TECHNOLOGY EXPLAINER
Single-Use Systems
— when the wall of the culture vessel changed from stainless steel to multilayer film
Single-use manufacturing means using culture bags, tubing, connectors and filters once and then discarding them. It removes the need for cleaning and cleaning validation, but in exchange the surface that touches the drug is now a polymer. In 2013 it was reported that a breakdown product of an antioxidant in that polymer inhibits cell growth. For materials engineers, this is the part of cell culture with the most points of contact.
- What a single-use system is, in three points
- The multilayer film of a culture bag: contact layer, gas barrier, outer layer
- Gamma sterilisation and antioxidants: the breakdown product that held back cell growth
- A materials engineer's view (1): antioxidants are necessary, but their breakdown products are the problem
- Extractables and leachables (E&L) and how they are assessed: ICH Q3E, USP <665> and <1665>, BioPhorum
- Our calculation: the smaller the vessel, the more polymer per millilitre
- Compared with stainless steel: cleaning, validation, waste
- The upper limit on volume: 2,000 L and 5,000 L
- A materials engineer's view (2): the supplier now owns part of the drug manufacturing process
- What could not be confirmed, and open problems
- Glossary / References / Claim-to-source audit
Sourced = stated in published material or a peer-reviewed paper (link given)
Our calculation = a value this article derived from assumptions it states
Not yet confirmed = a draft, plan or outlook that is not settled
Structural readings and materials-design interpretations are marked separately as Commentary.
Companies' technical documents are used only for facts about that company's own products.
1. What a single-use system is, in three points
A single-use system is an approach in which the wetted parts of culture vessels and piping are pre-sterilised polymer components that are replaced after every use. A stainless-steel tank is cleaned, steam-sterilised and reused; in single-use manufacturing the whole bag is swapped out.
- The benefit: a 1999 paper on a wave-type bioreactor notes that because it is disposable, it needs no cleaning or sterilisationSourced. Researchers at an equipment maker list a lower risk of cross-contamination and shorter lead times among the advantagesSourced
- How large: a film maker's documents show bags from 50 mL to 2,000 L made from the same film. Another equipment maker sells a 5,000 L single-use bioreactorSourced
- The new problem: because the surface touching the drug is now a polymer, chemical species that leach out of the polymer (leachables) have to be assessed. In 2013 researchers at Amgen reported that a breakdown product derived from an antioxidant in polyethylene strongly inhibits cell growthSourced
A bag maker itself states in its technical literature that as single-use systems replace traditional stainless-steel equipment, suppliers take on a more critical part of the drug manufacturing processSourced. The choice of film resin and additives has become part of the quality of the drug itself (our commentary).
2. The multilayer film of a culture bag: contact layer, gas barrier, outer layer
The wall of a culture bag is not a single polymer but a coextruded multilayer film. Here are two films whose layer structures the makers have published.
| Item | Aegis5-14 (Thermo Fisher) | S80 (Sartorius) |
|---|---|---|
| Structure | Five-layer, 14 mil cast film. Coextruded outer layer of polyester elastomer / EVOH barrier layer / low-density polyethylene contact layer | Coextruded PE | EVOH | PE structure. Contact layer LLDPE; backbone LLDPE and EVOH |
| Thickness | 0.356 mm (0.014 in.) | 400 µm |
| Oxygen transmission rate | 0.36 cc/m²/day (23 °C, 90% RH inside) | “High gas and water-vapour barrier” (no figure in the documents consulted for this article) |
| Water-vapour transmission rate | 0.35 g/m²/day (23 °C) | As above |
| Operating temperature range | −80 °C to 60 °C | Not stated in the documents consulted for this article |
| Sterilisation | Gamma irradiation at 25 to 40 kGy for a sterility assurance level of 10⁻⁶ (stated to comply with ANSI/AAMI/ISO 11137:2006). Physical properties measured after irradiation | Radiation sterilisation under ISO 11137, with gamma dose mapping stated to be carried out |
| Role of the contact layer (as stated by the maker) | Contact material: polyethylene | Film strength and flexibility, weld strength, low extractables, excellent biocompatibility |
All Sourced (Thermo Fisher Aegis5-14 document [Ref. 1], Sartorius Flexsafe document [Ref. 2]). These are each company's statements about its own products and are not representative of other companies' products or the industry as a whole.
- Layer thickness: with 1 mil = 25.4 µm, the Aegis5-14 contact layer is 10.4 × 25.4 ≈ 264 µm and the EVOH layer 1.0 × 25.4 ≈ 25 µm. The whole film, 14 mil ≈ 356 µm, matches the 0.356 mm in the documentOur calculation
- Oxygen passing through: converting 0.36 cc/m²/day as a gas at standard conditions (22.4 L/mol), about 0.016 mmol of oxygen passes through 1 m² per day
- Comparison: as calculated in our bioreactor explainer, CHO cells at 20 million cells/mL use about 4.58 mmol of oxygen per litre of culture per hour. 0.016 / 4.58 = 0.0035 h, or about 13 seconds' worth
Reading: the oxygen that passes through the film is orders of magnitude smaller than what cells in culture consume. What the EVOH gas barrier protects, it seems, is not the oxygen supply during culture but the quality of media, buffers and drug substance during long storage. Sartorius also describes S80's high gas and water-vapour barrier as suited to the long-term storage of media, buffers and drug substanceSourced (our commentary). Assumptions and limits: the transmission rate is a 23 °C value and will differ at the culture temperature of 37 °C. The 22.4 L/mol conversion is also an approximation.
3. Gamma sterilisation and antioxidants: the breakdown product that held back cell growth
Single-use bags arrive already sterilised by gamma irradiationSourced. This is where the question of polymer additives comes in. In 2013 researchers at Amgen (Hammond and colleagues) reported the following in the PDA JournalSourced.
- What was found: of the many chemical species extracted at low levels from bag materials, bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP) was strongly detrimental to cell growth
- Where it came from: it is a breakdown product of tris(2,4-di-tert-butylphenyl) phosphite (trade name Irgafos 168), a common antioxidant in many formulations of polyethylene, the polymer often used for contact layers
- At what level: detrimental effects appeared in several mammalian cell lines at concentrations well below the ppm level
- Under what conditions: migration from the film depended on time and temperature. Exposure of oxidised Irgafos 168 to ionising radiation such as gamma rays was suggested as an important condition for producing large amounts of bDtBPP
4. A materials engineer's view (1): antioxidants are necessary, but their breakdown products are the problem
Sartorius's 2020 white paper cites the report by Hammond and colleagues and then makes the following point.
Antioxidants are needed to keep the film stable, protecting the polymer from oxidation during extrusion and gamma irradiation. To ensure proper cell growth, it is critical to optimise the concentration of these additives and control it tightlySourced.
The company says that in its S80 film the concentration of phosphite antioxidant is limited, and that in a separate ethylene vinyl acetate film (S71) this antioxidant is not included in the contact layerSourced. It also shows in a figure that, if the additive formulation is not controlled, the age of a bag (its storage time after irradiation) can affect cell growthSourced.
The lesson is that gamma sterilisation is not just a step before use; it is a step that changes the chemistry of the material.
- Cut the antioxidant too far, and the polymer itself degrades during extrusion and irradiation
- Add too much, and irradiation produces more breakdown products that harm the cells
- And the amount of breakdown product changes with extrusion conditions, irradiation conditions and storage time
In another document the company also writes that transparency does not mean purity: there is no correlation between a film's clarity and its extractables and leachables profileSourced. A film that looks clean is not necessarily clean from the cells' point of view. Only suppliers able to manage additive formulation, extrusion, irradiation and storage as one system can meet the basic conditions of this market: that is how this reads (our commentary).
5. Extractables and leachables (E&L) and how they are assessed: ICH Q3E, USP <665> and <1665>, BioPhorum
(1) Definitions: the ICH Q3E draft
The draft of Q3E, the extractables and leachables guideline of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), defines the two terms as followsSourced.
- Leachables: chemical entities that migrate into the drug product from manufacturing components and systems, packaging, or delivery device components under the defined manufacturing conditions and labelled storage conditions
- Extractables: chemical entities deliberately extracted from the same components under defined laboratory test conditions, and therefore potential leachables
(2) What the ICH Q3E draft says about manufacturing components
For components and systems used in drug manufacturing (including polymeric manufacturing equipment components), the draft says the followingSourced.
- Its scope includes cell and gene therapy products
- Extraction studies on manufacturing components should be designed to represent the worst case of the manufacturing conditions (for example, the smallest scale with the longest contact time, and the highest temperature and pressure)
- The leachables risk from manufacturing components is recognised as lower than that from packaging, because contact times are relatively short and the volume of liquid relative to surface area is large
- Leachables introduced upstream may be removed in downstream steps
- Where several components of the same or similar materials are used, the leachables risk should be assessed cumulatively
- If all extractable peaks are at or below the analytical evaluation threshold (AET) and there are no Class 1 leachables, the risk may be considered minimal and acceptable
(3) USP <665> and <1665>
The United States Pharmacopeia (USP) has two chapters on plastic components and systems used in drug manufacturingSourced.
| Chapter | Title (as on the USP official page) | Status as confirmed for this article |
|---|---|---|
| <665> | Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products | The USP page states that it is an informational chapter and does not apply as a compendial requirement unless specified by a regulatory or enforcement authority |
| <1665> | Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products | A chapter on the characterisation and qualification of plastic components and systems (full text available to subscribers) |
All Sourced (the official USP page for each chapter [Refs. 6 and 7]). The full text of each chapter (details of test methods, solvents and conditions) is for subscribers and was not checked for this article. The official dates are also not stated, because the USP notice page could not be accessed and they could not be confirmed in primary sources.
(4) The BioPhorum extractables testing protocol
BioPhorum (formerly BPOG), an industry group of drug makers and suppliers of single-use components, updated its extractables testing protocol on 22 April 2020. According to the group's page, the main changes from the 2014 version were as followsSourced.
- 5 M sodium chloride and 1% polysorbate 80 were dropped from the extraction solvents (because their inherent extraction power was low)
- Time-zero sampling was dropped (because compounds detected then were detected at higher levels at later time points)
- Elemental analysis of the 50% ethanol extract was dropped
- The overall testing burden was cut by 30 to 50%, and an “extractables ecosystem” framework for sharing extractables data across the supply chain was introduced
Underlying all of this is a US federal regulation, 21 CFR 211.65, which requires that “surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements”Sourced. The requirement is the same for stainless steel and for polymer (our commentary).
6. Our calculation: the smaller the vessel, the more polymer per millilitre
The ICH Q3E draft names “the smallest scale” as a worst case, and gives “the large volume of liquid relative to surface area” as a reason why manufacturing components carry lower riskSourced. Both follow from the fact that the larger the vessel, the less polymer area there is per millilitre of liquid. Here is the calculation for a cubic vessel.
- Assumption: treat the vessel as a completely filled cube with all six faces wetted (real bags are not cubes)
- Formula: a cube of side L cm has an area of 6L² cm² and a volume of L³ mL, so area / volume = 6 / L (cm²/mL)
- 1 L (side 10 cm): 6 / 10 = 0.60 cm²/mL
- 100 L (side 46.4 cm): 6 / 46.4 = 0.13 cm²/mL
- 2,000 L (side 126 cm): 6 / 126 = 0.048 cm²/mL
Per millilitre of liquid, a 1 L vessel touches about 12.6 times as much polymer as a 2,000 L vesselOur calculation. A 1,000-fold increase in volume cuts area / volume to a tenth (one over the cube root of 1,000). Assumptions and limits: flat bags (2D bags) have a larger area / volume than a cube. The area of tubing, connectors and filters is not included.
7. Compared with stainless steel: cleaning, validation, waste
| Aspect | Stainless steel (reused) | Single-use (disposable) |
|---|---|---|
| Cleaning and sterilisation | Clean-in-place (CIP) and steam-in-place (SIP) are required. US federal regulations require written procedures for cleaning and maintenance (responsibility, schedules, methods, removal of previous batch identification and so on) [Ref. 12] | “Disposable, so no cleaning or sterilisation is needed” [Ref. 17]. Supplied gamma-sterilised [Ref. 1] |
| Water and energy | Water use is mainly associated with CIP and SIP and is a major environmental burden. Producing purified water and water for injection, and cleaning and sterilisation, are energy-intensive [Ref. 13] | (Water and energy use are expected to fall because CIP and SIP are no longer needed) [Ref. 13] |
| Speed of deployment | Fixed equipment is costly, and installing and qualifying tanks and their utilities takes long lead times [Ref. 14] | Shorter lead times and lower cross-contamination risk [Ref. 15]. Design changes can be implemented faster [Ref. 14] |
| Accumulated design knowledge | Because its geometry is well known and defined, it is described as “still the gold standard” [Ref. 15] | Shapes, agitation principles and aeration methods differ between products, which can make process transfer and scale-up a challenge [Ref. 15] |
| Assessment of the material | The wetted surface is metal. The requirement of 21 CFR 211.65 is the same [Ref. 11] | Extractables and leachables from the polymer must be assessed (Section 5) |
| Waste | The equipment is used again and again | Used bags, tubing and filters become solid waste (our commentary) |
Items with a source are Sourced. However, “water and energy use fall” is an expectation stated in the conclusion of Bunnak and colleagues (2016), who compared fed-batch and perfusion processesNot yet confirmed; that paper itself covers stainless-steel facilities and leaves the manufacture of consumables outside its scope [Ref. 13].
In the life-cycle assessment by Bunnak and colleagues, CIP and SIP accounted for more than 85% of total water use in a perfusion process under one set of conditionsSourced. The Amgen researchers also write, in the lay summary of their paper, that replacing stainless-steel tanks with disposable bags brings significant environmental and cost advantagesSourced. On the other hand, a comparison that includes the manufacture and disposal of the single-use components themselves could not be found in the primary sources consulted for this article.
8. The upper limit on volume: 2,000 L and 5,000 L
Thermo Fisher describes its single-use bioreactor as available from 5 L to 5,000 L and as the first 5,000 L single-use bioreactor on the market, and says Aegis5-14 film can be used for its bagsSourced. Stainless-steel tanks, meanwhile, have dominated large-scale manufacturing at 1,000 to more than 25,000 LSourced.
What sets the upper limit? The primary sources offer two clues.
- The weight of the liquid: Sartorius says bags for large-scale mixing and stirred tanks must withstand the high hydrostatic pressure generated by 2,000 to 3,000 L of liquidSourced
- How hard it can be stirred: in large single-use tanks, physical constraints are said to limit the maximum P/V to around 20 to 30 W/m³Sourced
Assumptions: a cylinder with height / diameter = 2 (the ratio of Dreher and colleagues' stirred tanks) and a liquid density of 1,000 kg/m³.
From volume V = (π/4)D² × 2D = (π/2)D³ = 2 m³, D ≈ 1.08 m and liquid depth H ≈ 2.17 m.
Hydrostatic pressure at the bottom = 1,000 × 9.81 × 2.17 ≈ 21 kPa (about 0.21 atm)Our calculation.
The pressure itself is not large, but it means that a film 0.36 to 0.4 mm thick is holding two tonnes of liquid while being pressed against a rigid outer container. That Sartorius lists, for large 3D bags, a point-of-use test that detects large leaks (100 to 200 µm) after installation in the appropriate containerSourced can be read as reflecting that some things can only be checked in that state (our commentary). Assumptions and limits: the shape and liquid depth of real tanks differ by product, and the stress on the film is set by how it bears against the outer container.
9. A materials engineer's view (2): the supplier now owns part of the drug manufacturing process
With stainless-steel equipment, a drug maker only had to specify the material (steel grade, surface finish) and validate the cleaning. With single-use, resin, additives, extrusion, welding, irradiation and storage all sit inside the supplier's process. Sartorius's document says it designs, manufactures, quality-controls and sterilises its bags under conditions that mirror drug manufacturing, meeting requirements close to cGMPSourced.
On top of that, the “language” of assessment is taking shape.
- The ICH Q3E draft: definitions of extractables and leachables, the worst-case approach, cumulative risk assessmentSourced
- USP <665> and <1665>: chapters on plastic components used in manufacturing (with <665> stated to be informational)Sourced
- The 2020 BioPhorum protocol: standard extraction testing with fewer solvents and time points, and a framework for sharing dataSourced
For materials makers, there are two implications (our commentary).
- Disclosing and controlling additives in a resin grade becomes a source of value. A resin whose antioxidant type and amount, and the behaviour of its breakdown products (particularly after gamma irradiation), can be explained is easier for film makers to build assessment data on
- Standardised data can be reused for the next customer. As BioPhorum's “extractables ecosystem” for sharing data across the supply chain makes clearSourced, data gathered once under the protocol gets reused. Conversely, changing the resin or additive means rebuilding that body of data
It is the same pattern as the “development standard material” discussed in our explainer on argyrodite solid electrolytes. A material that has become the basis of a body of assessment data is not easily displaced, even by a competitor with equal performance (our commentary).
Sartorius says that for 2D bags and 3D bags up to 500 L, pre-shipment integrity testing guarantees no defects larger than 2 µm, and gives detection limits for point-of-use testing of 10 µm for 2D bags and 100 to 200 µm for large leaks in large 3D bags after installationSourced.
The larger the volume, the coarser the defects that can be detected on site. This means that not creating defects in the film and welds in the first place matters more than inspection (our commentary). A multilayer film about 0.4 mm thick holds two tonnes of liquid while keeping it sterile. The uniformity of the material and the process capability of the welding become, in effect, the product's sterility assurance.
10. What could not be confirmed, and open problems
(1) The full text and official dates of USP <665> and <1665>
The chapter texts are for subscribers, and details such as test solvents and conditions were not checked for this article. The official dates are also not stated, because the USP notice page could not be accessed and they could not be confirmed in primary sources.
(2) ICH Q3E is still a draft
It is a draft published at Step 2 on 1 August 2025, and the work plan expects the final version (Step 4) in June 2027Not yet confirmed. The content may change before it is finalised.
(3) Layer structures of other companies' films
This article covers only the two products whose layer structures and thicknesses the makers show in published documents. Resin names and layer structures of other companies' products are not stated beyond what could be confirmed in primary sources.
(4) An overall comparison of environmental impact
Within the scope of this article, no primary source could be found comparing single-use and stainless steel with the manufacture and disposal of single-use components included.
- The wall of a culture bag is a multilayer film. In the published examples the contact layer is polyethylene, with an EVOH gas barrier at the coreSourced
- What the gas barrier protects is quality during storage. The oxygen passing through the film was orders of magnitude less than what cells consumeOur calculation
- A breakdown product of an antioxidant in polyethylene (bDtBPP) inhibits cell growth, as reported in 2013, with gamma irradiation suggested as an important conditionSourced
- The assessment framework is taking shape in the ICH Q3E draft, USP <665> and <1665>, and the BioPhorum protocol. Q3E is still a draftNot yet confirmed
- The smaller the vessel, the more polymer area per millilitre of liquid: 1 L is about 12.6 times 2,000 LOur calculation
- Single-use products have been published up to 2,000 L, and at most 5,000 L, while stainless steel reaches beyond 25,000 LSourced
11. Glossary
- Single-use system
- A manufacturing approach in which wetted parts are pre-sterilised polymer components replaced after each use.
- Coextruded film
- A single multilayer film made by extruding several polymers at the same time.
- Contact layer
- The layer of a film in direct contact with the culture or drug. Polyethylene in the published examples.
- EVOH
- Ethylene vinyl alcohol copolymer. Used as a gas-barrier layer that lets little oxygen through.
- LLDPE
- Linear low-density polyethylene. Flexible and easy to weld.
- Gamma sterilisation
- Sterilisation that kills microorganisms with radiation, to the ISO 11137 standard. It can also change the chemistry of polymers and additives.
- Sterility assurance level (SAL)
- The probability that a single microorganism survives sterilisation. 10⁻⁶ means one in a million.
- Extractables
- Chemical entities deliberately extracted from components under laboratory conditions. Potential leachables.
- Leachables
- Chemical entities that migrate into the drug under real manufacturing and storage conditions.
- bDtBPP
- A breakdown product of the antioxidant Irgafos 168, reported to inhibit cell growth.
- AET
- Analytical evaluation threshold. A guide above which extractables are identified and assessed.
- CIP / SIP
- Clean-in-place / steam-in-place. Cleaning and sterilising stainless-steel equipment without taking it apart.
- BioPhorum (formerly BPOG)
- An industry group of drug makers and suppliers. Publishes a standard protocol for extractables testing.
- Integrity testing
- Testing that confirms a bag has no leaks (pinholes and the like).
12. References (primary sources)
- Thermo Fisher Scientific “Thermo Scientific Aegis5-14 film — Five-layer, 14 mil cast film”, product document (COL01764, 2019) — documents.thermofisher.com
- Sartorius “Flexsafe 2D & 3D Pre-Designed Solutions for Storage and Shipping”, product document — sartorius.com
- Sartorius “Flexsafe New PE Film. New Benchmark.”, product document (SPT1503) — api.sartorius.com
- Sartorius (Lugari A, Schenk T) “Cell Growth Performance in Single-use Bags”, white paper (15 April 2020) — sartorius.com
- Hammond M et al. (Amgen) “Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers”, PDA J Pharm Sci Technol 67(2):123–134 (2013) (PubMed abstract) — pubmed.ncbi.nlm.nih.gov
- United States Pharmacopeia (USP) “<665> Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products”, official page — doi.usp.org
- United States Pharmacopeia (USP) “<1665> Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products”, official page — doi.usp.org
- ICH “Guideline for Extractables and Leachables Q3E”, draft (Step 2, 1 August 2025) — database.ich.org
- ICH “ICH Q3E EWG Work Plan” (8 February 2025) — database.ich.org
- BioPhorum “Disposables: Extractables testing of polymeric single-use components used in biopharmaceutical manufacturing” (22 April 2020) — biophorum.com
- US Code of Federal Regulations “21 CFR 211.65 Equipment construction” — ecfr.gov
- US Code of Federal Regulations “21 CFR 211.67 Equipment cleaning and maintenance” — ecfr.gov
- Bunnak P et al. (University College London and others) “Life-cycle and cost of goods assessment of fed-batch and perfusion-based manufacturing processes for mAbs”, Biotechnology Progress 32(5):1324–1335 (2016) — pmc.ncbi.nlm.nih.gov
- Li F et al. (Genentech) “Cell culture processes for monoclonal antibody production”, mAbs 2(5):466–479 (2010) — pmc.ncbi.nlm.nih.gov
- Dreher T et al. (Sartorius Stedim Biotech) “Design space definition for a stirred single-use bioreactor family from 50 to 2000 L scale”, BMC Proceedings 7(Suppl 6):P55 (2013) — pmc.ncbi.nlm.nih.gov
- Thermo Fisher Scientific “DynaDrive Single-Use Bioreactor”, product page — thermofisher.com
- Singh V “Disposable bioreactor for cell culture using wave-induced agitation”, Cytotechnology 30:149–158 (1999) — pmc.ncbi.nlm.nih.gov
- Lemire L et al. “Scale-up of a monoclonal antibody CHO fed-batch production in stirred tank bioreactors: Effect of hydrodynamic conditions and feeding regimen”, Biotechnology Progress (January–February 2026 issue, doi:10.1002/btpr.70073) — pmc.ncbi.nlm.nih.gov
13. Claim-to-source audit
| Claim in the text | Basis | Label |
|---|---|---|
| That Aegis5-14 is a five-layer, 14 mil cast film coextruded from a polyester elastomer outer layer, an EVOH barrier layer and a low-density polyethylene contact layer. The layer order in the schematic (polyester / tie / EVOH / tie / polyethylene) and values (0.8 / 0.9 / 1.0 / 0.9 / 10.4). Thickness 0.356 mm, oxygen transmission 0.36 cc/m²/day, CO2 transmission 1.35 cc/m²/day, water-vapour transmission 0.35 g/m²/day, −80 °C to 60 °C, gamma irradiation at 25 to 40 kGy for SAL 10⁻⁶ (ANSI/AAMI/ISO 11137:2006), polyethylene as the contact material, BPCs from 50 mL to 2,000 L, and supply in gamma-irradiated form | Thermo Fisher Aegis5-14 document. Reference 1 https://documents.thermofisher.com/TFS-Assets/BPD/Datasheets/aegis5-14-film-fact-sheet.pdf | Sourced |
| That S80 is a 400 µm coextruded PE | EVOH | PE structure (contact layer LLDPE; backbone LLDPE and EVOH). The role of the contact layer (strength, flexibility, weld strength, low extractables, biocompatibility). That the high gas and water-vapour barrier suits long-term storage of media, buffers and drug substance. ISO 11137 and dose mapping. Minimising antioxidants. That the company makes its bags under conditions that mirror drug manufacturing, and states that suppliers take on a more critical part of the drug manufacturing process. No defects above 2 µm in pre-shipment integrity testing (2D, and 3D up to 500 L); point-of-use detection limits of 10 µm (2D) and 100 to 200 µm for large 3D bags after installation | Sartorius Flexsafe 2D & 3D document. Reference 2 https://www.sartorius.com/download/340044/broch-flexsafe-2d-3d-bags-sp-1518-e-data.pdf | Sourced |
| That transparency does not mean purity (no correlation between film clarity and extractables and leachables profile). That bags for large-scale mixing and stirred tanks must withstand the hydrostatic pressure of 2,000 to 3,000 L of liquid | Sartorius Flexsafe New PE Film document. Reference 3 https://api.sartorius.com/document-hub/dam/download/5807/Flexsafe-New-PE-Film-Benchmark-Brochure-en-B-SPT1503-Sartorius.pdf | Sourced |
| That antioxidants are needed to keep the film stable and protect the polymer during extrusion and gamma irradiation; that optimising and tightly controlling their concentration is critical. That phosphite antioxidant is limited in S80 and not included in the contact layer of S71 (EVA-based). That without control of additives, bag age can affect cell growth. Extraction with water and ethanol, and cell-growth testing on irradiated bags | Sartorius white paper (2020). Reference 4 https://www.sartorius.com/download/13284/white-paper-cellgrowth-flexsafe-flexboy-spt1103-e-data.pdf | Sourced |
| That bDtBPP is strongly detrimental to cell growth. That it is a breakdown product of Irgafos 168 (tris(2,4-di-tert-butylphenyl) phosphite), which is present in many polyethylene formulations. That it is harmful at concentrations well below the ppm level. That migration depends on time and temperature. That exposure of oxidised Irgafos 168 to ionising radiation was suggested as an important condition. The statement that replacing stainless-steel tanks with disposable bags brings significant environmental and cost advantages | Hammond et al. 2013 (PubMed abstract). Reference 5 https://pubmed.ncbi.nlm.nih.gov/23569073/ | Sourced |
| The title of USP <665>, and the statement that it is an informational chapter that does not apply as a compendial requirement unless specified by a regulatory or enforcement authority | USP <665> official page. Reference 6 https://doi.usp.org/USPNF/USPNF_M11135_02_01.html | Sourced |
| The title of USP <1665> (characterisation and qualification of plastic components and systems) | USP <1665> official page. Reference 7 https://doi.usp.org/USPNF/USPNF_M11136_02_01.html | Sourced |
| The definitions of leachables and extractables. That the scope includes cell and gene therapy products. The three stages of risk assessment and example measures. That extraction studies on manufacturing components should be designed for the worst case (smallest scale, longest contact, highest temperature and pressure). That manufacturing components carry lower risk than packaging because of short contact times and a large volume relative to surface area. That upstream leachables may be removed downstream. Cumulative risk assessment. That risk is acceptable if all peaks are at or below the AET and there is no Class 1 leachable. That the draft was published at Step 2 on 1 August 2025 | ICH Q3E draft. Reference 8 https://database.ich.org/sites/default/files/ICH_Q3E_EWG_Step2_DraftGuideline_2025_0704.pdf | Sourced |
| That the final version of Q3E (Step 4) is scheduled for June 2027 | A schedule stated in the ICH Q3E work plan, not a settled fact. Reference 9 https://database.ich.org/sites/default/files/ICH_Q3E_EWG_WorkPlan_2024_0214.pdf | Not yet confirmed |
| That BioPhorum updated its protocol on 22 April 2020, dropping 5 M sodium chloride and 1% polysorbate 80, time-zero sampling and elemental analysis of the 50% ethanol extract, cutting the testing burden by 30 to 50%, and introducing the extractables ecosystem | BioPhorum published page. Reference 10 https://www.biophorum.com/download/extractables-testing-of-polymeric-single-use-components-used-in-biopharmaceutical-manufacturing/ | Sourced |
| The equipment-construction requirement that contact surfaces shall not be reactive, additive or absorptive | 21 CFR 211.65(a). Reference 11 https://www.ecfr.gov/current/title-21/chapter-I/subchapter-C/part-211/subpart-D/section-211.65 | Sourced |
| The requirements for equipment cleaning and maintenance and their written procedures (responsibility, schedules, methods, removal of previous batch identification and so on) | 21 CFR 211.67. Reference 12 https://www.ecfr.gov/current/title-21/chapter-I/subchapter-C/part-211/subpart-D/section-211.67 | Sourced |
| That water use is mainly associated with CIP and SIP and is a major environmental burden. That producing purified water and water for injection, and cleaning and sterilisation, are energy-intensive. That CIP and SIP accounted for more than 85% of total water use in one perfusion process. That the paper covers stainless-steel facilities and excludes the manufacture of consumables from its scope | Bunnak et al. 2016. Reference 13 https://pmc.ncbi.nlm.nih.gov/articles/PMC5082523/ | Sourced |
| That moving to disposable equipment is expected to reduce environmental impact | An expectation stated in the conclusion of Bunnak et al. 2016, not a result assessed in that paper. Reference 13 https://pmc.ncbi.nlm.nih.gov/articles/PMC5082523/ | Not yet confirmed |
| That stainless-steel tanks have dominated large-scale manufacturing (1,000 to more than 25,000 L). That fixed equipment is costly and slow to install and qualify. That disposables allow faster design changes | Li et al. 2010 review. Reference 14 https://pmc.ncbi.nlm.nih.gov/articles/PMC2958569/ | Sourced |
| Lower cross-contamination risk and shorter lead times as advantages of single-use tanks. That reusable tanks are regarded as the gold standard because their geometry is well understood. That single-use tanks differ in shape, agitation principle and aeration, which can make transfer and scale-up a challenge. The 50 to 2,000 L family, H/D 2:1 | Dreher et al. 2013. Reference 15 https://pmc.ncbi.nlm.nih.gov/articles/PMC3980816/ | Sourced |
| That DynaDrive runs from 5 L to 5,000 L, is described as the first 5,000 L single-use bioreactor on the market, and can use Aegis5-14 film for its bags | Thermo Fisher product page (a statement about its own product). Reference 16 https://www.thermofisher.com/us/en/home/bioprocessing/products/bioreactors/single-use-dynadrive.html | Sourced |
| That the wave-type bioreactor is disposable and needs no cleaning or sterilisation | Singh 1999 (abstract). Reference 17 https://pmc.ncbi.nlm.nih.gov/articles/PMC3449934/ | Sourced |
| That physical constraints limit the maximum P/V in large single-use tanks to around 20 to 30 W/m³ | Lemire et al. 2026. Reference 18 https://pmc.ncbi.nlm.nih.gov/articles/PMC12908111/ | Sourced |
| Layer thicknesses (contact layer about 264 µm, EVOH about 25 µm, contact layer about 74%). That the roughly 0.016 mmol of oxygen passing through 1 m² per day equals about 13 seconds' worth for 1 L of culture at 20 million cells/mL. Wetted area per mL of liquid (0.60 cm²/mL at 1 L, 0.048 cm²/mL at 2,000 L, about 12.6 times). Hydrostatic pressure of about 21 kPa at the bottom of a 2,000 L bag | Our calculation. 1 mil = 25.4 µm, 22.4 L/mol, oxygen uptake of 5.5 pmol/cell/day, a completely filled cube, a cylinder with H/D = 2 and a density of 1,000 kg/m³ are assumptions set by this article | Our calculation |
| The full text, test conditions and official dates of USP <665> and <1665>. The layer structures of other companies' films. An overall comparison of environmental impact including the manufacture and disposal of single-use components | Not stated because the text is for subscribers and was not checked, or because it could not be confirmed in primary sources within the scope of this article (commentary) | Commentary |
| The reading that the gas barrier protects quality during storage. The framing that gamma sterilisation is a step that changes the chemistry of the material. The framing that the supplier's process control is the price of entry. The reading that reuse of standardised data makes replacement harder. The reading that on-site detectable defects get coarser with volume, so the process capability of not creating defects matters. The point that the 21 CFR 211.65 requirement is the same for stainless steel and polymer. That used components become solid waste. The organisation of the comparison table | This article's own framing and commentary based on published content. Not views expressed by the companies or institutions | Commentary |
| That Figs. 1 to 5 are explanatory drawings, not real product cross-sections or measurement results. That the layer thickness ratios on the right of Fig. 1 are an equal-split schematic. That the hero image and Fig. 6 are AI-generated images | A note by this article (commentary) | Commentary |
Last updated 23 September 2026. Sources are limited to primary material (film and equipment makers' documents on their own products, peer-reviewed papers, and published material from ICH, USP, the US Code of Federal Regulations and BioPhorum). Makers' documents are treated only as statements about that company's own products. The full text and official dates of USP <665> and <1665>, the layer structures of other companies' films, and an overall comparison of environmental impact including the manufacture and disposal of single-use components are not covered, because they could not be confirmed in published primary sources. ICH Q3E is at the draft (Step 2) stage. All figures are explanatory concept graphics. Figs. 1 to 5 are vector drawings; the hero image and Fig. 6 are AI-generated images, and none of them shows a real product cross-section or an actual product.