METABOLIC AND KINETIC-STUDIES OF HYBRIDOMAS IN EXPONENTIALLY FED-BATCH CULTURES USING T-FLASKS

Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-fla sks were explored as a simple alternative experimental tool to chemost ats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode us ing an exponentially increasing and predetermined stepwise feeding pro file of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution r ates below the maximum growth rate. In particular, steady-state for gr owth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h(-1). For such a range, the glucose and lactate metabolic quotients and the ste ady-state glucose concentration increased, whereas total MAb, volumetr ic, and specific MAb production rates decreased 65-, 6-, and 3-fold, r espectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h(-1) , where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates bel ow 0.02 h(-1). No true washout occurred during operation above the max imum growth, as concluded from the constant viable cell number. Howeve r, growth rate decreased to as low as 0.01 h(-1), suggesting the requi rement of a minimum cell density, and concomitant autocrine growth fac tors, for growth. Chemostat operation drawbacks were avoided by EFBC i n T-flasks. Namely, simple and stable operation was obtained at diluti on rates ranging from very low to above the maximum growth rate. Furth ermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.