In the face of unprecedented competition within the industry, biopharma companies must now undergo significant transformation in order to meet the challenge of reducing costs while also providing safe and effective therapies. The adoption of single-use systems (SUS) is one key strategy for those companies actively involved in this transition (1). Compared to traditional manufacturing technology, SUS deliver many advantages, such as reduced requirements for process validation, higher manufacturing flexibility, etc., which ultimately translate into higher operating efficiency and reduced manufacturing costs (2).

In spite of these merits, the possibility exists that the plastic materials used for SUS may leach organic compounds, or inorganic substances, into the processing fluid or the final drug product; this remains a major concern. Such an undesired event could eventually compromise bioprocessing and/or significantly impact the safety, quality and purity of the drug product (3). Unfortunately, direct analysis of such leachates is difficult due to their low concentrations and their existence in complex matrices. Rather, indirect methods are normally used by extracting the component of interests under exaggerated conditions to produce an extractables profile— generally predictive of leachables (4). The capability to predict leachables from an extractable profile, however, depends to a large extent on the experimental conditions used. The most relevant information can be expected from an extractable profile generated with conditions close to those used in the processing or storage of the drug product.

Recently, researchers developed an effective extraction method in a study of extractable profiles of single-use biocontainers (SUBs), in which SUBs were extracted at 50°C for two days using an organoaqueous solvent containing 20% acetonitrile, 20% ethanol, and 60% water (5). The extraction conditions, along with a nontargeted analytical strategy, allowed for the generation and identification of an array of extractables compounds from the SUBs, including one later found harmful to mammalian cell growth (6).

Conditions Showcase Leaching Danger

The researchers selected an extraction solvent with 60% water due to the fact that SUBs are largely intended for use with aqueous-based systems such as cell cultures or process buffers. As a polar solvent, water permits discovery of potential leachables, but has limited extraction strength toward poorly water-soluble extractables (e.g., hydrophobic compounds, fatty acids, etc.). These may leach out in some application systems containing organic components, such as proteins, surfactants and/ or excipients, etc. The addition of 20% ethanol and 20% acetonitrile to water increased its extractive effectiveness, both in terms of the number of chemical species and the levels at which they are present in the extracts, as ethanol and acetonitrile are capable of solubilizing a wide range of organic compounds. An even larger number of organic compounds could be expected with a solvent of high organic content. More is not necessarily better, however; extractables generated from such solvents may not represent leachables generated from SUBs in aqueous-based applications, and would be of diminished value in predicting possible leachables.

Elevated temperatures and/or prolonged extraction are commonly used in extractables studies. A maximum temperature of 60°C is typically recommended by most SUB suppliers in order to avoid thermal degradation or loss of mechanical integrity. After preliminary testing with different temperatures, researchers considered 50°C as the most suitable extraction temperature that provided a large number of extractables without affecting the integrity of the system. With a temperature of 50°C, an incubation duration of two days was selected to approximate a condition of four days of cell culture at 37°C. This followed the Arrhenius-like time/temperature equivalence typically used in accelerated aging studies of polymers (7).

The RP-HPLC/UV (reversed-phase high performance liquid chromatography/ultraviolet) analysis of the extraction from a commercially available SUB shown in Figure 1 clearly demonstrated the advantages of using 40% organoaqueous solvents. Compared to water extraction, extraction with 40% organoaqueous solvent reproduced all the extractables, with most of the compounds extracted in much higher abundances. The higher levels of extractables produced provided significant advantages in their identification and quantification, especially when only instruments of low sensitivity were available. As expected, unique extractables were also observed with the 40% organoaqueous solvent. In fact, observation of the excessive amount of the compound corresponding to the most abundant peak in Figure 1 served as a critical clue to the discovery that excessive leaching of this compound, identified as (2,4-di-tert-butylphenyl) phosphate, into the cell culture significantly interfered with the growth of mammalian cells (6). Analysis of only the water extract from this SUB would give an incorrect impression that this compound would not present a problem for the SUB.

Nontargeted Approach Creates Results

In the study, a total of four commercially available SUB types, presterilized with gamma radiation, were extracted using the conditions described above. In total, 53 organic compounds (Table 1) were detected and identified using a nontargeted approach with an array of analytical methods, including GC (Gas chromatography)/MS (Mass spectrometry), RP-HPLC/MS and RP-HPLC/UV. The combination of these orthogonal analytical methods detected a wide range of volatiles, semivolatiles, and nonvolatile compounds of various physicochemical properties.

Of the 53 compounds, 28 were identified by GC/MS, 31 by RP-HPLC/UV and 34 by RP-HPLC/MS using a combination of commercial and in-house databases and structure elucidation. The majority of the 53 extracted compounds were confirmed against reference standards. For extractables generated with 40% organoaqueous solvent, RP-HPLC/MS appeared to be a very powerful analytical technique, since a large portion of the extracted compounds were polar, hydrophilic-like, thus falling into the category of compounds best suited for RP-HPLC/MS analysis.

Overall, the majority of the identified extractables were degradation products of polymers and their additives (Table 1). For example, the most abundant compounds detected in the four SUBs were degradants of (2,4-di-tert-butylphenyl) phosphite, an antioxidant commonly added to polyolefins and other plastics. Also evident were degradants from other antioxidants such as butylated hydroxyl toluene, octadecyl (3-(3,5-di-tert.butyl-4-hydroxyphenyl)-propionate) or pentaerythritol tetrakis (3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate). Intact additives were also observed such as plasticizers (e.g., fatty acids, fatty acid esters and phthalates) and slip agents (e.g., fatty acid amides).

Inorganic elements, especially some heavy metals, can influence the stability, safety, and efficacy of drug products and cell culture. Using ICP (Inductively Coupled Plasma)/MS, the following elements were detected in the SUB water extract: boron, sodium, silicon, calcium and potassium. The estimated levels of these inorganic elements were in the range of ng/cm2 levels and consistent across all four tested SUBs.

Conclusion

Selection of appropriate extraction conditions and analytical approaches is critical for an extractables profile to be predictive of leachables produced in a specific application. The extraction condition described herein has proven to be effective for the profiling of organic extractables from SUBs. The profusion of extractable information obtained from this extraction protocol, along with the nontargeted analytical approach, indicates its great usefulness in the study of extractables from SUBs and other SUS materials used primarily for aqueous-based bioprocesses.

References

1. Gorter, A., et al. "Production of bi-specific monoclonal antibodies in a hollow-fibre bioreactor." Journal of Immunological Methods 161 (1993): 145-150
2. Paust, T. "Technology integration through disposable - from components to systems." Presented at the ISPE Nordic conference on disposables in biopharma. Stockholm, Sweden, 2006.
3. Jenke, D. "Evaluation of the chemical compatibility of plastic contact materials and pharmaceutical products; safety considerations related to extractables and leachables." Journal of Pharmaceutical Sciences 96 (2007): 2566-2581
4. Jenke, D "Linking extractables and leachables in container/closure applications." PDA Journal of Pharmaceutical Science and Technology 59 (2005): 265-281
5. Marghitoiu, L., Liu, J., Lee, H., Perez, L., Fujimori, K., Ronk, M., Hammond, M.R., Nunn, H., Lower, A., Rogers, G., and Nashed-Samuel, Y. "Extractables analysis of single-use flexible plastic biocontainers." PDA Journal of Pharmaceutical Science and Technology 69 (2015): 49-58
6. Hammond, M., et al. "A Cytotoxic Leachable Compound from Single-Use BioProcess Equipment that Causes Poor Cell Growth Performance." Biotechnology Progress 30 (2014): 332–337
7. ASTM F1980 – "Standard Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices," ASTM international.

About the Authors

Jian Liu is currently a scientist at Amgen working on extractables/leachables assessment and nonconformance investigations related to clinical and commercial products.

Hans Lee has been with Amgen since 2003 and is responsible for extractables/leachables activities related to assessing product contact of manufacturing/infusion/device equipment and bulk/primary containers.

Kiyoshi Fujimori has been working for Amgen for 11 years in studies regarding extractables and leachables.

Mike Ronk is an analytical chemist with 30 years of experience in the structural characterization of proteins, peptides and small molecule pharmaceuticals.

Matthew R. Hammond’s work in the Materials and Systems Analytics group at Amgen focuses on correlating raw material properties to process outcomes or product quality attributes, specializing primarily in plastic or polymeric materials.

Yasser Nashed-Samuel, PhD, is currently a principal scientist at Amgen in the Attribute Science, Process Development, Operation group.