Extractables and Leachables Analysis for Single-Use Consumables: Technical Principles, Methods, and

Extractables and leachables (E&L) analysis is a critical quality and safety assessment for single-use consumables used in biopharmaceutical manufacturing, ensuring that polymeric materials such as films, tubing, and filters do not release harmful compounds into drug products. This technical guide provides a comprehensive overview of extractables testing samples, analytical items, validated methods, and regulatory applications for single-use systems. By understanding E&L study design and interpretation, manufacturers can accelerate process validation and satisfy regulatory expectations from the FDA, EMA, USP, and ICH Q3E.

Technical Overview

Single-use consumables—including bioprocess bags, transfer tubing, sterilizing-grade filters, connectors, and sampling systems—have become foundational to modern biopharmaceutical manufacturing. These components are constructed from polymeric materials such as polyethylene (PE), ethylene-vinyl acetate (EVA), silicone, thermoplastic elastomers (TPE), polypropylene (PP), polyethersulfone (PES), and polyvinylidene fluoride (PVDF). During product contact, these materials have the potential to release compounds originating from polymer additives, residual monomers and oligomers, curing agents, adhesives, and degradation products.

Extractables are compounds that can be released from a material of construction under exaggerated laboratory conditions, such as aggressive solvents, elevated temperature, and extended contact time. Leachables are the subset of compounds that actually migrate into the drug product under normal processing, storage, and use conditions. Extractables studies are used to characterize the worst-case chemical profile of a consumable, while leachables studies confirm which of those compounds appear in the final product at measurable levels.

Extractables and leachables analysis for single-use consumables is driven by a well-established regulatory and pharmacopeial framework:

• USP General Chapter <1663> — Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems
• USP General Chapter <1664> — Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems
• USP General Chapter <1665> — Plastic Components and Systems Used in Pharmaceutical Manufacturing
• BioPhorum (formerly BPOG) standardized extractables testing protocol for single-use systems
• ICH Q3E guideline on extractables and leachables, and ICH Q9 quality risk management
• ISO 10993 series for medical device biocompatibility, including ISO 10993-12, -17, and -18
• EU GMP Annex 1 and 21 CFR 600–610 expectations for sterile manufacturing support

A risk-based E&L program protects patient safety, prevents product quality issues such as protein aggregation or visible particles, and provides defensible scientific data for regulatory submissions and supplier qualification.

Testing Samples

Single-Use Bioprocess Bags and Films

Two-dimensional and three-dimensional bags used for storage, mixing, and bioreactor applications are among the most extensively tested single-use consumables. Multilayer contact films typically contain PE or EVA contact layers, EVOH or polyamide barrier layers, tie-layer adhesives, and antioxidants. Common extractables include hindered phenol antioxidants (for example, Irganox 1010 and Irgafos 168), fatty acid amide slip agents, cyclic oligomers, and low-molecular-weight hydrocarbons.

Tubing, Transfer Lines, and Pump Tubing

Silicone tubing (peroxide-cured or platinum-cured), TPE tubing, and braided or reinforced transfer lines are evaluated for cyclic and linear siloxanes, silanols, platinum catalyst residues, peroxide decomposition by-products, and plasticizers. Peristaltic pump segments warrant particular attention because mechanical stress and friction can generate elevated levels of extractables and subvisible particles.

Filter Membranes, Capsules, and Cartridges

Sterilizing-grade and bioburden-reduction filters constructed from PES, PVDF, PTFE, or nylon are tested for membrane casting residues, wetting agents, surfactants such as polyvinylpyrrolidone, and polypropylene housing extractables. Pre-use and post-use extractables characterization supports filter validation and extractables-related 21 CFR documentation.

Connectors, Fittings, Seals, and O-Rings

Sterile connectors, sanitary fittings, freeze-thaw connectors, and elastomeric seals made from silicone, EPDM, or fluoroelastomers are analyzed for organic acids, curing agents, per- and polyfluoroalkyl substances (PFAS)-related concerns, and metal oxide fillers.

Sampling Systems, Bottles, and Sensors

Sampling bags, bottles, crimpcaps, sensor flow cells, and in-line probe windows are product-contact components that require extractables characterization as part of a comprehensive single-use system assessment.

Testing Items

A complete extractables and leachables profile for single-use consumables typically includes the following categories of test items:

• Non-volatile organic compounds (NVOC): antioxidants, light stabilizers, oligomers, fatty acids and their esters, amides, waxes, and polymeric additives
• Semi-volatile organic compounds (SVOC): plasticizers (phthalates, adipates, citrates such as acetyl tributyl citrate), lubricants, siloxanes, and UV absorbers
• Volatile organic compounds (VOC): residual solvents, monomers, aldehydes, ketones, and low-molecular-weight siloxanes
• Elemental impurities: catalyst and additive-related metals including Si, Al, Ca, Zn, Mg, Ti, Sn, and toxic elements such as Pb, As, Cd, Hg, and Sb
• Inorganic ions: anions and cations determined by ion chromatography
• General physical and chemical parameters: total organic carbon (TOC), non-volatile residue (NVR), pH, conductivity, and UV absorbance
• Compounds of specific toxicological concern: nitrosamines, polycyclic aromatic hydrocarbons (PAHs), bisphenols, PFAS, and primary aromatic amines

Each reported compound is assigned an identifier, a confidence level of structural elucidation, a quantitative estimate, and a comparison against the Analytical Evaluation Threshold (AET) to determine whether toxicological qualification is required.

Testing Methods

E&L studies begin with a scientifically justified extraction design. In accordance with USP <1663> and the BioPhorum protocol, extractions are typically performed with multiple solvents of varying polarity and pH—for example, water or buffer, pH 3 buffer, pH 10 buffer, and a 50:50 ethanol-water mixture—to bracket the properties of process fluids. Extraction conditions commonly range from room temperature to 40–70 °C, with durations from several hours to several weeks, and controlled surface-area-to-volume ratios to simulate or exaggerate intended use. Controlled extraction studies are designed to avoid solvent-induced material degradation while still capturing the worst-case extractables profile.

Orthogonal analytical techniques are then applied to ensure broad compound coverage:

• Headspace GC-MS — volatile organic compounds and residual solvents
• Direct-injection GC-MS, often with BSTFA derivatization — semi-volatiles, carboxylic acids, and plasticizers
• LC-MS/MS and LC-QTOF high-resolution mass spectrometry — non-volatile additives, antioxidants, oligomers, and unknown identification
• HPLC with UV, CAD, or ELSD detection — complementary quantitation of non-volatile organics
• ICP-MS or ICP-OES — elemental impurities and metal catalyst residues
• Ion chromatography — inorganic anions, organic acids, and amines
• TOC, NVR gravimetry, pH, and conductivity — general extract characterization
• FTIR and GC-FTIR — confirmation of polymer-related unknowns

Data interpretation follows a structured workflow: chromatographic peak detection relative to the AET, tentative identification against spectral libraries, confirmation with authentic reference standards where needed, semi-quantitation using surrogate standards, and reporting in units such as μg per component or μg per mL. The AET is derived from the safety concern threshold (SCT) of 1.5 μg/day established by PQRI, the maximum daily dose of the drug product, and the number of component units in the process train. Compounds exceeding the AET are routed to toxicological risk assessment consistent with ICH Q3E and ISO 10993-17 methodology.

Applications

Extractables and leachables analysis for single-use consumables supports critical activities across the product lifecycle:

• Regulatory submissions: E&L data packages for IND, NDA, BLA, and MAA filings demonstrating product-contact material safety
• Supplier and material qualification: evaluation and comparability of films, tubing, and filters from different vendors, including film-equivalency studies
• Process validation: scientific justification that single-use systems used in upstream, downstream, and fill-finish operations do not impact product quality
• Change control: assessment of raw material, formulation, or manufacturing changes to polymer components
• Risk assessment: application of ICH Q9 and USP <1665> risk-based approaches to rank process steps and prioritize testing
• Biocompatibility programs: integration with ISO 10993 chemical characterization for combination products and medical devices
• Emerging modalities: E&L support for cell and gene therapies, vaccines, mRNA products, and sterile fill-finish operations where single-use contact is extensive
• Troubleshooting: investigation of visible or subvisible particles, discoloration, off-odors, and unexpected interactions with protein therapeutics

Where justified, supplier-generated extractables data may be leveraged in lieu of new testing, provided that a documented gap assessment confirms the data reflect the actual component, material of construction, and intended process conditions.

FAQ

What is the difference between extractables and leachables? Extractables are compounds released under exaggerated laboratory conditions designed to draw out the worst-case chemical profile, whereas leachables are the compounds that actually migrate into the drug product under real processing and storage conditions. Extractables studies are conducted first, and leachables studies are typically designed based on the observed extractables profile.

Which standards apply to single-use consumable E&L testing? Key references include USP <1663>, <1664>, and <1665>, the BioPhorum standardized extractables protocol for single-use systems, ICH Q3E, and the ISO 10993 series for devices. Regulatory agencies expect a risk-based rationale that ties the testing strategy to product contact, dose, and patient exposure.

How are extraction conditions selected? Extraction solvents are chosen to bracket the polarity and pH of the process fluids the component will contact, while temperature, duration, and surface-area-to-volume ratio are set to simulate or slightly exaggerate intended use without degrading the polymer matrix. The scientific justification must be documented in the study protocol.

What is the Analytical Evaluation Threshold (AET)? The AET is the concentration below which an extractable is considered too low to present a safety concern. It is calculated from the safety concern threshold of 1.5 μg/day, the patient's maximum daily dose, and the number of components in the process train, with an uncertainty factor applied to account for analytical response variability.

How long does an extractables study take? Controlled extraction incubations may run from several days to several weeks, and the complete workflow—including method development, multi-technique analysis, data processing, unknown identification, and reporting—typically requires several months, depending on the complexity of the component and the number of compounds requiring structural confirmation.

Can supplier extractables data be used instead of performing new studies? Yes. Supplier data can be leveraged when a documented gap assessment confirms that the tested component matches the material of construction, film formulation, and intended use conditions, and that the analytical coverage meets current expectations for compound classes and sensitivity.

When is a leachables or simulation study required? Leachables testing of the actual drug product, or a simulation study using a surrogate fluid, is generally expected for higher-risk scenarios such as long product hold times, extensive single-use contact, sensitive biotherapeutics, or when extractables exceed the AET and require toxicological qualification.