Extractables & Leachables in Single-Use Bioprocessing Systems: Risk Assessment, Testing, and USP <665> Compliance

What Are Extractables and Leachables?

Extractables are chemical compounds pulled out of a plastic, elastomer, or filter component under exaggerated laboratory conditions, elevated temperature, aggressive model solvents, and extended contact time, designed to reveal everything that could conceivably migrate out of the material. Leachables are the subset of those extractables that actually migrate into the drug substance or drug product under real, normal manufacturing and storage conditions. Every leachable starts life as an extractable; the extractables study is the ceiling, and the leachables study confirms what actually crosses into your product.

Single-use systems (SUS), bioreactor bags, tubing sets, connectors, filter capsules, and storage bags, are built from a stack of polymers, each formulated with additive packages: antioxidants, slip agents, plasticizers, catalysts, and mold-release compounds. None of these additives are inert forever. Under process conditions they can migrate, at trace but biologically relevant concentrations, into the fluid the polymer contacts. That migration is the entire reason extractables and leachables (E&L) programs exist.

Four variables control how much of an extractable becomes a leachable in a real process:

• Contact time — a media bag held for 30 days leaches far more than a transfer line used for 2 hours
• Temperature — migration rates roughly double for every 10–15°C increase, so autoclaved or steam-in-place components are higher risk
• pH and solvent strength of the process fluid — low pH and high ionic or organic content extract more aggressively than neutral aqueous buffer
• Surface-area-to-volume (SA:V) ratio — thin-walled tubing with a small bore has a much higher SA:V than a large storage bag, concentrating the leachable load per liter of product

An Analytical Evaluation Threshold (AET) is the concentration above which any detected extractable or leachable peak must be identified, quantified, and reported for toxicological review. Compounds that never cross the AET are, by definition, not a safety concern at the tested conditions and do not require further chemical identification, which is what makes AET-based screening tractable: analytical chemists don't have to fully characterize every trace peak, only the ones that matter.

Why E&L Matter for Single-Use Bioprocessing

Extractables and leachables matter because they can silently degrade cell growth, denature protein product, or introduce a patient safety signal, all without triggering any conventional in-process alarm. A bioreactor batch can look normal on pH, DO, and viable cell density trends while a leachable from the bag film quietly suppresses specific growth rate or titer.

The case that put E&L on every bioprocess engineer's radar is bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP). Hammond et al. (2013) traced unexplained CHO cell growth failures back to a degradation product of Irgafos 168, a phosphite antioxidant used to stabilize polyethylene film during gamma irradiation. bDtBPP formed when Irgafos 168 reacted with residual moisture and oxidative stress during sterilization, then leached from the bag film into culture medium at concentrations as low as a few micrograms per liter, enough to measurably inhibit CHO cell proliferation. A follow-up study, Hammond et al. (2014), documented a second cytotoxic leachable compound traced to single-use equipment, reinforcing that bDtBPP was not an isolated incident but part of a broader class of antioxidant-derived leachables.

The downstream consequences of an unmanaged leachable extend well beyond one failed batch:

• Reduced cell growth and titer — leachables can act as weak toxins or metabolic inhibitors at ppb-to-ppm concentrations
• Protein degradation — oxidizing leachables can drive methionine or tryptophan oxidation in the product
• Patient safety risk — leachables that survive purification and reach the final drug product are a direct toxicological concern
• Regulatory findings — an unqualified single-use component change without an E&L assessment is a common Form 483 and warning-letter observation

Dorival-García et al. (2018) performed the largest published survey of this risk, systematically extracting 34 commercial bag films from multiple suppliers and characterizing the resulting extractable chemical profiles by GC-MS and LC-MS. Their results confirmed that extractable identity and concentration vary substantially by film formulation, film supplier, and gamma irradiation dose, which is exactly why a generic "single-use bags are fine" assumption is not sufficient for a risk-based qualification program. More recently, Bossong et al. (2025) modeled how extractables and leachables accumulate across a multi-step bioprocess, from bioreactor bag through downstream hold bags, showing that cumulative leachable exposure across a process train can matter more than any single component's extractables profile in isolation.

USP <665> and <1665>: The New Regulatory Standard

USP General Chapter <665> becomes a mandatory, enforceable compendial standard on May 1, 2026. It defines standardized extraction conditions, analytical evaluation threshold methodology, and documentation requirements for plastic components used in biopharmaceutical manufacturing systems, closing a gap that the industry had previously filled only with voluntary industry protocols.

Before <665>, extractables testing for single-use systems was governed almost entirely by the BPOG (BioPhorum Operations Group) Standardized Extractables Testing Protocol, first published in 2014 and updated in 2020. BPOG is an industry consensus document, not a compendial requirement, and it remains the most detailed practical reference for study design: model solvents, contact conditions, and analytical method scope. USP <665> does not replace BPOG so much as formalize a compendial floor underneath it, while the companion informational chapter USP <1665> explains the risk-based framework manufacturers should use to scope, interpret, and document extractables and leachables studies under <665>.

The practical relationship between the two documents:

• USP <665> — mandatory, sets minimum extraction conditions, AET calculation requirements, and reporting thresholds for plastic components
• USP <1665> — informational guidance chapter explaining how to build a risk-based extractables and leachables program that satisfies <665>
• BPOG Protocol — industry-consensus testing methodology (model solvents, conditions, analytical scope) that most manufacturers use to generate the data <665> requires

Other regional and functional standards round out the regulatory landscape a single-use qualification program has to satisfy: the EMA Guideline on Plastic Immediate Packaging Materials in the EU, the FDA Process Validation Guidance for process-level risk justification, and the BPSA (Bio-Process Systems Alliance) Component Quality Test Reference Matrices, which define what baseline chemical and biological qualification testing a single-use component supplier should already have on file. For manufacturing sites shipping product into the US market, USP <665> compliance after May 1, 2026 is not optional; it becomes part of the compendial testing expectation that FDA inspectors will reference during facility and product reviews.

E&L Testing Workflow: From Risk Assessment to Qualification

A complete extractables and leachables qualification runs through eight sequential steps, starting with a paper-based component inventory and ending with a signed qualification report. USP <665> and the BPOG protocol intersect at two specific decision points in this workflow: model-solvent selection during extraction study design, and the AET comparison gate that decides whether a leachable confirmation study is required.

1. Component identification and inventory — catalog every product-contact plastic, elastomer, and filter material in the process train, including polymer type, supplier, and irradiation history
2. Risk ranking — score each component by contact time × temperature × pH/solvent strength to prioritize which get tested first
3. Extraction study design — select model solvents and conditions per BPOG (or an equivalent justified protocol) to simulate worst-case process exposure
4. Controlled extraction — expose coupons or intact assemblies to model solvents at exaggerated conditions, typically 40°C or 70°C for a fixed contact period
5. Analytical characterization — screen extracts by GC-MS, LC-MS, and ICP-MS to identify and quantify individual extractable compounds
6. AET comparison — compare every detected peak against the calculated Analytical Evaluation Threshold; peaks below AET require no further action
7. Leachable confirmation study — for any compound at or above AET, repeat the analysis using actual process fluid under real operating conditions to confirm real-world migration
8. Toxicological assessment and qualification report — confirmed leachables above AET are risk-assessed by a toxicologist (often against ICH M7), and the full package is documented in a component qualification report

Common Extractables by Polymer Type

Extractable identity and concentration depend heavily on which polymer, and which additive package, a single-use component is built from. Silicone tubing consistently produces the highest extractable load per unit surface area of any common bioprocess material, while EVOH barrier films produce the lowest, a roughly 25-fold spread across materials used in the same process train.

Every polyolefin film used in bioreactor and storage bags is stabilized with an antioxidant package to survive gamma irradiation, which is exactly what makes Irgafos 168 and its degradation product bDtBPP such a recurring finding in extractables surveys. Silicone tubing sheds low-molecular-weight cyclic and linear silicone oligomers continuously, independent of any sterilization event, because these species are a normal byproduct of the polymerization chemistry rather than a stabilizer additive. Nylon-containing components can release caprolactam, the nylon-6 monomer, while polycarbonate housings are watched for Irganox 1010 and mold-release residues.

Metals are a separate extractables category that ICP-MS screens for independently of the organic profile: zinc, barium, and calcium are the most frequently reported trace metals, typically originating from stabilizer salts or catalyst residues rather than the base polymer resin itself.

How to Calculate the Analytical Evaluation Threshold (AET)

The Analytical Evaluation Threshold is calculated by dividing the Safety Concern Threshold (SCT) by the maximum daily dose volume a patient receives, then dividing again by the number of product-contact components that contribute to a single dose. The result is a concentration, typically expressed in micrograms per milliliter, above which any extractable or leachable peak must be identified and toxicologically assessed.

AET (µg/mL) = SCT (µg/day) ÷ [Daily Dose Volume (mL/day) × Number of Contact Units]

The Safety Concern Threshold of 1.5 µg/day is the widely adopted default for parenteral biopharmaceutical products, derived from the Threshold of Toxicological Concern (TTC) framework, below which the probability of any compound posing a carcinogenic or systemic toxicity risk is considered negligible. Compounds with structural alerts for genotoxicity are excluded from this default and instead evaluated under the ICH M7 mutagenic impurity framework, which applies a far more conservative threshold.

In practice, AET values for bioprocess single-use systems typically fall between roughly 0.1 and 5 µg/mL, depending on dose, route of administration, and how many product-contact components a single dose passes through. Lower daily-dose-volume products (potent biologics dosed in single-digit milliliters) push the AET tighter; large-volume infused products with higher daily dose volumes allow a more permissive AET.

Analytical Methods for E&L Characterization

No single analytical technique covers the full chemical space of E&L compounds, so a complete study runs extracts through four complementary methods: headspace GC-MS for volatiles, direct-injection GC-MS for semi-volatiles, LC-MS in both ionization modes for non-volatile organics, and ICP-MS for trace metals.

• HS-GC-MS (headspace GC-MS) — screens volatile and semi-volatile organics by sampling the vapor phase above a heated extract, ideal for residual solvents and low-boiling-point compounds
• GC-MS (direct injection) — characterizes semi-volatile organics injected directly onto the column, the workhorse method for antioxidants, slip agents, and silicone oligomers
• LC-MS (positive and negative ionization modes) — covers non-volatile and polar organics that GC-MS cannot elute, run in both modes because a single ionization polarity misses roughly half the relevant chemical space
• ICP-MS — quantifies 15–20 trace elements simultaneously with detection limits of 0.01–1 ppb, the only practical method for the metals (zinc, barium, calcium) that stabilizer and catalyst residues introduce

The chart below compares total organic extractables measured across the three BPOG model solvents for each polymer type from Table 1, against the AET-equivalent surface loading of ≈1.2 µg/cm² derived in the worked example above.

Risk Matrix: Which Components Need Testing First?

Bioreactor bags and pump tubing carry the highest combined E&L risk score of common single-use components, driven respectively by large surface-area-to-volume ratio and continuous oligomer shedding, and should be first in line for extractables testing when qualification resources are limited.

Risk ranking combines the same four variables introduced in Section 1, contact time, temperature, surface-area-to-volume ratio, and process fluid aggressiveness, scored on a 1–5 scale per component and summed into a total priority score. This is the practical output of Step 2 in the testing workflow: it tells a validation team which components to test first, not which components to skip.

A high-risk score does not automatically mean a component fails qualification, it means the component should be tested early, with the full BPOG model-solvent panel, rather than deferred or covered by a read-across justification from a similar material. Lower-risk components, like sampling assemblies with brief, intermittent contact, are often appropriate candidates for a reduced testing scope or a documented risk-based waiver under USP <1665>.

Frequently Asked Questions

Related Tools

• Filtration Calculator — Size sterile and depth filtration trains using extractable-compatible membrane materials and Vmax scaling.
• Buffer Calculator — Prepare model solvents and process buffers with molarity, dilution, and pH calculations.
• Endotoxin Calculator — Calculate endotoxin limits and LAL sensitivity for single-use filtered drug products.
• Scale-Up Calculator — Model how contact time and surface-area-to-volume ratio shift when scaling single-use bioreactor volume.

References

1. Hammond M, Nunn H, Rogers G, et al. Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers. PDA J Pharm Sci Technol. 2013;67(2):123–134. doi:10.5731/pdajpst.2013.00905
2. Dorival-García N, Carillo S, Ta C, et al. Large-scale assessment of extractables and leachables in single-use bags for biomanufacturing. Anal Chem. 2018;90(15):9006–9015. doi:10.1021/acs.analchem.8b01208
3. Bossong M, Hauk A, Pahl I, Menzel R, Langguth P. Simulating extractables and leachables in biopharmaceutical manufacturing to support safety assessment. Eur J Pharm Sci. 2025;214:107262. doi:10.1016/j.ejps.2025.107262
4. Hammond M, Marghitoiu L, Lee H, et al. A cytotoxic leachable compound from single-use bioprocess equipment that causes poor cell growth performance. Biotechnol Prog. 2014;30(2):332–337. doi:10.1002/btpr.1869
5. Jenke D, Carlson T. A compilation of safety impact information for extractables associated with materials used in pharmaceutical packaging, delivery, administration, and manufacturing systems. PDA J Pharm Sci Technol. 2014;68(5):407–455. doi:10.5731/pdajpst.2014.00995