Alternative Solvents for Extractables and Leachables Evaluation

Extractables and leachables evaluation of packaging components and components of bioprocessing systems is a crucial regulatory requirement. Solvents used for evaluation of process components may include surfactants that can interfere with chromatographic detection and contaminate the chromatographic system. The authors examine alternative solvents that provide extraction equivalence and do not interfere chromatographically.

Since the FDA Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics was issued in May of 1999 (1), extractables and leachables evaluation of final packaging components has become an increasing priority of FDA. The regulation on equipment construction (applicable to bioprocessing system components) as per 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). Every new drug application is expected to include some form of extractables profiling and leachables evaluation for the components at highest risk and in closest contact with the drug. In addition to final container-closure systems, components associated with the bioprocessing system of biologics are considered at risk for leachables. Shelf life, storage temperature, and conditions of real-time use of a component under evaluation with the process stream, drug substance, or a final drug product are all key factors in designing appropriate extractables studies, simulation studies, and ultimately leachables studies (3).

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To demonstrate that a container-closure system or a bioprocessing component is suitable for its intended use, the components typically undergo an initial extractables screening. The design of the extraction experiment should appropriately exaggerate the conditions of real-time use without breaking down or degrading the polymeric component under testing. Although strong polar and strong nonpolar solvents, such as 100% isopropanol (IPA) and 100% hexanes, are commonly used for highly aggressive reflux or soxhlet extractions of final container-closure systems that typically contain the final drug product for extended periods of time, these solvents are not always appropriate for the components of bioprocessing systems, such as bioprocess bags, filters, tubing, O-rings, diaphragms, and gaskets (3, 4).

For bioprocessing components, initial extractables screening involves filling or immersing the component in a variety of model solvents that more closely represent the formulation and exaggerate the conditions of real-time use. The components are incubated in the solvent for a predetermined length of time at an elevated temperature, such as 40–60 °C for several days, weeks, or even months. This type of extraction is recommended over an aggressive reflux extraction because exposure of the formulation to bioprocessing components is usually very short, and the temperatures of real-time use are typically at or below 25 °C. Reflux extraction of bioprocessing components using strong polar or nonpolar solvents is commonly not recommended unless the real-time exposure of the formulation to the component is long or at temperatures greater than 25 °C and the component is compatible with the solvent (3, 4).

Many extractables studies have instead used model solvents that are comprised of the same excipients that are present in the process buffers, drug substances, or final drug-product formulations. These excipients often include surfactants, which are common ingredients in the formulations of biologics and are regarded as essential components of the model solvents to be used to generate extractables profiles. Surfactants, however, pose major chromatographic interferences when screening for nonvolatile organic compounds by high-performance liquid chromatography–mass spectrometry (HPLC–MS). The detection of extractable compounds may be masked by co-eluting surfactant peaks. In addition, high concentrations of these surfactants are problematic, as they can contaminate the HPLC–MS system. Dilution is not always a viable solution because sensitivity can be greatly affected. Therefore, alternative solvents that provide extraction equivalence and do not interfere chromatographically were examined.

Materials and methods

Polytetrafluoroethylene (PTFE)-lined polypropylene (PP) caps were extracted with 25 mL of each of the following surfactants: 1% nonionic, octylphenol ethoxylate surfactant (Triton X-100, Dow Chemical); 0.1% polysorbate 80 (PS 80); and 0.1% polysorbate 20 (PS 20). The same caps were also extracted with the following two alternative solvents: 60% IPA and 15% ethylene glycol monobutyl ether (EGMB). The caps were submerged in each solvent at 40 °C at ambient relative humidity for seven days. The resulting extracts were tested by gradient HPLC using a time-of-flight (TOF) LC–MS (Agilent 6500 series) equipped with a multimode source (electrospray and atmospheric pressure chemical ionization) using positive ionization. Data were acquired using scan mode with a range of 80 to 1500 m/z and then by extracting ions that corresponded to the compounds of interest.

The PP caps were chosen for this experiment due to the presence of known additives that could be easily tracked during extractables screening. Compounds previously observed through extractables studies that were targeted in this experiment include a di-tert-butyl(phenyl)phosphite (Irgafos 168, BASF); a phosphate oxidative degradant of Irgafos 168; ethylene bis(heptadecanamide); pentaerythritol tetrakis 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate (Irganox 1010, BASF); and erucamide. Determination of extraction efficiency equivalence was made by comparing the responses for each of the targeted extractables observed in each solvent.

Results and discussion

Figure 1a presents a visual representation overlay of LC–MS total ion chromatograms (TIC) of all the solvents used for the extraction study. The total ion chromatograms of the 60% IPA and 15% EGMB are similar; therefore, they cannot be distinguished in the figure but are shown as the gray/red line. Figure 1b presents a visual representation of just the overlay of 0.1% PS 80 and 60% IPA to show that any potential extractables would be masked by the PS 80 interference. Interferences are also observed with 0.1% PS 20 and 1% Triton X-100 as shown in Figure 1.

Figure 1: Liquid chromatography–mass spectometry (LC–MS) time-of-flight (TOF) multimode positive total ion chromatograms (visual representation). (a): 60% isopropanol (IPA) and 15% ethylene glycol monobutyl ether (EGMB) (gray/red), 0.1% polysorbate (PS) 20 (green), 0.1% PS 80 (blue), and 1% octylphenol ethoxylate surfactant (Triton X-100, Dow Chemical) (purple). (b): 0.1% PS 80 (blue) and 60% IPA (gray). (ALL FIGURES ARE COURTESY OF THE AUTHORS)

Figure 2 presents the concentration results in µg/mL of each extractable compound detected versus the type of solvent. Concentrations were estimated based upon the average of the responses of the reserpine system suitability standards. As Figure 2 indicates, not all of the compounds of interest were extracted in each of the solvents. Irgafos 168 and Irganox 1010 were extracted in both 60% IPA and 15% EGMB while ethylene bis(heptadecanamide) was only extracted in the 60% IPA solvent. Irgafos 168, Irganox 1010, and ethylene bis(heptadecanamide) were not extracted in the 0.1% PS 20, 0.1% PS 80, and 1% Triton X-100 solvents. Erucamide was extracted in all solvents. Irgafos 168 phosphate results were not presented because concentrations were similar to the blank concentrations.

Figure 2: Results for the seven-day extraction study on polypropylene (PP) caps showing extractables of common PP additives using five different types of extraction solvents; IPA is isopropanol, EGMB is ethylene glycol monobutyl ether, Triton X-100 (Dow Chemical) is a nonionic octlyphenol ethoxylate surfactant, Irgafos 168 (BASF) is di-tert-butyl(phenyl) phosphite, Irganox 1010 (BASF) is 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate.

Based on the study results, 60% IPA was shown to be the worst-case model solvent for the extraction study because it extracted all of the compounds of interest except for Irgafos 168 phosphate. Not only did the 60% IPA extract the same compounds as the surfactants, it also extracted additional compounds that the surfactants did not extract. These results were comparable to findings from a related study (5) performed in 2011 in which two types of bioprocessing bag films were submerged for seven days at 40 °C/ambient relative humidity in various types and strengths of solvents, as shown in Figure 3. Compounds that were targeted in the related study included bis(2,4-di-tert-butyl)hydrogen phosphate, erucamide, palmitamide, stearamide, and Irgafos 168 phosphate. The study also showed that 60% IPA had a greater extraction efficiency compared to the other solvents evaluated including 1% PS 20. Due to carryover issues associated with the 1% PS solutions, 0.1% PS solutions were used to perform the study using PP caps.

Figure 3: Comparison of extractables of common polymer additives using 60% isopropanol (IPA) and 1% polysorbate 20 extraction solvents in two types of polymeric bioprocessing bags; DTBHP is bis(2,4-di-tert-butyl) hydrogen phosphate, Irgafos 168 phosphate is a common degradant of di-tert-butyl(phenyl) phosphite (Irgafos 168, BASF).

In addition to having greater extraction efficiency, as demonstrated in two separate studies using two different types of material, 60% IPA was also shown to eliminate interferences observed in the sample chromatography of surfactants. The use of extraction solvents that do not pose significant chromatographic interferences is critical so that potential extractable compounds are not missed during the extractables screening. In this case, erucamide was extracted in all of the solvents and was tracked using extracted ion analysis based upon the total ion chromatogram of the 60% IPA solvent. To perform extracted ion analysis, the ion of interest must be known. If only the total ion chromatograms of the surfactant solvents were used to screen for potential extractable compounds, and IPA was not used as one of the extraction solvents, erucamide would have been missed in the chromatograms of the surfactants. Erucamide elutes at approximately 7.7 minutes in Figure 1.

Conclusion

When comparing extraction efficiency between the various solvents for extractable screening studies, it is recommended that IPA be used as a worst-case solvent in cases for which surfactants are of interest. In addition, the ions from the mass spectra of compounds detected in the IPA extraction solvent can then be used to perform extracted ion analysis on the extracts that contain surfactants. Use of an alternate solvent such as IPA ensures that potential extractable compounds are not missed in the surfactant extractions during initial extractables screening.

Jennifer M. Roark, Mai N. Jacques, PhD, Erica J. Tullo, PhD, Andrew T. Blakinger, and Thomas C. Lehman*, PhD are all in the Method Development & Validation group at Eurofins Lancaster Laboratories, 2425 New Holland Pike, Lancaster, PA 17601, USA, 1.717.656.2300, www.lancasterlabspharm.com.

*To whom all correspondence should be addressed.

References

1. FDA, Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics (Rockville, MD, May 1999).

2. CFR Title 21, Part 211.65 (Government Printing Office, Washington, DC, 2006).

3. Bio-Process Systems Alliance, Recommendations for Testing and Evaluation of Extractables from Single-Use Process Equipment, (Washington, DC, 2010).

4. Bio-Process Systems Alliance, BioProcess Intl. 5 (11) 36-49 (2007).

5. T. C. Lehman, "Evaluation of Model Solvents for Generating Extractable Compound Profiles from Single-Use Systems," presentation at AAPS Annual Meeting and Exposition (Washington DC, 2011).

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