1Fully automatic peptide synthesizerEquipment calibration and preparation

Installation and Debugging

Professional technicians are responsible for installing and debugging the equipment to ensure its proper operation.

Check if the solvent storage bottle is properly connected to the gas pipeline and if all parts of the instrument are intact.

Turn on the power switch and compressed air switch of the synthesizer, and check if the pressure is within the range (such as "AIR" gauge pressure greater than 60psi but less than 100psi); The pressure on the "N2" gauge is greater than 5psi but less than 10psi.

Calibration and Testing

According to the manufacturer's instructions, calibrate and test the equipment to ensure that all systems (such as pumps, valves, detectors, etc.) are functioning properly.

Conduct tests on temperature, barcode, reactor mixing and stirring, liquid sampling needle, and amino acid bottle pushing; Electromagnetic valve testing; Flow calibration of chemical reagent bottles and amino acid bottles; UV control calibration.

Choose the appropriate reaction vessel

Select a suitable reaction vessel based on the synthesis scale and ensure that the material of the vessel does not react with reactants or solvents.

2、 Preparation of reagents and materials

Reagent preparation

Prepare amino acids, activation reagents, solvents, etc. for synthesis, ensuring that all materials are fresh and pure to avoid the introduction of impurities.

According to the synthesis plan, weigh according to the required substance ratio to ensure accuracy.

Fill the reagents into the corresponding labeled storage bottles in sequence.

Resin preparation

Add the resin to the solid-phase synthesis apparatus, swell the resin with a suitable solvent (such as DCM), and then drain it.

Wash the resin with solvents such as DMF to remove impurities, and drain it after washing for later use.

3、 Synthesis operation

Set reaction conditions

Set appropriate reaction conditions based on the required peptide sequence and length, including temperature, pressure, time, etc.

deprotection

Remove the protective group of the previous amino acid so that it can react with the next amino acid.

For example, deprotection with a 40% trifluoroacetic acid/dichloromethane solution, followed by washing with dichloromethane, methanol, etc.

conjugation

Combine new amino acids with deprotected amino acids to form peptide bonds.

Dissolve the first amino acid (usually starting from the C-terminus) in a certain volume of DMF and add a condensing agent (such as DCC, HBTU, etc.) for activation.

Put the activated amino acid solution into a solid-phase synthesizer, add DMF to the appropriate reaction concentration, and stir for condensation reaction.

wash

Clean off unreacted substances and by-products to ensure the purity of the reaction.

After each step of synthesis, washing is required to remove the by-products and unreacted raw materials generated by the reaction, reducing the accumulation of impurities.

Repeat the steps

Repeat the deprotection and conjugation steps until the polypeptide chain reaches the predetermined length.

4、 Product cutting and purification

resin cracking

Prepare a cracking solution (usually containing strong acids such as TFA) and store it in a pre freezer for refrigeration.

Add peptide resin into the reaction vessel, add pre cooled lysis buffer, and stir the reaction to lyse the peptide from the resin.

After the cracking is completed, the reaction solution is released, the resin is removed by filtration, and washed with solvents such as TFA.

polypeptide purification

Transfer the lysate into a rotary evaporator and concentrate it at room temperature to a small volume.

Use an appropriate precipitant (such as methyl tert butyl ether) to precipitate the peptide, and obtain the crude peptide through steps such as centrifugation and washing.

Purification of crude peptides using high-performance liquid chromatography (HPLC) and other methods to remove impurities and isolate the target peptide.

Concentration, filtration, and freeze-drying

Spin evaporate the purified peptide solution to remove organic solvents and obtain a concentrated peptide solution.

Perform sterile filtration on the concentrated peptide solution.

Place the filtered peptide solution in a freeze dryer and set an appropriate temperature program for freeze-drying treatment.

5、 Quality Control and Records

Monitor the reaction process

Use appropriate detection methods (such as UV detection, mass spectrometry analysis, etc.) to monitor the reaction process and ensure that each step of the reaction proceeds as expected.

Record and Report

Record in detail the operating parameters and results of each step, including reagent usage, reaction time, temperature, pressure, etc.

Generate an experimental report summarizing key data and product purity information during the synthesis process.