URui Huang, uBo Xu
Isicelo se-R&D Centre
Intshayelelo
I-peptide yikhompawundi eyenziwe ngee-amino acids, nganye ineempawu ezikhethekileyo zomzimba nezekhemikhali ngenxa yeentlobo ezahlukeneyo kunye nolandelelwano lweentsalela ze-amino acid ezenza ukulandelelana kwayo.Ngophuhliso lwesigaba esiluqilima sokudityaniswa kweekhemikhali, ukudityaniswa kwemichiza yeepeptides ezahlukeneyo ezisebenzayo yenze inkqubela enkulu.Nangona kunjalo, ngenxa yokubunjwa okunzima kwe-peptide efunyenwe yi-synthesis yesigaba esiqinileyo, imveliso yokugqibela kufuneka ihlanjululwe ngeendlela ezithembekileyo zokwahlula.Iindlela ezisetyenziswa ngokuqhelekileyo zokuhlanjululwa kweepeptide ziquka i-ion exchange chromatography (IEC) kunye nesigaba esibuyiselweyo esiphezulu se-liquid chromatography (RP-HPLC), eneengxaki zomthamo ophantsi wokulayisha isampuli, iindleko eziphezulu zokwahlula imidiya, izixhobo eziyinkimbinkimbi kunye neendleko zokwahlula, njl. Ukucocwa ngokukhawuleza kwee-peptides ze-molecule encinci (MW <1 kDa), isicelo esiphumeleleyo sapapashwa ngaphambili yi-Santai Technologies, apho i-cartridge ye-SepaFlash RP C18 yayisetyenziselwa ukuhlanjululwa ngokukhawuleza kwe-thymopentin (TP-5) kunye Imveliso ekujoliswe kuyo ifunyenwe.
Umzobo 1. I-20 i-amino acids eqhelekileyo (eveliswe kwi-www.bachem.com).
Kukho iindidi ezingama-20 zee-amino acids eziqhelekileyo ekubunjweni kweepeptides.Ezi zi-amino acids zinokwahlulwa zibe ngamaqela alandelayo ngokwe-polarity kunye nepropathi esisiseko se-asidi: i-non-polar (i-hydrophobic), i-polar (i-uncharged), i-acidic okanye isiseko (njengoko kuboniswe kwi-Figure 1).Kulandelelwano lwepeptide, ukuba i-amino acids ezenza ulandelelwano ubukhulu becala zezo polar (njengoko ziphawulwe ngombala opinki kuMfanekiso 1), njengeCysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, etc. ke le peptide inokuba namandla I-polarity kwaye inyibilike kakhulu emanzini.Ngethuba lenkqubo yokuhlanjululwa kwezi sampuli ezinamandla ze-polar peptide nge-chromatography yesigaba esibuyiselweyo, into ebizwa ngokuba yi-hydrophobic phase collapse iya kwenzeka (bhekisa kwinqaku lesicelo elipapashwe ngaphambili nguSanta Technologies: I-Hydrophobic Phase Collapse, i-AQ i-Reversed Phase Chromatography Columns kunye nezicelo zabo).Xa kuthelekiswa neekholamu zeC18 eziqhelekileyo, iikholamu eziphuculweyo zeC18AQ zifaneleke kakhulu ukuhlanjululwa kweesampuli ezinamandla ze-polar okanye ze-hydrophilic.Kule post, i-peptide eqinile ye-polar isetyenziswe njengesampuli kwaye ihlanjululwe yikholomu ye-C18AQ.Ngenxa yoko, imveliso ekujoliswe kuyo ehlangabezana neemfuno yafunyanwa kwaye ingasetyenziswa kolu phando nophuhliso lulandelayo.
| Isixhobo | SepaBean™umatshini 2 | |||
| Iikhatriji | 12 g SepaFlash C18 RP flash cartridge (silica spherical, 20 - 45 μm, 100 Å, Order nunber:SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica spherical, 20 - 45 μm, 100 Å, inombolo Order: SW-5222-012-SP(AQ)) | ||
| Ubude bamaza | 254 nm, 220 nm | 214 nm | ||
| Isigaba esiphathwayo | Isinyibilikisi A: Amanzi Isinyibilikisi B: IAcetonitrile | |||
| Umgangatho wohambo | 15 mL/min | 20 mL/min | ||
| Ukulayisha isampulu | 30 mg | |||
| Umgangatho | Ixesha (CV) | Isinyibilikisi B (%) | Ixesha (min) | Isinyibilikisi 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 | |||
Iziphumo neengxoxo
Ukuthelekisa umsebenzi wokuhlanjululwa kwesampuli ye-polar peptide phakathi kwekholamu ye-C18 eqhelekileyo kunye nekholomu ye-C18AQ, sasebenzisa ikholamu ye-C18 eqhelekileyo yokuhlanjululwa kwe-flash yesampuli njengesiqalo.Njengoko kuboniswe kwi-Figure 2, ngenxa yokuwa kwesigaba se-hydrophobic yeentambo ze-C18 ezibangelwa umlinganiselo ophezulu we-aqueous ratio, isampuli ayizange igcinwe kwi-cartridge ye-C18 eqhelekileyo kwaye ikhutshwe ngokuthe ngqo kwisigaba seselula.Ngenxa yoko, isampuli ayizange ihlulwe ngokufanelekileyo kwaye ihlanjululwe.
Umzobo 2. I-chromatogram ye-flash yesampuli kwi-cartridge ye-C18 eqhelekileyo.
Emva koko, sasebenzisa ikholamu ye-C18AQ yokuhlanjululwa kwe-flash yesampuli.Njengoko kubonisiwe kuMfanekiso 3, i-peptide yagcinwa ngokufanelekileyo kwikholamu kwaye emva koko yakhutshwa.Imveliso ekujoliswe kuyo yahlulwa kwizinto ezingcolileyo kwisampuli eluhlaza kwaye iqokelelwe.Emva kwe-lyophilization kwaye emva koko ihlalutywe yi-HPLC, imveliso ehlanjululweyo inobunyulu be-98.2% kwaye ingasetyenziselwa ngakumbi kwisinyathelo esilandelayo sophando kunye nophuhliso.
Umzobo 3. I-chromatogram ye-flash yesampuli kwi-cartridge ye-C18AQ.
Ukuqukumbela, i-SepaFlash C18AQ RP flash cartridge edityaniswe ne-flash chromatography system SepaBean™Umatshini unokubonelela ngesisombululo esikhawulezayo nesisebenzayo sokucocwa kweesampulu ezinamandla zepolar okanye ze-hydrophilic.
Kukho uluhlu lwee-cartridges ze-SepaFlash C18AQ RP ezineenkcazo ezahlukeneyo kwi-Santai Technology (njengoko kuboniswe kwiThebhile 2).
| Inombolo yento | Ubungakanani beKholam | Umgangatho wohambo (mL/min) | Uxinzelelo olukhulu (psi/bar) |
| 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) | 33 g | 10-25 | 400/27.5 |
| I-SW-5222-040-SP(AQ) | 48 g | 15-30 | 400/27.5 |
| I-SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| I-SW-5222-120-SP(AQ) | 155 g | 30-60 | 300/20.7 |
| I-SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| I-SW-5222-330-SP(AQ) | 420 g | 40-80 | 250/17.2 |
Itheyibhile 2. SepaFlash C18AQ RP flash cartridges.Izinto zokupakisha: Ukusebenza okuphezulu kwe-spherical C18 (AQ) -i-silica edibeneyo, i-20 - 45 μm, 100 Å.
Ngolwazi oluthe vetshe ngeenkcukacha zomatshini weSepaBean™, okanye ulwazi lokuodola kwiikhatriji zeSepaFlash, nceda undwendwele iwebhusayithi yethu.
Ixesha lokuposa: Oct-12-2018