SOP for Operation, Cleaning, and Maintenance of Chromatography Skid

1. 0 Objective

To lay down the standardized procedure for the operation, monitoring, and cleaning of the In Vitro Transcription (IVT) Skid used for mRNA production in gene therapy manufacturing.

2.0 Scope

This SOP applies to all IVT skid units used in GMP production areas for the synthesis of mRNA via enzymatic transcription from DNA templates.

3.0 Responsibility

4.0 Definitions

• IVT (In Vitro Transcription): A biochemical reaction to synthesize RNA using DNA templates, NTPs, and RNA polymerase.
• Skid: An automated process system used for fluid handling, equipped with pumps, filters, sensors, and control software.
• CIP: Clean-in-Place.
• SIP: Steam-in-Place.

5.0 Materials and Equipment

5.1 Materials

• DNA Template (linearized, GMP-grade)
• rNTP mix (ATP, UTP, GTP, CTP)
• Reaction Buffer
• T7 RNA Polymerase Enzyme
• Pyrophosphatase
• DNase (for post-transcription template digestion)
• Water for Injection (WFI)
• 70% IPA
• Sodium Hydroxide (0.5N) / CIP solution

5.2 Equipment

• IVT Skid (with PLC/HMI interface)
• Temperature-controlled reaction vessel
• pH/DO sensors
• Peristaltic or diaphragm pumps
• UV conductivity sensor (optional)
• Filter assemblies (0.2 μm sterilizing grade)
• Single-use tubing or stainless-steel piping

6.0 Procedure

6.1 Equipment Preparation

1. Ensure the IVT Skid is installed and qualified (IQ/OQ/PQ completed).
2. Verify that the area is clean and classified for the operation.
3. Review the last cleaning log and maintenance records.

Ensure all consumables (bags, filters, tubing) are available and within expiry

SOP for Operation and Cleaning of Tangential Flow Filtration (TFF) System

1.0 Purpose

To define the procedure for the assembly, operation, monitoring, cleaning, and maintenance of the Tangential Flow Filtration (TFF) system used during downstream processing for the concentration and diafiltration of protein or viral drug substances.

2.0 Scope

This SOP is applicable to all trained personnel involved in the operation and cleaning of TFF systems in the GMP downstream processing area of biologics manufacturing.

3.0 Responsibilities

4.0 Definitions

• TFF: Tangential Flow Filtration – a filtration process used to concentrate or diafilter biomolecules.
• UF/DF: Ultrafiltration/Diafiltration.
• TMP: Transmembrane Pressure – the driving force for fluid across the membrane.
• CIP: Clean-in-Place.
• WFI: Water for Injection.
• HMI: Human Machine Interface.

5.0 Materials and Equipment

• TFF skid with pressure gauges, pump, and sensors
• UF/DF membrane cassettes or hollow fibers
• Tubing and connectors
• pH and conductivity meter
• Pre-use and post-use filters
• CIP solutions (NaOH, Acid, WFI)
• Sanitizing agents (e.g., peracetic acid)

6.0 Procedure

6.1 Pre-Operation Checks

• Ensure the TFF system is clean and idle.
• Verify calibration status of pressure sensors, conductivity, UV, and pH meters.
• Ensure availability of correct membrane (based on MWCO) and buffers.
• Verify equipment logbook entries for previous use and cleaning.
• Perform visual inspection of tubing, filters, and pump seals.

SOP for Operation, Cleaning, and Maintenance of Surge Vessel

1.0 Purpose

To describe the standard procedure for the operation, cleaning, sanitization, and maintenance of surge vessels used during downstream bioprocessing steps such as Tangential Flow Filtration (TFF), chromatography, and buffer holding operations.

2.0 Scope

This SOP applies to all stainless steel or single-use surge vessels (ranging from 5L to 500L) used in downstream processing of biologics, including gene therapy and cell therapy product manufacturing at [Facility Name].

3.0 Responsibilities

4.0 Definitions

• Surge Vessel: A process vessel used to dampen pulsation in flow or act as a buffer reservoir during filtration or transfer steps.
• CIP: Clean-In-Place
• SIP: Steam-In-Place
• SUS: Single-Use System
• WFI: Water for Injection

5.0 Materials and Equipment

• Stainless steel surge vessel or SUS bag with jacket
• Pressure gauge and rupture disk
• Level sensor (weight or ultrasonic)
• Sight glass or top-view port
• Inlet and outlet tubing with sanitary fittings
• Temperature sensor/RTD probe
• Nitrogen overlay setup (regulated pressure)
• Cleaning/sanitizing agents: NaOH, PAA, WFI

6.0 Procedure

6.1 Pre-Use Inspection

1. Verify vessel ID and status in the equipment logbook.
2. Confirm previous cleaning, sanitization, or SIP cycle completion.
3. Check calibration status of attached sensors (pressure, level, temperature).
4. Inspect vessel integrity, clamp fittings, and check for visible contamination.
5. Ensure appropriate gaskets, clamps, and vent filters are available.

6.2 Surge Vessel Setup

1. Connect vessel to TFF skid or chromatography system using validated hoses/tubing.
2. Install vent filters (0.2 µm hydrophobic) on designated ports.
3. Ensure nitrogen line is connected and pressure regulator is in place.
4. If required, perform SIP cycle using steam at 121°C for 30–60 minutes.
5. Allow vessel to cool before transferring product.

6.3 Operation During TFF or Chromatography

1. Fill surge vessel with feed solution (e.g., intermediate bulk, diafiltration buffer).
2. Apply gentle nitrogen overlay (0.2–0.4 bar) to prevent air ingress.
3. Maintain pressure and monitor for pulsation dampening.
4. Continuously monitor level via weight scale or level transmitter.
5. Prevent overflow or dry-out of vessel to protect integrity of filtration system.
6. Use sight glass or sampling port to take in-process samples if required.

SOP for Operation, Cleaning, and Maintenance of Lipid Nanoparticle (LNP) Formulation Skid

1.0 Purpose

To lay down the procedure for the operation, cleaning, sanitization, and maintenance of the Lipid Nanoparticle (LNP) formulation skid used in the preparation of lipid nanoparticles for drug delivery systems in gene therapy and biologics manufacturing.

2.0 Scope

This SOP applies to the formulation of LNPs using a continuous or semi-continuous LNP formulation skid, primarily used in the preparation of liposomal formulations for mRNA or gene-based therapies at the [Facility Name]. The SOP covers all associated equipment including lipid infusion pumps, homogenizers, and filtration systems.

3.0 Responsibilities

4.0 Definitions

• LNP (Lipid Nanoparticles): Nano-sized lipid particles used for encapsulating therapeutic agents, often for gene delivery.
• mRNA: Messenger RNA used in gene therapy products.
• CIP (Clean-in-Place): A method of cleaning the system without disassembling it.
• SUS (Single-Use System): Disposable system components used in LNP formulation.

5.0 Materials and Equipment

• LNP Formulation Skid with lipid infusion pump, homogenizer, and buffer dosing pump
• Lipid Solutions (e.g., cholesterol, phospholipids)
• Active Pharmaceutical Ingredient (API)
• Water for Injection (WFI)
• Solvents (e.g., ethanol, isopropanol)
• Sterile filters (0.2 µm)
• N2 or pressurized air supply
• Flow meters and temperature sensors
• pH sensors
• Conductivity meter

6.0 Procedure

6.1 Pre-Operation Setup

1. Verify Equipment Status:
   • Ensure the LNP formulation skid is cleaned and ready for use, with completed cleaning logs.
   • Verify that all components, including the lipid infusion pump, homogenizer, filtration unit, and tubing, are in proper working condition.
2. Check Calibration:
   • Ensure that the temperature sensors, pH probes, and flow meters are calibrated and within specification.
3. Prepare LNP Ingredients:
   • Prepare lipid formulations in compliance with the defined recipe (e.g., cholesterol and phospholipids) for LNP production.
   • Ensure that the Active Pharmaceutical Ingredient (API) is in solution at the required concentration and validated for use.
4. Install Membranes or Filters:
   • Install 0.2 µm sterile filters to the inlet and outlet lines to ensure sterility.

6.2 LNP Formulation Setup

1. Connect Lipid Reservoir and Pumps:
   • Connect the lipid solution reservoir to the lipid infusion pump.
   • Set the pump flow rate per formulation specifications (typically 1–5 mL/min for lipid infusion).
2. Connect API Reservoir:
   • Connect the API reservoir to the appropriate dosing system (e.g., peristaltic pump) ensuring precise control of API flow into the formulation system.
3. Connect Homogenizer:
   • Connect the homogenizer in-line with the lipid infusion pump and API delivery system. Ensure that the homogenizer is set to the required pressure (e.g., 10,000–20,000 psi) for efficient lipid nanoparticle formation.
4. Configure Flow Parameters:
   • Set the flow rate for the lipid and API components based on the process recipe (e.g., 10–50 mL/min for lipid and API mixing).
   • Set the temperature of the formulation system to the required temperature (typically 5°C–10°C).

SOP for Operation, Cleaning, and Maintenance of Chromatography Skid

1.0 Purpose

To lay down the procedure for operation, cleaning, sanitization, and maintenance protocols for the Chromatography Skid used in the purification of biomolecules, such as proteins, antibodies, and other biologics. This SOP ensures consistent system performance, regulatory compliance, and integrity of the final product.

2.0 Scope

Applies to the chromatography skid used in the downstream processing of biologics, such as monoclonal antibodies (mAbs), recombinant proteins, and gene therapy vectors. The skid is equipped with integrated pumps, valves, and detectors to perform various chromatography techniques like ion-exchange, affinity, and size-exclusion chromatography.

3.0 Responsibilities

4.0 Definitions

5.0 Materials and Equipment

• Chromatography System:
  Includes pumps, UV detectors, conductivity detectors, valves, pressure sensors, and chromatography columns.
• Buffers and Reagents:
  • Equilibration Buffer: Typically, a low-salt buffer (e.g., PBS, citrate) used to prepare the column for the sample.
  • Elution Buffer: A buffer that contains increasing ionic strength or pH gradients to elute the bound biomolecules.
  • Regeneration Buffers: Solutions like low pH or high salt to regenerate the chromatography resin.
• Other Required Materials:
  • WFI (Water for Injection) for cleaning and buffer preparation.
  • Single-Use Components: Including filters (0.2 µm), tubing, and connectors.

6.0 Procedure

6.1 Pre-Operation Setup

1. Verify System Integrity:
   • Check the Chromatography Skid: Ensure that all components are functional (e.g., pumps, valves, detectors). Perform a visual inspection for leaks or wear on gaskets and tubing.
   • Pumps Calibration: Ensure that flow rates are calibrated as per the manufacturer's specifications, with accuracy within ± 5% of the set value.
   • Check Pressure Sensors: Verify that pressure sensors are zeroed before running the system to ensure accurate pressure readings during operation.
2. Column Preparation:
   • Packing Verification: Confirm that the chromatography column is packed correctly. If necessary, perform a column packing procedure following the manufacturer's guidelines.
   • Column Wettability Test: Before use, ensure that the column is equilibrated with buffer. Check for air bubbles in the column, as these can interfere with flow.

SOP for Operation, Maintenance, and Calibration of Microplate Reader

1.0 Purpose

To define the procedure for the operation, routine maintenance, and calibration of the microplate reader used for spectrophotometric, fluorescence, and luminescence detection of samples in 96-well, 384-well, or custom microplates.

2.0 Scope

Applicable to all qualified analysts using the microplate reader in the Quality Control (QC) or analytical laboratories for methods such as ELISA, protein quantification (BCA/Bradford), DNA quantification, enzyme kinetics, cell viability, etc.

3.0 Responsibility

4.0 Definitions

• OD (Optical Density): A measure of absorbance at a specific wavelength.
• Monochromator/Filter-Based System: Wavelength selection systems used in plate readers for detection.
• Gain Adjustment: Setting optimal detector sensitivity in fluorescence/luminescence measurements.
• Background Subtraction: Removing signal noise from blank or negative control wells.

5.0 Equipment / Materials Required

• Microplate Reader (e.g., BioTek, Tecan, Molecular Devices)
• Desktop/Laptop with plate reader software (e.g., Gen5, SoftMax Pro)
• Calibrated pipettes and tips
• Flat-bottom 96/384-well microplates
• Plate sealing films (for incubated assays)
• Plate shaker (if integrated)
• Calibration Plate (Certified Optical or Fluorescence Reference Plate)
• Validated assay reagents (ELISA kits, BCA reagents, etc.)
• Wipe materials (lint-free wipes)
• 70% IPA or suitable surface disinfectant

6.0 Precautions

• Use gloves when handling plates to avoid smudges.
• Ensure no air bubbles in wells before reading.
• Plates must be oriented correctly in the reader tray.
• Avoid scratches or fingerprints on the plate bottom.
• Ensure the reader’s optics are clean before use.

7.0 Procedure

7.1 Equipment Startup

1. Turn ON the microplate reader using the power switch.
2. Start the software (e.g., Gen5 or SoftMax Pro) on the connected computer.
3. Allow the system to initialize (2–5 min) until “Ready” status is displayed.
4. Verify that the lamp/bulb status is OK (check in software diagnostics).
5. If using temperature control (e.g., for cell assays), preheat to required temperature (e.g., 37°C).

7.2 Pre-Run Checklist

7.3 Sample Reading

1. Load the plate into the reader (label facing outward or per orientation guide).
2. Run method or protocol as per assay SOP (pre-defined settings).
3. Monitor status on software interface.
4. Once reading is complete:
   • Review data for abnormal values or outliers.
   • Perform background subtraction if required.
   • Export results in PDF and Excel format.
   • Save project file for audit trail.