Cleaning Validation Protocol and Procedure for Tangential Flow Filtration System with Reusable Flow Paths in Biologics Manufacturing

Purpose and Scope

This document establishes a standardized cleaning validation protocol and cleaning procedure (SOP) for Tangential Flow Filtration (TFF) Systems equipped with reusable flow paths employed in the manufacturing of biologics and biosimilars productions. The validation aims to confirm that residues of product, cleaning agents, and microbial contamination are effectively removed to pre-defined acceptance limits, ensuring product quality, patient safety, and compliance with current Good Manufacturing Practices (cGMP) and regulatory guidance.

The scope applies to all TFF systems used for intermediate processing, concentration, and diafiltration of biopharmaceutical drug substances where reusable flow paths are involved. It encompasses system components that directly contact product and cleaning agents, including membranes, tubing, filters, housings, and connectors.

This protocol is intended for use by Quality Assurance (QA), Quality Control (QC), Validation, Production, and Engineering departments supporting biologics and biosimilars manufacturing environments.

Definitions and Abbreviations

Responsibilities

Safety and Personal Protective Equipment (PPE)

The cleaning operations involve chemicals and detergents with potential hazards; therefore, appropriate PPE must be worn at all times, including but not limited to:

• Laboratory coat or coveralls resistant to chemicals
• Chemical-resistant gloves suitable for detergent handling
• Safety goggles or face shield
• Closed-toe footwear
• Respiratory protection if specified by Safety Data Sheets (SDS) or site risk assessment

All personnel must be trained on chemical hazards, spill response, and emergency procedures prior to performing cleaning or sampling operations.

Equipment Overview and Product-Contact Parts

The Tangential Flow Filtration (TFF) system considered herein includes the following primary reusable and product-contact components:

• Membranes: Hollow fiber or flat sheet membranes used for filtration.
• Membrane Housings: Cartridges containing the membranes.
• Tubing and Hoses: Silicone, PVC, or other bio-compatible tubing connecting system components.
• Connectors and Clamps: Stainless steel or polymer fittings facilitating flow path connections.
• Pumps and Valves: Pumps and valves contacting product or cleaning fluids, including peristaltic or diaphragm pumps.
• Sensors and Probes: Inline sensors for process parameters, only included if contacting product.

All these parts require validated cleaning to ensure complete removal of residues before reuse.

Cleaning Strategy Overview

The cleaning approach for the TFF system’s reusable flow paths follows a scientifically established, risk-based strategy incorporating the following key components:

• Detergent Cleaning: Initiation of cleaning with an appropriately selected cleaning agent ([detergent_name]) capable of removing biopharmaceutical residues and biofilms.
• Rinsing: Multiple sequential rinses with purified water to remove detergent residues, monitored via conductivity and/or TOC.
• Sanitization/Disinfection: Where applicable, disinfection steps using validated chemistries to control microbial contamination risk.
• Defined Hold Times: Limits on the duration between processing, cleaning, and reuse to prevent microbial growth and residue fixation.
• Validated Cleaning Cycle: Controlled procedure parameters, including flow rates, temperatures, detergent concentration, and contact times to optimize residue removal.

This strategy aims to minimize residues consistent with patient safety and regulatory requirements, utilizing the Maximum Allowable Carryover (MACO) calculated from PDE/ADE toxicological principles.

Cleaning Agents and Tools List

Hold Times Definitions

Records and Forms List

Site-specific Inputs Required

• Detergent name, formulation, and concentration ([detergent_name])
• Purified water rinse volumes ([rinse_volume_L]) and conductivity or TOC limits
• Cleaning cycle parameters including temperature, flow rate, contact times
• Maximum allowable hold times for dirty and clean states ([max_dirty_hold_time], [max_clean_hold_time])
• Specific membrane types and materials of construction for validation relevance
• Sampling surface area for swab and rinse samples ([swab_area_cm2])
• Microbial limits if applicable based on risk assessment

Cleaning Procedure for Tangential Flow Filtration (TFF) System with Reusable Flow Paths

1. Pre-Clean Preparation
   1. Verify that the TFF system has completed the production run and isolate it from the production area.
   2. Wear appropriate personal protective equipment (PPE) in line with site safety and hygiene policies.
   3. Document batch details including product ID, batch number, and equipment ID in the cleaning log.
   4. Ensure availability of cleaning agents, rinse water (WFI or as specified), and necessary tools for disassembly.
   5. Perform a visual inspection to ensure no visible product residues or particulate matter on external surfaces.
   6. Confirm availability of sampling materials (swabs, sample containers, labels) for post-clean sampling.
2. Disassembly of Reusable Flow Paths
   1. Shut down the TFF system as per operational SOPs ensuring no residual pressure in the flow paths.
   2. Carefully dismantle all reusable flow path components including tubing, membranes, connectors, and filters.
   3. Inspect each component for visible fouling, damage, or wear.
   4. Label components clearly to maintain traceability during cleaning.
   5. Place components on a clean surface designated for cleaning preparation.
3. Cleaning/Washing Sequence
   1. Prepare cleaning solution as per site-specified concentration using [detergent_name]. Site-specific inputs required (detergent concentration, temperature, contact time).
   2. Fill the cleaning tank or flushing system with the prepared cleaning solution and circulate through the reusable flow paths for a minimum of [contact_time_minutes] at [temperature_C] °C.
   3. Ensure cleaning solution reaches all system parts by verifying flow rates and stroke volumes as per equipment specification.
   4. Flush and soak heavily soiled sections longer if visible residues persist.
   5. Drain cleaning solution and immediately commence first rinse.
4. Rinse Sequence
   1. Perform an initial rinse using [rinse_type, e.g., Purified Water] with a minimum volume of [rinse_volume_L] liters per flow path component to remove detergent residues.
   2. Subsequently, perform a final rinse with Water for Injection (WFI) of at least [final_rinse_volume_L] liters or as per validation requirements.
   3. Monitor rinse water conductivity (or TOC depending on site method) to verify removal of cleaning agents.
   4. Repeat rinses if cleaning solution residues exceed acceptance limits.
   5. Collect rinse water samples at designated points for off-line testing as per sampling plan.
5. Drying
   1. Dry disassembled components using validated methods such as compressed air drying (filtered, sterile if required) or drying ovens set at [drying_temperature_C] °C for [drying_time_minutes].
   2. Ensure all visible moisture is removed to prevent microbial growth or cross-contamination.
   3. Inspect components for dryness visually and document findings.
6. Reassembly
   1. Reassemble the cleaned and dried reusable flow path components following equipment SOPs.
   2. Use only cleaned and validated tools; ensure no contamination from reassembly process.
   3. Verify component integrity and correct installation by functional checks, e.g., pressure hold test or flow verification as applicable.
   4. Update equipment status to “ready for use” in tracking system.
7. Visual Inspection
   1. Conduct a final visual inspection of all reassembled flow paths and accessible system partes to detect any residual dirt, particles, or discoloration.
   2. Take photographs if required by site procedure for documentation.
   3. Record any deviations or findings in the cleaning log and initiate investigative actions if necessary.

Cleaning Parameters and Critical Control Points

Sampling Plan for Cleaning Validation of TFF System

Swab Sampling Methodology

1. Use sterile, validated swabs pre-moistened with appropriate solvent (e.g., purified water, buffer, or detergent extraction solution as specified by analytical method).
2. Swab defined area ([swab_area_cm2]) applying firm, consistent pressure horizontally and vertically to ensure full coverage.
3. Place swab immediately into sterile container and seal.
4. Label sample container clearly with unique sample ID, location, date/time, batch details, and operator initials.
5. Log sample details in chain-of-custody form to maintain traceability from collection to analysis.

Sample Handling and Transport

1. Store all swab samples at controlled temperature (typically 2-8 °C) unless otherwise specified to prevent degradation/contamination.
2. Transport samples to analytical laboratory within [holding_time_hours] hours from collection.
3. Maintain chain-of-custody forms signed by all handlers during transport and receipt in the laboratory.
4. Document environmental conditions during transport if warranted by analyte stability.
5. Samples not analyzed within hold time should be rejected and re-sampled.

Site-Specific Inputs Required

• Identification and concentration of detergent(s) used ([detergent_name], % w/v or molarity)
• Detergent contact time and temperature ([contact_time_minutes], [temperature_C])
• Rinse volumes for initial and final rinses ([rinse_volume_L], [final_rinse_volume_L])
• Maximum acceptable conductivity (µS/cm) or TOC limit (ppm) for rinse water
• Swab area in cm² for each location ([swab_area_cm2])
• Maximum sample holding time before analysis ([holding_time_hours])
• Drying temperature and duration ([drying_temperature_C], [drying_time_minutes])

Recovery, Limit of Detection (LOD), and Limit of Quantification (LOQ) Expectations

Accurate determination of residual contaminants in the Tangential Flow Filtration (TFF) system is essential for reliable cleaning validation. Method performance characteristics, including recovery, LOD, and LOQ, must be established during method development and verification phases to ensure analytical robustness.

• Recovery: Analytical recovery studies must demonstrate a minimum of 80–120% recovery of residues from representative stainless steel or polymeric surfaces used within TFF flow paths. Recovery validation involves spiking known quantities of product active, product-related impurities, cleaning agents, and microbial residues onto coupons or actual surfaces, followed by sampling using the designed sampling techniques (swabbing or rinsing) defined in Part B.
• Limit of Detection (LOD): The LOD for each residue or contaminant should be sufficiently sensitive to detect residues at concentrations well below the established acceptance limits. Typically, LOD values should be in the low ppb range, ensuring early detection of any residual contamination before it reaches a level of regulatory concern.
• Limit of Quantification (LOQ): LOQ must be established at or below the minimum acceptance limits derived from the MACO (Maximum Allowable Carryover) calculation to guarantee quantitative analysis with acceptable precision and accuracy. LOQ should generally be below 0.5x MACO for confident residue quantification.
• Site-specific Inputs Required: Analytical method validation reports, matrix-matched recovery data, spike concentration levels, sampling surface characteristics.

Acceptance Criteria Methodology (PDE/ADE-Based MACO Approach)

The cornerstone of acceptance criteria for TFF system cleaning validation is the application of a PDE (Permissible Daily Exposure) or ADE (Acceptable Daily Exposure)-based MACO model. This scientifically justified framework considers patient safety, product potency, cleaning agent toxicity, and batch sizes to establish meaningful residual limits.

Overview of MACO Concept

MACO represents the Maximum Allowable Carryover residue such that any residual contaminant transferred from equipment to the next batch does not pose a safety risk. It is calculated using the most stringent PDE or ADE value among relevant active pharmaceutical ingredients (APIs), residues, or cleaning agents.

MACO Calculation Structure

Implementation and Interpretation

Residue acceptance limits derived via MACO apply to assay results obtained from the Sampling Plan (Part B). SOPs must clearly define the link between PDE/ADEs, batch sizes, and residue limits. In instances where no PDE/ADE is available for a cleaning agent or impurity, a worst-case toxicological surrogate or default low-level threshold must be employed with justification.

Legacy Acceptance Criteria (Fallback)

Where PDE/ADE information is unavailable, legacy acceptance thresholds such as 10 ppm or 1/1000th of the therapeutic dose may be applied with explicit labeling as legacy limits. These criteria must be applied cautiously and supplemented with risk assessment to ensure patient safety is not compromised.

Detergent Residue Rationale and Acceptance

Detergent residues pose unique challenges due to their chemical nature and potential impact on product quality and patient safety. Acceptance criteria for detergent residues are based on validated analytical methods, predominantly Total Organic Carbon (TOC) analysis, conductivity measurements, or detergent-specific colorimetric/spectrophotometric assays.

• Rationale: Detergent residues must be controlled to prevent interference with subsequent manufacturing steps and to avoid patient exposure to harmful surfactants or cleaning agents.
• TOC-Based Acceptance: TOC provides a universal measure of organic contamination, including detergents. Acceptance limits are derived relative to the PDE of the detergent, or based on historical data indicating safe residual levels within the system. For example, acceptable TOC limits might be set at [TOC_limit] ppm or equivalent mg/cm² depending on system surface area.
• Conductivity Assessment: For ionic detergents, conductivity measurements offer rapid, in-process evaluation. Established limits correlate with detergent residues below the PDE or MACO limits.
• Specific Assays: Where applicable, detergent-specific analytical methods (e.g., MBAS for anionic surfactants) may be employed to quantify residues with higher specificity.
• Site-specific Inputs Required: Detergent chemical properties, validated TOC or assay LOD/LOQ, system surface area, detergent PDE or toxicological benchmark, typical rinse volumes.

Deviations and Corrective and Preventive Actions (CAPA)

Strict governance on deviations from established acceptance criteria or procedural failures ensures continued validation integrity and regulatory compliance.

1. Deviation Identification: Any batch failing cleaning validation criteria or deviations during sampling, analysis, or cleaning procedure execution must trigger a formal deviation report.
2. Cause Investigation: Root cause analysis must determine whether deviations are due to procedural non-compliance, analytical errors, equipment malfunction, or environmental factors.
3. Capa Implementation: Appropriate corrective actions include re-cleaning, re-sampling, retraining of operators, maintenance of equipment, or analytical method refinement.
4. Documentation: All deviations and CAPA steps must be comprehensively documented, with risk assessment on product impact and release decisions.
5. Preventive Measures: Reassessment of SOPs, cleaning agent selection, sampling methods, or schedule adjustments are critical preventive actions.

Continued Verification Plan

Cleaning validation maintenance requires periodic continued verification to monitor ongoing cleaning efficacy and detect process drift. The plan includes:

Revalidation Triggers

Revalidation of the TFF system cleaning process is mandated under the following conditions to uphold validation lifecycle management and regulatory compliance:

• Process Changes: Any modification to cleaning procedures, detergents, contact time, temperature, or rinse volumes that may influence cleaning efficacy.
• Equipment Alterations: Replacement, refurbishment, repositioning, or any change in system components or flow paths.
• Product Changes: Introduction of new biologics, biosimilars, or product variants with differing residue profiles or toxicological properties.
• Failure Events: Repeated deviations or out-of-specification results in cleaning validation or continued verification activities.
• Regulatory Requirements: Regulatory agency mandates or updated guidance necessitating revalidation.

Annexures and Templates

The following annexures and templates support the execution, documentation, and governance of TFF system cleaning validation:

Conclusion

The cleaning validation for the Tangential Flow Filtration (TFF) system with reusable flow paths is grounded on a scientific, risk-based approach leveraging PDE/ADE-derived MACO calculations to establish credible and patient-safe residue acceptance criteria. Accurate method performance characterization, including thorough recovery, LOD, and LOQ validation, underpins analytical reliability. Detergent residues are controlled using justified TOC and detergent-specific assays aligned to toxicological thresholds. Robust deviation management, CAPA, and a structured continued verification plan maintain ongoing process control and compliance. Revalidation protocols ensure adaptability to changes in equipment, process, or products, thereby sustaining validation integrity throughout the product lifecycle. Comprehensive annexures and templates facilitate standardization and audit readiness, aligning with pharmaceutical industry expectations and regulatory mandates.