Rui Huang, Bo Xu
Ntchito R&D Center
Mawu Oyamba
Peptide ndi chinthu chopangidwa ndi ma amino acid, omwe ali ndi mawonekedwe apadera akuthupi ndi makemikolo chifukwa cha mitundu yosiyanasiyana komanso dongosolo la zotsalira za amino acid zomwe zimapanga dongosolo lake.Ndi chitukuko cha kaphatikizidwe ka mankhwala olimba, kaphatikizidwe ka mankhwala a ma peptide osiyanasiyana ogwira ntchito apita patsogolo kwambiri.Komabe, chifukwa cha zovuta za peptide zomwe zimapezedwa ndi kaphatikizidwe kolimba, chomaliza chiyenera kuyeretsedwa ndi njira zodalirika zolekanitsa.Njira zomwe zimagwiritsidwa ntchito kwambiri poyeretsa ma peptide ndi monga ion exchange chromatography (IEC) ndi reversed-phase high performance liquid chromatography (RP-HPLC), zomwe zimakhala ndi zovuta zotsitsa zitsanzo zochepa, kukwera mtengo kwa ma TV olekanitsa, zida zovuta komanso zodula zolekanitsa, Kuti ayeretse mwachangu ma peptides ang'onoang'ono a molekyulu (MW <1 kDa), nkhani yabwino yogwiritsira ntchito idasindikizidwa kale ndi Santai Technologies, momwe katiriji ya SepaFlash RP C18 idagwiritsidwa ntchito poyeretsa mwachangu thymopentin (TP-5) ndi chandamale mankhwala kukwaniritsa zofunika anapezedwa.
Chithunzi 1. 20 ma amino acid omwe amapezeka (opangidwanso kuchokera ku www.bachem.com).
Pali mitundu 20 ya ma amino acid omwe amapezeka m'magulu a peptides.Ma amino acid awa akhoza kugawidwa m'magulu otsatirawa malinga ndi polarity ndi acid-base katundu: non-polar (hydrophobic), polar (uncharged), acidic kapena Basic (monga momwe tawonetsera pa Chithunzi 1).Mu ndondomeko ya peptide, ngati ma amino acid omwe amapanga ndondomekoyi amakhala ambiri a polar (monga momwe amasonyezera mtundu wa pinki mu Chithunzi 1), monga Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, ndi zina zotero ndiye peptide iyi ikhoza kukhala ndi mphamvu yolimba. polarity ndi kukhala kwambiri sungunuka m'madzi.Panthawi yoyeretsa zitsanzo zamphamvu za polar peptide pogwiritsa ntchito reverse-phase chromatography, chodabwitsa chotchedwa hydrophobic phase collapse chidzachitika (onani zomwe zalembedwa kale za Santai Technologies: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Their Applications).Poyerekeza ndi mizati ya C18 yanthawi zonse, mizati yabwino ya C18AQ ndi yoyenera kuyeretsa zitsanzo zamphamvu za polar kapena hydrophilic.Mu positi iyi, peptide yolimba ya polar idagwiritsidwa ntchito ngati chitsanzo ndikuyeretsedwa ndi gawo la C18AQ.Chotsatira chake, chinthu chomwe mukufuna kukwaniritsa zofunikira chinapezedwa ndipo chikhoza kugwiritsidwa ntchito mu kafukufuku ndi chitukuko chotsatira.
| Chida | SepaBean™makina 2 | |||
| Makatiriji | 12 g SepaFlash C18 RP flash cartridge (silica yozungulira, 20 - 45 μm, 100 Å, Nambala ya Order: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica yozungulira, 20 - 45 μm, 100 Å, Nambala ya Order: SW-5222-012-SP(AQ)) | ||
| Wavelength | 254 nm, 220 nm | 214 nm | ||
| Gawo la mafoni | Zosungunulira A: Madzi Zosungunulira B: Acetonitrile | |||
| Mtengo woyenda | 15 ml / min | 20 ml / mphindi | ||
| Kutsegula kwachitsanzo | 30 mg pa | |||
| Gradient | Nthawi (CV) | Zosungunulira B (%) | Nthawi (mphindi) | Zosungunulira 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 | |||
Zotsatira ndi zokambirana
Poyerekeza kuyeretsa kwa zitsanzo za polar peptide pakati pa gawo lokhazikika la C18 ndi C18AQ, tidagwiritsa ntchito gawo lokhazikika la C18 pakuyeretsa kwachitsanzo ngati poyambira.Monga momwe tawonetsera pa Chithunzi 2, chifukwa cha kugwa kwa hydrophobic gawo la maunyolo a C18 chifukwa cha kuchuluka kwa madzi amadzimadzi, chitsanzocho sichinasungidwe pa cartridge ya C18 yokhazikika ndipo chinatulutsidwa mwachindunji ndi gawo la mafoni.Chotsatira chake, chitsanzocho sichinapatulidwe bwino ndikuyeretsedwa.
Chithunzi 2. Chromatogram yowala ya chitsanzo pa cartridge ya C18 yokhazikika.
Kenako, tidagwiritsa ntchito gawo la C18AQ pakuyeretsa kwachitsanzo.Monga tawonetsera pa Chithunzi 3, peptide idasungidwa bwino pamzatiyo kenako idatulutsidwa.Chotsatiracho chinasiyanitsidwa ndi zonyansa mumtsuko waiwisi ndikusonkhanitsidwa.Pambuyo pa lyophilization ndikuwunikiridwa ndi HPLC, chinthu choyeretsedwacho chimakhala ndi chiyero cha 98.2% ndipo chitha kugwiritsidwanso ntchito pakufufuza ndi chitukuko china.
Chithunzi 3. Chromatogram yowala ya chitsanzo pa cartridge ya C18AQ.
Pomaliza, SepaFlash C18AQ RP kung'anima katiriji kuphatikiza ndi kung'anima chromatography dongosolo SepaBean™makina atha kupereka njira yofulumira komanso yothandiza pakuyeretsa zitsanzo zamphamvu za polar kapena hydrophilic.
Pali mndandanda wa makatiriji a SepaFlash C18AQ RP omwe ali ndi mawonekedwe osiyanasiyana ochokera ku Santai Technology (monga momwe tawonetsera mu Table 2).
| Nambala Yachinthu | Kukula kwa Mzere | Mtengo Woyenda (mL/mphindi) | Max.Pressure (psi/bar) |
| SW-5222-004-SP(AQ) | 5.4g pa | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g pa | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33 g pa | 10-25 | 400/27.5 |
| SW-5222-040-SP(AQ) | 48g pa | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 105 g pa | 25-50 | 350/24.0 |
| SW-5222-120-SP(AQ) | 155g pa | 30-60 | 300/20.7 |
| SW-5222-220-SP(AQ) | 300 g pa | 40-80 | 300/20.7 |
| SW-5222-330-SP(AQ) | 420 g pa | 40-80 | 250/17.2 |
Table 2. SepaFlash C18AQ RP flash cartridges.Zida zonyamula: Silika yolimba kwambiri yozungulira C18(AQ), 20 - 45 μm, 100 Å.
Kuti mumve zambiri zatsatanetsatane wamakina a SepaBean™, kapena kuyitanitsa ma cartridge a SepaFlash, chonde pitani patsamba lathu.
Nthawi yotumiza: Oct-12-2018