STABILITY OF PRODUCER HYBRIDOMA CELL-LINES AFTER CELL SORTING - A CASE-STUDY

Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface-associated antibody contents to identif y a significant nonproducer cell fraction in a murine hybridoma cell l ine that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface-associated antibo dy content were isolated by cell sorting on the basis of surface fluor escence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultiva ted for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line d uring passaging and after a freeze-thaw cycle was investigated. This c ell line was found to be highly unstable. A simple batch-growth model simulation was used as follows: (i) to demonstrate that nonproducers a ppear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy-chain gene and/or light-chain gene e xpression; (ii) to show that a high rate of conversion of producer hyb ridomas to nonproducer hybridomas cannot entirely explain the populati on dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 x 10(-5) h(-1); (iv) to show that, for this con version rate, the nonproducer cells' specific growth rates need only b e 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cell s are preferentially lost during eel freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analy zed and purified frequently to prevent overgrowth by nonproducers.