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-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth 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 steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, 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 below 0.02 h-1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h-1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.
Higareda
A E
AE
Department of Bioengineering, Universidad Nacional Autónoma de México, Morelos.
Possani
L D
LD
Ramírez
O T
OT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002455:Cell Division; D046508:Culture Techniques / Q000295:instrumentation / Q000379:methods; D005947:Glucose / Q000378:metabolism; D006825:Hybridomas / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D051379:Mice; D008962:Models, Theoretical; D009498:Neurotoxins / Q000378:metabolism; D012604:Scorpion Venoms / Q000276:immunology; D013997:Time Factors
D000911:Antibodies, Monoclonal; C035413:Centruroides toxin; D007773:Lactates; D009498:Neurotoxins; D012604:Scorpion Venoms; D005947:Glucose