Preparation of Immunoliposomes for Targeted Drug Delivery
1) Purpose
The purpose of this SOP is to describe the procedure for the preparation of immunoliposomes. Immunoliposomes are liposomes that are surface-modified with antibodies or antibody fragments for targeted drug delivery. These liposomes bind specifically to target cells, such as cancer cells, enhancing the efficacy of the delivered therapeutic agents while minimizing off-target effects. This SOP outlines the steps for antibody conjugation to liposomes, ensuring correct antibody attachment and functionality.
2) Scope
This SOP applies to personnel involved in the preparation of immunoliposomes for research or pharmaceutical purposes. It includes instructions for the conjugation of antibodies to liposomes, preparation of lipid films, hydration of liposomes, size reduction, and quality control testing to ensure proper antibody incorporation and targeting specificity.
3) Responsibilities
- Operators: Responsible for executing the procedure and ensuring proper conjugation of antibodies to liposomes.
- QA Team: Responsible for ensuring compliance with SOP guidelines and quality standards.
- QC Team: Responsible for conducting quality control tests to verify the stability, size, and targeting efficiency of immunoliposomes.
4) Procedure
4.1 Equipment Setup
The equipment required for the preparation of immunoliposomes must be properly cleaned and calibrated before use. The following equipment is necessary:
4.1.1 Required Equipment
- Rotary evaporator
- High-pressure homogenizer or sonicator
- Magnetic stirrer
- pH meter
- Temperature-controlled water bath
- Surface plasmon resonance (SPR) instrument (for antibody binding efficiency)
- Dynamic light scattering (DLS) instrument
4.1.2 Equipment Calibration
- 4.1.2.1 Ensure that the rotary evaporator is calibrated for temperature and vacuum pressure.
- 4.1.2.2 Verify that the sonicator or high-pressure homogenizer is calibrated for accurate size reduction of liposomes.
- 4.1.2.3 Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).
4.2 Lipid Film Formation
The first step in the preparation of immunoliposomes is the formation of a lipid film by dissolving lipids in an organic solvent, followed by solvent evaporation under reduced pressure.
4.2.1 Lipid Dissolution
- 4.2.1.1 Weigh the required amounts of lipid components (e.g., phospholipids, cholesterol, and DSPE-PEG or DSPE-Maleimide) based on the formulation protocol and record the weights in the Batch Manufacturing Record (BMR).
- 4.2.1.2 Dissolve the lipids in an organic solvent, such as chloroform or ethanol, in a round-bottom flask.
- 4.2.1.3 Use a magnetic stirrer to ensure complete dissolution of the lipids.
4.2.2 Solvent Evaporation
- 4.2.2.1 Attach the round-bottom flask to the rotary evaporator and set the water bath temperature slightly above the lipid phase transition temperature (e.g., 37°C).
- 4.2.2.2 Evaporate the solvent under reduced pressure to form a thin lipid film on the walls of the flask.
- 4.2.2.3 Once the solvent has been fully evaporated, dry the lipid film under vacuum for an additional 30 minutes to remove any residual solvent.
4.3 Hydration of Lipid Film
After the lipid film has been prepared, it is hydrated with an aqueous phase containing the therapeutic agent or drug. This step involves adding a buffer or drug solution to the lipid film and gently stirring the mixture.
4.3.1 Preparation of the Aqueous Phase
- 4.3.1.1 Prepare the aqueous phase according to the formulation protocol. This may be a drug solution or a buffer solution, such as phosphate-buffered saline (PBS).
- 4.3.1.2 Adjust the pH of the aqueous phase to match the optimal pH for antibody conjugation and liposome stability.
- 4.3.1.3 Warm the aqueous phase to the appropriate temperature (typically 37°C) in a temperature-controlled water bath.
4.3.2 Hydration Process
- 4.3.2.1 Add the warmed aqueous phase to the round-bottom flask containing the dried lipid film.
- 4.3.2.2 Stir the mixture gently for 30 minutes to ensure complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).
4.4 Size Reduction of Liposomes
To convert multilamellar vesicles (MLVs) into unilamellar vesicles (ULVs), size reduction techniques such as sonication or extrusion must be used.
4.4.1 Sonication Method
- 4.4.1.1 Transfer the liposome suspension to the sonicator and sonicate for 5 to 30 minutes, depending on the desired liposome size.
- 4.4.1.2 Monitor the temperature during sonication to prevent overheating, which may affect the stability of the liposomes.
- 4.4.1.3 After sonication, allow the liposome suspension to cool to room temperature before proceeding with antibody conjugation.
4.5 Antibody Conjugation to Liposomes
Antibodies or antibody fragments are conjugated to the liposome surface to enable specific binding to target cells. The conjugation process involves using cross-linking reagents to attach the antibodies to the liposomes.
4.5.1 Antibody Preparation
- 4.5.1.1 Prepare the antibody solution according to the formulation protocol. The antibody should be in an appropriate buffer, such as PBS or HEPES.
- 4.5.1.2 Determine the concentration of the antibody solution using UV spectrophotometry or other appropriate methods.
4.5.2 Conjugation Procedure
- 4.5.2.1 Activate the liposome surface using a cross-linking reagent, such as EDC/NHS or maleimide, to prepare the liposomes for antibody attachment. Incubate the liposome suspension with the cross-linker for 30 minutes to 1 hour at room temperature.
- 4.5.2.2 Add the prepared antibody solution to the liposome suspension and stir gently to ensure uniform conjugation. Allow the reaction to proceed for 1 to 2 hours at room temperature.
- 4.5.2.3 Purify the antibody-conjugated liposomes by removing any unbound antibody using dialysis or gel filtration.
4.6 Quality Control of Immunoliposomes
Once the antibodies are conjugated, immunoliposomes must undergo quality control testing to ensure proper antibody attachment and functionality. The following tests are recommended:
- 4.6.1 Measure the particle size of the immunoliposomes using dynamic light scattering (DLS) to confirm size distribution.
- 4.6.2 Test the antibody conjugation efficiency using surface plasmon resonance (SPR) or a similar assay to quantify the amount of antibody attached to the liposome surface.
- 4.6.3 Evaluate the targeting functionality by performing cell-binding assays to confirm that the conjugated antibodies are active and capable of binding to their target cells.
- 4.6.4 Assess the stability of the immunoliposomes by storing them under specified conditions and monitoring their size, morphology, and encapsulation efficiency over time.
4.7 Storage of Immunoliposomes
The prepared immunoliposomes must be stored under appropriate conditions to ensure stability. Store the immunoliposomes in sterilized, airtight containers at 4°C or as specified in the formulation protocol. Label all containers with the batch number, preparation date, and storage conditions. Perform periodic stability testing to assess the long-term performance of the immunoliposomes.
5) Abbreviations, if any
- DLS: Dynamic Light Scattering
- SPR: Surface Plasmon Resonance
- EDC/NHS: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide
- ULV: Unilamellar Vesicle
- QA: Quality Assurance
- QC: Quality Control
6) Documents, if any
- Batch Manufacturing Record (BMR)
- Particle Size Analysis Report
- Antibody Conjugation Efficiency Report
- Targeting Functionality Test Report
7) References, if any
- FDA Guidelines for Targeted Drug Delivery Systems
- ICH Q7: Good Manufacturing Practice Guide
8) SOP Version
Version 1.0
Annexure
Annexure 1: Batch Manufacturing Record Template
| Batch No. | Lipid Type | Weight | Antibody Type | Conjugation Method | Size Reduction Technique | Operator Initials | QA Signature |
|---|---|---|---|---|---|---|---|
| Batch Number | Lipid Name | Weight in grams | Antibody/Fragment | EDC/NHS/Maleimide | Sonication/Extrusion | Operator Name | QA Name |
