Cell cycle‐ and growth phase‐dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Abstract

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G₁ cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO₂) is also a function of cell cycle, being minimum for cells in G₀ and G₁ phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01–0.05 to 0.5–0.7 mmol O₂/L h as the cells went from the lag to the midexponential phase. The qO₂ increased from 0.3 × 10⁻¹⁰−0.9 × 10⁻¹⁰ mmol O₂/cell h at inoculation to 3.3 × 10⁻¹⁰−3.7 × 10⁻¹⁰ mmol O₂/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO₂ rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO₂ occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.