What does USP chapter 665 require for single-use bioprocessing systems?

USP chapter 665, which became mandatory on May 1, 2026, requires that plastic components and systems used in pharmaceutical and biopharmaceutical manufacturing be characterized for extractables using standardized testing conditions and analytical methods. The chapter establishes the analytical evaluation threshold below which compounds need not be individually identified, defines the extraction conditions required for different material and contact condition categories, and requires that a formal risk assessment be conducted by the drug manufacturer for all product-contact plastic components. The companion chapter 1665 provides the risk assessment framework and additional guidance for implementing chapter 665 in practice.

Are supplier extractables data packages sufficient for regulatory compliance?

Supplier extractables data packages are a necessary input to regulatory compliance but are not sufficient on their own. The supplier can characterize the compounds that can be extracted from their component under standardized conditions. They cannot assess whether those compounds, at the concentrations present in the drug manufacturer's specific product under the manufacturer's specific process conditions, pose a patient safety risk. That assessment is the drug manufacturer's responsibility and must be documented as a formal leachables risk assessment available for regulatory review. Using supplier data as a compliance endpoint without a sponsor-conducted risk assessment is a common deficiency.

What is the BioPhorum extractables protocol and should I use it?

The BioPhorum standardized extractables testing protocol is a guidance document developed by the BioPhorum industry consortium that provides a standardized approach to extractables characterization for single-use components in biopharmaceutical manufacturing. It is widely used by both suppliers and drug manufacturers as a rigorous, science-based framework for generating extractables data. It is not a regulatory requirement, but it is accepted by regulators as a scientifically valid approach when properly implemented. For facilities conducting their own extractables testing or evaluating supplier data packages, alignment with the BioPhorum protocol provides a strong baseline for regulatory defensibility.

When is direct leachables testing required rather than a risk assessment based on extractables data?

Direct leachables testing in the drug substance or drug product is required when the extractables-based risk assessment cannot exclude a patient safety concern. This occurs when an identified extractable compound exceeds the safety concern threshold in the risk assessment, when the compound is uncharacterized or lacks an established toxicological threshold, when the drug substance formulation includes conditions (extreme pH, high temperature, long contact time) that increase leaching potential beyond what extractables data can conservatively bound, or when the regulatory reviewer requests additional confirmatory data. In most cases, a thorough extractables risk assessment with conservative process condition assumptions will not require direct leachables testing.

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Leachables and extractables in single-use bioprocessing: a compliance guide

The plastic touching your drug product must be proven safe for patients. USP Chapter 665 became mandatory in May 2026. Here is what compliance now requires.

Written byTrevor J Henderson

| 6 min read

A laboratory scientist reviews extractables testing results alongside a single-use bioreactor bag, with analytical chemistry equipment visible in a pharmaceutical testing laboratory.

USP Chapter 665, the mandatory US standard for extractables testing of plastic components and systems used in pharmaceutical manufacturing, became enforceable in May 2026. For biopharmaceutical manufacturers using single-use systems, compliance requires a documented risk assessment for every product-contact component, supported by extractables characterization data.

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Highlights in this article

What is the difference between leachables and extractables?
The regulatory framework: USP 665, EMA, and FDA
How are extractables testing studies designed and conducted?
What do supplier data packages cover, and what do they not cover?
What are the common compliance gaps in single-use E&L programs?

Every single-use component in direct contact with a biopharmaceutical drug substance must be characterized for chemical compounds that can migrate from the plastic into the product. Regulatory agencies require this characterization as part of the marketing authorization submission, and USP chapter 665, which became mandatory on May 1, 2026, now defines the enforceable US standard for how that testing must be conducted. Gaps in leachables and extractables documentation are among the most common technical deficiencies cited in regulatory submissions for biologic drug products manufactured using single-use systems.

Key takeaways

USP chapter 665 became mandatory on May 1, 2026, establishing the enforceable US standard for extractables testing of plastic components and systems used in pharmaceutical manufacturing. Single-use biomanufacturing facilities that have not yet aligned their testing programs to USP 665 are now out of compliance.

Extractables and leachables are distinct: extractables are compounds identified by aggressive laboratory testing; leachables are the subset that actually migrate into the drug product under normal process conditions. The regulatory compliance obligation covers both.

Supplier extractables data packages are an input to the drug manufacturer's risk assessment, not a substitute for it. The drug manufacturer bears the compliance responsibility for demonstrating that leachables from their specific components, under their specific process conditions, pose no risk to product safety or patient safety.

The BioPhorum extractables testing protocol is the industry-standard best practice for extractables characterization, widely used by both component suppliers and drug manufacturers, though it is not a formal regulatory document.

Risk assessment under USP 665 is not a pass/fail test. It is a documented analysis of leaching propensity, process contact conditions, downstream dilution, and analytical evaluation against patient safety thresholds for each compound identified.

This article covers the extractables and leachables compliance framework for single-use bioprocessing systems. For the supply chain considerations that affect supplier E&L data package availability, see mitigating supply chain risk in single-use manufacturing. For the full single-use facility regulatory context, see the blueprint for single-use biomanufacturing facilities.

What is the difference between leachables and extractables?

Extractables and leachables are related but distinct concepts that are often conflated in practice, creating compliance gaps when the distinction is not understood precisely.

	Extractables	Leachables
Definition	Chemical compounds that can be released from a plastic material under aggressive or exaggerated laboratory conditions (elevated temperature, extreme solvents, extended contact time)	Chemical compounds that actually migrate into the drug substance or drug product under real process conditions (normal temperature, contact time, and solvent composition)
How identified	Systematic extraction studies conducted in the laboratory using standard solvents and conditions defined by USP 665 or the BioPhorum protocol	Analytical testing of the actual drug substance or product after contact with the component under real process conditions, or risk-based modeling from extractables data
Who generates the data?	Typically generated by the single-use component supplier and provided in an extractables characterization data package	Responsibility of the drug manufacturer; may be based on supplier extractables data (risk assessment) or on direct drug substance testing
Regulatory status	Required testing under USP 665 (mandatory May 2026); extractables data must be generated for all plastic components in drug manufacturing contact	Risk assessment required for all product-contact components; direct leachables testing required when risk assessment indicates patient safety concern cannot be excluded
Key threshold	Analytical evaluation threshold (AET): the concentration below which a compound need not be individually identified in the extractables study	Safety concern threshold (SCT): the concentration in the drug product above which a leachable requires toxicological evaluation for patient safety

The regulatory framework: USP 665, EMA, and FDA

USP general chapter 665, entitled "Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products," became mandatory on May 1, 2026. It defines the enforceable US standard for extractables characterization of plastic components used in pharmaceutical manufacturing, establishing standardized extraction conditions, analytical evaluation thresholds, and documentation requirements. USP chapter 1665 is the companion guidance chapter that provides a risk assessment framework and additional analytical guidance for implementing chapter 665. USP chapters 665 and 1665 are available through the United States Pharmacopeia.

For EU-regulated programs, EMA guidelines for biologics manufacturing require E&L characterization as part of the marketing authorization application. EMA Annex I Part 8.131 specifically addresses the requirements for single-use systems in sterile medicinal product manufacturing. ISO 10993-18, the standard for biological evaluation of medical devices addressing chemical characterization of materials, is also referenced in some regulatory contexts for the characterization of drug-contact materials.

FDA's foundational guidance on container closure systems for packaging human drugs and biologics provides the primary US regulatory framework for demonstrating that materials in contact with drug products are safe and suitable for their intended use. Although primarily directed at finished product packaging, its principles for chemical suitability, protection, and compatibility testing apply directly to single-use manufacturing systems in contact with drug substances. For EU programs, the 2022 revision of EMA GMP Annex I: Manufacture of Sterile Medicinal Products substantially expanded the requirements for single-use systems, requiring manufacturers to demonstrate that all product-contact single-use materials are compatible with the drug product and that E&L risks have been systematically assessed.

BioPhorum's practical roadmap for implementing USP 665 and E&L risk assessments (April 2026) provides timely, implementation-focused guidance for end-users and suppliers navigating the now-mandatory USP 665 requirements. It addresses how to use the USP 665 risk evaluation matrix in conjunction with supplier extractables data to build a defensible leachables risk assessment package suitable for regulatory submission.

The BioPhorum standardized extractables testing protocol, published by BioPhorum (formerly the BioPhorum Operations Group), is the industry-standard best practice for extractables characterization. It is widely used by both single-use component suppliers and drug manufacturers as the framework for generating extractables data packages. The BioPhorum protocol is not a regulatory document, but it is accepted by regulators as a scientifically rigorous approach to extractables characterization when properly implemented and documented.

How are extractables testing studies designed and conducted?

Extractables testing for single-use systems uses standard solvents and extraction conditions designed to identify all compounds that could plausibly migrate from the plastic under process conditions, not just compounds that are expected. The extraction conditions are typically more aggressive than actual process conditions (higher temperature, longer contact time, more aggressive solvents) to ensure that all potential leachables candidates are identified in the extraction study.

Extraction solvents typically include aqueous solutions at both acidic and basic pH, water for injection, and an organic solvent such as ethanol to capture lipophilic compounds. Extracts are analyzed by a battery of analytical techniques including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and inductively coupled plasma-mass spectrometry (ICP-MS) for elemental impurities. Compounds detected above the analytical evaluation threshold must be identified and, for organic compounds, assessed for toxicological risk relative to patient exposure through the drug product.

The analytical evaluation threshold is the compound concentration below which the compound is considered to pose a negligible toxicological concern and need not be individually characterized for patient safety. Compounds detected above the threshold require individual identification and, where the compound is unknown or has no established safety threshold, further toxicological evaluation.

What do supplier data packages cover, and what do they not cover?

Major single-use bioreactor and component suppliers provide extractables characterization data packages for their product portfolios. These packages typically include: extraction study results using standardized solvents and conditions, analytical data for compounds detected above the threshold, compound identification for unknown peaks in the chromatographic data, and in some cases, a preliminary leachables risk assessment based on conservative assumptions about process contact conditions.

What supplier data packages do not cover is the drug-specific leachables risk assessment for the specific drug substance, at the specific concentration and process conditions of the sponsor's manufacturing process. The supplier can characterize what can be extracted from their component; they cannot determine whether those compounds, at the concentrations that would be present in the sponsor's specific drug product under the sponsor's specific process conditions, pose a patient safety risk. That assessment is the responsibility of the drug manufacturer and must be documented and available for regulatory review as part of the marketing authorization application.

For the GMP validation framework within which E&L documentation is generated and maintained, see Lab Manager's guide to GMP compliance and equipment validation efficiency.

What are the common compliance gaps in single-use E&L programs?

The most common E&L compliance gaps identified in regulatory submissions for biologic drug products manufactured with single-use systems are:

Risk assessment not performed: E&L data from the supplier was reviewed but no formal documented risk assessment was conducted to determine whether identified extractables present a leachables concern under the sponsor's process conditions
Process conditions not adequately represented: the risk assessment used conservative default assumptions rather than the sponsor's actual process parameters (temperature, contact time, drug substance concentration, buffer pH) to assess leaching potential
Data coverage incomplete: extractables data is available for some single-use components but not all product-contact components; the risk assessment therefore cannot address the full product-contact surface
Changed components not reassessed: the drug manufacturer switched to an alternative single-use component supplier without conducting a new extractables review or updating the leachables risk assessment for the changed material
USP 665 alignment not demonstrated: extractables testing was conducted using a methodology not aligned to USP 665 standards, and the regulatory reviewer requires supplementary testing data to demonstrate equivalence

This article was produced under Drug Discovery News' AI Editorial Guidelines

Frequently Asked Questions (FAQs)

What is the difference between leachables and extractables?
Extractables are all chemical compounds that can be released from a plastic material under aggressive laboratory extraction conditions. They are identified by systematic laboratory testing using standard solvents at elevated temperatures and extended contact times. Leachables are the subset of extractables that actually migrate into the drug substance or product under normal manufacturing process conditions. The regulatory compliance obligation requires both: extractables testing to identify all candidate compounds, and a leachables risk assessment (and in some cases direct leachables testing) to determine whether those compounds pose a patient safety risk at the concentrations present in the drug product.
What does USP chapter 665 require for single-use bioprocessing systems?
USP chapter 665, which became mandatory on May 1, 2026, requires that plastic components and systems used in pharmaceutical and biopharmaceutical manufacturing be characterized for extractables using standardized testing conditions and analytical methods. The chapter establishes the analytical evaluation threshold below which compounds need not be individually identified, defines the extraction conditions required for different material and contact condition categories, and requires that a formal risk assessment be conducted by the drug manufacturer for all product-contact plastic components. The companion chapter 1665 provides the risk assessment framework and additional guidance for implementing chapter 665 in practice.
Are supplier extractables data packages sufficient for regulatory compliance?
Supplier extractables data packages are a necessary input to regulatory compliance but are not sufficient on their own. The supplier can characterize the compounds that can be extracted from their component under standardized conditions. They cannot assess whether those compounds, at the concentrations present in the drug manufacturer's specific product under the manufacturer's specific process conditions, pose a patient safety risk. That assessment is the drug manufacturer's responsibility and must be documented as a formal leachables risk assessment available for regulatory review. Using supplier data as a compliance endpoint without a sponsor-conducted risk assessment is a common deficiency.
What is the BioPhorum extractables protocol and should I use it?
The BioPhorum standardized extractables testing protocol is a guidance document developed by the BioPhorum industry consortium that provides a standardized approach to extractables characterization for single-use components in biopharmaceutical manufacturing. It is widely used by both suppliers and drug manufacturers as a rigorous, science-based framework for generating extractables data. It is not a regulatory requirement, but it is accepted by regulators as a scientifically valid approach when properly implemented. For facilities conducting their own extractables testing or evaluating supplier data packages, alignment with the BioPhorum protocol provides a strong baseline for regulatory defensibility.
When is direct leachables testing required rather than a risk assessment based on extractables data?
Direct leachables testing in the drug substance or drug product is required when the extractables-based risk assessment cannot exclude a patient safety concern. This occurs when an identified extractable compound exceeds the safety concern threshold in the risk assessment, when the compound is uncharacterized or lacks an established toxicological threshold, when the drug substance formulation includes conditions (extreme pH, high temperature, long contact time) that increase leaching potential beyond what extractables data can conservatively bound, or when the regulatory reviewer requests additional confirmatory data. In most cases, a thorough extractables risk assessment with conservative process condition assumptions will not require direct leachables testing.

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About the Author

Trevor J Henderson
Trevor Henderson is the Creative Services Director for the Laboratory Products Group at LabX Media Group. With over two decades of experience, he specializes in scientific and technical writing, editing, and content creation. His academic background includes training in human biology, physical anthropology, and community health. Since 2013, he has been developing content to engage and inform scientists and laboratorians.
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