EXTRACTABLES AND LEACHABLES
Figure 1 - Time dependent extraction of bDtBPP for 40°C incubation in water.
Bis(2,4-di-tert-Butylphenyl) Phosphate
A prominent extractable from single-use bioprocess containers has been identified as being highly detrimental to cell growth.9 The compound, bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), is derived from the breakdown of tris(2,4-di-tert-butylphenyl) phosphite (trade name Irgafos 168®). Irgafos 168® is a secondary antioxidant added to the formulation of polyolefin plastics, such as polyethylene.10
In polymer formulations, a
primary antioxidant (such as Irganox 1076®) is one that acts to deactivate radicals, and a secondary antioxidant acts to inactivate hydroperoxides.11,12
The secondary anti-
oxidants are necessary to protect the polymers during high-temperature processing or sterilization techniques.
The reaction of
the secondary antioxidant protects the polymer but oxidizes Irgafos 168®, leaving the molecule vulnerable to chemical breakdown. In extractable studies of single-use bioprocess containers, Irgafos 168® and its breakdown products are commonly observed. The most commonly observed compounds are bDtBPP and Irgafos 168® phosphate (tris [2,4-di-tert- butylphenyl] phosphate).
Figure 2 - Time dependent extraction of bDtBPP for 40°C incubation in 60% IPA.
A related compound, di-tert-butylphenyl (DtBP), has been shown to be toxic to fish13 and mammalian cells.14
These data beg the
question: Are the Irgafos breakdown products cytotoxic, given the structural similarities? The large majority of the breakdown products are not.9
Most of the commonly seen Irgafos
168® breakdown products show no toxicity to CHO cells at concentrations up to 1 μg/mL.9 In contrast, bDtBPP causes strong inhibition of cell growth at even lower concentrations. Cell growth experiments using several mammalian cell lines and growth media spiked with bDtBPP show harmful effects at concentrations down to 0.1 μg/mL.9
Cellular
response to bDtBPP is rapid and results in a significant decrease in mitochondrial membrane potential.9
The migration of
bDtBPP from bioprocess containers has been shown to be time and temperature dependent with significant amounts of bDtBPP continuing to be extracted after weeks of incubation.9, Personal Observations
Further,
numerous experiments suggest that exposure of oxidized Irgafos 168® to ionizing radiation (such as gamma irradiation) can generate significant amounts of leachable bDtBPP.9,15
The example of bDtBPP should be a stark reminder to all manufacturers that
3 American Pharmaceutical Review | Biopharmaceutical Supplement 2014
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