Peptide Purification Technology
  • Peptide compounds exist widely in nature, and the research on peptides with certain biological activities has been a main direction of drug development.The known active peptides in vivo are mainly produced or obtained from endocrine glands, secretory cells and body fluids. Cell differentiation, neurotransmitter regulation, tumor pathological changes and immune regulation in life activities are closely related to active peptides.

     

    Generally the concentration of crude peptide is low and the composition is complex, especially the physical and chemical properties of impurities are very similar to the target peptide, so the traditional purification methods have not achieved the ideal separation effect, and all have some disadvantages more or less.Based on more than 10 years of synthesis experience, we summarized the properties and types of various peptides in the synthesis process, established a database of peptide separation and purification methods (including HPLC, electrophoresis etc),made a prediction software for peptide purification, and used a combination of multiple purification methods to obtain a higher purity target peptide.

     

    HPLC & RP-HPLC

    The emergence of HPLC provides a beneficial method for peptide separation, because compared with other compounds, the application of HPLC in protein and peptide can not only complete the separation purpose in a short time, but also produce bioactive peptides on a large scale.

     

    In the purification of proteins and peptides, RP-HPLC is the most commonly used method. And also the main peptide purification method adopted by Aurora Peptide at present.This method is suitable for the separation and purification of components with molecular weight less than 5000D, especially small peptides with molecular weight less than 1000D.In the process of using, it has the following characteristics:

     

    •Fast speed: It only takes 15-30 minutes to analyze a sample generally, and some samples can be completed within 5min.

     

    •High resolution ratio: Stationary and mobile phases can be selected to achieve the best separation effect.

     

    •High sensitivity: 0.01ng for UV detector, 0.1pg for fluorescence and electrochemical detection.

     

    •High efficiency: the column efficiency can be up to 5000 trays / m, 100 components can be separated in one column at the same time, and the flow rate of high-pressure pump can be over 150ml / min, which can be used for mass production of samples

     

    •The chromatographic column can be used repeatedly: different compounds can be separated with one column.

     

    Sequence :KSHTPGSAETNPLADTRRSVKTPLSAC  27AA,Purity:98.579%

    Peptide Purification Technology

     

    Iron-Exchange chromatography (IEXC)

    Ion exchange chromatography is a method of separation and purification of substances by ion exchange between the stationary phase and the separated components.IRXC can be used to separate and purify bioactive peptides under neutral conditions with different charged properties.

     

    The adsorption, diffusion, van der Waals force and electrostatic attraction of different molecules and ion exchange resins were eluted in turn to achieve the purpose of separation. It can be used for the separation of biological macromolecules (such as proteins and peptides) with similar polarity.In general, ion exchange will combine with RPC and SEC to separate complex samples, such as peptide fragments from enzymatic hydrolysis or synthetic peptides, and peptides from natural sources.Ion exchange packings for polishing purification , such as HP, Source, Mono, Mini, are very suitable for peptide separation.

     

    At present, ion exchange chromatography is more suitable for industrial production due to its advantages of good repeatability, simple operation and wide elution range.

      

    Size exclusion chromatography (SEC/GPC)

    SEC is a method to separate peptide substances according to the size and shape difference of peptide molecule,especially convenient for some molecules with larger aggregated state.It provides a method to purify samples quickly and measure the size and dispersion of samples.In the field of drug development, the benefits of advanced SEC are self-evident: it can provide direct and abundant measurement data to solve multiple applications related to proteins, polymers and excipients.

     

    In SEC experiments, when the components to be separated enter the column, the macromolecular components can not enter the gel particles, only flow with the eluent from the gap of the gel particles, so the elution speed is faster; the small molecular components can directly enter the gel particles, the speed is slow.so according to the different moving speed in the chromatographic column, the macromolecular components are eluted first, and the small molecules are eluted later to achieve the purpose of separation and purification.

     

    Omizzur uses a high resolution molecular sieve gel specially designed for the separation of peptide products, with a molecular weight range of 100-7000Da, high chemical stability, PH1-14, compatibility with organic solvents (acetonitrile + TFA)

     

    Separation of standard peptides

     Peptide Purification Technology

    Other Purification Methods:

    • Chromatography of Membrane Protein(CMP)

    • High-Performance Displacement Chromatography(HPDC)

    • Hydrophobic interaction chromatography(HIC)

    • Perfusion Chromatography(PC)

    • Affinity Chromatography(AC)

    • Capillary Zone Electrophoresis(CZE)

     

    For most peptides one-step purification or one technology application is not enough. Omizzur usually combines the RP-HPLC, IEXC and SEC technologies for multi-step purification, and uses other targeted purification methods for special situation of peptides, ultimately achieving a excellent level of purification:

     

    Name:PY574a  14AA  pl>10                 Name: Thymosin a1  28AA NW:3108 pl4.2 

              Purity<90%                                                        Purity<80%

                    ↓                                                                        ↓

         Crude purification                                            Crude purification

    (IEXC:SP SEPHAROSE XL)                        (IEXC:DEAE SEPHAROSE FF)

                    ↓                                                                         ↓

       Peptide desalination                                       Peptide desalination

    (SEC: SEPHADEX G10)                                (SEC: SEPHADEX G10)

                   ↓                                                                          ↓

      Polishing purification                                      Polishing purification

    (RP-HPCL:SOURCE 15RPC)                    (RP-HPCL:SOURCE 15RPC)    

                   ↓                                                                         ↓

          Purity>99.68%                                                   Purity>99.58%