Chromatography System Cleaning Validation Protocol for Reusable Flow Paths in Biologics and Biosimilars Manufacturing

Purpose and Scope

This protocol establishes the foundational framework for validating the cleaning process of chromatography systems with reusable flow paths used in the manufacturing of biologics and biosimilars. The purpose is to ensure effective and reproducible removal of product residues, cleaning agents, and potential contaminants to maintain system integrity, prevent cross-contamination, and comply with regulatory requirements for cleaning validation.

This document applies to all chromatography systems utilized within the facility that employ reusable flow paths, including but not limited to preparative and analytical chromatography columns, tubing, valves, and associated hardware contacting product solutions during downstream purification processes. The protocol covers equipment cleaning validation from a quality assurance and manufacturing perspective, ensuring the validated cleaning process meets established acceptance criteria based on a scientifically justified risk and residue-based approach.

Definitions and Abbreviations

Responsibilities

Safety and Personal Protective Equipment (PPE)

Personnel involved in cleaning procedures and validation activities must adhere to established safety protocols and utilize appropriate PPE to minimize exposure to chemical cleaning agents and biological residues. The following guidelines apply:

• Wear chemical-resistant gloves compatible with the used detergents and disinfectants.
• Use safety goggles or face shields to protect eyes from splashes.
• Don protective clothing such as lab coats, gowns, or coveralls as specified.
• Utilize respiratory protection where ventilation is inadequate or exposure limits may be exceeded.
• Employ proper training for handling chemical agents and biohazard materials.
• Follow Material Safety Data Sheet (MSDS) instructions for each cleaning agent used.
• Maintain good hygiene practices, including handwashing after handling residues or chemicals.

Equipment Overview and Product-Contact Parts

The chromatography system comprises multiple components, which form the product-contact flow path. Below is an overview of key equipment and parts relevant to cleaning validation:

Cleaning Strategy Overview (High Level)

The cleaning strategy for chromatography systems focuses on validated procedures to ensure effective removal of product residues, cleaning agents, and microbial contaminants where applicable. The approach includes the following elements:

• Selection of cleaning agents: Use of detergents and sanitizers compatible with the product and equipment materials to facilitate thorough residue removal.
• Cleaning methods: Combination of automated flush cycles, manual cleaning (where required), and use of validated cleaning-in-place (CIP) systems to minimize contamination risks.
• Validated hold times: Defined maximum times equipment can remain idle with product residues or cleaned without quality degradation.
• Residue monitoring: Sampling techniques including rinse and swab sampling coupled with analytical methods targeting protein residues, detergents, and total organic carbon.
• Microbial control (risk-based): Assessment of microbial bioburden and implementation of sanitization as part of cleaning where necessary.
• Reproducibility and robustness: Cleaning protocols developed to yield consistent, repeatable cleaning outcomes compliant with acceptance criteria across all equipment.

Cleaning Agents and Tools List

Hold Times Definitions

Records and Forms List

Site-Specific Inputs Required

• Cleaning detergent name and composition ([detergent_name])
• Detergent concentration and contact times for cleaning procedure
• Volume of purified water used for rinsing ([rinse_volume_L])
• Microbial risk assessment data to decide microbial limits applicability
• Sampling area size for swab sampling ([swab_area_cm2])
• Maximum allowable dirty hold time ([maximum_dirty_hold_hours])
• Maximum allowable clean hold time ([maximum_clean_hold_hours])
• Analytical testing methods to be employed for residue detection
• Details of chromatography system material construction and configuration

Chromatography System Cleaning Procedure for Reusable Flow Paths

1. Pre-Clean Preparation
   1. Ensure the chromatography system is shut down and depressurized per manufacturer’s instructions.
   2. Confirm availability of appropriate personal protective equipment (PPE) including gloves, goggles, and lab coats.
   3. Prepare cleaning agents: [detergent_name], rinse water, and sanitizing solutions as per site validation parameters.
   4. Document system run batch details, time of last use, and prior cleaning events in the cleaning logbook.
   5. Verify that all required cleaning tools and utensils (brushes, lint-free cloths, swabs) are clean and ready for use.
2. Disassembly
   1. Disconnect the chromatography system flow paths as per system-specific disassembly instructions.
   2. Separate all reusable tubing, column hardware, frits, valves, seals, and connectors for individual cleaning.
   3. Inspect parts for visible soils, deposits, or damage; record in maintenance log if abnormalities found.
3. Cleaning Steps
   1. Initial Rinse: Rinse disassembled parts with [rinse_volume_L] of purified water to remove loose residues and buffer salts.
   2. Detergent Wash:
      1. Immerse parts in an aqueous solution of [detergent_name] at site-specific concentration and temperature [temp °C] for [duration] minutes.
      2. Perform manual brushing or appropriate mechanical cleaning of intricate flow paths and seals to remove bound contaminants.
      3. Ensure detergent penetrates difficult geometries; repeat immersion if visible residues persist.
   3. Intermediate Rinse: Rinse with fresh purified water for [rinse_volume_L] to remove detergent residues from components.
   4. Sanitation Step (if applicable):
      1. Soak parts in sanitizing solution (e.g., 70% isopropanol or site-specified agent) for [duration] minutes.
      2. Allow contact time per sanitizing agent validation data.
   5. Final Rinse: Thoroughly rinse all parts with [rinse_volume_L] of purified water to eliminate sanitizing agent residues.
   6. Drying:
      1. Air dry parts in a contamination-controlled environment or use filtered compressed air with [pressure] as per site standard.
      2. Verify parts are visibly dry; avoid residual moisture to prevent microbial growth or analytical interference.
4. Reassembly
   1. Reassemble chromatography system components carefully adhering to manufacturer’s torque and fitting specifications.
   2. Replace any seals or gaskets as per preventive maintenance schedules or if damage is observed.
   3. Perform a system leak test and pressure integrity check prior to system restart.
5. Visual Inspection
   1. Visually inspect all reassembled flow paths and visible components for cleanliness, absence of particulates, and dry surfaces.
   2. Document visual inspection results and any anomalies on the cleaning record log.
   3. Authorize system for next batch operation only upon satisfactory cleaning verification.

Cleaning Parameter Table

Sampling Plan for Chromatography System Cleaning Validation

Sampling Locations and Rationale

Sampling Methodology

1. Utilize validated sterile swabs moistened with purified water or site-approved extraction solvent specifically selected for residue recovery efficiency.
2. Swab the defined area ([swab_area_cm2]) with controlled pressure and technique to ensure representative sample collection.
3. Collect swabs immediately post-cleaning and prior to system reassembly or use to avoid recontamination.
4. Label each sample with unique identifiers including system id, location, date/time of sampling, and batch number.
5. Follow chain-of-custody procedures:
   1. Record personnel responsible for sampling, transport, and receipt in analytical laboratory.
   2. Use tamper-evident seals and transport samples under controlled temperature and time conditions.
6. Forward samples immediately to the Quality Control (QC) laboratory for analysis with complete documentation.

Sample Handling and Storage

Site-Specific Inputs Required

• Name and concentration of detergent ([detergent_name], [detergent_concentration])
• Detergent contact time and temperature ([duration_minutes], [temp °C])
• Rinse volume per step ([rinse_volume_L])
• Swab area for sampling ([swab_area_cm2])
• Sanitizing agent type and contact time if used ([sanitizing_agent_name], [sanitizing_duration_minutes])

Sampling Plan for Chromatography System Cleaning Validation

Sampling Locations and Rationale

1. Flow Path Surfaces: Swab sampling of inner surfaces of tubing, valves, and connectors identified as most prone to residue retention and difficult to clean.
2. Column Hardware: Swab or rinse sampling of column frits, seals, and hardware that contact process fluids directly.
3. Waste Lines: Rinse sample collection from drain and waste tubing to assess overall system cleanliness.
4. Critical Contact Points: Swab sample at seals and O-rings where product contact occurs and potential for residue trapping exists.

Site-specific inputs required:

• Swab area size [swab_area_cm2]
• Number of sampling points per component
• Sampling technique specifications (swab type, rinse volume)

Sampling Methodology and Frequency

1. Perform swab sampling immediately after completion of the cleaning cycle and prior to reassembly, ensuring aseptic sampling technique to avoid contamination.
2. Rinse samples to be collected from system lines after final rinse but before drying, allowing collection of eluates potentially containing residual detergent or drug substance.
3. Sampling frequency aligns with validation protocol plan: typically three consecutive cleaning cycles per equipment set evaluated to demonstrate cleaning consistency.
4. Additional sampling during routine campaigns as required by risk assessment or change control.

Analytical Methods and Residue Limits

Acceptance Criteria Based on PDE/ADE and MACO

The Maximum Allowable Carryover (MACO) shall be calculated using the following equation:

MACO = (PDE or ADE) × (Batch Size of Next Product) / (Batch Size of Previous Product)

Where:

• PDE/ADE: Permitted Daily Exposure or Acceptable Daily Exposure of the previous product /detergent based on toxicological assessment.
• Batch Sizes: Use representative batch sizes of previous and subsequent products for MACO calculation.

The residue acceptance limit in parts per million (ppm) is derived as:

Residue Limit (ppm) = (MACO × Dilution Factor) / (Swab or Rinse Sample Volume or Area)

Site-specific inputs required:

• PDE/ADE values for prior product and cleaning agents
• Batch sizes of products
• Sample volume or swab area normalization factors

Analytical Methods for Residue Detection

Legacy Acceptance Criteria (Fallback)

If PDE/ADE data or MACO calculation is unavailable, apply legacy cleaning limits as follows:

• Detector limit of ≤10 ppm for residual proteins or detergents
• Limit of 1/1000 of the therapeutic dose of previous product for carryover residues

Note: Legacy limits are only acceptable temporarily until toxicological justification or PDE/ADE data is established.

Documentation and Traceability

1. Record all cleaning and sampling activities with timestamps, operator signatures, and equipment used in validated logbooks or electronic systems.
2. Attach analytical test results with method validation certificates and chain of custody forms.
3. Document any deviations from the protocol and corrective actions taken.
4. Maintain traceability of cleaning validation batches and associated test reports for regulatory audit preparedness.

Recovery, LOD, and LOQ Expectations

Validation of analytical methods used for chromatography system cleaning residue assessment requires robust investigation into recovery rates as well as determination of limits of detection (LOD) and quantification (LOQ). Recovery studies must be conducted using representative swab and rinse samples from the drug contact surfaces defined in the Sampling Plan (Part B). Expected recovery for targeted residues, including active pharmaceutical ingredient (API), process impurities, and cleaning agents, should ideally be ≥ 80%. This threshold ensures method accuracy and avoids false-negatives that could jeopardize cleaning validation integrity.

The LOD for each analyte should be sufficiently sensitive to detect residues at or below the defined acceptance criteria, typically 10–30% of the permissible residual concentration (PDE/ADE-based MACO). LOQ must provide reliable quantification down to the PDE/ADE threshold, with precision and accuracy within ± 20%. Method sensitivity should be demonstrated through spiking blank matrices at multiple concentrations near the acceptance limit and establishing calibration curves with correlation coefficients (R²) ≥ 0.99.

Common practices for LOD and LOQ determination include signal-to-noise ratio calculations (LOD at S/N ≥ 3; LOQ at S/N ≥ 10) or standard deviation of response and slope techniques according to ICH Q2(R1). These studies must be conducted for each residue assay, including total organic carbon (TOC), specific detergents (e.g., detergents measurable by conductivity or HPLC), and, where applicable, biological contaminants or microbial endotoxins.

Acceptance Criteria Methodology: PDE/ADE-Based MACO Approach

The cornerstone of acceptance criteria for cleaning validation in biologics and biosimilars manufacturing chromatography systems is the PDE (Permitted Daily Exposure) / ADE (Acceptable Daily Exposure)-based Maximum Allowable Carryover (MACO) methodology. This approach aligns with regulatory expectations (FDA, EMA, ICH) and scientific risk assessment principles, facilitating risk-based residue limits tailored to patient safety and product risk profile.

The MACO is calculated per the following formula:

Within the protocol, the PDE/ADE values should be defined based on toxicological evaluations, supported by literature, regulatory databases, or health authority submissions. The batch size for the subsequent product is the conservative estimate of maximum product produced before equipment cleaning.

When analytical detection is based on surface swab or rinse samples, acceptance limits must translate MACO values into measurable concentrations (μg/cm² or μg/L), considering sampling recovery. The validated method’s limit of quantification (LOQ) must be below these calculated limits to ensure reliable detection and quantification.

Legacy Acceptance Criteria (Fallback): If PDE/ADE data is unavailable, legacy thresholds of 10 ppm residue or 1/1000 of the minimum therapeutic dose may be used temporarily with justification as a risk mitigation measure. Such limits are typically conservative but less scientifically precise than PDE/ADE MACO and should be replaced as soon as relevant toxicological data is available.

Detergent Residue Rationale and Limits

Detergent residues pose a significant risk in chromatography system reusable flow paths due to potential product contamination and adverse patient reactions. The selection of acceptance criteria for these cleaning agents must be method-specific and risk-based.

Detergent residue limits are frequently established using Total Organic Carbon (TOC) analysis, conductivity measurements, or specific analytical assays (e.g., HPLC quantification of surfactant components). TOC is the preferred screening method, given its sensitivity to organic residues including detergents; however, specificity limitations require confirmatory testing when indicated.

For TOC-based limits, the acceptable detergent residue threshold is usually linked to the maximum permitted TOC residual concentration derived from cleaning agent toxicity and rinse volume used during cleaning. For conductivity or specific surfactant quantification, limits correspond to analytically established LOQ levels aligned with toxicological safety margins.

Site-specific detergent acceptance limits should incorporate the following:

• The toxicological profile of the detergent (e.g., dermal/ingestion irritation levels, sensitization potential)
• Analytical sensitivity and specificity achievable by validated assay method
• Maximum rinse volume and residual amount expected to remain post-cleaning
• Cross-validation with TOC and specific detergent assays to ensure consistency

Residual detergent data must be interpreted in conjunction with process risk assessment outputs. The cleaning validation protocol must justify the detergent limits by referencing historical cleaning performance data combined with toxicological and analytical method rationales.

Deviations and Corrective Action/Preventive Action (CAPA)

Any deviations observed during cleaning validation activities—including sample collection, analytical testing, or acceptance criteria breaches—must be documented, investigated, and managed under the site’s deviation handling and CAPA systems.

The deviation investigation should include, but is not limited to:

1. Identification of root cause(s): improper cleaning procedure, analytical method limitations, sampling errors, or environmental factors
2. Impact assessment on product quality and patient safety
3. Immediate corrective actions: re-cleaning, re-sampling, re-testing, or analytical re-validation
4. Preventive measures: personnel training, SOP revisions, equipment maintenance, or process redesign
5. Effectiveness checks through subsequent validation runs or periodic monitoring

CAPA documentation must integrate learning from deviations to continuously improve the cleaning procedure and validation framework, ensuring compliance with regulatory GMP and supporting sustained manufacturing quality.

Continued Verification Plan

Cleaning validation is not a one-time exercise but requires ongoing monitoring and verification to maintain validated status throughout the equipment lifecycle. A systematic continued verification plan shall be included in the protocol and SOP, consisting of:

1. Routine cleaning efficacy checks via reduced frequency sampling per a risk-based schedule
2. Periodic analytical method requalification including recovery and sensitivity assessment at defined intervals or upon changes
3. Review of cleaning batch records, deviation reports, and maintenance logs for trends indicating possible cleaning failures
4. Verification following any changes in product formulation, detergent type, cleaning method, or equipment modifications
5. Microbiological monitoring if applicable as a risk mitigation for bioburden control

The schedule and scope of continued verification will be defined based on historical process consistency, product risk categorization, and regulatory guidance.

Revalidation Triggers

Revalidation of cleaning procedures and systems shall be performed whenever specific triggers occur. Triggers include but are not limited to:

• Change in drug product or intermediate processed through the chromatography system
• Modification in cleaning agents or detergents used, including concentration or formulation changes
• Significant equipment maintenance or replacement of flow path components
• Analytical method failures or substantial process deviations indicating potential impact on cleaning efficiency
• Regulatory audit findings or warning letters requesting validation reassessment
• Implementation of new cleaning technology or protocols

Upon a revalidation trigger, an impact assessment shall determine the scope and extent of revalidation required—from full cleaning validation to targeted verification studies.

Annexures and Templates

The following annexures and templates are included or referenced within this Cleaning Validation Protocol to facilitate implementation, documentation, and review:

Additional documents may be developed site-specifically to support training, equipment qualification, and management reviews related to cleaning validation governance.

Conclusion

An effective cleaning validation strategy for chromatography systems with reusable flow paths in biologics and biosimilars manufacturing hinges on scientifically sound acceptance criteria rooted in PDE/ADE-based MACO methodology. Robust validation of analytical methods—demonstrating recovery, LOD, and LOQ commensurate with these limits—is essential to ensure reliable detection of residual product and detergents. Rational detergent residue limits linked to validated assays protect product purity and patient safety.

Governance measures, including deviation management, CAPA implementation, and planned continued verification, sustain validated status and ensure compliance with evolving regulatory expectations. Clear documentation using standardized annexures and templates facilitates inspection readiness and supports continuous improvement of cleaning processes.

Revalidation policies anchored on well-defined triggers provide assurance that changes impacting cleaning efficacy are promptly addressed. Collectively, these justification and governance components deliver a robust framework ensuring the chromatography system cleaning validation program meets the highest pharmaceutical quality standards.