Cleaning Validation Protocol and Procedure for Mixing Vessels in Ophthalmic Dosage Manufacturing

Purpose and Scope

This document establishes the foundational framework for the cleaning validation protocol specific to mixing vessels utilized in the manufacture of ophthalmic dosage forms. The primary objective is to ensure that the cleaning procedures effectively remove residues of active pharmaceutical ingredients (APIs), excipients, and cleaning agents from product-contact surfaces of the mixing vessel to prevent cross-contamination and ensure patient safety.

This protocol covers the definition of cleaning processes, responsibilities, equipment overview, cleaning agents, and cleaning strategies applied to the mixing vessels used in ophthalmic formulations, including solutions, suspensions, and emulsions.

The scope encompasses all mixing vessels dedicated to ophthalmic product manufacturing within the facility, focusing on validated cleaning methods suitable for sterile or non-sterile environments as applicable, and aligned with regulatory expectations for cleaning validation.

Definitions and Abbreviations

Responsibilities

Quality Assurance (QA): Approves cleaning validation protocols, reviews data, and ensures compliance with regulatory standards.

Quality Control (QC): Performs sampling, sample analysis, and documents cleaning verification results.

Validation Team: Designs and executes cleaning validation studies, develops acceptance criteria, and compiles validation reports.

Production/Operations: Performs cleaning activities as per established procedures and documents them accurately.

Engineering: Supports maintenance of equipment cleanliness and ensures calibration and preventive maintenance of cleaning tools.

Safety Officer: Ensures PPE availability and compliance with safety guidelines during cleaning operations.

Safety and Personal Protective Equipment (PPE)

Personnel involved in cleaning activities and validation studies must wear appropriate PPE to protect themselves from chemical exposure, contamination, and injury. The recommended PPE includes:

• Chemical-resistant gloves suitable for the cleaning agents used.
• Protective goggles or face shields.
• Laboratory coats or coveralls.
• Respiratory protection if aerosols or harmful vapors are expected.
• Closed-toe, non-slip footwear.

All employees must be trained in the safe handling and disposal of cleaning agents and waste materials. Emergency procedures for chemical spills and exposure are to be strictly followed as per site safety guidelines.

Equipment Overview and Product-Contact Parts

Mixing Vessel Description:

• Type: Stainless steel jacketed mixing vessel designed for aseptic or non-aseptic ophthalmic formulations.
• Capacity: [Site-specific input: e.g., 500 L, 1000 L]
• Material of Construction: 316L stainless steel, electropolished finish (Ra < 0.5 microns).
• Agitator Type: Turbine/anchor agitator, dismantlable for cleaning.
• Seals and Gaskets: FDA-grade elastomer or PTFE-lined, cleanable by SIP/CIP as applicable.

Product-Contact Components Include:

• Vessel interior surfaces.
• Agitator blades and shaft.
• Manway covers and sight glasses.
• Spray balls or cleaning-in-place (CIP) nozzles (if available).
• Drain valves and lines connected to the vessel.
• Instrumentation—temperature probes, pH probes (if product-contact).

All product-contact surfaces are designed to avoid dead legs and areas that are inaccessible to cleaning tools or cleaning solutions.

High-Level Cleaning Strategy Overview

The cleaning approach for the mixing vessels used in ophthalmic dosage form production is designed to ensure elimination of residues from both the API and cleaning agents, with particular emphasis on the prevention of endotoxin and particulate contamination which can compromise product safety in ophthalmic formulations.

• Cleaning Methodology: Combination of manual cleaning and Cleaning-In-Place (CIP) systems where applicable.
• Detergent Use: Utilization of validated pharmaceutical-grade detergent [detergent_name], compatible with vessel materials and effective against product residues.
• Cleaning Tools: Non-shedding brushes, lint-free swabs, and specialized cleaning pads that reach all product-contact surfaces.
• Rinse Process: Multiple rinses with purified water with conductivity monitoring to ensure detergent removal.
• Validation Samples: Swab and rinse samples collected from critical product-contact surfaces per the Sampling Plan described in Part B.
• Cleaning Frequency: Cleaning is performed immediately after batch completion (within defined dirty hold time) and verified to maintain viral, microbial, and chemical product quality.

Cleaning Agents and Tools List

Definitions of Hold Times

Records and Forms

• Cleaning Validation Protocol Document
• Cleaning Procedure (SOP) for Mixing Vessels
• Cleaning Batch Records
• Cleaning Validation Sampling Log
• Sample Analysis Reports (TOC, HPLC, Conductivity)
• Equipment Cleaning Checklists
• Personal Protective Equipment Use Log
• Deviation Reports, if any anomalies occur during cleaning or validation

Site-specific Inputs Required

• Detergent product name and specifications ([detergent_name])
• Cleaning solution concentrations and preparation methods
• Mixing vessel specific dimensions including volume and critical surface area
• Hold time limits (dirty hold time and clean hold time)
• Rinse volume for CIP or manual rinses ([rinse_volume_L])
• Swabbing area for residue sampling ([swab_area_cm2])
• Authorized cleaning agents and tool list based on site availability
• Environmental conditions applicable to cleaning and hold time controls
• Analytical methods to be used for detergent and residue detection (TOC, HPLC, Conductivity)

Mixing Vessel (Ophthalmics) Cleaning Procedure

1. Pre-Clean:
   • Drain any residual product from the mixing vessel and associated piping.
   • Perform a visual inspection to ensure no visible residues remain.
   • Flush the vessel with potable water at ambient temperature to remove loose debris and bulk residues.
2. Disassembly:
   • Disassemble all removable parts of the mixing vessel including agitator blades, seals, gaskets, and inspection ports as per the equipment manual.
   • Lay out components on a clean, sanitized surface assigned for cleaning activities.
   • Ensure all parts are accounted for and inspected for damage before cleaning.
3. Cleaning (Wash):
   • Prepare cleaning solution using [detergent_name] at the recommended concentration as per supplier guidelines.
   • Immerse the disassembled parts in the cleaning solution or apply via automated CIP system ensuring complete coverage of all surfaces.
   • For the fixed vessel shell and internal surfaces, circulate the cleaning solution maintaining temperature between [temp_range_Celsius] °C and agitation for [time_minutes].
   • Use brushes as needed on detachable parts to remove stubborn residues.
4. Rinse Sequence:
   

   Rinse Step	Rinse Type	Parameters	Comments
   First Rinse	Potable Water	Volume: [rinse_volume_L] Temperature: Ambient Agitation: Yes, moderate	Removes detergent and loosened residues
   Second Rinse	Purified Water	Volume: [rinse_volume_L] Temperature: Ambient or 25–30°C Agitation: Moderate	Removes residual detergent and particulate matter
   Final Rinse	Purified Water	Volume: [rinse_volume_L] Temperature: Ambient Agitation: Thorough circulation or spray rinse	Ensures no detergent or residue remains

5. Drying:
   • Allow all cleaned components and vessel interior to air dry in a clean environment or use filtered compressed air as per site procedures. Avoid contamination during drying.
   • Ensure moisture removal particularly from crevices and threaded areas to prevent microbial growth.
6. Reassembly:
   • Reassemble the mixing vessel components following equipment manufacturer instructions.
   • Verify that all fasteners, seals, and gaskets are correctly positioned and secured.
   • Perform a final visual inspection for cleanliness and integrity of assembly.
7. Visual Inspection:
   • Inspect the interior surfaces of the vessel and all reassembled parts under appropriate lighting conditions to detect residues, stains, or discoloration.
   • Document findings; any visual residue requires repeat cleaning and inspection.

Cleaning Process Parameters

Sampling Plan for Cleaning Validation

Sampling Procedures and Considerations

1. Swabbing shall be performed using validated sterile swabs moistened with an appropriate extraction solvent or diluent compatible with the analytical method.
2. Following swabbing, each swab shall be placed into an individually labeled sterile container to prevent cross-contamination.
3. Each sample must be documented carefully with batch number, date of sampling, location, and the person performing the sampling to ensure traceability.
4. Chain-of-custody forms will be maintained from sample collection through to receipt in the QC laboratory to document sample integrity and secure handling.
5. Samples should be transported in temperature-controlled containers (2–8 °C) to preserve analyte stability until analysis.
6. Where feasible, sampling should occur immediately after the drying phase, but before reassembly to prevent recontamination.
7. In case of inaccessible surface areas for direct swabbing, rinse samples may be collected and analyzed as secondary verification.

Site-Specific Inputs Required

• Detergent name and concentration ([detergent_name], [detergent_concentration])
• Cleaning temperature range and time ([temp_range_Celsius], [time_minutes])
• Rinse volumes for each rinse step ([rinse_volume_L])
• Swab sampling areas ([swab_area_cm2])
• Validated extraction solvent for swabs

Sampling Plan

Sampling Locations

Samples shall be collected from critical surfaces most prone to product and detergent residues as well as possible microbial harborage sites. The sampling plan includes:

1. Vessel interior surfaces, including bottom and sidewalls
2. Agitator blades and hub
3. Seals and gaskets
4. Wetted piping and spray balls (if applicable)
5. Any fixed internal components such as baffles

Site-specific inputs required: Specify exact surface areas for sampling [swab_area_cm2] and any unique parts requiring additional sampling.

Sampling Methods

1. Swab Sampling: Use pre-moistened swabs suitable for residues and detergent detection to sample defined surface areas following established solvent and swabbing techniques.
2. Rinse Sampling: Collect rinse water samples from final rinse cycle output for detergent residue and TOC analysis.
3. Microbial Sampling (Risk-based): Where justified by risk assessment, include environmental and surface microbial sampling using contact plates or swabs.

All samples shall be clearly labeled with date, location, and time. Sampling personnel must wear appropriate PPE and follow aseptic techniques where applicable.

Analytical Methods and Test Parameters

Detergent Residue Analysis

Detergent residue levels shall be monitored using site-validated methods such as Total Organic Carbon (TOC) or specific detergent assays (e.g., HPLC, conductivity) according to the detergent chemistry. The method must have demonstrated sensitivity below the established acceptance limit.

• TOC Analysis: Suitable if detergent, product components, and process fluids have distinct organic carbon signatures.
• Specific Assay: When detergent possesses unique marker compounds allowing specific detection.

Product Residue Analysis

Product residues shall be quantified using validated analytical methods appropriate for the ophthalmic formulation, e.g., HPLC, UV-Vis spectroscopy, or other applicable assays depending on the active ingredient and formulation excipients.

Microbial Limits (If Applicable)

Microbiological limits shall be based on the risk assessment of the ophthalmic product and the cleaning environment. Typical limits include:

• Total viable count (TVC) ≤ [site-specific_limit] CFU/cm²
• Absence of specific pathogens as per pharmacopeial and regulatory guidelines

Acceptance Criteria

MACO-Based Approach for Product Residue

The acceptance limit for product residue is derived from the Maximum Allowable Carryover (MACO) calculated using the Permitted Daily Exposure (PDE) or Acceptable Daily Exposure (ADE) as follows:

MACO Calculation:

MACO (mg/cm²) = (PDE or ADE × batch size of current product) / (batch size of previous product × maximum dose × surface area)

The residue limit is adjusted accordingly and expressed in mg/cm². Swab and rinse sample results must meet levels below this value to be acceptable.

Detergent Residue Acceptance

Detergent residues must be below limits established by the supplier’s safety data and analytical method detection capabilities. Using TOC or specific assays, the limit is typically defined as:

• [TOC_limit] ppm TOC or corresponding specific assay threshold
• Complete absence of visible foam or odor after final rinse

Results exceeding limits require corrective action and re-cleaning.

Legacy Acceptance Criteria (Applicable Only if Required)

• Product Residue: ≤ 10 ppm or 1/1000th of the therapeutic dose (whichever is lower)
• Detergent Residue: ≤ 10 ppm or below method detection limit

This approach is considered less precise and should only be used if PDE/ADE data are unavailable.

Documentation and Reporting

1. Complete sampling records including location, time, operator, and sampling method
2. Analytical test reports for product residue, detergent residue, and microbiological tests (if applicable)
3. Calculation worksheets for MACO and acceptance criteria derivations
4. Deviations and non-conformances documented and investigated
5. Final approval signatures from QA, QC, and Validation teams

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

To ensure robust assessment of cleaning efficiency, method validation for sampling and analytical techniques must include determination of recovery, limit of detection (LOD), and limit of quantification (LOQ). Recovery studies shall be conducted by spiking representative surfaces or swabs corresponding to the mixing vessel material with known quantities of residual active pharmaceutical ingredients (APIs), formulation excipients, and cleaning agents such as [detergent_name]. A minimum acceptance recovery criterion of 80-120% is recommended to demonstrate method suitability.

The Limit of Detection (LOD) is defined as the lowest analyte amount that can be reliably distinguished from background noise, typically corresponding to a signal-to-noise ratio of 3:1. The Limit of Quantification (LOQ) is the lowest analyte concentration that can be quantitatively measured with acceptable precision and accuracy, often at a signal-to-noise ratio of 10:1. Both values shall be established for each analytical method employed, including assay of API residues, detergent residues, and total organic carbon (TOC) analysis if applicable.

These validated parameters ensure that analytical methods applied in the cleaning validation of mixing vessels for ophthalmic dosage forms are sensitive and accurate enough to reliably detect and quantify residue levels expected post-cleaning.

Acceptance Criteria Methodology: PDE/ADE-Based MACO Approach

The primary approach for setting acceptance criteria for residual residues in the mixing vessel cleaning validation utilizes the Permitted Daily Exposure (PDE) or Acceptable Daily Exposure (ADE) based Maximum Allowable Carryover (MACO) methodology.

Concept of PDE/ADE Based MACO: The PDE or ADE is the established limit of residual daily intake for a particular API or related compound, derived from toxicological data and regulatory guidance. The MACO represents the maximum allowable residual content of a substance that may carry over into the next product batch without posing safety or efficacy concerns.

This approach aligns with current industry best practices and regulatory expectations, ensuring patient safety, minimizing cross-contamination, and incorporating risk-based scientific rationale.

Procedure for Applying MACO:

1. Identify PDE/ADE values for all relevant APIs and excipients involved in the manufacturing process.
2. Determine worst-case batch size and maximum daily dosage of subsequent product.
3. Calculate MACO values per API using the formula above.
4. Apply calculated MACOs to convert into surface concentration acceptance criteria (e.g., µg/cm²). Use the total contact surface area of the mixing vessel defined in the Cleaning Validation master plan.
5. Compare analytical results against these acceptance limits to determine pass/fail of the cleaning process.

Fallback Legacy Acceptance Criteria: In cases where PDE/ADE data is unavailable or insufficient, legacy acceptance criteria may be applied for conservative justification. These include:

• Residue limit of 10 ppm (10 µg/g) in product or on equipment surfaces.
• 1/1000^th of the lowest therapeutic dose or toxic dose, whichever is lower.

This fallback approach is clearly documented as legacy and replaced once PDE/ADE data becomes available as part of ongoing continual improvement.

Detergent Residue Rationale and Acceptance

Cleaning agents such as [detergent_name] are commonly used in pharmaceutical cleaning processes. To assess the effectiveness of rinse steps and confirm removal of detergents, analytical methods such as Total Organic Carbon (TOC) analysis, conductivity, or specific detergent assays (e.g., colorimetric or HPLC-based tests) are employed.

The acceptance criteria for detergent residues must be scientifically justified and linked to analytical method sensitivity and potential impact on product quality or patient safety. Typical TOC limits may range between 10-25 ppm depending on detergent composition and regulatory guidance, but site-specific and product-specific evaluations must be conducted.

Justification for setting detergent residue limits includes:

• Demonstrated recovery and specificity of the chosen analytical method for detergent components.
• Assessment of toxicological profiles of detergent ingredients.
• Compatibility and stability studies showing no adverse impact of residual detergent on product quality.
• Historical data supporting effective removal using the validated cleaning procedure.

Where applicable, conductivity measurements serve as rapid in-process verification tools, but final compliance must be verified by validated chemical assays.

Deviations, Corrective and Preventive Actions (CAPA)

Any deviations identified during cleaning validation execution—including sampling, analytical results exceeding acceptance criteria, or procedural non-compliance—must be documented and investigated thoroughly to determine root cause.

Common deviation scenarios include:

• Residual API or detergent exceeding MACO or detergent acceptance limits.
• Analytical method or equipment failure leading to invalid data.
• Missed or improper sampling locations as per the Sampling Plan defined in Part B.
• Deviations in cleaning process parameters such as temperature, contact time, or detergent concentration.

CAPA processes must:

• Implement root cause analysis (e.g., Ishikawa, 5 Whys) to identify underlying issues.
• Define immediate containment measures such as batch holds or re-cleaning.
• Correct underlying procedural or training gaps.
• Update cleaning procedure, SOPs, or validation protocols if necessary.
• Monitor effectiveness of CAPA implementation through follow-up audits or sampling.

Continued Verification and Revalidation Plan

Cleaning validation is not a one-time event; continuous monitoring is essential to maintain validated status over the lifecycle of the mixing vessel used for ophthalmic drug manufacturing.

Continued Verification includes:

• Periodic routine swab and rinse sample analysis as per established frequency (e.g., quarterly or annually).
• Trend analysis of residue and microbial data to identify process drift or emerging risks.
• Periodic review of cleaning procedure adherence and training effectiveness.
• Verification of equipment condition, surface integrity, and maintenance records.

Triggers for Revalidation include:

• Change in product formulation or cleaning agent.
• Process equipment modifications such as changes in vessel lining, seals, or agitators.
• Significant deviations or failures discovered during routine verification sampling.
• Change in regulatory requirements or updated toxicity data impacting PDE/ADE values.
• Introduction of new manufacturing technologies or cleaning methods.

Revalidation scope and protocols will be risk-based and proportionate to the nature and extent of changes or findings.

Annexures and Templates List

• Annexure 1: Sampling Plan (referenced in Part B)
• Annexure 2: Analytical Method Validation Reports (Recovery, LOD, LOQ data)
• Annexure 3: PDE/ADE Justification Documentation
• Annexure 4: MACO Calculation Worksheets (including example calculations with placeholders)
• Annexure 5: Cleaning Procedure Standard Operating Procedure (SOP) for Mixing Vessel
• Annexure 6: Detergent Residue Analytical Method SOP
• Annexure 7: Deviation Reporting and CAPA Form Templates
• Annexure 8: Continued Verification Sampling Schedule and Reporting Template
• Annexure 9: Risk Assessment Documents for Cleaning Validation Revalidation Triggers

Conclusion

The cleaning validation acceptance criteria for ophthalmic mixing vessels rely on a scientifically justified PDE/ADE-based MACO methodology that ensures cross-contamination risk is minimized while optimizing operational efficiency. Recovery studies and method validation parameters such as LOD and LOQ must be rigorously established for both API residues and detergent residues to provide accurate residual quantification. The detergent residue limits are anchored to validated analytical methods, including TOC or specific assays, with robust rationale addressing patient safety and product integrity.

Deviations from validation criteria will follow a structured CAPA approach, emphasizing root cause analysis and corrective measures to maintain compliance and process reliability. A continued verification and risk-based revalidation plan ensures sustained control and alignment with evolving regulatory expectations throughout the equipment lifecycle.

This structured approach, supported by comprehensive annexures and templates, provides pharmaceutical manufacturing stakeholders with a clear, auditable framework to validate and maintain cleaning efficacy, supporting patient safety and product quality in the manufacture of ophthalmic dosage forms.