Rui Huang, Bo Xu
Isicelo se-R&D Center
Isingeniso
I-peptide iyinhlanganisela eyakhiwe ama-amino acid, ngayinye enezakhiwo ezihlukile zomzimba namakhemikhali ngenxa yezinhlobo ezahlukene nokuhleleka kwezinsalela zama-amino acid akha ukulandelana kwayo.Ngokuthuthukiswa kokuhlanganiswa kwamakhemikhali esigaba esiqinile, ukuhlanganiswa kwamakhemikhali kwama-peptide asebenzayo ahlukahlukene kuye kwathuthuka kakhulu.Kodwa-ke, ngenxa yokwakheka okuyinkimbinkimbi kwe-peptide etholwe ngokuhlanganisa kwesigaba esiqinile, umkhiqizo wokugcina kufanele uhlanzwe ngezindlela ezithembekile zokuhlukanisa.Izindlela zokuhlanza ezivame ukusetshenziswa zama-peptide zifaka i-ion exchange chromatography (IEC) kanye ne-reverse-high performance liquid chromatography (RP-HPLC), ezinobubi bomthamo ophansi wokulayisha wesampula, izindleko eziphezulu zemidiya yokuhlukanisa, imishini yokuhlukanisa eyinkimbinkimbi nebizayo, njll. Ukuze kuhlanzwe ngokushesha ama-peptides ama-molecule amancane (MW <1 kDa), ikesi lesicelo eliyimpumelelo lanyatheliswa ngaphambilini yi-Santai Technologies, lapho i-cartridge ye-SepaFlash RP C18 yasetshenziselwa ukuhlanzwa okusheshayo kwe-thymopentin (TP-5) kanye umkhiqizo okuhlosiwe ohlangabezana nezidingo watholwa.
Umfanekiso 1. Ama-amino acid angama-20 ajwayelekile (akhiqizwe kabusha ku-www.bachem.com).
Kunezinhlobo ezingama-20 zama-amino acid ajwayelekile ekwakhiweni kwama-peptide.Lawa ma-amino acid angahlukaniswa ngamaqembu alandelayo ngokuya nge-polarity kanye ne-acid-base impahla: i-non-polar (i-hydrophobic), i-polar (engashajiwe), i-acidic noma isisekelo (njengoba kuboniswe kuMfanekiso 1).Ngokulandelana kwe-peptide, uma ama-amino acid akha ukulandelana ngokuvamile kuyi-polar (njengoba imakwe ngombala ophinki kuMfanekiso 1), njengeCysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, njll. khona-ke le peptide ingase ibe namandla aqinile. polarity futhi incibilike kakhulu emanzini.Phakathi nenqubo yokuhlanza lawa masampuli aqinile e-polar peptide nge-reverse-phase chromatography, into ebizwa ngokuthi i-hydrophobic phase collapse izokwenzeka (bheka inothi lesicelo elishicilelwe ngaphambilini le-Santai Technologies: I-Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns kanye Nezicelo Zabo).Uma kuqhathaniswa namakholomu avamile e-C18, amakholomu athuthukisiwe e-C18AQ afaneleka kakhulu ukuhlanzwa kwamasampuli aqinile e-polar noma e-hydrophilic.Kulokhu okuthunyelwe, i-polar peptide eqinile isetshenziswe njengesampula futhi yahlanzwa yikholomu ye-C18AQ.Ngenxa yalokho, umkhiqizo ohlosiwe ohlangabezana nezidingo watholwa futhi ungasetshenziswa ocwaningweni nasekuthuthukisweni okulandelayo.
| Insimbi | SephaBean™umshini 2 | |||
| Amakhatriji | 12 g SepaFlash C18 RP flash cartridge (silica eyindilinga, 20 - 45 μm, 100 Å, Inombolo ye-oda:SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica eyindilinga, 20 - 45 μm, 100 Å, inombolo ye-oda:SW-5222-012-SP(AQ)) | ||
| Ubude begagasi | 254 nm, 220 nm | 214 nm | ||
| Isigaba seselula | I-solvent A: Amanzi I-solvent B: I-Acetonitrile | |||
| Izinga lokugeleza | 15 mL/min | 20 mL/min | ||
| Isampula iyalayisha | 30 mg | |||
| I-Gradient | Isikhathi (CV) | I-solvent B (%) | Isikhathi (imizuzu) | I-solvent B (%) |
| 0 | 0 | 0 | 4 | |
| 1.0 | 0 | 1.0 | 4 | |
| 10.0 | 6 | 7.5 | 18 | |
| 12.5 | 6 | 13.0 | 18 | |
| 16.5 | 10 | 14.0 | 22 | |
| 19.0 | 41 | 15.5 | 22 | |
| 21.0 | 41 | 18.0 | 38 | |
| / | / | 20.0 | 38 | |
| 22.0 | 87 | |||
| 29.0 | 87 | |||
Imiphumela nengxoxo
Ukuqhathanisa ukusebenza kokuhlanza kwesampula ye-polar peptide phakathi kwekholomu evamile ye-C18 nekholomu ye-C18AQ, sisebenzise ikholomu evamile ye-C18 ukuze sihlanze isampula njengesiqalo.Njengoba kuboniswe ku-Figure 2, ngenxa yokuwa kwesigaba se-hydrophobic samaketanga e-C18 okubangelwa isilinganiso esiphezulu se-aqueous, isampula ayizange igcinwe ku-cartridge evamile ye-C18 futhi yakhishwa ngokuqondile isigaba seselula.Ngenxa yalokho, isampula ayizange ihlukaniswe ngokuphumelelayo futhi ihlanzwe.
Umfanekiso 2. I-flash chromatogram yesampula ku-cartridge evamile ye-C18.
Okulandelayo, sisebenzise ikholomu ye-C18AQ yokuhlanzwa kwe-flash yesampula.Njengoba kukhonjisiwe kuMfanekiso 3, i-peptide yagcinwa ngempumelelo kukholamu yabe isikhishwa.Umkhiqizo okuhlosiwe wahlukaniswa nokungcola kwesampula eluhlaza futhi waqoqwa.Ngemuva kwe-lyophilization futhi yabuye yahlaziywa yi-HPLC, umkhiqizo ohlanziwe unobumsulwa obungama-98.2% futhi ungasetshenziswa futhi esinyathelweni esilandelayo socwaningo nokuthuthukiswa.
Umfanekiso 3. I-flash chromatogram yesampula ku-cartridge ye-C18AQ.
Sengiphetha, i-SepaFlash C18AQ RP flash cartridge ehlanganiswe nohlelo lwe-flash chromatography i-SepaBean™umshini unganikeza isixazululo esisheshayo nesisebenzayo sokuhlanzwa kwamasampula aqinile we-polar noma we-hydrophilic.
Kukhona uchungechunge lwama-flash cartridges e-SepaFlash C18AQ RP anemininingwane ehlukene evela ku-Santa Technology (njengoba kukhonjisiwe kuThebula 2).
| Inombolo Yento | Usayizi Wekholomu | Isilinganiso sokugeleza (mL/min) | Ubukhulu.Ingcindezi (i-psi/ibha) |
| I-SW-5222-004-SP(AQ) | 5.4 g | 5-15 | 400/27.5 |
| I-SW-5222-012-SP(AQ) | 20 g | 10-25 | 400/27.5 |
| I-SW-5222-025-SP(AQ) | awu 33g | 10-25 | 400/27.5 |
| I-SW-5222-040-SP(AQ) | awu 48g | 15-30 | 400/27.5 |
| I-SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| I-SW-5222-120-SP(AQ) | awu 155g | 30-60 | 300/20.7 |
| I-SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| I-SW-5222-330-SP(AQ) | awu 420g | 40-80 | 250/17.2 |
Ithebula 2. I-SepaFlash C18AQ RP flash cartridges.Izinto zokupakisha: I-silica eyisiyingi esebenza kahle kakhulu i-C18(AQ)-eboshiwe, 20 - 45 μm, 100 Å.
Ukuze uthole ulwazi olwengeziwe mayelana nemininingwane eningiliziwe yomshini we-SepaBean™, noma imininingwane yoku-oda kuma-flash cartridge ochungechunge lwe-SepaFlash, sicela uvakashele iwebhusayithi yethu.
Isikhathi sokuthumela: Oct-12-2018