Extractables and leachables (E&L) in pharmaceutical and biopharmaceutical products: A comprehensive review of science, regulation, and analytical practice

Abstract

Extractables and leachables (E&L) represent one of the most complex and rapidly evolving challenges at the interface of analytical chemistry, materials science, toxicology, and pharmaceutical regulation. This review provides a comprehensive account of the E&L field, covering: (i) fundamental definitions and the physicochemical drivers of migration; (ii) the evolving global regulatory landscape, including the publication of the ICH Q3E draft guideline (August 2025); (iii) risk-based study design for both container closure systems and single-use bioprocessing equipment; (iv) current analytical methods including GC-MS, LC-MS/MS, LC-HRMS, and ICP-MS; (v) the role of certified reference materials (CRMs) in method development and validation; and (vi) safety assessment frameworks anchored to the threshold of toxicological concern (TTC) and analytical evaluation threshold (AET). Certified reference materials (CRMs), specifically the 21-component LC screening standard (95636) and the 14-component GC screening standard (01829), are discussed as tools that support robust E&L programs. The review concludes with perspectives on emerging challenges, including nitrosamine co-testing requirements, digital traceability, and the transition toward implementation of ICH Q3E.

Section overview

• Introduction
• Definitions and terminology
• Regulatory landscape
• Sources of extractables and leachables
• E&L study design: A risk-based framework
• Analytical methods for E&L characterization
• Certified reference materials in E&L practice
• Safety assessment of E&L findings
• Analytical method development and validation
• Emerging challenges and future directions
• Conclusion
• Related products

Introduction

The safety, quality, and efficacy of pharmaceutical and biopharmaceutical drug products depend not only on the active pharmaceutical ingredient (API) and its formulation, but also on the materials that come into contact with the product during manufacture, storage, and administration. Any chemical entity that migrates from a contact material, including primary packaging, secondary closures, single-use bioprocessing components, and drug delivery devices into the drug product constitutes a potential safety risk to the patient. The identification, quantification, and safety assessment of these migrating species form the basis of extractables and leachables (E&L) science.

Historically, E&L evaluation was guided by scattered product-specific regulatory requirements, most notably the U.S. food and drug administration (FDA) guidance on container closure systems (1999)¹ and the product quality research institute (PQRI) recommendations (2006).² The rapid adoption of single-use systems (SUS) in biopharmaceutical manufacturing from the mid-2000s onward, together with high-profile safety incidents associated with leachable contamination of drug products, elevated the importance of E&L science and underscored the need for a harmonized international framework.

The publication of the ICH Q3E draft guideline in August 2025, the first internationally harmonized regulatory guideline dedicated to extractables and leachables (E&L) for pharmaceutical products, represents a significant milestone in the field.³ Once finalized (projected June 2027), Q3E is expected to establish a structured, risk-based framework applicable to all drug product types, building upon the existing ICH impurity guidelines (Q3A–Q3D) harmonizing existing regional guidance for E&L assessment.⁴

This review examines the current state of E&L science across four key dimensions: regulatory landscape, study design, analytical methodology, and safety assessment. It is intended for pharmaceutical scientists, analytical chemists, quality assurance professionals, and regulatory affairs specialists involved in the design, execution, or review of E&L programs. Particular attention is given to the role of certified reference materials in supporting analytical rigor, with specific reference to two TraceCERT^® CRMs: the E&L screening standard for LC (95636, 21 components in acetonitrile) and the E&L screening standard for GC (01829, 14 components in tert-butyl methyl ether).

Definitions and terminology

Precise terminology is fundamental to E&L science, as the distinction between extractables and leachables has direct implications for study design, analytical thresholds, and regulatory submission strategies.

Extractables

Extractables are chemical compounds that can be removed from a material under exaggerated laboratory conditions, typically involving elevated temperature, prolonged contact time, and/or aggressive solvent systems. The extraction is intentionally exhaustive and is designed to generate a worst-case chemical inventory of all species that could potentially migrate from the material under foreseeable use conditions. Extractables studies serve as a predictive risk assessment tool, with the resulting profile used to identify compounds that may subsequently be detected as leachables and to inform safety evaluations before product contact studies are performed.

Leachables

Leachables are chemical compounds that migrate from a contact material into the drug product under actual or simulated conditions of storage and use. Unlike extractables, leachables represent the clinically relevant exposure scenario, as they are the chemical species to which patients may be exposed during administration of the drug product. Leachables studies are conducted using the final drug product formulation under real-time or accelerated storage conditions and require quantitative analytical methods with detection limits at or below the applicable regulatory threshold.

Key distinction and extractable-leachable (E–L) correlation

A fundamental principle of E&L science is that extractables represent the pool of compounds with the potential to become leachables. In principle, every leachable should be detectable in the corresponding extractables profile. However, not all extractables become leachables. The extent of migration under conditions of use is influenced by multiple factors, including the physicochemical properties of the compound (e.g., polarity, molecular weight, and vapour pressure) the characteristics of the drug formulation (e.g., pH, organic solvent content, ionic strength) as well as contact conditions (e.g., contact area, temperature, and contact duration). Establishing a scientifically defensible extractable–leachable (E–L) correlation is a regulatory expectation that supports risk assessment, reduces the extent of confirmatory leachables testing and facilitates lifecycle management of changes to container closure systems.

The key terminology used throughout E&L studies, together with the associated study conditions, is summarized in Table 1.

Regulatory landscape

Until August 2025, the global regulatory framework for E&L consisted of a combination of regional guidance documents, pharmacopeial chapters, and industry-led standards. The publication of the ICH Q3E draft guideline, as the first internationally harmonized guideline dedicated to E&L, represents a significant milestone and is expected to reduce the need for duplicate studies for products seeking approval across multiple jurisdictions.

United States food and drug administration (FDA)

The FDA's approach to E&L has historically been product class-specific. Key guidance documents include the 1999 Guidance for Industry on 'Container closure systems for packaging human drugs and biologics', which established the conceptual framework for extractables and leachables evaluation based on dosage form and route of administration.¹ This guidance introduced a risk-based approach with parenteral and inhalation products receiving the highest level of scrutiny, followed by topical, nasal, and oral solid dosage forms.

The FDA is a member of the ICH Q3E expert working group and published the draft guideline in the federal register (December 2025) for public comment. The agency also maintains product-specific guidance that addresses E&L considerations, including recent guidance for orally inhaled and nasal drug products, as well as combination products.

European medicines agency (EMA)

The EMA's key guidance documents include the 'Guideline on plastic immediate packagingmaterials' (CPMP/QWP/4359/03) and the 'Guideline on the qQuality of inhalation products'. The EMA endorsed the ICH Q3E draft guideline in August 2025 and opened a public consultation period that closed in December 2025, followed by publication of a summary of comments in January 2026. The draft guideline provides a structured framework for E&L assessment, introducing systematic approaches to threshold derivation and documentation requirements.

ICH Q3E draft guideline (August 2025): A new regulatory framework

The ICH Q3E draft guideline, 'Guideline for extractables and leachables', reached Step 2b on August 1, 2025, marking the beginning of the public consultation period, which concluded in December 2025. Finalization is targeted for June 2027. Once adopted, ICH Q3E will become the first internationally harmonized guideline dedicated to E&L assessment for pharmaceutical products.^3,25

The ICH Q3E draft guideline introduces a structured, risk-based framework with the following principal elements:

• Scope: Applicable to all pharmaceutical drug products, including small molecule and biologics, across all dosage forms, routes of administration, and contact materials, including packaging components, drug delivery devices, manufacturing equipment.
• Risk-based tiering: A tiered approach to E&L assessment based on route of administration, drug product characteristics, and material properties, analogous to the principles described in ICH Q3D for elemental impurities.
• Analytical evaluation threshold (AET): A scientifically derived threshold, calculated from the safety concern threshold (SCT), above which extractables and leachables must be identified, quantified, and assessed. In practice, an uncertainty factor is often applied during AET derivation to account for analytical variability, particularly when semi-quantitative methods are used. For example, some laboratories establish the AET at 50% of the SCT.
• Safety concern threshold (SCT): Defined as the lower of the threshold of toxicological concern (TTC) for mutagenic compounds and the qualification threshold (QT) for non-mutagenic systemic toxicity. Compounds below the SCT generally do not require further toxicological evaluation.
• E–L correlation requirement: The guideline emphasizes correlating leachables with the corresponding extractables profile to support a science-based justification when expected leachables are not detected.
• Class 3 leachable monographs: Supporting documentation includes draft Class 3 leachable monographs that provide a structured framework for the toxicological assessment of commonly encountered leachables, including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), Irganox 1076, and others that appear in the two reference standard mixes described in this article.
• Extractable–leachable lifecycle: The guideline promotes the use of E&L data throughout the product lifecycle, including post-approval changes such as modifications to container closure systems, manufacturing sites, and single-use systems.

USP chapters <661>, <665>, <1031>, <1663>, <1664>, and <1665>

The United States Pharmacopeia (USP) provides a series of chapters that support E&L assessment throughout the pharmaceutical product lifecycle. The principal chapters include:

• USP <661>: Establishes requirements for the evaluation of plastic packaging systems and their materials of construction for pharmaceutical use.
• USP <665>: Provides requirements for the chemical suitability assessment of plastic components and single-use systems used in pharmaceutical manufacturing, including standardized extractables testing.⁵
• USP <1031>: Provides guidance on the biological evaluation of materials used in pharmaceutical packaging and medical devices.
• USP <1663>: Provides a framework for the assessment of extractables associated with pharmaceutical packaging and delivery systems^.6
• USP <1664>: Describes the framework for the design, justification, and implementation of leachables assessments for pharmaceutical packaging and delivery systems.⁷
• USP <1665>: Provides guidance on the characterization, qualification, and risk assessment of plastic manufacturing components and complements the requirements of USP <665>.

Collectively, these chapters provide a comprehensive framework for the characterization of packaging materials and manufacturing components, the design of extractables and leachables studies, and the assessment of material suitability for pharmaceutical applications.

BPOG/BioPhorum for single-use systems

BioPhorum, formerly the BioPhorum operations group (BPOG), has published two key guidance documents for the evaluation of extractables and leachables in single-use systems (SUS), 'Best practice guide for evaluating leachables risk from polymeric single-use systems' and  'Standardised extractables testing protocol for single-use systems in biomanufacturing'.^8,9 These BPOG documents have been widely adopted by suppliers of single-use systems (SUS) and biopharmaceutical manufacturers, and have contributed to the standardization of extractables testing.

The TraceCERT^® E&L screening standard for LC and the TraceCERT^® E&L screening standard for GC discussed in this review have been developed using the BPOG extractables testing protocol as the methodological basis.

Sources of extractables and leachables 

E&L species can originate from virtually any material that comes into contact with a drug product during manufacturing, storage, or administration. Consequently, a systematic inventory of these materials provides the foundation for identifying potential sources of extractables and leachables and conducting an effective risk-based assessment.

Primary container closure systems

Primary packaging components that come into direct contact with the drug product represent the most extensively studied and regulated source of extractables and leachables, owing to their direct and prolonged contact with the drug product. Representative extractables associated with the principal material categories are summarized below, as these profiles form the basis for subsequent leachables assessments.

Single-use bioprocessing systems

The widespread adoption of single-use systems (SUS), including bioreactor bags, tubing, filter assemblies, mixing vessels, connectors, and transfer lines has significantly expanded the scope of E&L assessment in biopharmaceutical manufacturing. Unlike traditional stainless-steel equipment, which is cleaned and qualified for repeated use, each SUS component introduces a new set of potential extractables that must be characterised by the supplier and evaluated as part of the drug manufacturer's E&L risk assessment.

The polymer materials most commonly used in single-use systems and their representative extractables are summarized in Table 4. The extent of migration depends on the duration and conditions of product contact, ranging from brief processing operations to extended incubation periods. Consequently, extractables studies and migration assessments are essential components of the E&L risk assessment for single-use systems.

Drug delivery devices

Drug delivery devices, including metered-dose inhalers, dry powder inhalers, nebulisers, prefilled syringes, auto-injectors, nasal sprays, ophthalmic droppers, and transdermal patches present unique E&L challenges. The drug product come into contact with multiple polymeric and elastomeric components under a range of conditions, including mechanical stress, temperature cycling, and long shelf lives. Inhalation and parenteral drug products require the most rigorous E&L assessment because they present the greatest potential for direct systemic or pulmonary exposure.

E&L study design: A risk-based framework

Effective E&L study design integrates material knowledge, product risk tier, and analytical capability into a structured assessment programme. A risk-based approach, as described in BPOG guidance and the ICH Q3E draft guideline, avoids unnecessary testing while ensuring that all safety-relevant species are detected and characterised.

Risk assessment

The initial step in an E&L study is a formal risk assessment that considers the following factors:

1. Route of administration and intended patient population, including pediatric patients, immunocompromised patients, and products intended for chronic use.
2. Nature and quantity of contact materials, including packaging components, manufacturing equipment, and single-use systems.
3. Drug product characteristics, including aqueous, organic, surfactant-containing, protein-based formulations
4. Process conditions, including temperature, contact duration, and pH
5. Existing supplier data and material knowledge, including available extractables data and prior knowledge of material composition.

For single-use systems, the BPOG risk assessment framework evaluates each component based on the type and duration of product contact, together with formulation characteristics, to prioritize components for detailed extractables characterization.

Extraction conditions

Extraction conditions for extractables studies are designed to be sufficiently exhaustive to recover a broad range of extractable compounds regardless of their physico-chemical properties. A standard extraction regime, as described in BPOG and USP <665>, employs solvents representing a range of polarities.

Leachables study design

Leachables studies are conducted using the final drug formulation in contact with the primary container closure system under real-time or accelerated storage conditions, as appropriate and in accordance with ICH Q1A(R2) and, where applicable, ICH Q1B. The study duration typically encompasses the proposed shelf life, with samples collected at appropriate intervals to evaluate changes in leachables over time. Sample preparation and analytical methods must be validated to demonstrate adequate recovery of all target leachables from the drug formulation matrix, without introducing artificial artefacts.

The analytical evaluation threshold (AET)

The AET defines the minimum analytical sensitivity required for an analytical method. In extractables or leachables studies, compounds detected above the AET, must be identified and assessed, in accordance with the applicable risk assessment framework. The ICH Q3E guidance provides AET calculation examples for multiple product types, taking into account factors such as daily dose and route of administration that influence the permissible exposure level and, consequently, the calculated threshold.

Analytical methods for E&L characterization

No single analytical technique can comprehensively detect and characterize all extractables and leachables with adequate sensitivity and structural resolution. A comprehensive E&L programme therefore employs a complementary suite of techniques, chosen based on the physicochemical properties of target analytes and the analytical sensitivity required to meet the AET. These techniques are typically used in combination, with GC-MS applied to volatile and semi-volatile organic compounds, LC-MS to polar and semi-polar compounds, and ICP-MS to elemental (metal) impurities.

Gas chromatography–mass spectrometry (GC-MS)

GC-MS is the primary analytical technique for the screening of volatile and semi-volatile organic extractables and leachables. Its high chromatographic resolving power, extensive mass spectral libraries (e.g., NIST), and high sensitivity make it ideal for the analysis of antioxidants, UV stabilizers, slip agents, plasticizers, processing aids, and cyclic siloxanes that dominate the extractables profiles of polyolefin- and silicone-based SUS components.

The TraceCERT^® E&L screening standard for GC (01829, 14 components in MTBE) was developed based on the BPOG GC screening protocol and provides a certified reference material for method development, validation, and performance verification in GC-MS-based E&L analysis. The CRM was certified using a GC-MS method based on a Supelco^® SLB^®-5MS capillary GC column (30 m × 0.25 mm × 0.25 µm) coupled to an electron ionization (EI) mass spectrometer operated in selected ion monitoring (SIM) mode (70 eV), achieving certified concentrations of 50.0 mg/L per analyte (67.4–67.8 mg/kg).¹⁰ The principal analytical parameters used for certification are summarized in Table 6.

Liquid chromatography–mass spectrometry (LC-MS/MS and LC-HRMS)

LC-MS addresses the analytical gap for compounds that are not amenable to GC analysis, including, polar, ionic, thermally labile, and high-molecular-weight species. In E&L studies, LC-MS is particularly suited for the analysis of rubber vulcanisation accelerators (e.g., 2-mercaptobenzothiazole, MBT), amine-based species (e.g., dibenzylamine), high-molecular-weight antioxidants (e.g., Irganox 1010, pentaerythritol tetrakis), UV stabilizers (e.g., drometrizole), and non-volatile degradation products of polymer additives.

The TraceCERT^® E&L screening standard for LC (95636, 21 components in acetonitrile) was developed based on the BPOG LC screening protocol to support method development and performance verification for LC-based E&L analysis.¹¹ Two complementary were used to accommodate compounds with different physicochemical properties and detection requirements. The key analytical conditions are summarized in Table 7.

For targeted quantification of known leachables, triple quadrupole (QqQ) mass spectrometers operated in multiple reaction monitoring (MRM) mode offer the best sensitivity and selectivity. High-resolution mass spectrometry (HRMS), including orbitrap and quadrupole time-of-flight (Q-TOF) platforms, is increasingly used during discovery-phase extractables studies to assign molecular formulae and support the tentative identification of previously uncharacterized compounds in the absence of authentic reference standards.

Inductively coupled plasma–mass spectrometry (ICP-MS)

Metal extractables and leachables originating from glass containers, stainless steel equipments, rubber closures, and residual metal catalysts used during the manufacture of polymeric materials typically require elemental analysis by ICP-MS or ICP-OES. Representative elements of concern in E&L studies include:

• Tungsten (W): Residual tungsten originating from tungsten pins used during the manufacture of prefilled syringes and associated with protein aggregation in certain biopharmaceutical formulations.
• Barium (Ba): May originate from glass formulations where it is used as a stabilizer.
• Tin (Sn): May arise from organotin stabilizers used in certain polymer formulations, such as PVC.
• Platinum (Pt): Residual catalyst associated with platinum-catalyzed silicone curing.
• ICH Q3D elemental impurities: Including Class 1 elements (As, Cd, Hg, Pb), Class 2A elements (Co, Ni, V), and representative Class 2B elements (Ba, Cr, Cu, Li, Mo), which require assessment based on the route of administration and risk.

ICP-MS achieves sub-ppb detection limits, making it well-suited for the determination of elemental impurities at concentrations relevant to the permitted daily exposures (PDEs) specified in ICH Q3D, including the stringent limits established for parenteral drug products.

Nuclear magnetic resonance (NMR) and other analytical techniques

Quantitative ¹H NMR (qNMR) plays a specialized role in E&L studies. It serves as a primary technique supporting the characterization and value assignment of certified reference materials, including the TraceCERT^® E&L screening standards (95636 and 01829), which are traceable to NIST PS1 benzoic acid and NIST SRM 841 potassium hydrogen phthalate (KHP).^10,11 In addition, qNMR is used as a complementary structural elucidation tool for unknown extractables when MS alone is insufficient for confident compound identification.

Additional techniques that support E&L characterization include:

• Headspace GC-MS: Analysis of volatile compounds, including cyclic siloxanes
• Ion chromatography (IC): Determination of inorganic anions
• Total organic carbon (TOC): Non-specific screening for total organic contamination)
• Fourier-transform infrared spectroscopy (FTIR): Polymer identification and semiquantitative profiling.

Certified reference materials in E&L practice

The accuracy, reliability, and regulatory defensibility of E&L analytical programmes depend on the use of well-characterized certified reference materials (CRMs). CRMs support method development, system suitability testing, calibration, and the evaluation of recovery and accuracy during method validation. The TraceCERT^® E&L screening standards are certified reference materials that are characterized by using complementary analytical techniques, including gravimetric preparation, quantitative ¹H NMR (qNMR), and chromatographic verification. They are produced under ISO 17034 accreditation and are metrologically traceable to NIST primary standards.

E&L screening standard for LC (95636)

The TraceCERT^® E&L screening standard for LC (95636, Lot BCCL4865) is a certified reference solution comprising of 21-components in acetonitrile with certified concentrations of approximately 50.0 mg/L per analyte (57.1–63.9 mg/kg). The CRM (Lot BCCL4865) was certified on November 11, 2024, and has an expiry date of July 2028.¹¹ The reference material is supplied in an amber glass ampule and should be stored at −20 °C ± 5 °C. The mixture a broad range of chemical functionalities representative of compounds expected in an LC-based E&L screening programme.

E&L screening standard for GC (01829)

The TraceCERT^® E&L screening standard for GC (01829, Lot BCCN8984) is a certified reference solution comprising of 14-components in tert-butyl methyl ether (MTBE), with certified concentrations of 50.0 mg/L per analyte (67.4–67.8 mg/kg). The CRM (Lot BCCN8984) was certified on March 6, 2026 (version 02) and has an expiry date of September 2029. The reference material is supplied in an amber glass ampule under argon and stored at −20 °C ± 5 °C. The 14 components represent commonly encountered extractables in GC-MS-based E&L profiling. 12 components are shared with the LC standard, enabling orthogonal analysis by both GC-MS and LC-MS.

• Unique to the GC standard: 2,6-Di-tert-butylphenol (CAS 128-39-2, t_R 7.6 min), an antioxidant isomer that can be resolved by GC-MS but co-elutes under the LC conditions used for the LC screening standard.
• Components shared with the LC standard: 13 components are common to both screening standards, including Irganox 1076, ε-caprolactam, MBT, BPA, BHT, 1,3-di-tBubenzene, oleamide, DEHP, stearic acid, cis-13-docosenoamide, tris(2,4-ditBuphenyl)phosphate, 2,4-DTBP, palmitic acid.
• Certification: Gravimetric preparation, qNMR traceable to NIST PS1 (benzoic acid) and NIST SRM 841 (potassium hydrogen phthalate, KHP), GC-MSD confirmation, and a certified density of 741.0 kg/m³ ± 0.4 kg/m³ (k=2).

Certification methodology and metrological traceability

Both CRMs are produced by an ISO 17034:2016-accredited reference material producer (Swiss Accreditation Service, SAS; SRMS 0001).^12,13 Their certification is based on a combination of orthogonal analytical techniques and metrological traceability to internationally recognized reference standards, ensuring the accuracy and reliability of the certified values. The principal elements of the certification workflow include:

• Gravimetric preparation: Certified values are assigned by gravimetric preparation with traceability to the SI unit of mass (kilogram) using DKD-certified balances and OIML Class E2/F2 weights. This high-precision gravimetric preparation using calibrated balances minimizes the contribution of weighing to the uncertainty of the certified values.^14,15
• qNMR characterisation: The purity of the starting materials is independently verified by quantitative ¹H NMR using NIST PS1 (benzoic acid) or NIST SRM 841 (potassium hydrogen phthalate, KHP) as reference materials. This provides metrological traceability to national measurement standards and enables multiple analytes to be characterized using the same primary material.
• Double isotope dilution mass spectrometry (IDMS) for dibenzylamine: Dibenzylamine in 95636 is additionally characterized by double IDMS, providing an independent orthogonal analytical approach to complement gravimetric preparation and qNMR during certification.¹⁶
• Chromatographic verification: The packaged CRMs are independently analyzed by LC (95636) or GC (01829) using internal standard calibration to confirm agreement between the measured and certified values within the stated measurement uncertainty.¹⁷
• Homogeneity and stability: Evaluated in accordance with ISO Guide 35 (95636)¹⁸ and ISO 33405:2024 (01829)¹⁹ using analysis of variance (ANOVA) for between-unit homogeneity and stability studies conducted under multiple storage conditions.

Safety assessment of E&L findings

Once extractables and leachables have been identified and quantified, a safety assessment is performed to determine whether the observed levels pose an unacceptable risk to patients. The assessment is conducted on a compound-by-compound basis, taking into account the route of administration, duration of treatment, patient population, and the intrinsic toxicological profile of each compound.

Threshold framework

The modern threshold framework for E&L safety assessment is built on tiered threshold approach that guides the reporting, identification, and toxicological evaluation of extractables and leachables.

• Reporting threshold (RT): The analytical level above which detected compounds are reported. Depending on the study objective and applicable guidance, the reporting threshold may correspond to the Analytical Evaluation Threshold (AET). In discovery or screening studies, all detectable peaks above the instrument noise threshold are typically reported.
• Analytical evaluation threshold (AET)The analytical level above which compounds should be structurally identified and evaluated to support a toxicological risk assessment.
• Safety concern threshold (SCT): The SCT is derived using the Threshold of Toxicological Concern (TTC) concept. For non-mutagenic compounds, the SCT may be established using the Cramer classification (Class I: 30 µg/kg body weight/day; Class II: 9 µg/kg body weight/day; Class III: 1.5 µg/kg body weight/day). For mutagenic impurities, the ICH M7 TTC framework is applied (generally 1.5 µg/day, with lower thresholds for compounds of exceptionally high potency, including Cohort of Concern compounds such as aflatoxin-like, azoxy, and N-nitroso compounds). Below the SCT, no further toxicological evaluation is generally required.
• Qualification threshold (QT): For species above the SCT, a compound-specific toxicological assessment is required, analogous to the ICH Q3B qualification of drug product degradation products. The QT is established based on the route of administration, daily exposure, and the available toxicological data.

Compound-specific safety assessment

For leachables identified above the SCT, a compound-specific evaluation is required. The assessment typically follows a structured approach that includes:

• Literature and database review: Review available toxicological information from sources such as ToxCast, REACH dossiers, IARC monographs, and publicly available FDA and EMA assessment reports.
• In-silico assessment (ICH M7, DEREK, SARAH): For compounds lacking sufficient published toxicological data, quantitative structure-activity relationship (QSAR) tools may be used to predict mutagenic potential in accordance with the principles described in ICH M7.²⁰
• Read-across and structural analogues: Toxicological data from structurally related compounds may be used to support the safety assessment when compound-specific data are unavailable.
• Risk characterization: Compare the estimated margin of exposure with the available toxicological reference values (e.g., NOAEL, PDE, or TTC) as appropriate, to determine whether the identified leachable presents an acceptable risk.
• Class 3 leachable monographs (ICH Q3E): he draft ICH Q3E supporting documentation includes Class 3 monographs for commonly encountered leachables, including BHT, Irganox 1076, and 2,4-DTBP, which are present in products 95636 and 01829.

Compounds of special concern

Certain leachables attract heightened regulatory scrutiny because of their toxicological properties or regulatory status, regardless of their concentration:

1. Bisphenol A (BPA): An endocrine disruptor associated with polycarbonate plastics and epoxy resins; restricted in EU under REACH and included in both products 95636 and 01829.
2. Di(2-ethylhexyl) phthalate (DEHP): Reproductive toxicant, SVHC under EU REACH, and restricted in medical devices under the EU MDR; included in both CRMs.
3. 2-Mercaptobenzothiazole (MBT): Rubber vulcanization accelerator with skin sensitization potential.
4. Nitrosamine precursors (e.g., dibenzylamine): Where amine-based rubber accelerators or other nitrosamine precursors are present, co-testing for nitrosamines in accordance with FDA and EMA guidance is performed.^21,22

Single-use systems: The evolving E&L challenge

The biopharmaceutical industry's transition from traditional stainless-steel manufacturing to disposable, single-use systems represent one of the most significant operational changes of the past two decades. SUS offers compelling advantages, including the elimination of cleaning validation, reduced risk of cross-contamination, faster product changeovers, and scalable, modular manufacturing. However, every SUS component also introduces potential sources of extractables and leachables that must be systematically characterized and assessed.

SUS-specific challenges

• Material complexity: A typical single-use bioreactor may contaiN 15-20 distinct polymeric components, each with its own additive package, processing aids, and adhesive layers, generating a highly complex extractables profile.
• Scale-up effects: Surface area-to-volume ratios differ dramatically between small-scale process development (where E&L concentrations may be higher per unit volume) and commercial-scale manufacturing.
• Film-to-film variation: Multilayer film formulations vary between manufacturers and, in some cases, between production lots, making extractables variability assessment a continuous requirement.
• Drug substance sensitivity: Biopharmaceutical drug substances, particularly proteins and nucleic acids, may interact with leachables through aggregation, oxidation, deamidation, or adsorption mechanisms that may not be detected by standard concentration-based assays.
• Downstream carry-over: Leachables introduced during early upstream processing steps may be concentrated or structurally modified through subsequent downstream processing steps (e.g., protein A purification, UF/DF), complicating the correlation between extractables and leachables.

BPOG standardized protocol

The BPOG standardized extractables testing protocol for single-use systems (SUS) defines a tiered approach to extractables characterization and served as the methodological basis for the development of the TraceCERT^® E&L CRMs. The protocol specifies three extraction solvent systems, (1) water (pH 7), (2) 50% ethanol/water, (3) 100% ethanol as a worst-case organic simulant, together with standard extraction conditions (40 °C for 24 hours for most of the components and 70 °C for the aggressive materials). The protocol recommends the use of GC-MS for semi-volatile organics, using an SLB^®-5MS or equivalent, LC-UV/MS for polar organics, and ICP-MS for metals as analytical methods. The 14 GC-target analytes in 01829 and the 21 LC-target analytes in 95636 were selected to represent the most frequently encountered extractables across a broad survey of commercial SUS materials, providing a practical compound library for systematic E&L programmes.

Analytical method development and validation

Method development strategy

E&L method development must address two distinct analytical challenges: (1) broad-spectrum screening to detect and identify the full range of extractables associated with a material, and (2) targeted quantitative analysis of identified leachables in the drug product matrix to demonstrate compliance with applicable regulatory thresholds.²³ The certified reference standard mixes (95636 and 01829) are particularly suited to the latter, enabling construction of multi-point calibration curves that are metrologically traceable to NIST primary standards.^10,11

The use of these CRMs for the purpose of method development must follow the recommended workflow outlined below:

• Step 1 (System suitability): Inject the CRM solution at a concentration corresponding to 100% of the AET and verify peak area precision (≤2% RSD), retention time reproducibility, and resolution between closely eluting analyte pairs (e.g., 2,4-DTBP and 2,6-DTBP in the GC method, t_R = 8.1 and 7.6 min respectively).
•  Step 2 (Calibration curve): Prepare 5 to 8 calibration levels from the stock CRM spanning the AET to 150 % of the target leachable limit. Use an isotope-labelled internal standard where available.
• Step 3 (Matrix spiking): Fortify blank drug product matrix at 50%, 100%, and 150% of the AET. Validate extraction recovery and assess matrix effects.
• Step 4 (Selectivity): Confirm the absence of interfering peaks from the drug product matrix at the retention times of all 21 (LC) or 14 (GC) target analytes.
• Step 5 (Stability): Demonstrate the stability of prepared standards and spiked samples under the intended storage conditions.

Validation parameters

Emerging challenges and future directions

Nitrosamine risk assessment and testing

The global nitrosamine crisis in pharmaceutical manufacturing (NDMA/NDEA in sartan-class antihypertensives, ranitidine, and metformin from 2018 onwards) has created an important intersection with E&L science. Rubber closure systems and elastomeric components containing secondary amine-based vulcanisation accelerators (such as dibenzylamine, component 5 in 95636) are potential sources of nitrosamine leachables under oxidative or acidic drug product conditions. FDA's 2024 guidance revision and ICH Q3E guideline recognize nitrosamines as a special class of leachables requiring  evaluation under nitrosamine impurity frameworks (ICH M7 and the FDA Control of Nitrosamine Impurities in Human Drugs guidance).^3,21,22 E&L programmes for parenteral and inhalation products containing rubber components must now include specifically a nitrosamine risk assessment and, where appropriate, nitrosamine testing.

Advanced materials in drug delivery

The proliferation of novel drug delivery systems, including mRNA lipid nanoparticles (LNPs), antibody-drug conjugate (ADC) formulations, cell and gene therapies, introduces new E&L challenges. LNP formulations contain organic solvents (ethanol, DMSO), ionizable lipids, and PEGylated excipients that may interact with product-contact materials differently from conventional aqueous biopharmaceutical formulations. The physicochemical complexity of these drug products demands advanced extraction models and analytical methods capable of operating in high-organic, surfactant-containing matrices.

In silico extractables prediction

Migration modelling, which applies established polymer physics frameworks (e.g., the Piringer model, Crank diffusion equation) to predict the maximum theoretical leachables concentration from a packaging system, is increasingly accepted as supporting evidence for E–L correlation and as a complement to analytical testing.²⁴ ICH Q3E is expected to incorporate migration modelling provisions, particularly for oral solid dosage forms where analytical sensitivity requirements are less demanding.

Digital traceability and data integrity

The use of certified reference materials with metrological traceability (such as the NIST-traceable TraceCERT^® standards) is not merely an analytical best practice but is increasingly becoming a regulatory expectation for traceability and data integrity. ICH Q3E, together with current regulatory guidance on data integrity (21 CFR Part 11 and EU Annex 11), requires that the traceability of calibration standards be documented and auditable. The move towards electronic certificates of analysis (eCOAs), blockchain-enabled supply chain traceability, and LIMS integrated reference standard management is accelerating, driven by both regulatory expectations and the operational demands of global E&L programmes.

Artificial intelligence and non-targeted screening

High-resolution mass spectrometry combined with AI-driven spectral deconvolution and in silico fragmentation prediction is transforming non-targeted extractables screening. Tools such as SIRIUS/CSI:FingerID, MS-DIAL, and commercial platforms (e.g., Agilent MassHunter PCDL and Waters UNIFI) enable tentative structural assignments for novel extractables without authentic reference standards, substantially expanding the range of compounds that can be identified. However, confirmatory identification still requires comparison with authenticated CRMs, underlining the continued and non-negotiable role of certified reference standards in regulatory E&L submissions.

Conclusion

Extractables and leachables science has matured from a reactive, crisis-driven discipline into a proactive, risk-based component of pharmaceutical product lifecycle management. The landmark ICH Q3E draft guideline (August 2025) represents the culmination of nearly three decades of scientific and regulatory development and is expected to deliver global of E&L assessment.^3,25 When finalized, Q3E will establish a clear, structured, and internationally consistent framework for E&L assessment across all drug product types and routes of administration.

Analytically, the field is supported by a mature toolkit: GC-MS for volatile and semi-volatile organics, LCMS/MS and LC-HRMS for polar and thermally labile species, and ICP-MS for elemental impurities. The TraceCERT^® certified reference materials, including the 21-component LC screening standard (95636) and the 14-component GC screening standard (01829), provide a metrologically traceable solution for E&L method development and validation. Certified by gravimetric preparation and qNMR against NIST primary standards, accredited under ISO 17034 and ISO/IEC 17025, and covering the most analytically significant E&L target compounds identified in BPOG and regulatory guidance, these standards provide the traceability foundation that regulatory submissions require.^12,13 In addition, CCS specific impurities can be incorporated into the analytical workflows to better address the objectives of individual E&L studies.

Looking ahead, the convergence of E&L science with nitrosamine risk-assessment and testing, the rise of novel drug delivery modalities (LNPs and gene therapies), and the growing role of AI-driven non-targeted screening will continue to shape the field. The consistent use of well-characterized, metrologically traceable reference materials will remain fundamental to robust, scientifically-defensible, and regulatory-ready E&L program, regardless of how the technology and the regulatory landscape evolves.