those obtained by static culture is normally achieved with hollow fibre.
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 quantitation ELISA. Following the peak of antibody production, cultures are harvested 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 and immunoblotting. Supernatant is then aseptically distributed into aliquots at a range of different volumes, and stored at -30°C until required. The activity of the frozen material is monitored.