Performing process-specific filter validation for low volumes

Sterile filtration is a critical control point in fill‑finish when terminal sterilization is not feasible. Regulators expect process‑specific validation of sterilizing‑grade (≤ 0.2 µm) filters under worst‑case, realistic conditions.

Early clinical programs increasingly involve low volume, high value drug product (DP) (e.g., high-concentration mAbs, adeno-associated viral DP, and mRNA/lipid nanoparticles [LNPs]), leaving limited material for validation studies. Some fluids (especially lipidic/low surface tension systems) carry higher penetration propensity. Validation teams therefore need approaches that conserve DP yet still generate audit-ready evidence.

A science-driven, risk-based approach works best. This includes (1) a documented design space, (2) ≥ 1 × 10⁷ colony-forming units (CFU)/cm² bacterial retention, and (3) a defensible integrity testing (IT) strategy with pre-use, post-sterilization integrity testing (PUPSIT) and post‑use IT. This delivers representative worst-case exposure while mapping cleanly to the European Union (EU) Annex 1: Manufacture of Sterile Medicinal Products guidelines (Annex 1) and the Parenteral Drug Association (PDA) Technical Report No. 26: Sterilizing Filtration of Liquids (TR 26) (1,2). A filter validation program includes, but may not be limited to, a range of studies under worst-case process conditions (Fig 1).

Fig 1. Many variables play into whether a filter validation test is performed.

Cytiva Fast Trak™ validation services offer a scalable, regulatory-compliant strategy for validating Cytiva sterilizing-grade filters that minimizes product consumption while aligning with regulatory expectations and customer workflows. Methods are aligned with TR 26 and documented to Annex 1 expectations, enabling Phase I/II execution without compromising sterility assurance or auditability. This white paper outlines how validation can be effectively executed across clinical phases, even with limited product volumes.

Tests that do not require actual drug product sample

Extractables test

The study of extractables characterizes potential compounds released from the filter under exaggerated conditions. The data inform toxicological risk assessments and help decide whether a leachables study is warranted during late-phase, process-level runs. This enables early filter selection and risk mitigation without consuming drug product.

Cytiva provides BioPhorum Operations Group (BPOG) and USP <665> data for many filters. When these reports are not representative of the drug formulation, a model solvent approach simulates worst-case conditions using aggressive solvents—no drug product required. This process-specific extractables study enables early risk assessments and supports regulatory filings without consuming precious drug material.

Workflow insight: Early extractables data supports filter selection and toxicological risk assessments, aligning with regulatory expectations for Phase I/II filings.

Product-wet integrity test

Product-wet integrity test (PWIT) assesses filter integrity when wetted with the drug product. Cytiva performs Knee limit (KL) studies using forward flow tests at increasing pressures. When performed with larger‑area capsules, integrity testing using product as the wetting fluid may require significant volume; if product volume is limited, Cytiva recommends using a reference fluid for integrity testing. However, this test is not mandatory for completing a sterilizing-grade filter validation.  

Workflow insight: Manufacturing science and technology (MSAT) or validation teams should consider cost-benefit of performing integrity testing with the drug product instead of a reference fluid.

Note: The KL value is a calculated value taken from data used to perform a forward flow spectrum. A series of forward flow measurements is collected at increasing pressures, until the bubble point of the filter is exceeded. Using linear regression, the intersection where the extrapolated line from the stable portion meets the extrapolated portion of the line defines the bulk flow region of the curve. At the intersection of these two lines, a line perpendicular to the x axis is drawn to show the pressure of the KL point.

Tests recommended during late clinical phase

As product volumes increase in late-phase development, more comprehensive validation activities can be conducted under representative manufacturing conditions, and process parameters are better established.

Leachables test

Leachables are compounds that migrate under normal processing conditions. If extractables exceed safety thresholds, a leachables study may be required. Cytiva recommends a system-level approach, collecting samples during pilot-scale runs at the manufacturing site to capture real-world data. Where needed, Cytiva supports customized leachables protocols to match site-specific assemblies and sampling plans. This approach ensures that all product contact materials, not just the filter, are evaluated for potential leachables.

Workflow insight: MSAT engineers can integrate leachables testing into tech transfer activities, ensuring that final process conditions are validated before commercial launch.

Filter adsorption test

A filter adsorption test evaluates how much product is needed to saturate adsorptive sites on the filter, ensuring formulation components remain within specifications. It is typically performed with smaller filter configurations and extrapolated to commercial scale. Some preliminary adsorption insights may be available from early filterability studies or literature; however, a system level adsorption assessment is best reserved for late clinical phase using pilot batch samples. While the sterile filter with its large surface area is the main contributor to component adsorption, drug manufacturers are expected to understand the adsorptive effects from all product contact materials. Cytiva recommends performing this study during late clinical phase using pilot batch samples and supports protocol development and saturation volume estimation.

Workflow insight: Adsorption data helps ensure product potency and consistency, supporting final formulation decisions and fill-finish operations.

Optimized, low volume tests available at any phase

Cytiva has developed qualified, small-scale methods for three essential validation tests using as low as 100 mL of product. The actual drug volume depends on the product properties and process conditions and is communicated by the Cytiva project manager after the customer completes a technical questionnaire. These methods enable early-phase execution and provide real-world evidence that the filtration process retains bacteria to regulatory standards designed to ensure patient safety. These methods are available in both standard volume and low volume formats to fit batch size and risk.

Chemical compatibility test

A chemical compatibility test assesses filter integrity post-exposure to the drug product at process worst-case temperature and maximum contact time. It involves pre- and post-filtration integrity tests using small-volume capsules. The filter is exposed to the product, then flushed and post-integrity tested. This test may not require additional drug volume if performed in the same scope of work as the bacterial challenge test.

Workflow insight: Compatibility testing supports early formulation decisions and helps avoid late-stage surprises during scale-up.

Bacterial viability test and filter flush

Both the bacterial viability test (BVT) and filter flush are prerequisites for conducting a bacterial challenge study. The bacterial viability test assesses the ability of Brevundimonas diminuta (B. diminuta) inoculated into the drug product to remain viable over time. A defined flush regime is implemented to remove potential inhibitory residues from the recovery membrane, ensuring B. diminuta can grow in these conditions and colonies will be visible after seven days of incubation. The outcomes of these tests guide the design of the bacterial challenge protocol. By optimizing equipment configuration, the drug sample volume required for these tests can be reduced by up to 90%.

Workflow insight: With only few milliliters of drug volume, we enable early-phase execution, even in pre-clinical stage, streamlining the design of the bacterial challenge study.

Note: Final sample volume is dependent on product composition and process conditions. Final volume to be communicated after evaluation by the validation services project manager.

Bacterial challenge test

The bacterial challenge test (BCT) demonstrates bacterial retention in product under worst-case process conditions. Our scaled-down filter disc method meets TR 26 standards and regulatory expectations. The method uses three membrane lots, including one near the lower integrity limit, and simulates worst-case conditions for volume throughput, pressure, flux, temperature, and contact time. The actual drug sample volume needed depends on the filtration process conditions, with potential to reduce volume requirements by up to 90%.

Workflow insight: BCT is the cornerstone of filter validation. Our approach enables early-phase execution, accelerating readiness for Investigational New Drug (IND) or Investigational Medicinal Product Dossier (IMPD) submissions.

Conclusion: Fast Trak™ validation services offer phase-appropriate, regulated tests

Cytiva Fast Trak™ validation services empower MSAT and validation engineers to execute robust, phase-appropriate filter validation strategies—even when product volumes are limited. Our validation services are delivered from five Fast Trak™ labs across the US, United Kingdom (UK), India, Japan, and China, using standard operating procedures (SOPs). Reports map to Annex 1, TR 26 (2025), and ASTM F838 expectations for bacterial retention and integrity testing. Whether you are initiating early development or preparing for commercial launch, Cytiva can help you build confidence in your sterile filtration process.

About Cytiva validation services for low and ultra-low volumes

Cytiva validation services enable companies to accelerate clinical development with our robust and effective filter validation packages. Our validation services for low and ultra-low volumes, including bacterial viability and retention, compatibility, and extractables, offer regulatory-compliant, process-specific studies using as low as 100 mL sample volumes—ideal for costly or limited-supply drugs. With project management and end-to-end regulatory support, we enable first-time success in regulatory review and accelerate your time to market.

Do you have more questions about our low and ultra-low volume validation services? Read our article covering frequently asked questions.

Are you ready to speak with one of our specialists about your low volume validation program? Request a consultation today.

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