A Study of Leachables for Biopharmaceutical Formulations Stored in Rubber-Stoppered Glass Vials

ABSTRACT

This article describes a systematic approach to evaluating the leachables profile from various biopharmaceutical formulations stored in rubber-stoppered glass vials. We evaluate leachables from rubber stoppers in 11 different formulations containing typical biopharmaceutical excipients to assess how differences in various formulation excipients affect leachables. The information obtained from this type of study is critical to support the Quality by Design paradigm of incorporating product quality into the product design process and using risk-based approaches to managing quality.

Biopharmaceutical packaging materials (container/closure systems) must be evaluated for compatibility with the drug formulation in the early stages of development to ensure the product is safe for use throughout its shelf life. This requirement also applies to the development of biopharmaceutical manufacturing processes including single-use components used in manufacturing equipment and in-process production conditions.¹ Although compendial tests provide some preliminary extractables information about the container/closure systems, they do not identify or quantitate individual extractables. Therefore, they cannot correlate extractables from the container/closure systems to the leachables in the drug products. This article describes a systematic approach to determining extractables and leachables based on the concepts proposed by the Product Quality Research Institute's Leachables and Extractables Working Group for the development of orally inhaled and nasal drug products in concert with relevant regulatory guidelines.^2–4 The study design focuses on identifying and elucidating the correlation between leachables caused by biopharmaceutical formulations stored in rubber-stoppered glass vials and its potential application in supporting Quality by Design (QbD) in the biopharmaceutical development process.

(PPD, INC.)

Glass vials and rubber stoppers are widely used as container/closure systems for biopharmaceutical products and other drug formulations. Organic compounds in the stoppers, such as oligomers, antioxidants, and curing agents, can leach out into drug formulations and affect drug safety and efficacy. In addition, typical biopharmaceutical drug product formulation ingredients, such as co-solvents, surfactants, chelating agents, bulking agents, and pH modifiers, can alter the physicochemical properties of the drug formulation itself and also may have an impact on the leaching of organic compounds from rubber stoppers. This paper evaluates leachables from rubber stoppers in 11 different formulations containing typical biopharmaceutical excipients, and addresses how differences in various formulation excipients affect leachables.

In this study, typical biopharmaceutical formulation components were chosen to create a variety of test formulations. The extractables profiles of chlorobutyl rubber stoppers were determined. The major extractables were then chosen as targets for a leachable assessment in which the test formulations were stored at 40 °C and 75% relative humidity (RH) for one month. The following formulation variations were evaluated: pH (5.0, 6.8, 8.2), chelating agent (0.1 to 0.5 mM EDTA), surfactant concentration (0.1–0.5% Tween 80), bulking agent (sucrose, mannitol, or trehalose), and the presence of a co-solvent (2% glycerol).

Table 1. Aqueous formulations used for leachables evaluations

STUDY DESIGN

Eleven aqueous placebo formulations were evaluated in this study (Table 1). The various test formulations included a simple phosphate buffer at pH 6.8, a buffer with a glycerol co-solvent, three formulations in which the pH was varied from slightly acidic to slightly alkaline, two formulations with different amounts of ethylenediaminetetraacetic acid (EDTA) included as a chelating agent, two formulations with different amounts of Tween 80 included as a surfactant, and two formulations with either mannitol or trehalose added as bulking agents. Commercially available glass vials and rubber stoppers were used in the study.

Table 2. Headspace GC–MS chromatographic conditions

Separate glass vials were filled with one of the 11 formulations listed in Table 1 and crimp-sealed with rubber stoppers. The stoppered vials containing various formulations were inverted and then stored at 40 °C and 75% RH. After one month, the formulations were tested for volatile, semi-volatile, and nonvolatile leachables using headspace gas chromatography–mass spectrometry (GC–MS), direct injection GC–MS, and gas chromatography–ultra violet mass spectrometry LC–UV–MS, respectively. The portions of each sample to be analyzed were transferred to headspace GC vials and high performance liquid chromatography (HPLC) vials without further treatment for headspace GC–MS and LC–UV–MS analysis, respectively. For direct injection GC–MS analysis, the samples were back extracted into methylene chloride, which is a more suitable solvent for this analysis. Control samples (portions of each formulation that were stored in glass volumetric flasks at 5 °C for the same time duration as the samples) also were analyzed.

Table 3. GC–MS chromatographic conditions

To correlate the leachables with the extractables from the stoppers, the stoppers were extracted with water and isopropanol and analyzed with the same headspace GC–MS, direct injection GC–MS, and LC–UV–MS conditions. For headspace GC–MS analysis, the stoppers were extracted in headspace GC vials at 90 °C for 24 h, and the vials were directly used for volatile extractables analysis, without opening the vial cap. All volatile extractables, including water insoluble extractables, were detectable using this approach. For direct injection GC–MS and LC–MS analyses, the stoppers were extracted by refluxing with water and isopropanol for 16 h. Tables 2–4 show the chromatographic conditions used for each analysis.

Table 4. LC–MS chromatographic conditions

RESULTS AND DISCUSSION

Headspace GC–MS Analysis Extractables Evaluation

The water extracts of stoppers yielded seven extractables peaks (Figure 1). Six of the seven peaks were identified as 2-methylpentane (4.24 min), 3-methylpentane (4.62 min), hexane (5.14 min), methylcyclopentane (6.24 min), cyclohexane (7.93 min), and butylated hydroxytoluene (BHT, 33.26 min). The peak at 28.60 min was not identified; the GC–MS library search and manual spectral interpretation did not produce a good match or a tentative identification. The amounts for each peak are summarized in Table 5. The peak at 28.60 min was quantitated using cyclohexane as the surrogate standard. Because the relative response factor of the unknown peak against cyclohexane is not determined, the amount reported for this peak is only considered a semi-quantitative estimate.

Table 5. Quantitation of extractables/leachables with headspace GC–MS

Leachables Evaluation

Six leachables peaks were observed in the headspace GC–MS analysis from the formulations: 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, cyclohexane, and BHT. These leachables peaks correlate to stopper extractables. The unknown extractables at 28.60 min were not observed as leachables. This may be because of insolubility of the compound in the aqueous media. The amount of leachables in different formulations is summarized in Table 5. The effect of various formulation ingredients on the leachables profile is discussed in detail below.

Figure 1

Phosphate Buffer with or without Glycerol Co-Solvent

Five leachables peaks were observed in the pH 6.8 phosphate buffer: 3 methylpentane, hexane, methylcyclopentane, cyclohexane, and BHT. All of the peaks were very small. Methylcyclopentane was the largest leachables peak, probably because it is more soluble in aqueous media than the other compounds. The addition of 2% glycerol in the phosphate buffer as a co-solvent did not significantly affect the amount of leachables observed.

Formulation pH

Formulation pH had an impact on leachables. The neutral pH (pH 6.8) provided slightly lower amounts of leachables compared to the slightly acidic (pH 5.0) and the slightly alkaline (pH 8.2) formulations (Figure 2). The acidic and alkaline formulations had similar leachable profiles.

Figure 2

EDTA Chelating Agent

Based on the leachables profiles of formulations containing 0, 0.1, and 0.5 mM EDTA, EDTA did not significantly affect the amount of organic leachables.

Tween 80

The excipient Tween 80 had the most significant effect on leachables compared to other formulation ingredients. The addition of Tween 80 significantly increased the leached amount of 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, and cyclohexane. The leached amounts of these compounds in the formulation with 0.5% Tween 80 is five to 10 times higher than in formulations without Tween 80. This is because the surfactant increases the solubility of these compounds in the aqueous formulation (Figure 3). However, the addition of Tween 80 did not affect the amount of leached BHT because no BHT was detected in the formulations with 0.5% or 0.1% Tween 80.

Figure 3

Bulking Agents: Sucrose, Mannitol, and Trehalose

The use of bulking agents affected the amount of leachables observed. Higher amounts of leachables were observed in the formulation with 7% mannitol compared to the formulation with 7% trehalose, in which few leachables were observed (Figure 4). The formulation with 7% sucrose had leachables levels between those seen in formulations with mannitol and trehalose.

Figure 4

Direct Injection GC–MS Analysis Extractables

There was only one semivolatile extractable peak observed from the water extracts. The peak was identified as BHT and is very small because of its limited solubility in water.

There were many extractables peaks observed in the isopropanol extracts (Figure 5). The two dominant peaks are BHT (12.18 min) and an unknown hydrocarbon at 13.66 min. Other compounds observed were palmitic acid (15.35 min) and stearic acid (16.62 min). All other peaks appear to be various hydrocarbons.

Figure 5

Leachables

Leachables were determined by comparing the stressed formulations to the formulation blanks and the stopper extractables profile. There is no appreciable interference from the formulation blanks for the main extractables peaks. The only leachables observed was BHT, which was reported in formulation 3 (pH 5.0), formulation 6 (0.1 mM EDTA), and formulation 7 (0.5 mM EDTA). This result was consistent with headspace GC–MS data. The BHT peak is very small. No other peaks observed in the isopropanol extract were observed in any of the formulations. This can be explained by the insolubility of these compounds in the aqueous formulations.

There were two additional peaks observed in formulation 1 (pH 6.8 phosphate buffer) and formulation 2 (phosphate buffer with 2% glycerol). The first elutes at 11.27 min and the second elutes at 12.11 min. The peak at 12.11 min is tentatively identified as 2,4-t-butylphenol, while the peak at 11.27 min was not identified. For all other formulations, the peak at 11.27 min was not observed. For the peak at 12.11 min, there is a co-eluting interference peak from all the formulation blanks except formulations 1 and 2, so it is not conclusive if the peak observed in formulations 1 and 2 was also present in those formulations. The source of the two peaks requires further evaluation.

Figure 6

LC–UV–MS Analysis

No extractables were observed with LC–MS from the water extracts of rubber stoppers. The isopropanol extract of the stoppers contained BHT (5.02 min) and three fatty acids (myristic acid, 5.21 min; palmitic acid, 5.80 min; and stearic acid, 6.30 min).

The leachables were evaluated by comparing the stability samples to the formulation blanks and the stopper extractables profile. There is no appreciable interference from the formulation blanks for the main extractables peaks. There are no leachables observed in the LC–MS analysis of the 11 formulations. The observation of BHT in the GC–MS analysis but not in the LC–MS analysis is likely because of the increased sensitivity of GC–MS over LC–MS.

CONCLUSION

Eleven different typical formulations of biopharmaceutical products were evaluated to determine their effect on the leachables profile of a common rubber stopper after storage at 40 °C, 75% RH for one month. The key findings are as follows:

• Different formulation excipients have a significant impact on leachables. Especially of note is Tween 80, which significantly increased the amount of leachables as compared to other formulations.

• The impact of different formulation excipients does not always affect all leachable components to the same extent. For example, Tween 80 significantly increased the leachables 2-methylpentane, 3-methylpentane, hexane, methylcyclopentane, and cyclohexane. However, it does not appear to have an impact on BHT. The bulking agents used, such as sucrose, mannitol, and trehalose, lead to different leachables levels, with trehalose being a superior bulking agent from the standpoint of minimizing leachables.

• The volatility of the extractables in the stopper material and the solubility of those compounds in the formulations are the determining factors for leachables. All leachables observed in this study were volatile compounds and observed in headspace GC–MS analysis. Volatile compounds migrate more easily than nonvolatile compounds and are more likely to leach out into formulations. The relative solubility of leachables in formulation is directly proportional to the amount that will be present in the formulation as a leachable from the stopper material.

• This study demonstrates that biopharmaceutical excipients, formulation storage conditions, and to some extent the container/closure material, can have a significant impact on the success of a potential commercial formulation.

A well-designed study can help a biopharmaceutical company build quality and efficiency into its biopharmaceutical product design and in-process development and commercialization. This approach provides a knowledge base for a better selection of product formulation components, with respect to leachables.

Scientific understanding based on data obtained from this particular study can be used to support other pharmaceutical product development processes. For instance, for different rubber stopper materials or other types of container/closure materials, the potential leachables profile can be predicted based on its extractables profiles and the intended formulation and excipients regime with greater accuracy for the model systems studied. The knowledge and understanding accumulated will in turn aid the product development process and make it possible to develop safer and more efficacious medicines faster.

Based on our study, changes in the components in a biopharmaceutical formulation can lead to appreciable changes in the leachable profiles. A thorough understanding of how various formulation components may affect a leachables profile can aid in selecting appropriate formulation constituents based on the container/closure materials and storage conditions. Such an understanding overcomes the limitations of a traditional selection process and allows the early adoption of a risk-based approach and QbD at the beginning of product development. Consequently, drug development duration can be shortened significantly while product quality, safety, and efficacy are maintained.

Xiaochun Yu, PhD, is a senior research scientist, Don DeCou, PhD, is a senior research scientist, Derek Wood is a laboratory manager, Steven Zdravkovic is a senior scientist, Heather Schmidt is an associate research scientist, Laurie Stockmeier is a scientist, Robert Piccoli, PhD, is an associate research scientist, Daniel Rude is a research scientist, and Xiaoya Ding, PhD, is the director of scientific and technical affairs, all at the cGMP laboratory, PPD, Inc., Wilmington, NC, 910.558.7585, xiaoya.ding@ppdi.com

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