Biowaste Management During Biopharmaceutical Plant Start-Up: From Regulatory Guidance to Verified Inactivation Methods - - BioPharm International

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Biowaste Management During Biopharmaceutical Plant Start-Up: From Regulatory Guidance to Verified Inactivation Methods


BioPharm International


At commercial production-scale (12,500 L) the recombinant CHO cells are grown in either protein-containing or protein-free media, in flasks or stirred-tank reactors. At various stages of the process the cells are present at densities ranging from 3.0x105 to 4.5x107 cells/ml. A strategy of "out-of-place" NaOCl inactivation of flask cultures and in situ NaOH inactivation of stirred-tank cultures was planned. Flask cultures signify cells growing in shake- or spinner-flasks, in protein-containing media, and at densities up to 3x106 cells/ml. Inactivation was planned to involve the transfer of the cells to a reservoir and the addition of concentrated NaOCl stock solution to a pre-defined concentration of active chlorine and for a predetermined duration. GMM inactivation by NaOH addition to a predetermined concentration and for a defined time was planned for cells grown in stirred-tank reactors. As the cells are maintained in a variety of conditions in stirred-tank reactors, a single inactivation strategy that would encompass all conditions was essential. Although all stirred-tank reactors contain mixing and pH-monitoring capabilities, under specific circumstances inactivation of cultures at volumes lower than the level of the pH probe, impeller, or sample port could be required. To counteract the potential inability to monitor online pH or to easily recover a sample for off-line analysis, the inactivation method was designed to achieve a predetermined volumetric concentration of NaOH, rather than a specific pH.

MATERIALS AND METHODS

Recombinant CHO cells were grown as suspension culture in flasks (shake-flasks and spinner-flasks) and glass (Applikon) reactors, in either protein-containing or protein-free media. Flask cultures were grown without external pH control, while the pH of cultures grown in reactors was controlled at a set point of pH 7.2, by the addition of CO2 and 1 M sodium carbonate.


Figure 2. Schematic of the Bench-scale Inactivation Experiments Small-scale experiments were conducted on recombinant CHO cells to establish inactivation conditions for site-wide implementation. Viable cell density (VCD) was measured using a Cedex automatic cell counter.
Prior to cell inactivation, the density of the recombinant CHO culture was determined and cells were transferred to 60 ml sample tubes. Cell densities were adjusted by the addition of phosphate-buffered saline (PBS) or growth medium, if required. Concentrated NaOH (10 M) or NaOCl (13 percent active chlorine) was added to the cells to a predetermined final concentration. A constant volume and cell density was maintained in all sample tubes by the appropriate addition of PBS. Sample tubes were inverted to incorporate the inactivation solution into the cultures, which were then maintained at room temperature. Samples were withdrawn routinely, from 1 minute up to 90 minutes post-addition of the inactivating agent, and analyzed for viable cell numbers. Viable cell density was assessed using a Cedex automatic cell counter (from Innovatis AG), with viability determined by trypan blue exclusion. All test conditions were assessed in duplicate. The inactivation trial process is outlined in Figure 2. One hour after the addition of the inactivating agent, the pH of the NaOH-inactivated culture was measured. Statistical analysis was performed by analysis of variance (ANOVA), using Design-Expert software (from Stat-Ease, Inc.). The probability values (p-values) less than 0.05 indicated that test parameters were statistically significant.

Cell inactivation in both protein-free and protein-containing media was analyzed in the NaOH inactivation study. Culture inactivation at high (5.0x107 cells/ml; a cell density approximately 10 percent higher than the maximum density expected in the commercial process) and low (5.0x106 cells/ml) densities was assessed. NaOCl inactivation was also assessed at high and low cell densities (3x107 cells/ml and 3x106 cells/ml) but only in protein-containing medium, reflecting the planned use of this inactivation process.


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