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EXTRACTABLES AND LEACHABLES


extractables should be considered in a risk assessment for any manufacturing process.


Extractables Profiles of Three Common Bioprocess Containers


Three types of single use bioprocess containers were filled with either 60% IPA or water and incubated at 40°C/Ambient Relative Humidity for 20 days. Each bag and corresponding control had 1.0 mL removed at 12, 24, 48, and 72 hours, and 5, 10, 15, and 20 days. The resulting extracts were tested by gradient HPLC using an LC/MS-Time of Flight equipped with a multimode source (electrospray and atmospheric pressure chemical ionization) using positive ionization and negative ionization. Table 1 lists the compounds observed in the extractables profiles for each of the bags used in this study with a breakdown by solvent type.


In water, the major extractable was bDtBPP from all three bag types. In the 60% IPA extracts, Bag 1 had the most extensive profile.


Bag 1 also contained a The


extraction profile included three Irgafos 168® breakdown products: 3,5-di-tert-Butyl-4-Hydroxybenzaldehyde, Bis(2,4-di-tert-Butylphenyl) Phosphate, and Irgafos 168 Phosphate.


number of slip agents incorporated into polymer films to reduce their coefficient of friction, including Ethylene Bis Palmitamide, Ethylene Bis Heptadecanamide, Erucamide, and Ethylene Bis Stearamide along with their breakdown products Palmitamide and Steamide.


In the 60% IPA extracts of Bag 2, the same three Irgafos 168® breakdown products were observed as in Bag 1 as well as Ethylene Bis Heptadecanamide.


Finally, the 60% IPA extracts of Bag 3 contained


the same three Irgafos 168® breakdown products as well as Ethylene Bis Heptadecanamide and Ethylene Bis Palmitamide along with its breakdown product Palmitamide.


Figures 1 and 2 show the time dependent extraction of bDtBPP from the three bioprocess bags.


This time dependence appears to be


nonlinear. Although the data was not fit using a nonlinear algorithm, this observation is consistent with what has been previously published for this compound where extracted bDtBPP concentration initially increased rapidly, but the extraction slowed down significantly after 5 to 10 days.9


In our experiment, after nearly three weeks


of incubation, the bDtBPP extraction rate seemed to continue to increase. Maximal levels of water-extracted bDtBPP from the three bags were Bag 1 = 0.5 μg/mL; Bag 2 = 2.1 μg/mL; and Bag 3 = 3.8 μg/mL (Figure 1). Maximal levels of IPA-extracted bDtBPP from the three bags were Bag 1 = 1.5 mg/mL; Bag 2 = 10.5 mg/mL; and Bag 3 = 12.6 mg/mL (Figure 2). Published EC50


values for bDtBPP range from 0.12 to 0.73 mg/mL.9 Two of the three bags exceed the upper limit by


three- to five-fold with the third falling just shy of the upper limit in water where as all three bags exceed the upper limit when extracted with IPA.


Conclusion


The comparison of extractable compounds observed from three bioprocess bags demonstrates the need for biomanufacturers to carefully evaluate their components in order to ensure their process is safe and robust. The presence of bDtBPP at levels above the EC50


of the


compound, for at least two of the bags evaluated, is a serious concern for biopharmaceutical manufacturers.


In order to reduce the levels


of bDtBPP, these bags could be formulated and/or processed in such a way that, at the time when the component is sent for sterilization,


4 American Pharmaceutical Review | Biopharmaceutical Supplement 2014 13. 9. 7. 8. 5. 6. 2.


a minimum of oxidized Irgafos 168® is present in the film. This could be accomplished by reducing the content of Irgafos in the initial formulation and/or increasing the content of other antioxidants in the formulation.


Finally, deciding whether or not leached bDtBPP will affect the biomanufacturing process will depend not only on the leachable profile of the chosen bioprocess container but also on a number of other process parameters, including the sensitivity of the cell line in use.


In


the end, all single use components need to be evaluated on a case-by- case basis to ensure that bDtBPP or other leachables will not have an adverse impact on the overall manufacturing process.


References 1.


U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER). Guidance for Industry Container Closure Systems for Packaging Human Drugs and Biologics. May, 1999.


U.S. Food and Drug Administration. Equipment Construction. Code of Federal Regulations, Food and Drugs Title 21, Part 211.65. Available at: http://www.accessdata. fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=211.65. Last updated September 1, 2014. Accessed September 16, 2014.


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Bio-Process Systems Alliance. Recommendations for Testing and Evaluation of Extractables from Single-Use Process Equipment. 2010.


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Pang J, Blanc T, Brown J, Labrenz S, Villalobos A, Depaolis A, et al. Recognition and identification of UV-absorbing leachables in EPREX® pre-filled syringes: an unexpected occurrence at a formulation–component interface. PDA. J Pharm Sci Technol. 2007;61(6):423–432.


Hammond M, Nunn H, Rogers G, Lee H, et al. Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers. PDA. J Pharm Sci Technol. 2013;67:123-134.


10. Brandolini AJ, Garcia JM, Truitt RE. Spectroscopic characterization of the degradation products of phosphorus-containing polymer additives. Spectroscopy. 1992;7(3):34-39.


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Djouani F, Richaud E, Fayolle B, Verdu J. Modelling of thermal oxidation of phosphite stabilized polyethylene. Polym Deg Stabil. 2011; 96(7):1349-1360.


Peacock AJ. Handbook of Polyethylene: Structures, Properties, and Applications. New York: Marcel Dekker, Inc. 2000:385-388.


Tollefsen KE, Blikstad C, Eikvar S, Farmen Finne E, Gregersen KI. Cytotoxicity of alkylphenols and alkylated non-phenolics in a primary culture of rainbow trout (Onchorhynchus mykiss) hepatocytes. Ecotoxicol Environ Saf. 2008;69(1):64-73.


14. Moridani MY, Siraki A, O’Brien PJ. Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes. Chem-Biol Interact. 2003;145(2):213-223.


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Carlsson DJ, Krzymien ME, Deschenes L, Mercier M, Vachon C. Phosphite additives and their transformation products in polyethylene packaging for gamma-irradiation. Food Addit Contam. 2001;18(6):581-591.


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