SOP for Viral Filtration and Polishing Filtration Procedures for Final Drug Substance

1.0 Objective

To provide a comprehensive and controlled procedure for performing viral filtration and polishing filtration of monoclonal antibody (mAb) drug substance using validated virus retentive filters and downstream-grade membrane filters to ensure safety and purity of the biopharmaceutical product prior to final formulation or fill-finish.

2.0 Scope

This SOP applies to all downstream purification processes in the Biologics Manufacturing Facility where monoclonal antibodies or recombinant proteins require removal of potential viral contaminants and sub-visible particulates using 20 nm virus filters and 0.22 μm sterilizing-grade filters.

3.0 Responsibility

• DSP Technicians: Perform setup, priming, viral filtration, polishing filtration, and flushing of the system. Document activities in batch records.

• Validation & Engineering: Ensure validated filter integrity and filtration parameters.

• QA Department: Review and approve documentation, deviations, and final batch release.

• QC Microbiology: Perform post-filtration bioburden and integrity tests.

• Warehouse: Ensure availability of filters and materials as per bill of materials (BOM).

4.0 References

• PDA Technical Report 26: Sterilizing Filtration of Liquids

• ICH Q5A (R1): Viral Safety Evaluation of Biotechnology Products

• Manufacturer’s Instructions for Use (IFU) for virus filters (e.g., Millipore Viresolve®, Sartorius Virosart®)

• JSA/SOP/BIO/DP/006 – Buffer Exchange and Concentration using TFF

• ISO 13408-2: Aseptic Processing – Filtration

• 21 CFR Part 610.122 – Filter integrity testing

5.0 Definitions

• Viral Filtration: Nanofiltration step designed to remove viruses ≥ 20 nm by size exclusion.

• Polishing Filtration: A 0.2/0.22 μm sterile filtration step for removal of particulates and bioburden.

• LRV: Log Reduction Value (measurement of virus removal efficacy).

• Flush Volume: The buffer volume passed through the filter before use to condition the membrane.

• Pre-Use Post-Sterilization Integrity Test (PUPSIT): Filter integrity test performed before product contact.

6.0 Materials and Equipment

• Sterile Virus Filter (20 nm cutoff) e.g., Viresolve® Pro or Virosart® HF

• 0.22 μm Sterile Polishing Filter (e.g., Sartopore® 2, Millipore Express®)

• Pre-filter (optional, 0.45/0.2 μm dual layer)

• Filter holders or housings

• Stainless steel filtration skid or disposable single-use flowpath

• Sanitized and calibrated pressure gauges (0–3 bar range)

• Integrity tester (e.g., Sartocheck®, Palltronic®)

• pH and conductivity meter

• WFI (Water for Injection) or buffer solution (as per batch record)

• Pressure-rated containers for filtrate collection

• Sterile tubing and aseptic connectors

• Sanitized laminar airflow hood or cleanroom suite (Grade B/A)

• Batch-specific viral clearance validated filters as per Process Validation Protocol

SOP for Low pH Viral Inactivation Procedure for Monoclonal Antibody Purification

1.0 Objective

To describe the standard procedure for performing low pH viral inactivation during the downstream purification of monoclonal antibodies (mAbs), ensuring effective removal or inactivation of enveloped viruses following the Protein A capture step.

2.0 Scope

This SOP applies to the viral inactivation step performed after Protein A chromatography in the purification of monoclonal antibodies derived from mammalian cell culture, specifically Chinese Hamster Ovary (CHO) cell lines.

3.0 Responsibilities

Downstream Processing (DSP) Technicians:

• Prepare buffers, set up equipment, execute the inactivation step, and maintain process integrity.

Quality Assurance (QA):

• Review the batch records and approve process execution.

Quality Control (QC):

• Analyze pre- and post-inactivation samples for pH, protein concentration, and virus clearance verification (if applicable).

Validation Team:

• Support viral clearance studies and maintain required documentation.

4.0 References

• ICH Q5A(R1): Viral Safety Evaluation of Biotechnology Products
• EMA CHMP/BWP/398498/2005
• WHO TRS 978 – Annex 3
• Manufacturer’s Instructions for Protein A resin
• JSA/SOP/BIO/DP/003 – MabSelect SuRe™ Column Use
• JSA/SOP/BIO/DP/010 – A280 Protein Quantification

5.0 Definitions

Low pH Inactivation:
Acidifying the eluate to pH 3.5–3.7 for ≥60 minutes to inactivate enveloped viruses.

Eluate:
Product collected post Protein A chromatography.

Neutralization:
Adjusting the pH of the inactivated pool back to 6.8–7.5 using a basic buffer.

6.0 Materials and Equipment

• Protein A eluate
• 1 M Acetic Acid or Citric Acid (sterile)
• 1 M Tris Base (pH 8.8–9.0)
• pH meter (calibrated)
• Magnetic stirrer or overhead mixer
• Sterile, endotoxin-free vessel
• A280 reader or spectrophotometer
• 0.2 µm sterile filter
• Sampling tubes

7.0 Procedure

7.1 Preparation

1. Verify availability and volume of Protein A eluate (quantify via A280).
2. Cool eluate to 20–25°C.
3. Transfer to a sterile reaction vessel with stirring capabilities.

7.2 Buffer Preparation

1. Prepare acid buffer (1 M Acetic or Citric Acid) using WFI.
2. Prepare neutralization buffer (1 M Tris Base, pH 8.8–9.0).
3. Label and record lot numbers and expiry dates.

7.3 pH Adjustment & Inactivation

1. Measure initial pH of eluate (should be ~5.5–6.0).
2. Add acid buffer slowly and dropwise while mixing.
3. Monitor until pH reaches 3.5–3.7.
4. Start timer when target pH is achieved.
5. Maintain gentle mixing for at least 60 minutes at 20–25°C.

SOP for Regeneration and Storage of Affinity and Ion Exchange Chromatography Columns

1.0 Objective

To provide a comprehensive procedure for the regeneration, sanitization, storage, and reuse of affinity (e.g., Protein A) and ion exchange (e.g., Q and S) chromatography columns used in the downstream purification of monoclonal antibodies (mAbs). This ensures consistent column performance, prevents cross-contamination, and maintains resin integrity and binding capacity across multiple cycles.

2.0 Scope

Applicable to all pre-packed and self-packed chromatography columns used in downstream processing for mAb purification, including Protein A, Q Sepharose™, and SP Sepharose™ columns operated in GMP production suites.

3.0 Responsibility

4.0 References

• Manufacturer’s instructions for chromatography resins (Cytiva, Bio-Rad, Sartorius)
• ICH Q7 – Good Manufacturing Practice for Active Pharmaceutical Ingredients
• ICH Q5C – Stability Testing of Biotechnological/Biological Products
• JSA/SOP/BIO/DP/004 – Ion Exchange Chromatography Procedure
• JSA/SOP/BIO/DP/002 – Operation and Cleaning of Protein A Columns

5.0 Definitions

• Regeneration: Restoration of column performance by removing bound impurities, proteins, and fouling agents after use.
• Sanitization: Use of agents (e.g., NaOH) to control microbial growth and bioburden on column resin.
• CIP: Clean-in-place; automated internal cleaning without disassembling the system.
• Reuse Cycle: One complete use of a column in a batch process, followed by regeneration and storage.

6.0 Materials and Equipment

• Buffers:
  • Regeneration Buffer (e.g., 1 M NaCl, 1 M Acetic Acid, 6 M Urea)
  • Sanitizing Agent (0.1–0.5 M NaOH)
  • Storage Buffer (20% Ethanol or 0.1 M NaOH as per resin type)
• Column(s) (e.g., MabSelect SuRe™, Q Sepharose™ FF, SP Sepharose™ FF)
• Chromatography Skid with CIP capability
• pH and conductivity meter
• Peristaltic pump (if manual)
• Column performance test kit (for periodic evaluation)
• Logbook for column use history

SOP for Use of Nanodrop or Spectrophotometer for A280 Analysis of Protein Eluates

1.0 Objective

To establish a standardized procedure for determining the protein concentration of monoclonal antibody (mAb) eluates using A280 nm absorbance measurement via Nanodrop™ spectrophotometer or UV-visible spectrophotometer. This SOP ensures accurate quantitation based on Beer-Lambert Law for in-process and final purification samples.

2.0 Scope

This SOP applies to all in-process eluate samples collected from Protein A, ion exchange, or size-exclusion chromatography steps during the downstream purification of monoclonal antibodies at the biologics manufacturing site.

3.0 Responsibility

4.0 References

• Beer-Lambert Law: A = ε × c × l

• Nanodrop 2000/One User Manual (Thermo Fisher Scientific)

• USP <853> – Spectrophotometry and Light Scattering

• ICH Q6B – Specifications for Biotech/Biological Products

• JSA/SOP/BIO/QC/007 – Calibration and Maintenance of UV-Vis Spectrophotometer

5.0 Definitions

• A280: Absorbance measured at 280 nm to estimate protein concentration.

• ε (Epsilon): Extinction coefficient of the protein (typically ~1.4 for mAbs at 1 mg/mL).

• c: Concentration of protein in mg/mL.

• l: Path length of the cuvette or Nanodrop pedestal (usually 1 cm).

6.0 Materials and Equipment

• Nanodrop 2000 or Nanodrop One Spectrophotometer

• OR UV-Vis Spectrophotometer with 1 cm quartz cuvettes

• Sample eluates from downstream purification

• WFI or 1x PBS (as blank)

• Lint-free tissue for Nanodrop pedestal cleaning

• Protein concentration worksheet or validated LIMS entry form

SOP for Cleaning and Sanitization of Chromatography Skid and Associated Systems

1.0 Objective

To define a standardized, validated procedure for the cleaning and sanitization of chromatography skids and associated fluid paths used in downstream processing, ensuring removal of process-related and microbial contaminants, and compliance with cGMP requirements.

2.0 Scope

This SOP is applicable to all automated and semi-automated chromatography systems (skids) used for purification of biologics, particularly monoclonal antibodies (mAbs), including all connected tubing, sensors, sample loops, column housings, and flow paths within the downstream purification suites.

3.0 Responsibility

4.0 References

• Equipment Operation Manual (GE ÄKTA™, Sartorius, Bio-Rad NGC, etc.)
• FDA 21 CFR Part 211 – cGMP for Equipment Cleaning
• USP <1072> – Disinfectants and Antiseptics
• JSA/SOP/BIO/QA/009 – Cleaning Validation Master Plan
• JSA/SOP/BIO/QC/022 – Endotoxin Testing (LAL Method)

5.0 Definitions

• CIP: Clean-in-Place – Automated internal cleaning of the skid and fluid paths.
• SIP: Sanitize-in-Place – Sanitization of system components using chemical agents.
• Product Contact Surfaces: Any skid surface (e.g., tubing, valves, pressure sensors, flow paths) exposed to process fluids.

6.0 Materials and Equipment

• Purified Water (PW) or Water for Injection (WFI)
• 1 M Sodium Hydroxide (NaOH)
• 20% Ethanol or 0.5 N Sodium Hydroxide (for sanitization)
• 1 M Hydrochloric Acid (if needed for pH neutralization)
• Peracetic Acid (0.2%) for final sanitization, if applicable
• Chromatography skid (GE ÄKTA™, Bio-Rad NGC, or equivalent)
• Pressure gauges, pH & conductivity meters
• Waste containers (dedicated for alkaline and acidic waste)

7.0 Procedure

7.1 Pre-Cleaning Checks

7.1.1 Ensure the skid is idle and disconnected from columns or product reservoirs.

7.1.2 Confirm all tubing is connected securely to flow paths and waste containers.

7.1.3 Verify drain valves are open and skid is properly grounded.

7.1.4 Review previous batch record and confirm cleaning requirement (e.g., Type A: post-campaign, Type B: post-batch).

7.2 Cleaning Procedure (CIP)

7.2.1 Flush with WFI or PW (10 CV):

• Remove residual product and buffer.
• Monitor UV, pH, and conductivity to confirm clearance.

7.2.2 Alkaline Cleaning with 1 M NaOH (5–10 CV):

• Circulate NaOH through all product contact surfaces.
• Hold static for 30–60 minutes (depending on soil load).
• Maintain NaOH contact temperature between 25–30°C.

7.2.3 Post-Caustic Flush (10 CV WFI):

• Remove NaOH residues thoroughly.
• pH should be < 9.0 before proceeding.

7.2.4 Acid Rinse (Optional, 1 M HCl):

• Used only if NaOH neutralization or metal scale prevention is required.
• Flush with 5 CV, then rinse with WFI until pH ~7.0.

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SOP for Protein Quantitation Using BCA and Bradford Assay During Downstream Processing

1.0 Objective

To describe the standardized procedure for quantifying total protein concentration in process samples using Bicinchoninic Acid (BCA) and Bradford protein assays as part of downstream processing (DSP) in biologics manufacturing.

2.0 Scope

This procedure applies to in-process, intermediate, and final drug substance samples obtained during purification of monoclonal antibodies (mAbs) and recombinant proteins in a GMP-regulated downstream processing environment.

3.0 Responsibility

4.0 References

• Thermo Scientific BCA Protein Assay Kit Manual
• Bio-Rad Bradford Reagent Kit Instructions
• ICH Q2(R1) – Validation of Analytical Procedures
• JSA/SOP/BIO/QC/010 – General Laboratory Practices
• JSA/SOP/BIO/QC/022 – Spectrophotometer Operation & Calibration

5.0 Definitions

• BCA Assay: A colorimetric assay based on reduction of Cu²⁺ to Cu¹⁺ by protein in an alkaline medium, followed by BCA complexation forming a purple color.
• Bradford Assay: Dye-binding assay using Coomassie Brilliant Blue G-250, which binds to protein and shifts absorbance maximum from 465 nm to 595 nm.
• Standard Curve: Calibration plot constructed from known concentrations of bovine serum albumin (BSA).

6.0 Materials and Equipment

7.0 Procedure

7.1 Preparation of Standards

1. Thaw BSA standard and prepare a dilution series (e.g., 0, 25, 125, 250, 500, 750, 1000 µg/mL) using assay diluent (e.g., PBS or 0.9% NaCl).
2. Vortex and label each dilution tube clearly.

7.2 BCA Assay Procedure

1. Reagent Preparation:
   • Mix BCA Reagent A and Reagent B in a 50:1 ratio as per kit instructions. Prepare fresh before use.
2. Sample Loading:
   • Add 25 µL of standards and samples into wells of a microplate or cuvette.
3. Reagent Addition:
   • Add 200 µL of working BCA reagent to each well or cuvette.
   • Mix gently by tapping or brief vortexing.
4. Incubation:
   • Incubate at 37°C for 30 minutes or RT for 2 hours (depending on kit protocol).
5. Reading:
   • Measure absorbance at 562 nm.
6. Calculations:
   • Plot standard curve and interpolate sample concentrations.
   • Acceptable linearity: R² > 0.98.

7.3 Bradford Assay Procedure

1. Reagent Preparation:
   • Equilibrate Bradford reagent to RT. No dilution needed.
2. Sample Loading:
   • Pipette 5–20 µL of sample or standard into clean tubes.
3. Reagent Addition:
   • Add 1.0 mL Bradford reagent to each tube.
   • Vortex gently for uniform mixing.
4. Incubation:
   • Incubate at RT for 5–10 minutes (avoid light exposure).
5. Reading:
   • Measure absorbance at 595 nm.
6. Calculations:
   • Generate standard curve, determine concentration by interpolation..

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SOP for Inline pH and Conductivity Monitoring in Chromatography and UF/DF Processes

1.0 Objective

To provide a standardized procedure for inline monitoring of pH and conductivity during chromatography and ultrafiltration/diafiltration (UF/DF) steps in downstream purification of biological drug substances.

2.0 Scope

This SOP applies to all DSP operations involving real-time control and monitoring of critical process parameters—pH and conductivity—during anion/cation exchange chromatography, polishing filtration, buffer exchange, and viral inactivation steps.

3.0 Responsibility

4.0 References

• USP <791> pH
• USP <645> Water Conductivity
• ICH Q8(R2) – Pharmaceutical Development
• JSA/SOP/BIO/ENG/014 – Probe Calibration and Maintenance
• JSA/SOP/BIO/DP/004 – Ion Exchange Chromatography
• JSA/SOP/BIO/DP/006 – Buffer Exchange & TFF

5.0 Definitions

6.0 Materials and Equipment

7.0 Procedure

7.1 Sensor Calibration (Prior to Batch Start)

1. Perform 2-point or 3-point calibration for both pH and conductivity probes using certified standards.
2. Rinse sensor with deionized water before and after each calibration point.
3. Record calibration results in the calibration logbook.

7.2 System Setup

1. Verify that sensors are properly installed into their respective flow cells or process tubing.
2. Confirm electrical connection to the transmitter or SCADA interface.
3. Initialize software interface to capture real-time pH and conductivity readings.
4. Set alarm limits based on the buffer or step-specific target values.

7.3 Inline Monitoring During Chromatography

1. Start monitoring immediately after column equilibration.
2. During sample loading, observe pH and conductivity stability; deviation indicates improper binding.
3. During wash steps, expect conductivity to remain stable while pH reflects the buffer profile.
4. During elution, expect a conductivity increase during salt gradient and a minor pH shift.
5. Record real-time trends and correlate with UV280 peaks and fraction collection.

7.4 Inline Monitoring During UF/DF (TFF)

1. Confirm sensors are installed post-pump or within the retentate line.
2. Monitor conductivity trends to ensure effective buffer exchange (e.g., drop from 150 mM NaCl to <10 mM).
3. Ensure pH remains within specified limits during diafiltration (e.g., 6.5–7.5).
4. Record values at:
   • Start of diafiltration
   • Every 2 DVs (Diafiltration Volumes)
   • End of process

5. If set-points are not met, continue additional buffer exchanges as per protocol

SOP for Endotoxin Control and Clearance in Downstream Purification Steps

1.0 Objective

To define a standardized procedure for controlling, monitoring, and validating the removal of bacterial endotoxins during the downstream purification of monoclonal antibodies and other biologics, ensuring the final drug substance meets regulatory limits.

2.0 Scope

This SOP applies to all downstream purification operations where endotoxin presence is a critical quality attribute (CQA), including Protein A capture, ion exchange polishing, viral filtration, and ultrafiltration/diafiltration steps.

3.0 Responsibility

4.0 References

• USP <85> Bacterial Endotoxins Test
• ICH Q6B – Specifications for Biotechnological/Biological Products
• EudraLex Volume 4, Annex 1 – Manufacture of Sterile Medicinal Products
• FDA Guidance for Industry: Pyrogen and Endotoxin Testing
• JSA/SOP/BIO/QC/023 – Limulus Amebocyte Lysate (LAL) Assay
• JSA/SOP/BIO/DP/003 to DP/013 – Relevant DSP SOPs

5.0 Definitions

6.0 Materials and Equipment

7.0 Procedure

7.1 Pre-Processing Controls

1. Verify equipment cleaning using depyrogenation logs and rinse water BET testing.
2. Use only validated low-endotoxin buffers, prepared with Water for Injection (WFI).
3. Ensure all tubing, bags, and containers are certified as endotoxin-free or have undergone dry heat depyrogenation.

7.2 In-Process Endotoxin Control Points

a. Protein A Capture Chromatography

• Acts as first significant clearance step.
• Collect flow-through and eluate samples for BET.
• Expected LRV: 2–3 logs.

b. Ion Exchange Polishing (Q and S Columns)

• Removes host cell proteins and residual endotoxins via charge interactions.
• Collect post-load and eluate samples.
• Expected LRV: 1–2 logs.

c. Viral Filtration (0.2 µm or nanofiltration)

• Removes bacteria and endotoxin aggregates.
• Monitor pre- and post-filter samples.
• Expected LRV: 2–3 logs, depending on filter type.

d. UF/DF and Final Concentration

• Use membranes with <10 EU/mL release criteria.
• Perform diafiltration using LAL-tested buffer.
• Monitor final pool for endotoxin content.
• Expected LRV: 1 log or more if using large volumes of low-endotoxin buffer.

7.3 In-Process Testing and Documentation

1. Collect triplicate in-process samples at each critical point for endotoxin testing.
2. Record sample ID, process stage, time, and operator initials in the batch record.
3. Submit to QC Microbiology within 2 hours of sampling for LAL testing.
4. Maintain sample chain-of-custody.

7.4 Acceptance Criteria

Note: Limits may vary based on product type and dose. Refer to product-specific specifications.

SOP for Calibration and Qualification of Chromatography Columns and Skids

1.0 Objective

To establish a standardized procedure for calibration and qualification of chromatography skids and columns used in downstream purification, ensuring accuracy, reliability, and regulatory compliance during biologics manufacturing.

2.0 Scope

This SOP applies to all chromatography systems and associated columns used in the downstream processing of monoclonal antibodies and other recombinant proteins at [Facility Name].

3.0 Responsibility

• DSP Production Operators: Execution of calibration, qualification, and performance verification.
• Engineering Team: Execution of calibration activities, documentation, and scheduling.
• QA Department: Review and approval of calibration records, qualification protocols, and deviations.
• QC Department: Analysis of in-process samples and analytical support for PQ.

4.0 References

• USP <1058> – Analytical Instrument Qualification
• ICH Q8 (R2), Q9, Q10 – Pharmaceutical Development, Risk Management, Quality System
• Manufacturer Manuals (e.g., Cytiva ÄKTA™, Pall, Bio-Rad systems)
• JSA/SOP/BIO/DP/013 – Inline pH and Conductivity Monitoring

5.0 Definitions

6.0 Materials and Equipment

• Chromatography skid (e.g., ÄKTA™, Pall, or equivalent)
• Columns (Protein A, Ion Exchange, SEC, etc.)
• Calibration standards:
  • pH 4.0, 7.0, 10.0 buffers
  • Conductivity standards (e.g., 1.413 µS/cm and 15.0 mS/cm)
  • Certified pressure and flow calibrators
  • Temperature validation bath and thermometer
• BSA or IgG protein standard for PQ
• Data acquisition software and control system
• Qualification protocol templates and calibration logbooks

7.0 Procedure

7.1 Installation Qualification (IQ)

1. Confirm the chromatography skid and columns are installed per manufacturer’s specifications.
2. Verify component labeling, serial numbers, and traceability.
3. Review vendor-supplied documentation (FAT, SAT, calibration certificates).
4. Confirm electrical connections, utility hookups (WFI, compressed air, etc.), and drain systems.
5. Log all verification steps and discrepancies in the IQ protocol.

7.2 Operational Qualification (OQ)

1. Power on the system and initialize all components.
2. Verify the following functional tests:
   • Pump flow accuracy (±5%)
   • Detector signal stability (UV, conductivity, pH)
   • Valve switching response
   • Alarm system functionality
   • System communication with software
3. Conduct a test run with water to verify baseline parameters.
4. Document results and sign off OQ checklist.

7.3 Performance Qualification (PQ)

7.3.1 Purpose

To verify reproducible system performance using a standard protein under simulated production conditions.

7.3.2 Preparation

1. Equilibrate column per standard method using appropriate buffer.
2. Prepare BSA or IgG solution (1–5 mg/mL in loading buffer).
3. Validate pH, conductivity, and volume of buffer prior to use.

7.3.3 Procedure

Perform three independent purification runs under identical conditions.

For each run:

1. Equilibrate column with 5 CV of buffer A.
2. Load standard protein solution at controlled flow rate.
3. Wash column with 5 CV of wash buffer.
4. Elute with appropriate elution buffer or gradient.
5. Collect fractions and monitor UV280, pH, and conductivity inline.
6. Regenerate column post-run as per SOP JSA/SOP/BIO/DP/009.
7. Save chromatograms and sensor data from each run.

7.3.4 Data Collection

For each PQ run, document and compare:

• Yield (% Recovery): Compare load vs eluate concentration
• Pressure Profile: Monitor pressure increases across the column
• Flow Accuracy: Validate system flow rate with manual measurement
• pH/Conductivity Control: Inline sensors should reflect expected ranges
• System Alarms/Deviations: Record and investigate any anomalies
• Chromatographic Consistency: Overlay chromatograms and analyze peak uniformity

7.3.5 Acceptance Criteria

• Yield consistency: ≥ 95% ± 2%
• Pressure variation: ±10% across runs
• Chromatographic reproducibility: Peak retention time within ±0.5 min

SOP for Cleaning Validation and Verification of Downstream Chromatography Systems

1.0 Objective:

To establish a validated procedure for cleaning and verification of chromatography systems (including columns, skids, tubing, and inline sensors) used in downstream purification to prevent cross-contamination and maintain system performance.

2.0 Scope:

This SOP is applicable to all chromatography systems used in the purification of monoclonal antibodies (mAbs) and recombinant proteins in the downstream processing areas of the biologics manufacturing facility.

3.0 Responsibility:

• DSP Operators: Perform cleaning and verification as per protocol.
• QA: Review cleaning records and approve validation status.
• QC Microbiology and QC Chemistry: Perform microbial and chemical residue testing.
• Validation Team: Design and execute cleaning validation protocols and reports.

4.0 References:

• EMA Guideline on Cleaning Validation (EMEA/INS/GMP/173701/2012)
• FDA Guide to Inspections of Cleaning Validation (1993)
• WHO Annex 7 (TRS 1019) – Cleaning validation
• JSA/SOP/BIO/QA/CL/001 – Cleaning Validation Master Plan
• JSA/SOP/BIO/QA/VAL/002 – Analytical Method Validation
• Equipment manuals for chromatography skids and columns

5.0 Definitions:

• Cleaning Validation: Documented evidence that a cleaning procedure consistently removes residues to predetermined acceptable levels.
• Cleaning Verification: Testing performed after individual cleaning events to verify adequacy.
• ARL: Acceptable Residual Limit based on PDE (Permitted Daily Exposure) values.
• MACO: Maximum Allowable Carryover.
• TAC: Total Organic Carbon – indicator of organic residue presence.

6.0 Materials and Equipment:

• 0.5 M NaOH, 1 M NaCl, WFI, 70% IPA
• CIP and SIP systems or manual cleaning set-up
• TOC Analyzer
• Swabs and rinse sample bottles (validated for recovery)
• Conductivity and pH meters
• Chromatography system with cleaning loop
• LAL test kits (for endotoxin)
• Bacterial endotoxin-free WFI

7.0 Procedure:

7.1 Cleaning Strategy:

1. Determine cleaning approach – manual or automated (CIP).
2. Select worst-case product for validation based on:
   • Lowest solubility
   • Highest toxicity
   • Highest batch size
3. Define shared equipment trains requiring cleaning validation.

7.2 Cleaning Procedure:

For Automated Cleaning (CIP):

1. Flush system with WFI for 5 CV.
2. Introduce 0.5 M NaOH at 1 CV/min for 20 minutes.
3. Hold for 30 minutes (static exposure).
4. Flush with WFI for 5 CV.
5. Neutralize with 1 M NaCl if required.
6. Final rinse with WFI until conductivity < 1.3 µS/cm and pH ~7.

For Manual Cleaning:

1. Disassemble flow path components (tubing, connectors, column).
2. Soak in 0.5 M NaOH for 30–60 minutes.
3. Rinse thoroughly with WFI.
4. Visually inspect for residues or discoloration.
5. Final rinse until conductivity and pH meet acceptance criteria.

7.3 Cleaning Validation:

7.3.1 Protocol Design:

1. Select three consecutive cleaning runs for validation.
2. Choose locations for sampling: worst-case, hard-to-clean areas (e.g., outlet tubing, valve joints).
3. Perform swab and rinse sampling after each cleaning.
4. Analyze samples for:
   • TOC
   • Specific active protein (e.g., mAb)
   • Conductivity
   • pH
   • Microbial contamination
   • Endotoxins

7.3.2 Acceptance Criteria:

7.4 Performance Qualification (PQ):

Perform three purification runs using a model protein (e.g., BSA or IgG) post-cleaning.

Monitor and Document:

• Yield (% recovery)
• Pressure profile during each step
• pH and conductivity profiles during equilibration, load, wash, and elution
• Flow rate accuracy and stability
• System alarms (if any)
• Chromatogram analysis for peak reproducibility

Acceptance Criteria:

• Yield variance < 5%
• Pressure deviation < 10%
• Chromatographic profile should match validation baseline.

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