Antifoams are added with defined shots (e.g., amount and times) to bioreactors during bioprocess cell culture steps to control foaming typically associated with the use of culture media in bioreactors, including both seed bioreactors and production bioreactors if heavy foam is an issue. Also, Larger sparger holes may generate more bubbles and require more antifoams compare to sparger with smaller holes. Simethicone (Anifoam A, α-(Trimethylsilyl)-ω-methylpoly[oxy(dimethylsilylene)] mixture with silicon dioxide) is one of the most commonly used antifoam, it is a non-ionic emulsion and is hydrophobic in nature, with molecular formula of C2H6OSi. The multi-compendial grade materials (e.g., USP, EP, and JP) are available to meet all monograph requirements.

Simethicone is also acceptable under FDA Regulation 21 CFR 332.10 as a safe and effective drug to alleviate symptoms of gas with heartburn, sour stomach and acid ingestion. Simethicone is used in bioprocess ubiquitously and generally considered to be safe. In current market, the non-animal origin formulation, gamma-irradiated, ready-to-use products are available from several suppliers, including Merck, GE, Corning, and ThermoFisher, etc.

The maximum allowable amount of antifoam has to be established during process development and validation stage to demonstrate the process clearance capability and product safety. Simethicone antifoam can be removed and during downstream purification process, including chromatography column steps and UF/DF step. Justification for Simethicone usage can be addressed based on following considerations:

1.Clearance through chromatography columns: Simethicone is inert and not charged, it doesn’t bind to the resin and is cleared during loading of the product. Further impurity clearance can be achieved during column washing. The clearance factor can be calculated through the following equations:

Clearance factor = eN, where N is number of complete buffer exchanges.

Clearance through UF/DF: UF/DF filter membrane has large molecular cut off (e.g., 30-50 KDa), small molecule (<1/10 MWCO) compounds can pass through the membrane. The clearance factor can be calculated by the following equation:

Clearance factor = eN * S. where N is number of defiltration volume (DF), S is sieving coefficient (1).

However, Simethicone can form micelles with potential larger size than 3 kDa. It is difficult to claim clearance from UF/DF step alone.

The concentration of Simethicone in Bulk Drug Substance (BDS), amount of impurity per amount of BDS ([C]BDS µg/mg), can be calculated through below equation:

[C]BDS µg/mg = [Amount of Simethicone [g]/Clearance factor]/volume of BDS(L)/concentration of BDS (g/L)* 1000 µg/mg.

The calculated amount can be compared to ICH M7 (R1) to assess safety concerns, 1.5µg/day is a threshold of acceptable lifelong daily intake limit of a mutagenic impurity. If the limit is below this level, the simethicone antifoam usage is considered safe.

Amount of Simethicone per dose [µg/dose] = amount of Simethicone per amount of BDS (µg/mg) * maximal dose [mg]

Reference:

ICH M7: Guideline for the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit carcinogenic risk