I. Nanofiltration Process for Peptide Concentration

1. Raw Material Pretreatment

• Filtration to Remove Impurities: Use microfiltration membranes with pore sizes of 0.1-0.45 μm to remove suspended particles, bacteria, and large molecular impurities.
• pH Adjustment: Adjust the solution pH away from the isoelectric point (pI) of the peptides (typically pH 7-9 or 3-5) to prevent peptide aggregation or adsorption on the membrane surface.
• Temperature Control: Maintain the solution temperature between 25-40℃ to avoid denaturation of peptides due to high temperatures.

2. Nanofiltration System Configuration

• Membrane Module Selection: Use spiral-wound or flat-sheet nanofiltration membranes with a molecular weight cut-off (MWCO) of 500-1000 Da (selected based on the target peptide molecular weight).
• Membrane Material Recommendation: Polyamide (PA) composite membranes are recommended due to their strong resistance to fouling and suitability for peptide systems.
• System Mode: Employ a recirculating cross-flow filtration mode to reduce concentration polarization, equipped with a pressure pump, flow meter, and temperature controller.
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3. Concentration Operation Steps

• Initial Feeding: Pump the pretreated peptide solution into the nanofiltration system and adjust the operating pressure to 10-15 bar.
• Recirculating Concentration:
  • Continuously retain peptides and discharge the permeate containing small molecular impurities (salts, monosaccharides, etc.).
  • Monitor the volume of the concentrated solution and stop when the target concentration factor (e.g., 5-10 times) is reached.
• Constant Volume Dialysis (Optional): Continuously add deionized water and discharge the permeate to further desalt/purify the solution.

4. Post-Treatment

• Concentrate Collection: Adjust the pH to conditions that stabilize peptide storage (e.g., pH 7.0) and temporarily store at 4℃.
• Membrane Cleaning:
  • Water Rinse: Rinse with deionized water for 10 minutes.
  • Chemical Cleaning: Circulate 0.1-0.5% NaOH or 0.1% HCl for 30 minutes (depending on membrane tolerance).
  • Storage: Soak in a 1% sodium bisulfite solution to prevent microbial contamination.

5. Key Parameter Optimization

• Transmembrane Pressure (TMP): 10-20 bar (too high causes membrane fouling, too low results in insufficient efficiency).
• Flow Rate: 3-5 L/min (higher flow rates reduce concentration polarization).
• Concentration Endpoint Determination: Monitor peptide concentration through UV absorption (280 nm) or HPLC.

II. Simplified Scheme (Laboratory Level)

1. Equipment and Reagents

• Equipment: Small-scale nanofiltration apparatus (e.g., spiral-wound membrane module), peristaltic pump, pressure gauge, liquid storage tank.
• Reagents: Pretreated peptide solution, deionized water, pH adjusters (NaOH/HCl).

2. Operating Steps

• Pretreatment: Filter the peptide solution through a 0.45 μm filter membrane and adjust the pH to 7.5.
• System Assembly: Connect the membrane module (MWCO 500 Da) and remove any air bubbles from the piping.
• Concentration Operation:
  • Start the peristaltic pump, adjust the pressure to 12 bar, and initiate the recirculation mode.
  • Take samples every 30 minutes to monitor concentration and concentrate to the target volume (e.g., 1/5 of the original volume).
• Finalization:
  • Collect the concentrate and store at 4℃.
  • Clean the membrane module with a 0.1% NaOH solution and store it properly.

3. Precautions

• Prevent Membrane Drying: Keep the membrane moist during operation and fill with preservative solution when the system is not in use.
• Prevent Contamination: Incomplete pretreatment can lead to a rapid decline in membrane flux.
• Temperature-Sensitive Peptides: Maintain the temperature at ≤30℃ throughout the process.

III. Technical Advantages

• Gentle Concentration: Low-temperature, low-pressure operation to protect the biological activity of peptides.
• Simultaneous Purification: Removal of small molecular impurities (such as salts, solvents) to increase product purity.
• Energy-Efficient: Lower energy consumption compared to evaporation concentration, suitable for heat-sensitive substances.

IV. Application Scenarios

• Laboratory: Desalting and concentration after peptide synthesis.
• Industrial Production: Recovery and refining of peptides from bioprocess fermentation broth.
• Pharmaceutical Field: Preparation of biological formulations such as vaccines and antibody fragments.