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

A systematic approach facilitates formulation component selection.


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.

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.

Table 1. Aqueous formulations used for leachables evaluations
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 2. Headspace GC–MS chromatographic conditions
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 3. 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 4. LC–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.