Extractables and Leachables Analysis of Single-Use Products: Methods, Standards, and Applications

Extractables and leachables (E&L) analysis of single-use products is a critical quality and safety assessment for pharmaceutical, bioprocess, and medical applications, ensuring that polymer-based components do not release harmful compounds into final products. This technical guide explains the scope of extractables and leachables testing, the sample types involved, analytical methods, regulatory standards, and typical applications. Understanding E&L study design helps manufacturers mitigate contamination risks and meet the requirements of USP, ICH, and ISO frameworks.

Technical Overview

Single-use (disposable) products used in pharmaceutical manufacturing, bioprocessing, and medical applications are predominantly fabricated from polymeric materials such as polyethylene, polypropylene, ethylene-vinyl acetate, silicone, thermoplastic elastomers, and multilayer films. These materials contain additives, including antioxidants, plasticizers, slip agents, colorants, curing agents, and oligomers, which can migrate out of the material under processing, storage, or sterilization conditions. Extractables and leachables (E&L) analysis is the systematic scientific study of these migrant compounds to evaluate their potential impact on product quality and patient safety.

Extractables versus Leachables

Extractables are compounds that can be released from a material of construction under exaggerated conditions, such as aggressive solvents, elevated temperatures, or extended contact times. They represent the worst-case chemical profile of the material. Leachables are compounds that actually migrate into the drug product, buffer, or therapeutic formulation under normal conditions of use. Leachables are typically a subset of extractables, although they can also arise from interactions between the formulation and the container closure system.

Regulatory Framework and Industry Standards

E&L studies for single-use products are guided by a well-established set of compendial and regulatory references:

• USP <1663> Assessment of Extractables Associated with Pharmaceutical Packaging and Delivery Systems
• USP <1664> Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging and Delivery Systems
• ICH Q3E Guideline on Extractables and Leachables for pharmaceutical products
• ISO 10993 series for biological evaluation of medical devices, including chemical characterization (ISO 10993-12 and ISO 10993-18)
• USP <87> and <88> for in vitro cytotoxicity and biological reactivity testing of plastic materials
• The BioPhorum (formerly BPOG) standardized extractables protocol for single-use systems in biophrocessing
• PQRI safety thresholds and the Safety Concern Threshold (SCT) concept, typically 1.5 micrograms per day for parenteral drug products

These frameworks collectively require a risk-based approach in which the analytical evaluation threshold (AET), route of administration, dose, and duration of patient exposure determine the required analytical sensitivity.

Testing Samples

E&L analysis applies to a broad range of single-use and disposable product categories. Each sample type requires a tailored extraction design based on its material composition, surface area, and intended contact conditions.

Single-Use Bioprocess Containers and Bags

Flexible 2D and 3D bags used for media holding, buffer preparation, cell culture, harvest, bulk drug substance storage, and cryopreservation. Multilayer films may contain polyamide, EVOH oxygen barriers, and adhesive tie layers, all of which contribute unique extractables profiles.

Tubing and Transfer Lines

Silicone, thermoplastic elastomer (TPE), and PVC tubing used for fluid transfer, peristaltic pumping, and filling operations. Common extractables include siloxanes, cyclic silicones, curing agents such as 2,4-dichlorobenzoyl peroxide residues, and plasticizers such as DEHP.

Filtration and Purification Devices

Membrane filters, depth filters, and chromatography columns constructed from polyethersulfone, polyvinylidene fluoride, nylon, and regenerated cellulose, along with their housings and support layers.

Primary Packaging and Delivery Components

Pre-filled syringes, cartridges, vials, stoppers, aluminum seals, and components of closed system transfer devices (CSTDs). Key extractables include butyl rubber vulcanization agents, phenolic antioxidants, and silicone lubrication coatings.

Medical and Laboratory Disposables

Iv sets, catheters, blood bags, dialysis cartridges, pipette tips, well plates, and single-use mixing systems used in clinical and laboratory environments.

Testing Items

A comprehensive E&L study characterizes multiple chemical classes of extractables, because no single analytical technique can capture the entire chemical space. Typical testing items include:

• Volatile organic compounds (VOCs), such as residual solvents and low molecular weight hydrocarbons
• Semi-volatile organic compounds (SVOCs), including plasticizers, antioxidant stabilizers such as Irganox 1010 and Irgafos 168, and their degradation products like 2,4-di-tert-butylphenol
• Non-volatile organic compounds (NVOCs), including oligomers, polymerization catalysts, fatty acid amide slip agents such as oleamide and erucamide, and dye or pigment related species
• Elemental impurities and metal ions, such as zinc, titanium, silicon, aluminum, cobalt, and tin derived from catalyst residues and inorganic additives
• Inorganic anions and cations, including nitrate, sulfate, chloride, ammonium, and organic acids such as acetate and formate
• Non-volatile residue (NVR) and total organic carbon (TOC) as global screening measurements
• Particular target compounds of toxicological concern, including bisphenol A, phthalate esters, nitrosamines, polycyclic aromatic hydrocarbons (PAHs), and per- and polyfluoroalkyl substances (PFAS)

Each detected extractable is assigned a level of structural confidence according to established identification categories, and concentrations are compared against the calculated analytical evaluation threshold to determine whether toxicological assessment is required.

Testing Methods

The analytical strategy combines orthogonal chromatographic, spectrometric, and spectroscopic techniques to achieve broad chemical coverage with the sensitivity required by the AET.

Extraction Study Design

Extraction conditions are selected to simulate or accelerate product contact scenarios. Standard practice includes multiple solvent systems of varying polarity, for example water, pH 3 acidic buffer, pH 10 basic buffer, and 50 percent ethanol/water for aqueous formulations, or isopropanol and hexane for non-aqueous or lipophilic products. Extractions are performed at elevated temperatures such as 40 to 60 degrees Celsius over defined periods, or by reflux and Soxhlet extraction for aggressive worst-case studies. Sterilization pretreatments, including gamma irradiation and autoclaving, are frequently applied because they can significantly increase the extractables burden through polymer degradation.

Chromatographic and Mass Spectrometric Analysis

• Headspace GC-MS for volatile organic compounds and residual solvents
• Direct injection GC-MS with derivatization options for semi-volatile compounds
• LC-MS/MS and high-resolution LC-QTOF-MS for non-volatile and thermally labile compounds, with accurate mass data supporting unknown identification
• GC-FID and HPLC-UV/CAD for orthogonal quantification of specific targets
• ICP-MS or ICP-OES for elemental impurities in accordance with ICH Q3D principles
• Ion chromatography (IC) for inorganic anions, cations, and small organic acids

Supporting Characterization Techniques

• FTIR spectroscopy for identification of polymeric and insoluble particulate species
• Nuclear magnetic resonance (NMR) spectroscopy for definitive structural elucidation of unknowns
• TOC analysis and gravimetric non-volatile residue determination
• Particle counting and microscopy when subvisible particulate shedding is a concern

Data Evaluation and Reporting

Quantitation is performed using surrogate standards with response factor corrections, and an uncertainty factor of 50 percent is commonly applied to the AET to account for response variability. Reports include total ion chromatograms, extracted mass chromatograms, extracted compound tables with proposed identities and concentrations, an assessment against safety thresholds, and, where warranted, a toxicological risk assessment conducted by qualified toxicologists.

Applications

E&L analysis of single-use products supports quality, regulatory compliance, and process development across multiple industries:

• Biopharmaceutical manufacturing: qualification of single-use systems for upstream cell culture, downstream purification, and bulk drug substance storage, including supplier qualification under the BioPhorm standardized protocol
• Pharmaceutical packaging: demonstrating compatibility of container closure systems for liquid and lyophilized dosage forms in support of regulatory filings
• Medical devices: chemical characterization under ISO 10993-18 in support of biocompatibility evaluations and regulatory submissions to agencies such as FDA and notified bodies
• Cell and gene therapy: evaluating patient-contact disposables where high sensitivity and strict contamination control are essential
• Food contact materials: verifying compliance of disposable food packaging with migration limits established by FDA and EU regulations
• Change control and supplier management: comparing extractables profiles when materials, suppliers, or manufacturing processes change

By integrating extractables screening, simulated-use leachables studies, and toxicological evaluation, manufacturers can select appropriate single-use components, define acceptable use conditions, and protect both product quality and patient safety throughout the product lifecycle.

FAQ

What is the difference between extractables and leachables?

Extractables are compounds released under exaggerated laboratory conditions using aggressive solvents, heat, or time, representing a worst-case chemical profile. Leachables are the compounds that actually migrate into the product under normal storage and use conditions. Extractables studies are used to predict and prioritize potential leachables.

Which regulatory guidelines govern E&L testing of single-use products?

Key references include USP <1663> and <1664>, ICH Q3E, the ISO 10993 series for medical devices, and the BioPhorum standardized extractables protocol for single-use bioprocess equipment. Product-specific guidance from FDA and EMA also applies depending on the route of administration.

How long does an E&L study take?

A typical extractables screening study takes approximately six to twelve weeks, including extraction, multi-technique analysis, data interpretation, and reporting. A confirmatory leachables study on stability samples requires the duration of the product stability program, often several months to years.

What is the analytical evaluation threshold (AET)?

The AET is a concentration threshold derived from the safety concern threshold (SCT), the daily dose of the drug product, and the number of doses per container. Any extractable detected above the AET must be identified and evaluated toxicologically.

Why is gamma irradiation included in extraction studies?

Gamma sterilization can cleave polymer chains and degrade additives, generating new extractables such as antioxidants fragments and oxidized species. Pretreating samples with the actual sterilization dose ensures the study reflects the real-world worst case.