GMM inactivation was dependent on both NaOH concentration and media composition. In protein-containing media, NaOH addition
to a final concentration ≤ 0.025 M resulted in negligible cell kill over a one-hour period. At concentrations ≥0.05 M, cell
viability fell to below detectable levels within one minute of contact time( Figures 4a and 4b). However, in protein-free
media, culture inactivation was achieved at lower NaOH concentrations; addition of 0.0125 M NaOH resulted in viable cell density
(VCD) falling to below detectable levels within 30 minutes (Figures 4c and 4d).
Culture inactivation in both protein-containing and protein-free media was independent of cell density. In protein-containing
media, inactivation of recombinant CHO cells by 0.05 M NaOH was comparable at both 5x106 and 5x107 cells/ml (Figures 4a and 4b). In protein-free media, cell inactivation was also comparable at both high and low cell densities
but inactivation was achieved at a lower NaOH concentration (0.0125 M) (Figures 4c and 4d).
Following NaOH addition to the cultures, an increase in solution viscosity was noted. The increase in viscosity is a consequence
of cell lysis, as the detection of both viable and dead cells decreased with increasing culture viscosity. From visual inspection,
culture viscosity was broadly dependent on NaOH concentration, initial culture density, and the duration of exposure to NaOH.
Increased NaOH concentrations, cell densities, and exposure time resulted in the generation of more viscous solutions. The
viscosity of the solution prevented the accurate determination of cell densities. However, when the solution was examined
microscopically, no cells (viable or dead) were observed.
The pH of the NaOH-inactivated culture was dependent on both the final concentration of NaOH (p-value = 0.0020) and the culture density (p-value = 0.0029), but was independent of the media composition (p-value = 0.9710) (Figure 5). The pH of the high-density control cultures (cultures unexposed to NaOH for 60 minutes) was significantly
lower than the pH of low-density cultures. This may be accounted for by cellular metabolism during the study hold-time, such
as lactate production by the viable culture. The finding was observed for cultures suspended in both protein-containing and
protein-free media. Following inactivation, the extent of the pH increase in the culture was dependent on the amount of NaOH
added. However, the effect of cell lysis and the associated release of intracellular components on the final pH was not accounted
for in this study. In generating low-density cultures, high-density cultures were diluted with either PBS or culture medium.
To minimize disparities in results due to the diluents, the pH of the PBS and media were identical and the same as the culture
media in which the cells were grown, pH 7.2. PBS has an effective pH buffering range of 5.8 to 8.0. Both the protein-containing
and protein-free culture media contain 15mM HEPES, which at room temperature has a pKa of 7.5 and an effective buffering range
of 6.8 to 8.2. The similarities in solution pH and buffering range would indicate that cultures should behave similarly in
response to NaOH addition, whether the cultures were diluted with PBS or media, or undiluted.
Figure 5. Increase in Culture pH Following NaOH Addition to Recombinant CHO Cells Suspended in Protein-containing and Protein-free
Medium. The average pH of the inactivated cultured is graphed against final NaOH concentration.