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SPPS Synthesis Reactor — Standard Operating Procedure

Official SOP

This standard operating procedure governs operation of SPPS synthesis reactors. For product inquiries, visit the SENO Biotechnology Official Website.

1. Safety Precautions

Hazard Source Required PPE Engineering Controls
Chemical exposure TFA, piperidine, DMF, DCM Nitrile gloves (double for TFA), safety goggles, lab coat, chemical apron Fume hood required for TFA handling
TFA burns Concentrated acid Acid-resistant gloves (neoprene or butyl) over nitrile, face shield Dedicated TFA station with spill tray
N₂ asphyxiation Compressed N₂ supply — Adequate room ventilation, O₂ monitor recommended
Piperidine toxicity Deprotection reagent Nitrile gloves, goggles Closed system transfer
Flammable solvents DCM, ether, ACN Flame-resistant lab coat No open flames, grounded containers

2. Pre-Operation Check List

Item Check Acceptable
N₂ supply Pressure gauge reading 2.0 – 4.0 bar
N₂ purity Certificate verified ≥99.999%
Reagent vessels Sufficient volume for synthesis ≥2× estimated requirement
Frit integrity N₂ back-pressure test (0.5 bar) Pressure holds steady
Temperature sensor Comparison with reference thermometer ±0.5 °C at current temp
Drain valve Manual verification Fully closed
Waste container Capacity check ≥50% empty
Batch record All fields completed to current step No gaps
Emergency stop Visual inspection Unobstructed, accessible

3. Resin Loading and Swelling

  1. Weigh required resin mass within ±1% of target using analytical balance
  2. Transfer resin to reactor vessel with DMF rinse
  3. Add sufficient DMF to cover resin (3 – 5 bed volumes, approximately 10 mL/g resin)
  4. Initiate N₂ bubbling at low flow rate (0.5 L/min, adjust as needed)
  5. Allow swelling for 30 – 60 min at 25 °C with gentle agitation
  6. Drain DMF through frit, retain resin
  7. Record initial resin mass and swelling time in batch record

4. Fmoc Deprotection Cycle

  1. Add 20% (v/v) piperidine in DMF (10 mL/g resin)
  2. Agitate with N₂ bubbling for 5 min at 25 °C
  3. Open drain valve; collect deprotection solution (optional UV monitoring at 301 nm)
  4. Add fresh 20% piperidine in DMF (10 mL/g resin)
  5. Agitate for 10 min at 25 °C
  6. Drain fully
  7. Wash resin with DMF (10 mL/g resin, 3 – 5 min each, repeat 5×)
  8. Collect and discard washes appropriately

5. Amino Acid Coupling Cycle

  1. Prepare amino acid solution:
  2. Dissolve Fmoc-AA-OH (3.0 – 5.0 eq. relative to resin loading) in minimal DMF
  3. Add coupling reagent (3.0 – 5.0 eq.): HBTU, HATU, or DIC + Oxyma Pure
  4. Add activator base (6.0 – 10.0 eq.): DIPEA or NMM
  5. Vortex or stir until fully dissolved (2 – 5 min)
  6. Add activated amino acid solution to reactor
  7. Initiate N₂ bubbling at moderate flow (1.0 L/min)
  8. Couple at 25 – 40 °C for 30 – 60 min
  9. Drain coupling solution
  10. Wash resin with DMF (10 mL/g resin, 3 – 5 min each, repeat 5×)

6. Kaiser Test (Ninhydrin Monitoring)

Component Preparation
Solution A 40 g phenol in 10 mL ethanol
Solution B 65 mg KCN in 100 mL H₂O, dilute 2 mL to 100 mL with pyridine
Solution C 0.5 g ninhydrin in 10 mL ethanol

Procedure: 1. Transfer 5 – 10 resin beads to a small glass test tube 2. Add 2 – 3 drops each of Solutions A, B, and C 3. Heat at 100 °C for 5 min 4. Colorless/yellow = coupling complete (negative test) 5. Blue/purple = free amines present (positive test → repeat coupling) 6. Document result in batch record

7. Capping Step (Optional)

  1. Prepare capping solution: Acetic anhydride (10 eq.) + DIPEA (10 eq.) in DMF
  2. Add to reactor
  3. Agitate for 10 – 15 min at 25 °C
  4. Drain
  5. Wash with DMF (3×, 10 mL/g resin)

8. Final Wash Before Cleavage

  1. Wash resin with DCM (10 mL/g resin, 3 – 5 min each, repeat 3×)
  2. Drain thoroughly
  3. Record final resin mass (optional: dry small sample for gravimetric yield check)

9. Peptide Cleavage and Precipitation

  1. Prepare cleavage cocktail fresh:
  2. 95% (v/v) TFA
  3. 2.5% (v/v) triisopropylsilane (TIS)
  4. 2.5% (v/v) water
  5. Add cleavage cocktail (10 mL/g resin) to reactor
  6. Agitate at 25 °C for 2 – 4 h (longer for Arg-rich sequences: 3 – 5 h)
  7. Filter cleavage solution through frit into round-bottom flask
  8. Wash resin with minimal TFA (2 × 2 mL); combine filtrates
  9. Concentrate filtrate under reduced pressure (rotary evaporator) if volume >50 mL
  10. Add cold diethyl ether (−20 °C, 10× volume of filtrate) to precipitate peptide
  11. Centrifuge at 3000 – 4000 × g for 10 – 15 min at 4 °C
  12. Decant supernatant carefully
  13. Wash peptide pellet with cold ether (2 × 20 mL)
  14. Dry under N₂ stream or vacuum desiccator (30 min – 2 h)
  15. Transfer crude peptide to pre-weighed vial; record yield

10. Cleaning Procedure (Post-Batch)

Step Solvent Volume Time Repeat
Pre-rinse DMF 500 mL 5 min 1×
Main wash DMF 500 mL 10 min with N₂ agitation 2×
Organic wash DCM 500 mL 10 min 1×
Alcohol wash Methanol 500 mL 5 min 1×
Aqueous wash DI Water 500 mL 5 min 1×
Drying Acetone 250 mL 5 min 1×

11. Troubleshooting Guide

Symptom Possible Cause Corrective Action
Low coupling yield Insufficient AA excess Increase to 5 eq.
Kaiser test positive after coupling Steric hindrance or difficult sequence Use HATU, increase temperature to 40 °C, extend time to 90 min
N₂ bubbling too vigorous Resin splashing on vessel walls Reduce flow rate, check frit condition
N₂ bubbling absent Clogged frit Back-flush N₂ from drain side; replace frit if persistent
Temperature deviating Sensor drift or circulator failure Recalibrate PT100; check circulator fluid level
Frit blockage Resin fines accumulation Back-flush with DMF under pressure; sonicate if needed
Incomplete cleavage Insufficient time or scavenger Extend to 4 h; verify TFA quality