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


A direct correlation between the pH of the inactivated GMM culture and the final NaOH concentration could not be established due to the influence of cell density and media composition. Addition of NaOH to GMM cultures suspended in protein-containing media to a final concentration of 0.025 M NaOH resulted in a final pH of 8.9 and 9.5, for high- and low-density cultures respectively. In protein-free media, addition of NaOH to a final concentration of 0.025 M resulted in a final pH of 7.5 and 11.9 for high- and low-density cultures respectively. However, 0.025 M NaOH resulted in low or negligible cell kill in the protein-containing culture, with 100 percent cell kill achieved in the protein-free culture.

CONCLUSIONS

To implement an effective inactivation system and to ensure inactivation in all circumstances, parameters were selected that significantly exceeded the conditions concluded from inactivation trials. GMM inactivation with NaOCl has been restricted to low-density cultures, where 0.25 percent (active chlorine) resulted in complete inactivation within one minute. To increase confidence in the inactivation process, the contact time between the CHO cells and NaOCl was extended. A NaOCl-inactivation protocol consisting of a room-temperature incubation of CHO cells, up to a maximum concentration of 3.0x106 cells/ml with a volume of NaOCl ≥ a final concentration of 0.25 percent active chlorine and for a contact time equal to or greater than 30 minutes, has been implemented. When densities in excess of 3.0x106 cells/ml were encountered, the culture was diluted to below this limit prior to inactivation.

In the inactivation study, 0.05 M NaOH was shown to effectively destroy the CHO cell line within one minute of contact with the culture, under all culture conditions employed. Again, to increase confidence in the inactivation procedure, the contact time between NaOH and cells was extended to 30 minutes. For routine NaOH inactivation, a strategy of a room-temperature incubation of a maximum concentration of 5.0x107 cells/ml with a minimum concentration of 0.05M NaOH and for a minimum duration of 30 minutes was implemented. NaOH inactivation was based on attaining a predetermined final concentration of NaOH as opposed to attaining a target pH, as a direct correlation between the pH and the NaOH concentration of the inactivated GMM culture could not be established. The inability to easily assess the pH of inactivated culture in all circumstances, due to low culture volume in some instances and the positioning of probes and sample ports on the stirred-tank reactors, also contributed to the adoption of this strategy. The generation of highly viscous solutions during NaOH-induced cell inactivation should not be ignored when implementing inactivation strategies, as it may present a challenge when removing cultures from stirred-tank reactors.

SUMMARY

In formulating the inactivation strategy for GMM at our biopharmaceutical manufacturing facility, a combination of inactivation procedures was selected, reflecting the nature of the waste and phase of the commercial operation.

  • Autoclave inactivation was employed for solid waste.
  • A heat "kill" system was employed as the principal means of liquid-waste inactivation. Due to the availability of literature citing the use of "kill" systems, a scheme of commissioning following an annual verification of its effectiveness was adopted.
  • Cell inactivation based on NaOCl addition was employed for small culture volumes and used routinely in the quality control and development laboratories. Inactivation consists of exposing cultures at a maximum density of 3x106 cells/ml to a minimum concentration of 0.25 percent (active chlorine) NaOCl for at least 30 minutes.
  • During start-up operations and prior to commissioning of the "kill" system, GMM inactivation by NaOH addition was implemented for large-volume liquid waste. NaOH inactivation of GMM would also be available as a backup for the "kill" system in instances of unforeseen equipment breakdown. Inactivation consists of exposing a maximum of 5x107 cells/ml to a minimum concentration of 0.5 M NaOH for at least 30 minutes.
  • Due to the dearth of published literature on chemical inactivation of GMM, the NaOH- and NaOCl-inactivation procedures were verified at bench-scale prior to their site-wide implementation. Following establishment of a chemical-inactivation regime, the procedure was implemented with a commitment to an annual verification of its efficacy.


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