1. Preparation:

   – **Equipment and Tools:**
     – **Sterile Workbench (Laminar Flow Hood):** Provides a sterile environment for operations.
     – **Sterile Vials:** Used for filling lyophilized peptides.
     – **Sterile Syringe:** Used for extracting solution.
     – **Solution Filter (e.g., 0.22 micron filter):** Filters the solution to remove any particulate or impurities.
     – **Lyophilizer (Freeze Dryer):** Used for freeze-drying process to remove water from the solution and form lyophilized powder.
     – **Rubber Stoppers and Aluminum Caps:** Used to seal the vials to prevent contamination.
     – **Sterile Gloves, Mask, Disinfecting Wipes:** For personal protection and to ensure sterility.
   
   – **Auxiliary Solutions:**
     – **Sterile Water:** Used for dissolving peptide raw materials.
     – **Buffer Solution (e.g., PBS or pH-balanced solution):** Used to dissolve or adjust pH.
     – **Ethanol or Isopropyl Alcohol:** For disinfecting equipment and environment.

2. Dissolving Peptide Raw Materials:

   – **Equipment:**
     – **Sterile Beaker or Container:** Used for dissolving peptide raw materials.
     – **Magnetic Stirrer (optional):** Helps to dissolve the peptide, but avoid excessive stirring to prevent damage to the peptide structure.
   
   – **Procedure:**
     – In a sterile environment (e.g., under laminar flow hood), weigh the peptide raw materials.
     – Accurately measure the peptide powder using a **sterile syringe** or **electronic scale**.
     – Add the appropriate amount of **sterile water** or **buffer solution** based on the desired concentration and gently shake the container to aid dissolution.
     – If necessary, gently heat the container to ensure uniform dissolution, but the temperature should not exceed the stability range for the peptide.

3. Filtering the Solution:

   – **Equipment:**
     – **0.22 Micron Filter:** Used to filter the solution, removing bacteria, particulates, and impurities.
     – **Sterile Syringe:** Used to extract and filter the solution.
   
   – **Procedure:**
     – Filter the dissolved solution through a **0.22-micron filter** to ensure it is sterile and free of particles.
     – Ensure that the filter and syringe are both sterile during the process.

4. Filling the Vials:

   – **Equipment:**
     – **Sterile Syringe or Sterile Transfer Device:** Used to extract the solution and transfer it into the vials.
     – **Sterile Vials:** Used for filling the final peptide solution.
     – **Sterile Rubber Stoppers:** Used to seal the vials to prevent contamination.
   
   – **Procedure:**
     – Carefully transfer the filtered solution into the **sterile vials** using a sterile syringe or transfer device.
     – Ensure the amount of solution filled in each vial is consistent, according to the desired lyophilized powder amount and concentration.

5. Freeze Drying Process:

   – **Equipment:**
     – **Lyophilizer (Freeze Dryer):** Used for the freeze-drying process, which removes water from the solution to form lyophilized powder.
     – **Temperature Sensor:** Monitors the temperature during the freeze-drying process.
     – **Vacuum Pump:** Creates the vacuum environment necessary for freeze-drying.
   
   – **Procedure:**
     – Place the vials with the solution into the **lyophilizer**.
     – Set the lyophilizer with appropriate temperature and time parameters. The process typically involves freezing and sublimation stages.
     – **Freezing Stage:** The solution is rapidly frozen to -40°C or lower to ensure no ice formation.
     – **Sublimation Stage:** The frozen water is removed under vacuum, resulting in lyophilized powder.

6. Sealing and Packaging:

   – **Equipment:**
     – **Sterile Rubber Stoppers and Aluminum Caps:** Used to seal the vials.
     – **Vial Sealing Equipment:** Used to securely seal the vials with the caps and stoppers.
   
   – **Procedure:**
     – After the freeze-drying process is complete, remove the vials and inspect the lyophilized powder for any contamination or damage.
     – Seal the vials with **sterile rubber stoppers** and use **aluminum caps** to securely close them.
     – Ensure that the vials are sealed in a sterile manner to prevent contamination and to maintain the powder’s stability.

7. Storage:

   – **Equipment:**
     – **Cold Storage (Refrigerator or Freezer):** Used to store the lyophilized peptide to ensure its stability.
   
   – **Procedure:**
     – Store the sealed vials in **cold storage**, typically at -20°C or lower.
     – Ensure that the storage environment is dry and the temperature is stable to maintain peptide integrity and prevent degradation.

8. Quality Control and Record Keeping:

   – **Equipment:**
     – **Electronic Record System or Manual Record Sheets:** Used for tracking each step of the process, including batch numbers, concentrations, temperatures, and times.
   
   – **Procedure:**
     – Record each step of the process, including equipment cleaning, solution preparation, filtration, lyophilization time and temperature, and storage conditions for traceability and quality control.