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Two case studies illustrate a systematic approach.
To help maintain product safety, quality, and efficacy, a general approach to designing extractables and leachables studies should include evaluating and qualifying bioprocess materials used in a disposable manufacturing environment. This paper presents a systematic approach to the evaluation, identification, quantitation, and specification setting of extractables and leachables that builds product quality into the manufacturing process. It also discusses analytical thresholds of extractables for the evaluation of disposable materials.
The evaluation of extractables and leachables has become an increasingly important aspect in the Quality by Design (Qbd) initiative of FDA in the area of drug product design, including the selection of materials used in the drug product production process and the selection of container and closure systems used for product packaging to help maintain product safety, quality, and efficacy. This paper describes a general approach to designing extractables and leachables studies—based on the Product Quality Research Institute (PQRI) recommendations to FDA for Orally Inhaled and Nasal Drug Products (OINDP)—to evaluate and qualify the bioprocess materials used in a disposable manufacturing environment.1
In the method described here, disposable materials are extracted with solvents of various polarities, using various extraction techniques. The resulting respective extractions then are evaluated with various techniques, including liquid chromatography/mass spectrometry (LC/MS), gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography (HPLC), gas chromatography (GC), and inductively coupled plasma/mass spectrometry (ICP/MS). This paper also presents analytical thresholds of extractables for the evaluation of disposable materials. In addition, case studies are presented to exemplify the specific study designs and procedures that can be used to fully evaluate, characterize, and qualify certain specific disposable materials used in the bioprocess industry.
As pharmaceutical and biological technologies move into the twenty-first century, emerging global regulations are focusing on risk management and integrated quality systems.2–5 The FDA, the International Conference on Harmonization (ICH), and other regulatory bodies have joined forces to promote and facilitate discussions about and the implementation of risk management and integrated quality systems into pharmaceutical and biological manufacturing processes.6–8
The consideration and evaluation of extractables and leachables is a key aspect in the development of disposable bioprocess materials because these compounds, which can originate from the pharmaceutical container and closure packaging's processing and bulk storage supply systems, may adversely affect the safety or efficacy of the final drug product. Extractables are compounds that can be extracted from source materials using appropriate solvents under vigorous laboratory conditions. Leachables are compounds present in drug products caused by leaching from container, closure, or processing components. Therefore, leachables can be considered a subset of extractables.
The process begins with the judicious selection of manufacturing components, and with the proactive design of process qualifications and validation requirements for materials in a disposable bioprocess. The majority of disposable materials used in single-use bioprocesses are various types of plastics which tend to leach out foreign materials over time; therefore, a detailed extractables and leachables study that is designed from an analytical point of view is needed. This paper presents a roadmap to guide the manufacturing process. This roadmap begins with the selection of components in terms of the extractables and leachables (E/L) reports and documentation. Reports and documentation are required to support viable, valid, and qualified processes that meet current good manufacturing practice (cGMP) requirements for a disposable bioprocess.
In its component quality test matrices,9 the Bio-Process Systems Alliance's classification identifies consensus quality tests for each of the four most commonly used classes of disposable components:
Selecting the right material acceptable for use in a disposable bioprocess for components in any of these subclasses can eliminate most potential extractables and leachables because of the removal of materials that fail to satisfy such standards as listed in the US Pharmacopeia (USP) 27, monograph c88: Biological Reactivity Tests, In Vivo: Classification of Plastics, Class VI.
In addition to wisely selecting components in a disposable bioprocess, fully qualified or validated single-use systems are imperative. Core validations should be performed for all essential components in a single-use system, i.e., filters, bags, tubing, and connectors. A critical part of process qualification or validation involves data about the component's qualification for a complete sterilized system. The data comes from a complete extractables and leachables study design or experiment based on the actual manufacturing process, including specific filling (surface area:solvent ratio), storage time or temperature, and formulation components. Table 1 shows typical extractables for acceptable materials used in a disposable process.
Table 1. Typical extractables for acceptable materials used in disposable bioprocess manufacturing systems
Although PQRI's recommendations to the FDA are specific to orally inhaled and nasal drug products, the general approach presented therein can be applied to materials used in disposable bioprocess manufacturing systems. Figure 1 shows a step-by-step approach for performing extractables and leachables studies for materials used in disposable bioprocess manufacturing systems.
Figure 1. A step-by-step approach for performing extractables and leachables studies for materials used in disposable bioprocess manufacturing systems
Stage One: Compiling and Evaluating Information
Because studies of extractables and leachables (E/L) require a thorough understanding of the materials and the drug to construct an effective study plan, the first stage entails compiling and evaluating the following information:
1. Drug dosage form and formulation composition, including excipients and the active pharmaceutical ingredient.
2. Understanding the manufacturing process, including process conditions; evaluating which materials the drug formulation comes into contact with during the manufacturing process; and assessing the duration and types of interactions with these materials.
3. Understanding vendor-supplied disposable product components and the compositions of those materials; evaluating information from the manufacturers of these materials as to material matrix compositions; and assessing any extractables data that already may have been generated about these vendor-supplied materials.
The dosage regimen and the volume of product administered per unit time are the critical information required in calculating an analytical evaluation threshold (AET). The AET defines the required sensitivity of the analytical methodologies (instrumentation) for acceptably monitoring E/L at a specified safety threshold. PQRI defines a safety concern threshold (SCT) for OINDP of 0.15 µg leachable per day. (Note that the SCT for other delivery systems has not been specified by PQRI.) Therefore, based on the amount of product exposed to the patient per day, the leachable quantity per material can be calculated and the method sensitivity requirements in µg/mL of extracted solvent can be obtained based on the extraction study design.
Stage Two: Designing the Extraction Methods
The next stage involves designing the extraction methods for the applicable process and disposable product materials. Typically, at least three extraction solvents that bracket the polarity of the drug are chosen. For some studies it is advisable to include the drug vehicle or placebo, itself a fourth extraction solvent. Extraction conditions such as temperature, pressure, extraction time, and material-to-solvent ratios should be chosen to provide a thorough, complete extraction profile for the materials under evaluation, and to provide sufficient sensitivity per the analytical evaluation threshold (AET) calculations performed. The most common extraction technique is refluxing. Alternatives include Soxhlet extraction, which exposes the sample to less extreme temperatures and can be chosen for materials that may degrade under standard reflux conditions; and time-abbreviated techniques, such as the use of an accelerated solvent extractor, sonication, or extraction in a sealed container placed in an oven or autoclave.
As noted above, the calculated AET is based on the total daily intake (TDI) of the potential leachables or extractables in the drug product. Depending on the dosage regimen and administered amount, achieving the required instrument sensitivity may be a challenge. Sample extracts may also require treatment or modification to be appropriate for analysis using the specified analytical technique.
Stage Three: Designing the Sample Preparation
The third stage in constructing E/L studies is designing the sample preparation to achieve the AET, and the possible conversion of extracts to appropriate solvents for instrumental analysis. Liquid to liquid extraction is often used to make solvents more appropriate for GC analysis (volatiles and semivolatiles). Extracts may also be concentrated by evaporation for improved sensitivity, or, for solvent exchange, dried and reconstituted in an appropriate solvent for the analysis.
Stage Four: Analyzing Extracts, and Evaluating and Compiling Data
The next stage involves analyzing material solvent extracts, and evaluating and compiling data to identify extractable compounds. Multiple analytical techniques are typically used to fully profile the material extract solutions. Headspace GC/MS is used to analyze the extracts for volatile compounds, direct liquid injection GC/MS for semivolatiles, and LC/MS for nonvolatiles (typically by both atmospheric pressure chemical ionization [APCI] positive and negative modes of detection). If applicable, metals screening is performed by ICP/MS. Compounds of special concern to the FDA because of their carcinogenicity, such as formaldehydes, polyaromatic hydrocarbons, mercaptobenzothiozole, and nitrosamines, are analyzed using specialized instrumentation and detection, sample derivatization, or other techniques. The headspace and direct injection GC/MS extractables data are evaluated by a library compound software search, and these data, as well as the LC/MS spectral pattern data, are reviewed and evaluated for tentative compound identification. Tentative peak confirmations and semi-quantitative amounts typically are confirmed by obtaining commercial standards of the known extractable compounds (if available), and by injecting these compounds to confirm retention times and spectral patterns.
A toxicological evaluation of extractables data is critical in determining which compounds need to be tracked as potential leachables in the drug product. Typically, TDI values calculated for the confirmed, known extractable compounds are provided by the analytical laboratory to the board-certified reviewing toxicologist. The toxicologist evaluates the compounds and amounts observed for toxicological concern or structure alert. The resulting information, along with knowledge of the intended use of the product, is applied when deciding which of the identified extractable compounds should be tracked as potential leachables in the drug product, and what extractable compounds should be tracked in routine methods for raw material quality control screening (quality control release methods for vendor-supplied materials).
Methods are developed next for monitoring leachables. The methods are selective for identified leachables and for expected, unidentified leachables. The methods separate these peaks from drug product or process impurity chromatographic peaks. The leachable methods are developed to provide sufficient sensitivity, accuracy, precision, and linearity across a suitable range, to quantitatively determine the amount of leachable components present. Because these procedures are for routine, quality control (QC) use, they typically use high-performance liquid chromatography/ultraviolet visible (HPLC/UV) and gas chromatography/flame ionization detector (GC/FID) techniques, as opposed to mass spectrometric detection.
At this stage, it is also advisable to develop methods for monitoring extractables in incoming vendor raw material batches before these are used in the build process for a final product. To develop the most efficient process, the results of the controlled extraction studies should be used to optimize the routine extraction procedures applied to the incoming materials testing.
Disposal bags intended for bioprocessing must be qualified for extractables and leachables before use to support manufacturing process qualification and validation. Data collected for the respective materials also can be used to compile a knowledge base and enhance scientific understanding which, in preparation for a future paradigm shift into quality by design per ICH Q8 (pharmaceutical development), will support ICH Q9 (quality risk management) and ICH Q10 (pharmaceutical quality systems). The entire extractables and leachables qualification for a disposable bag is described here for a one-liter solution disposable bag with a two-doses-per-day regimen.
Step 1: Information Gathering (Component selection and final formulation determination). An appropriate bag should be chosen for the intended storage and use of the disposable drug product. The properties of the material the bag is made of (such as pH tolerance, thermal resistance, tensile strength, and other related chemical and physical properties), and the processes and additives used in the bag-making process, including how the plastics polymer is made, are crucial information to gather.
The final formulation and the bioprocess conditions (holding time, temperature, pH, etc.) are gathered when they are determined. For example: the drug placebo is a saline solution; the bags hold 1 L of the drug solution for at most 3 months; the bags are processed at 35 °C; and the dosage for the final drug product is 1 mL/dose and 2 doses/day.
Step 2: Calculation of AET. Based on information collected in Step 1, the AET can be calculated for each extractable per bag (the SCT of 0.15 µg/day is being used per PQRI recommendation for OINDP; however, the value could be different for other dosage forms):
Extractables above the AET should be evaluated, but extractables below the AET are of no significant safety or toxicity concern.
Step 3: Design Extraction Conditions. Extraction solvents should include a placebo buffer, a buffer with a lower pH (such as pH 3), and a buffer with a higher pH (such as pH 9). In addition, an organic solvent (such as methanol, ethanol, or isopropyl alcohol) should be used to cover a worst-case scenario. For analysis of metals, a dilute acidic solution should be used.
The extraction temperature and time equal 50 °C for 24 hours. The extraction technique is as follows: for the aqueous extraction, a sealed container is used; for the methanol extraction, reflux is used. For the material/solvent ratio, the important consideration is the AET. For example, if one bag is extracted with 500 mL of solvent, the AET for the extraction solution is 75 µg/500 mL = 0.15 µg/mL, which is within the analytical detection and quantitation limit of the GC/MS and LC/MS techniques.
Extraction solutions should be analyzed with headspace GC/MS, direct injection GC/MS, and LC/MS (APCI in both positive and negative modes), as well as with ICP/MS to screen trace metals extractables. Aqueous extractions should be converted to suitable solvents, such as methylene chloride and hexane for the direct injection GC/MS analysis. The amounts of extractables are quantified with appropriate standards.
Step 4: Extractables Evaluation. The analytical evaluation should include extractables identity, amount, and potential TDI, as well as a toxicological evaluation. These evaluations can qualify or disqualify the bag, based on extractables or leachables, from its intended use as a bioprocess disposable bag.
Step 5: Leachables Method Development and Validation. Based on the extractables profile and the profile evaluation, potential leachables are determined and one or more methods for analyzing the potential leachables are developed. These methods must separate the potential leachables from drug products such as the active pharmaceutical ingredient (API), additives, and degradants. The methods are validated per ICH guidelines in terms of method selectivity and specificity, accuracy, precision, linearity, range, and sensitivity.
Step 6: Leachables Analysis of Stability Samples. Leachables are monitored in at least three lots of stability samples at time points of initial, 1, 3, 6, and 12 months, if needed.
Step 7: Leachables Trending, and Extractables and Leachables Correlation Analysis. At the end of the leachables test program, a trending analysis of the leachables should be performed. Additionally, a correlation analysis of the leachables amount and the extractables amount should be demonstrated. If the leachables are more than the extractables, the extractables must be revisited.
Step 8: Routine Extractables Method Development and Validation. Based on the extractables profiles, routine extractables methods, which are usually GC/FID or HPLC/UV, are developed and validated for purposes such as sample preparation. The routine extractables methods should be robust, efficient, exhaustive, and simple to operate.
Step 9: Bag Release for Use Through Routine Extractables Testing. Different lots of incoming bags should be tested before they are filled with drug solutions. This will ensure that the bags meet the criteria for potential extractables, and that the bags are suitable for their intended use.
Like disposable bags, the tubing used in the bioprocess also requires qualification for extractables and leachables. However, because the tubing has a short contact time with the drug product formulation during the manufacturing process, leachables can be evaluated in parallel with a controlled extraction study, as a short-term compatibility study with drug formulation. An E/L study design is presented here.
Step 1: Information Gathering. Information regarding the tubing should be collected in the same manner as for the bag in case study 1. In the present case study, the drug placebo is an aqueous solution, the batch size is 10 liters, and the dosage for the final drug product is a one-milliliter dose at three doses per day.
Step 2: Calculation of Analytical Evaluation Threshold. Based on information collected in Step 1, the AET can be calculated for each extractable per tubing (the SCT of 0.15 µg/day is used here per PQRI recommendation for OINDP; however, the value could differ for other dosage forms.):
Extractables above the AET should be evaluated, but extractables below the AET are of no significant safety or toxicity concern.
Step 3: Design Combined Extractables and Leachables Study. For extraction solvents, because the tubing has a relatively short contact time with the drug solution, only a placebo buffer and the product formulation are needed, plus an organic solvent, such as 20% ethanol, to cover a worst-case scenario. For analysis of metals, a dilute acidic solution is used.
The extraction temperature is 50 °C for the placebo buffer and the 20% ethanol; the extraction time for both is 24 hours. Ambient conditions for the drug product formulation are for 1, 4, 8, and 24 hours (to obtain short-term compatibility and leachables information).
The remaining procedures for Step 3 are the same as those listed in case study 1.
Step 4: Extractables Evaluation. The analytical evaluation should include extractables and short-term leachables identity, amount, and potential TDI, as well as a toxicological evaluation. These evaluations can qualify or disqualify the tubing, based on extractables or leachables, from its intended use as bioprocess disposable tubing.
Step 5: Leachables Method Development and Validation. Leachables method development and validation may not be necessary because the tubing has a short contact time with the drug product formulation during the manufacturing process.
Step 6: Leachables Analysis of Stability Samples. Leachables analysis of long-term stability samples for tubing is not necessary. On the other hand, short-term tubing leachables information should be acquired and evaluated in parallel with extractables as described in Step 3.
Step 7: Leachables Trending, and Extractables and Leachables Correlation Analysis. Data acquired from Step 3 can be used for a leachables trending analysis and for the correlation of extractables and leachables.
The remaining steps (8 and 9) are the same as described in case study 1.
E/L studies should be designed to achieve their intended purposes as demonstrated in case studies 1 and 2 for bags and tubing used in a disposable bioprocess. The data obtained through E/L studies designed for different materials and manufacturing processes can be compiled into a well-organized system, such as a library system, to increase scientific understanding and the knowledge base for future use.
All materials chosen for use in a disposable (single-use) bioprocess must pass the specifications in USP 27, monograph c88: Biological Reactivity Tests, In Vivo: Classification of Plastics, Class VI. If the disposable materials require a very long holding time, those materials may need to meet the requirements set forth in ISO 10993–1:2003, Biological Evaluation of Medical Devices Part 1: Evaluation and Testing or in the FDA Blue Book memorandum, Required Biocompatibility Training and Toxicology Profiles for Evaluation of Medical Devices. In addition, materials used in a disposable bioprocess should be fully evaluated in terms of extractables profiles and potential leachables. Routine QC extractables methods should be developed and validated to qualify incoming material components of the disposable bioprocess.
Extractables and leachables studies are only one of many aspects of qualifying materials. Other aspects such as impurity, potency, and mechanical properties, etc., should also be evaluated to ensure that materials used in a disposable bioprocess are compatible with drug product formulations and manufacturing process parameters.
Product quality control can be achieved through well-designed process development, evaluation, and characterization, which begin with the selection of materials used in a manufacturing process. In this article, a roadmap has been presented for a comprehensive extractables and leachables evaluation to establish the suitability of materials used in a disposable bioprocess. Different analytical technologies used to support extractables and leachables studies have their own capabilities and limitations. Depending on the materials used in the disposable bioprocess, specific requirements are needed for the E/L study designs to support the manufacturing process qualification and validation from an E/L perspective. Furthermore, disposable containers designed for low-temperature storage, holding, and processing such as plastic bags made from ultra-low temperature film used in the freeze-and-thaw cycling process-can be evaluated, characterized, and quantified, as demonstrated in the case studies.
New global regulations such as ICH Q8, Q9, and Q10 are steering the pharmaceutical and biopharmaceutical industries to move toward the new paradigm of QbD. The study design approach presented here is based on the importance of a scientific understanding of compounds that could be extracted or leached from disposable materials that make contact with a drug product during manufacturing and product use; the approach is, thus, a means of incorporating quality by design into a disposable biopharmaceutical manufacturing process.
The authors wish to acknowledge the support of their employer, PPD, Inc., and to acknowledge, in particular, Magdalena Mejillano, Tom Rosanske, Donald F. DeCou, Bert Kittner, Louise Caudle, and Joel Galang for their productive input.
Xiaochun Yu is a senior research scientist, Derek Wood is a laboratory manager, and Xiaoya Ding is the director of scientific and technical affairs, all at the cGMP laboratory, PPD, Inc., Wilmington, NC, 910.558.7585, firstname.lastname@example.org.
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