Sterile filtration is crucial in aseptic manufacturing when terminal sterilization is not possible because it will destroy or degrade the drug product (DP). Increasingly, regulatory bodies expect process-specific validation of sterilizing-grade (≤ 0.2 µm) filters under worst-case, realistic conditions. Currently, an increasing number of early-stage clinical programs involve small to very small clinical batch sizes; these include high-concentration mAbs, adeno-associated viruses, and mRNA-based lipid nanoparticle (LNP) drugs. As such, this leaves limited material for validation studies, requiring validation approaches that conserve drug product.
Cytiva Fast Trak™ validation services are designed to support biomanufacturers with process-specific sterilizing-filter validation, from as low as 100 mL of drug product. Our low volume (0.5–1.5 L) and ultra-low volume (< 0.5 L) offering enables critical validation studies, using minimal drug product sample volumes. In this article, we address frequently asked questions about our validation services for low and ultra-low volumes, for customers considering this service.
1. Cytiva offers multiple options for sterilizing-grade filter validation: standard studies, low and ultra-low volume studies, and the SOAR program? Which option is the best for my project?
Each project has unique objectives and requirements, so the optimal approach depends on your product characteristics, development stage, and available drug supply. As part of our offering, a Fast Trak™ validation services project manager can advise on which is your best option.
As highlighted in recent industry publications (1,2) and acknowledged in industry standards such as Parenteral Drug Association (PDA) Technical Report No. 26: Sterilizing Filtration of Liquids (TR 26) (3) and ISO 13408-2, certain drug products pose a higher risk of bacterial penetration. These include DP containing liposomes or LNPs. For these modalities, bacterial challenge studies, which demonstrate bacterial retention in product under worst-case process conditions, are often performed early in clinical development to reduce the risk of late-stage process changes that could impact timelines. For new drug modalities with limited prior data, early bacterial challenge testing can also help maximize patient safety and minimize the risk of negatively affecting clinical outcomes.
In these scenarios, two main approaches are available:
- Low or ultra-low volume validation
Ideal when drug supply is limited and you have a good understanding of the future full-scale manufacturing process. This approach minimizes material use while meeting regulatory requirements. If process parameters and product composition remain stable throughout development, repeating the validation may not be necessary. - Sterility optimization by assessment of risk (SOAR) program
A process development approach that removes the constraints of a formal validation study (e.g., triplicate runs under worst-case conditions). Using Design of Experiments (DoE), SOAR identifies critical process parameters and defines a design space for successful bacterial retention. This option typically requires 1–2 L of DP, depending on scope (e.g., multiple filter types, process conditions, product variants). SOAR is well-suited for high-risk fluids and processes with limited prior knowledge. A formal validation study is still required later for the selected worst-case conditions.
When the goal is to execute a formal validation study to document the sterile filtration step, regardless of clinical phase, the choice between standard, low, and ultra-low volume approaches primarily depends on drug availability and manufacturing cost.
- Standard option is most economical for low cost drugs produced in large batches.
- Low and ultra-low options provide significant cost savings for expensive drugs (> $20/mL)
manufactured in small batches (< 5L).
2. How does low and ultra-low volume validation testing affect standard lead times for completing sterilizing-grade filter validation studies?
There is no impact on lead time, as the low and ultra‑low volume offering is fully incorporated into the existing Cytiva validation services standard workflow. The main milestones remain the same:
- After receiving a complete product and process questionnaire (PPQ) describing your process and validation needs, a Fast Trak™ project manager reviews it.
- The project manager assesses scale-down options and identifies the minimal product sample required for each approach.
- You will be presented with these options, along with a statement of work (SOW) document and a quote prepared in collaboration with our commercial team.
- Upon receipt of your purchase order (PO), we share a detailed protocol and sample shipment instructions.
- Once the sample and approved protocol are received in our validation lab, the lab work is scheduled and executed.
- Finally, the report is issued and submitted for your approval.
We always strive to minimize lead times; however, the overall timeline also depends on your responsiveness in providing the PO, reviewing and approving the SOW and protocols, and shipping the drug product on time. Close collaboration ensures projects stay on track.
3. What factors influence the drug sample volume required for low and ultra-low volume validation methods?
The exact volume for each project is determined by the Fast Trak™ validation services project manager after reviewing your product and process information provided in the PPQ. The project manager designs an appropriate scale-down model that accurately reflects worst case process conditions.
| Parameters | Impact |
| Control outside room-temperature | Low: may require use of a water bath during testing, impacting the hold-up volume of the test setup |
| Shear-sensitive, drug-limiting recirculation of product | Moderate: batch-size per membrane area must be achieved, possibly requiring single-pass bacterial challenge approach |
| Short shelf-life product | Moderate: may require fresh samples for each test |
| Filter reference | Moderate: can affect compatibility study and minimum hold-up volume of the test filter |
| Drug properties | Low: viscosity and surface tension may need to be assessed to select a suitable surrogate for bacterial challenge with a bactericidal drug |
4. Can you demonstrate the compliance of these low and ultra-low volume methods with regulatory guidance?
Yes. The methods applied for bacterial viability, filter flush, bacterial retention, and chemical compatibility—while minimizing drug product usage—follow the same principles as our standard validation methods. These methods are compliant with applicable regulatory guidance, including PDA TR 26, ISO13408-2, ASTM F838, European Union (EU) Annex 1: Manufacture of Sterile Medicinal Products guidelines (4), European Medicines Agency (EMA) Guideline on the sterilization of the medicinal product, active substance, excipient and primary container (5), and United States (US) Food and Drug Administration (FDA) Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice guidance (6). The acceptance criteria remain identical whenever a high volume, low volume or ultra-low volume method is employed.
5. What qualification activities have been conducted to confirm that the low and ultra-low volume methods are equivalent to industry-accepted approaches?
To demonstrate equivalence, optimized low volume methods were evaluated in side-by-side comparisons with standard industry practices across all critical validation tests. These studies confirmed that the alternative methods deliver comparable performance while reducing drug product consumption.
A summary of the qualification activities is provided below (Table 1).
Table 1: Qualification activities for different validation tests
| Viability test |
Standard: Brevundimonas diminuta (B. diminuta) inoculated in 50 mL stirred Erlenmeyer flask B. diminuta exposed to :
|
Result: Log reduction profiles were consistent with the nature of the fluid independently of the setup used. Conclusion: No impact of the viability setup and equivalent performance demonstrated with the standard |
| Flush regime | Standard: 47 mm filter disc Test discs exposed to the 3 process fluids mentioned above and then flushed with a buffer. B. diminuta exposed to the latest fraction of the buffer to demonstrate non-inhibitory effect. |
Results: For all conditions, bacterial counts recovered from the 3 process fluid test > 70% of the control (i.e, buffer not exposed to the test disc) Conclusion: Flushing regime not impacted by the setup and equivalent performance demonstrated with the standard |
| Bacterial challenge | Standard: 47 mm disc filter in a stainless steel disc holder. Side by side comparison: Test fluid:
| Results: Penetrative fluid: B. diminuta observed on at least one recovery for each condition tested. Buffer: 100% B. diminuta retention for all conditions tested. Conclusion: Challenge outcomes consistent with standard method and aligned with expected design space performance |
| Compatibility | Standard: mini-Kleenpak™ capsule Side by side comparison with various filter capsules exposed to test fluids:
|
Results: Compatible fluid: Pass integrity test post-exposure Non-compatible fluid: Fail integrity post-exposure observed across all designs. Conclusion: Compatibility results were consistent with standard method expectations. |
Additional qualifications:
All equipment and materials used in the low and ultra-low methods were characterized to define their operating range and ensure suitability for validation studies. Validated range is summarized in the table below (Table 2).
Table 2: Equipment and materials validated range
| Test duration | 0–48 h |
| Temperature | 4°C–40°C |
| Flux range | 200–4050 LMH |
| Upstream pressure range | 1–40 psi |
| Viscosity range | Water-like to 30 centipoise (cP) |
6. Have these low and ultra-low methods been reviewed by regulatory authorities?
Cytiva sterile filter validation services are designed in alignment with globally accepted regulatory guidance, including FDA aseptic processing principles, EMA requirements, and industry best practices such as PDA TR 26 and ISO 13408-2. While regulatory authorities do not formally endorse specific vendor methods, our approach has been applied in numerous customer validation programs that have undergone successful regulatory review.
We have executed 50+ bespoke validation projects across diverse modalities, including:
- monoclonal antibodies (mAbs)
- peptides
- proteins
- RNA-based drugs
- small molecules
- viral vectors
Importantly, at least three of these projects have achieved marketing authorization in multiple regions around the world and are currently commercialized by leading pharmaceutical companies, demonstrating that our validation strategies meet regulatory expectations in real-world submissions.
Conclusion: Fast Trak™ validation services offers tailored standard, low, and ultra-low volume filter validation
Cytiva Fast Trak™ validation services enable filter validation strategies of choice, from standard testing to low volumes, with as little as 100 mL of drug product. Our low volume (0.5–1.5 L) and ultra-low volume (< 0.5 L) sterile filter validation testing enables critical validation studies using minimal sample volumes—ideal for costly or limited-supply drugs.
References
- Folmsbee M, Moussourakis M. Sterilizing filtration of liposome and related lipid-containing solutions: enhancing successful filter qualification. PDA J Pharm Sci Technol. 2012;66(2):161-7. doi:10.5731/pdajpst.2012.00771.
- Folmsbee M. Evaluation of the Effect of the Volume Throughput and Maximum Flux of Low-Surface-Tension Fluids on Bacterial Penetration of 0.2 Micron-Rated Filters during Process-Specific Filter Validation Testing. PDA J Pharm Sci Technol. 2015;69(2):307-16. doi:10.5731/pdajpst.2015.01026.
- Technical Report No. 26 (Revised 2025): Sterilizing Filtration of Liquids. Parenteral Drug Association (PDA). November 2025. Accessed March 27, 2026. https://www.pda.org/bookstore/product-detail/8489-tr-no-26-revised-2025-sterilizing-filtration
- EudraLex - Volume 4 - Good Manufacturing Practice (GMP) guidelines; Annex 1 – Manufacture of Sterile Medicinal Products. European Commission. August 22, 2022. Accessed March 27, 2026. https://health.ec.europa.eu/document/download/e05af55b-38e9-42bf-8495-194bbf0b9262_en?filename=20220825_gmp-an1_en_0.pdf
- Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container. European Medicines Agency. 2019. Accessed March 27, 2026. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-sterilisation-medicinal-product-active-substance-excipient-and-primary-container_en.pdf
- Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. United States Food and Drug Administration. September 2004.
Accessed March 27, 2026. https://www.fda.gov/media/71026/download
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