Aseptic Sampling in Bioprocessing Explained: Closed Single-Use Sampling and Sample Integrity

SINGLE-USE BIOPROCESSING & PROCESS SAMPLING

Aseptic sampling must protect both the process and the sample

Process sampling provides the analytical link between a bioprocess and the measurements used to understand, control and release it. In pharmaceutical and biotechnology manufacturing, however, withdrawing a sample creates two simultaneous engineering requirements.

The sampling operation must protect the process from contamination while also delivering a sample that accurately represents the process condition being investigated.

A closed or controlled single-use sampling assembly can help maintain a defined fluid path between the process and collection container, but the quality of the result still depends on sampling-point location, hold-up volume, tubing geometry, sample volume, carryover, timing and subsequent sample handling.

Example of a configurable single-use bottle manifold. Bottle assemblies can form part of a controlled sampling fluid path using application-specific tubing and connection technology.

What is aseptic bioprocess sampling?

Aseptic sampling is the withdrawal of process material using a procedure and sampling path designed to minimise contamination of the process and the sample.

In bioprocessing, samples may be collected to evaluate parameters such as:

• cell concentration and viability;
• metabolite concentrations;
• substrate consumption;
• product titre;
• pH, osmolality or conductivity;
• bioburden or microbiological status;
• protein concentration and purity;
• process impurities;
• critical quality attributes during development or manufacturing.

The analytical method may be highly accurate, but its result is only meaningful if the sample reaching the analyser still represents the process state that existed at the intended sampling time.

Process integrity and sample integrity are different questions

Why representative sampling matters

Representative sampling has been recognised for decades as a fundamental part of bioprocess monitoring. Mattiasson and Håkanson described sampling and sample handling as crucial steps in process monitoring because the sampled material must allow the state of the bioprocess to be assessed reproducibly and reliably.

Later work on single-use bioreactors reaches the same practical conclusion: sampling ports must preserve sterility while providing a representative sample stream for frequent analysis.

Representativeness can be influenced by several factors:

• position of the sampling point within the process;
• local mixing conditions;
• cell or particle distribution;
• sample-line hold-up volume;
• residual liquid from a previous sample;
• required flush or displacement strategy;
• sample withdrawal rate;
• sample volume;
• time between withdrawal and measurement;
• temperature and storage conditions after withdrawal.

Sampling location is part of the measurement system

A sample can only represent the process region from which it is withdrawn. Poor mixing, settling, flotation, local concentration gradients or the presence of cells and particles can make some sampling locations less representative than others.

This is particularly relevant for cell-containing samples, suspensions and other heterogeneous process streams.

There is therefore no universal sampling-point geometry that is optimal for every process. The location should be selected according to the process dynamics, the property being measured and the purpose of the analytical result.

Closed sampling and EU GMP terminology

EU GMP Annex 1 defines a closed system as one in which sterile product is not exposed to the surrounding environment. The definition explicitly includes disposable bag and manifold systems as examples of architectures that can be used to create closed processing systems.

In practice, a single-use sampling assembly may connect a process vessel or bioreactor to one or more preconfigured sample containers using tubing, connectors and isolation devices.

Whether a particular installation qualifies as a closed system depends on the complete configuration and operating procedure rather than on the presence of one individual component.

Hold-up volume: the hidden variable in a sampling line

Between the process sampling point and the final collection container there is usually a finite internal fluid volume.

This is commonly described as hold-up volume or, depending on context, sampling-line dead volume.

For a simple straight circular tube, the geometric liquid volume can be estimated from:

In a real assembly, however, the complete hold-up volume can also include connectors, valves, manifolds, reducers and other internal cavities.

This matters because the liquid initially entering the collection vessel may not be fresh process material. It can include material that remained in the line following a previous sampling event.

Why there is no universal flushing multiple

It is sometimes tempting to specify that every sampling line should be flushed with a fixed multiple of its calculated internal volume.

That is not universally defensible.

Fluid exchange depends not only on nominal volume but also on:

• line geometry;
• branches and poorly swept regions;
• flow regime;
• valve cavities;
• fluid viscosity;
• required analytical sensitivity;
• acceptable process loss.

The appropriate displacement or conditioning strategy should therefore be established for the actual sampling assembly and analytical objective.

Carryover between sequential samples

A multi-sample system introduces another question: how independent is each sample from the one collected before it?

Residual material in a common sampling line can cause cross-sample carryover. This becomes especially relevant when:

• process conditions are changing rapidly;
• samples are small;
• analytical sensitivity is high;
• one common fluid path is used for multiple collection events;
• the previous sample differs substantially in concentration from the next.

Recent automated-sampling research has specifically examined priming, washing, purging and low-dead-volume designs to reduce cross-talk between consecutive samples.

A 2024 A*STAR study developed a single-use sampling unit for cell and gene therapy manufacturing capable of collecting volumes between 0.02 and 1.00 mL while minimising dead volume. The authors also evaluated repeated microbial ingress and cross-sample carryover.

A subsequent 2025 study involving the Singapore-MIT Alliance for Research and Technology, A*STAR, MIT and National University of Singapore demonstrated an automated aseptic platform capable of accurately sampling volumes down to 30 µL in the tested system.

These values describe those specific experimental systems and should not be interpreted as universal performance specifications for single-use sampling assemblies.

Small sample volume can be critical in cell and gene therapy

In large production bioreactors, withdrawing several millilitres may represent a negligible fraction of the process volume.

In small-scale cultures, microbioreactors and autologous cell-therapy processes, repeated sampling can remove a meaningful proportion of the available culture.

Sampling-system design may therefore need to balance:

• the minimum analytical volume required by the assay;
• the volume required to condition or flush the sampling path;
• the amount of valuable process material removed;
• sampling frequency;
• analytical sensitivity;
• carryover requirements;
• process sterility.

Tubing dimensions influence more than connection size

Tubing inner diameter and length influence the internal volume of the sampling path and therefore affect the amount of process material required to fill, condition or displace the line.

Tubing selection can also interact with:

• required sample flow rate;
• available pressure or pumping method;
• peristaltic-pump compatibility;
• connector dimensions;
• required sample volume;
• hold-up-volume target;
• material compatibility;
• sterilisation method.

Filtra Custom Single-Use Tubing Assemblies can be configured as sampling assemblies as well as transfer lines, manifolds, filtration sets and other defined disposable fluid paths.

Bottle, bag or manifold: selecting the collection architecture

The choice of sample container depends on what will happen to the sample after withdrawal and how the sampling operation is performed.

Filtra's Custom Single-Use Bottle Assemblies can be configured specifically for process sampling, including individual bottle arrangements and multi-container manifolds.

Single-use bags can form part of the wider sampling architecture

Flexible bags are not limited to large-volume storage. Depending on the configuration, single-use bag systems can also form part of sampling, intermediate collection and controlled transfer operations.

The same principle applies at larger scale: the process container, ports, tubing and connectors should be treated as one integrated fluid path rather than as unrelated components.

Filtra Custom Single-Use Bioprocess Bag Assemblies can incorporate tubing, connectors, ports, filters and other process components according to the required fluid path.

Aseptic connection and disconnection are part of sampling design

The sample container may need to be attached before sterilisation, connected during operation or removed after sample collection.

These scenarios place different requirements on the connection technology.

Academic literature on single-use coupling and sampling describes several approaches, including thermoplastic tubing welding, dedicated aseptic connectors, fittings and other connection/disconnection technologies used to create closed or controlled disposable process steps.

The appropriate approach depends on:

• whether the connection is made before or after sterilisation;
• tubing material;
• required process closure;
• operator procedure;
• available connection equipment;
• sterilisation compatibility;
• how the filled sample container will be isolated and removed.

The sample continues to change after it leaves the process

Sample quality does not end at the collection point.

Once material has been removed from a bioreactor or process vessel, biological and chemical changes can continue. Cells may continue to metabolise substrates, consume oxygen and produce metabolites. Proteins may adsorb to surfaces, precipitate or degrade. Temperature changes can alter reaction rates and some analytes may be sensitive to light, oxidation or prolonged storage.

Busse and colleagues specifically note that after sampling from disposable bioreactors, cellular metabolic activity may need to be stopped to prevent the sample composition from changing.

The correct stabilisation method is analyte- and process-specific. It may involve rapid analysis, temperature control, cell removal, chemical stabilisation or another validated handling procedure.

Time-to-analysis is therefore a process parameter

If two samples are collected identically but one is analysed immediately and the other remains at uncontrolled temperature for an extended period, the analytical results may no longer be directly comparable.

Sampling procedures should therefore define, where relevant:

• collection time;
• sample identification;
• required sample temperature;
• maximum hold time before analysis;
• mixing before sub-sampling;
• protection from light or oxygen where required;
• centrifugation or filtration requirements;
• storage or freezing conditions;
• transport requirements between manufacturing and analytical areas.

Manual sampling versus automated sampling

Manual sampling remains appropriate for many manufacturing processes. Automation becomes more attractive when the number of sampling events increases, when very small volumes are required or when consistent timing and reduced operator intervention are important.

Research published in 2024 and 2025 demonstrates why automation is attracting particular interest in cell and gene therapy manufacturing: frequent sampling, limited available process volume and the need to minimise contamination risk can make conventional manual workflows difficult to scale.

These research systems should not be interpreted as universal production standards. They demonstrate engineering principles that can inform the design of future sampling platforms.

Aseptic sampling and Process Analytical Technology

Process Analytical Technology does not necessarily mean that every measurement occurs directly inside the process vessel.

Some measurements rely on an extracted sample that is transferred to an at-line or other analytical instrument. In such cases, the sampling system becomes part of the measurement chain.

The time between the process and the analyser, sample conditioning and the physical behaviour of the sampling line may therefore affect the usefulness of the measurement for process monitoring or control.

Automated small-volume sampling research has shown how sampling platforms can be coupled with metabolite analysis for parameters such as glucose, lactate, glutamine and glutamate in cell-culture processes.

Material compatibility and extractables & leachables

Single-use sampling assemblies place polymeric materials directly in the sample fluid path.

Depending on the final assembly, wetted components may include:

• bag or bottle materials;
• tubing;
• connectors;
• gaskets and seals;
• valves and manifolds;
• filters;
• other integrated process components.

Material suitability should therefore be considered against the actual fluid and operating conditions rather than assuming that all polymers behave identically.

Relevant parameters include:

• fluid composition and pH;
• organic solvents or surfactants;
• contact time;
• temperature;
• surface-area-to-volume ratio;
• sterilisation method;
• sample sensitivity and analytical purpose.

A large-scale 2018 study involving NIBRT and University College Dublin evaluated extractables and leachables from 34 single-use bag films and illustrates why polymer composition and process conditions are important considerations in single-use biomanufacturing.

Sterilisation compatibility applies to the complete assembly

Single-use sampling systems may be supplied in different sterilisation configurations depending on their components and intended use.

Filtra's current single-use platform can be configured, depending on the selected materials and components, for gamma irradiation, compatible autoclave processing, sterile ready-to-use supply or non-sterile applications.

This should not be interpreted as meaning that every bottle, bag, tubing, connector and filter combination is compatible with every sterilisation method.

Sterilisation suitability must be confirmed for the complete specified assembly.

How to specify a single-use sampling assembly

A useful sampling-system specification begins with the process and analytical requirement rather than with a catalogue component.

Single-use sampling throughout the bioprocess

Sampling requirements differ depending on the process stage.

Scientific and technical references

1. Schirmer, C., Rothe, S., Jenness, E. & Eibl, D. (2019). Systems for Coupling and Sampling. In: Eibl, R. & Eibl, D. (eds.), Single-Use Technology in Biopharmaceutical Manufacture. Wiley.
2. Mattiasson, B. & Håkanson, H. (1993). Sampling and sample handling — crucial steps in process monitoring and control. Trends in Biotechnology, 11(4), 136–142.
3. Busse, C., Biechele, P., de Vries, I., Reardon, K. F., Solle, D. & Scheper, T. (2017). Sensors for disposable bioreactors. Engineering in Life Sciences, 17(8), 940–952.
4. Dan, L., Ying Ying, W., Prabhu, A. V., bin Abdul Rahim, A. A. & Jia Sheng, Z. L. (2024). Device for automated aseptic sampling: Automated sampling solution for future cell and gene manufacturing. Frontiers in Bioengineering and Biotechnology, 12, 1452674.
5. Chan, Z. X., Chelvam, S. P., Sin, W.-X., Teo, D. B. L., Abdul Rahim, A. A. B., Wu, Y. Y., Liu, D., Birnbaum, M. E., Yong, D. & Ram, R. J. (2025). Automated, aseptic sampling with small-volume capacity from microbioreactors for cell therapy process analysis. Frontiers in Bioengineering and Biotechnology, 13, 1612648.
6. Dorival-García, N. et al. (2018). Large-Scale Assessment of Extractables and Leachables in Single-Use Bags for Biomanufacturing. Analytical Chemistry, 90(15), 9006–9015.
7. European Commission. EudraLex Volume 4 — EU Guidelines for Good Manufacturing Practice, Annex 1: Manufacture of Sterile Medicinal Products.
8. U.S. Food and Drug Administration. Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice.

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