The Risks of Single-Use Bioprocess Containers

Monday, November 24, 2014

Extractables and Leachables

The screening of the final packaging components of human drug products for extractables and leachables has become commonplace in the 15 years since the FDA released their Container Closure Systems for Packaging Human Drugs and Biologics. With the increase in prevalence of single-use systems for biomanufacturing, these components require the same scrutiny. The regulation applicable to bio-processing/manufacturing components (CFR Part 211.65) states: “Equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.”1,2 Drug applications to the FDA are expected to provide a safety evaluation, based on the extractables and leachables profile, of the highest risk components that are in closest contact with the drug. For biologics these components not only include the final container/ closure system but also components associated with the biomanufacturing process.

In an article in BioProcess International, Bestwick and Colton defined extractables and leachables as:

  • Extractables: Chemical compounds that migrate from any product contact material when exposed to an appropriate solvent under exaggerated conditions of time and temperature.3
  • Leachables: Chemical compounds, typically a subset of extractables, that migrate into the drug formulation from any product contact material—including elastomeric, plastic, glass, stainless steel or coating components—as a result of direct contact with the drug formulation under normal process conditions or accelerated storage conditions and are found in the final drug product.3

As will be discussed in this article, the containers used in biomanufacturing can themselves be a source of contamination as a result of leachable compounds. These leachables may result from anti-oxidants, stabilizers, or plasticizers used in the manufacture of the single use system. In addition, since the materials are polymeric in nature, residual monomers, oligomers or polymeric fragments may also be observed as leachables.4

To demonstrate that a bioprocessing/manufacturing component will not adversely affect the manufacturing process, an initial extractables screening study is typically performed. The extraction experiment is designed to exaggerate the conditions of real-time use. Due to the shorter contact time with the drug product overly aggressive conditions are not always appropriate for extracting the components of bioprocess systems, such as bioprocess bags, filters, and tubing.5,6 Use of aggressive extraction techniques may result in the breakdown of the polymer or component (eg, blistering the film of a bioprocess bag).

Single-use Systems

Most single-use products are constructed of polymeric materials that can introduce leachables into the manufacturing process. These leachables can affect the safety and efficacy of the product under manufacture. Assessing the risk posed by any given leachable can be challenging, be it from a container closure system or the biomanufacturing process. Single-use components are often constructed of numerous functional components such as bioprocess bags, tubing, and filters, each potentially made from different types of plastic and/or rubber. Additionally, sterilization, the addition of wetting agents, and other pretreatments of such assemblies can create leachables that are unrelated to the material making up the assemblies.7

The evaluation of extractables and leachables to reduce patient safety risks should not be underestimated.3 A study published in the PDA Journal of Pharmaceutical Science and Technology8illustrates how substituting human serum albumin with polysorbate 80 in a parenteral drug led to a new leachable that resulted in a high incidence of antibody positive pure cell aplasia.3 This leachable compound was a small-molecule, aromatic compound originating from uncoated rubber syringe stoppers. This example from a final container/closure system serves as an effective illustration of the potential for leachables to be a major safety concern. Additionally, some leachable compounds can affect the efficacy and stability of a product.3

In a 2013 article in the Journal of Pharmaceutical Science and Technology, the authors looked at the effects of leachable compounds from single-use bioprocess containers on cell viability.9 The study found that a common leachable compound found in many bioprocess containers could inhibit cell growth. The compound from the study was a breakdown product of the common anti-oxidant Irgafos 168® (CAS # 31570-04- 4). The authors of the paper found that the degradation product of Irgafos 168® inhibited cell growth of a number of Chinese hamster ovary (Cho) cell lines, which are commonly used in biomanufacturing. The issue of leachables inhibiting cell growth could cause variability in batch-to-batch yields, loss of expensive cell lines, and low yields in the manufacturing process. The experiments conducted in this paper highlight the need to understand the impact of the extractables not only from a patient safety standpoint but also from a biomanufacturing standpoint.

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 antioxidants 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-tertbutylphenyl] phosphate).

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 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.

Total Extractables from the Three Bag Types

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

Figure 1 - Time dependent extraction of bDtBPP for 40°C incubation in water.
Figure 2 - Time dependent extraction of bDtBPP for 40°C incubation in 60% 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, 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-bycase 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.
  2. 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.
  3. Bestwick D, Colton R. Extractables and Leachables from Single-Use Disposables. BioProcess International. 2009;7(2).
  4. Jenke D. Extractable/Leachable Substances from Plastic Materials Used as Pharmaceutical Product Containers/Devices. PDA. J Pharm Sci Technol. 2002; 56:332-371.
  5. Bio-Process Systems Alliance. Recommendations for Testing and Evaluation of Extractables from Single-Use Process Equipment. 2010.
  6. Bio-Process Systems Alliance. Recommendations for Extractables and Leachables Testing. Bioprocess International. 2007;5(11):36-49.
  7. Killian P. Assessing the Risk of Leachables from Single-Use Systems, BioPharm International. 2012;25(2):32-40.
  8. 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.
  9. 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.
  11. Djouani F, Richaud E, Fayolle B, Verdu J. Modelling of thermal oxidation of phosphite stabilized polyethylene. Polym Deg Stabil. 2011; 96(7):1349-1360.
  12. Peacock AJ. Handbook of Polyethylene: Structures, Properties, and Applications. New York: Marcel Dekker, Inc. 2000:385-388.
  13. 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.
  15. 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.

Dr. Charles Ducker is a Principal Chemist for Eurofins Lancaster Laboratories’ Method Development and Validation group where he performs extractables and leachables testing using LC/MSTOF, LC/MS/MS, GC/MS, and ICP-OES technology. Specializing in LC/MS analysis, Dr. Ducker has 13 years of experience in the biotech industry and has served as the lead scientist for two significant drug discovery and development programs during his career. Dr. Ducker has 10 peer reviewed publications and earned a PhD in Biochemistry/ Molecular Biology from The Pennsylvania State University, as well as a BS in Biology from Millersville University.

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