U LQC VA SAC KY DAO PHA (RP- HPLC)

HOI NGHI KHOA HQC CONG N G H t biwri n v - i^f >

NGHIEN CLTU LOAI B 6 NOI DOC T 6 K H 6 I S A N P H A M INTERLEUKIN-2 NGLfdl TAI T O HQP BANG PHUONG PHAP S | £ U LQC VA SAC KY D A O PHA (RP- HPLC)

LS P h w n g HoSng Anh, Dao Trpng Khoa, Nguyen Hong Thanh, Phung Thu Nguyet. T r w n g Nam Hai van Cdng ngh^ smh hpc. V^n Hdn LAm Khoa hoc va Cdng nghi Vi$t Nam

T 6 M TAT

Nfii dfic tfi cfi nguOT gfic tii VI khuan Gram fim, ttiuong nhilm vao djch protein tfii tfi hgp trongqna triah tixh chilt vfi tinh dil tuii bfio E. coli. Dfic tinh cua n9i dfic tfi ttiufing cao, chinh vi v ^ v i ^ kilm sofit, loai bo^nfii dfic ffi khoi cfic sfin [Aim d?ng tiftn lirfi cin diilt. Trong nghien ciru nay, chiing tfii phfii ttien phuong p h ^ lo^i bd n^i dfic to khfii sfin phfim biteTlcukin-2 ngudi tfii Id bgp bang v i ^ sii dyng phuong phfip sSc ky dfio pha RP HPLC kit hpp vdi mfing lpc Milhpore kich ttiuoc 30 kDa da dugc idj ly hing CaCliCl M. Hfim lugng nfii dfic tfl dugc xfic djnh bing Kit Limulus amebocyte lysate do 6 budc sfing tir 405-410 nm 4 m^do quang phfi. Kit qufi cho ttifiy >94% nfii dfic t^ trong san phfim dl dugc loai bfi sau khi sii dung mfing sibi lgc vfi CaClj 0,1 M. Hn kit hgp phuong p h ^ trrai vdi phuong p h ^ sic ky dfio pha RP-HPLC thi nfii dfic Id dS giam xuong chi cdn 0,04 EU/mL, tti^ bon ngudng cho p h ^ ciia FDA cho sfin jiJdn d^ng tiem la 0,25 EU/mL.

Tu khda: Endotoxin, Intericukin'2, nfii dfic t^, sfic k^ dao pha, sieu lgc

MdoAu

NOi ddc t6 (endotoxin) hay c6n gpi a pyrogen, co ngu6n g6c tir vi khuin Gram am, khi xSm nh5p vio ca Hie (m°cqf\

dirdng tidm vdi m$t hdm lirgng nhit dinh s§ g^y s6t, th$m chi ti> vong. Vi^c kiSm sodt ndi d^c t6 trong i ^ ste phlnl^

td tidm tn/c tilp vdo co ttil ngu^ cd tlm quan ttpng I6n d6i v6i cfing ngh$ san xuat dugc phlm.Vi^c p h ^ h i ^ ^ ^ i tra hdm lupng c6 trong quy ttlnh sdn xuit vd bong sSn phim cu6i cClng 6d difgc th^rc hi^n tir nhiaj ndm tnrdii^

phu'ang phdp logi bd n^i ddc t& dd vd dang du^c nghidn ci>u d6i vdi tCmg sdn phim cy ttil. lntef1eukin-2 (IL-2) cda n g u ^ ttiudc nhdm lymphokine vd Id mfit polypeptide cd 133 g6c axit amin vdi khdi lumig phdn ti> Itrpng I d i o ^ ISiCli cd hidu qud cao troiig vide h6 bpr iTidu tti bdnh ung fiiu' tti^n vd u hic t6 dc tinh (Chavez ef aL 2009). Tnsng linh v\rc cfing nghd sinh hpc, ddc bi$t Id ddi vdi cdng nghd gene, vi khuin Gram dm (nhif E. cdi) Id vdt chij thif^mg difgrc si> d^ng de sdn xuit cdc sdn phim protein tdi t l hpp (cdc protein chi>c ndng, enzyme, cdc cytoldne...} Tuy nhidn nhiing san piwn c6 nguAn glc tir vi khuin ndy ludn chOa hdm lijpng npi ddc t l cao. Ngodl ra, n$i dOc to cdn xuit hidn hlu nhir d Oilp mpi noi, tt> nhOng v ^ lidu bong phdng thi nghidm den cdc hda chit mdy mdc... nhd khd ndng phdt tdn trong khdng kM.

Ngdy nay, theo quy djnh cua cpc qudn ly Th^rc [^Sm vd Difpc phim Hoa Ky (FDA) ham lifpng n$i ddc t l cho ph^ dfi vdl cdc sdn phim d^ng tidm phdi dudi 0,25 EU/mL. Do ddc tinh cd mpt dau tfa nirirc vd m$t diu ky nirdc cOa endotoxin, ndn dilu d l hilu Id chiing cd khd ndng lien ket vdi cdc chit khdc bao gom cd protein. Cdc chit cd ttiS liSn kit vdl nOI ddc t l cd ttil Id nhOng khdng ttil khdng endotoxin vd ttiy ttil nhdn bilt endotoxin (vl dg nhu CD14, CD16, GDIS) (Morrison et al. 1993). Endotoxin (bdn chit Id lipopolysaccharide) xuit hidn d mdng ngodi cOa Idp mdng ddi - mdng bdo vd vi khuin gram dm (Rietschel at al. 1984). Endotown rit bin nhidt, chl khi du^c xi> ly d 250 "C ttong han 1 gid, ho|c 180 "C trong hon 3 gid ttil ndi ddc t l mdi bj bit hoat Cdc dung djch add hogc bazcr viri ning dO 0,1 M trd Idn c6 ttii dirpc si> dvng d l bit hogt ndi ddc t l d cdc dgng cg mdy mdc ttong phdng ttii nghipm (Gixbet vd Sefton 2005).

VI Id sdn phim dgng tidm ndn cdc phuang phdp bit hoat ndi ddc t l d nhidt dd cao, cflng nhu su dpng hda chit id khfing ttiich hpp vl cd ttil phdn huy hodc Win tlnh sdn phim, vd vide logi bd cdc hda chit ra khdi sdn phim khd khdn, khfing ttiudn tidn. Trong bdi bao ndy chiing tdi tiln hdnh nghidn ci>u ddnh gid cung nhu loai bd nOi ddc t l khdi djch IL-2 ngudi tdi t l hpp bing phirang phdp sic k;^ ldng cao dp kit hpp vdi mdng sidu Ipc d l nhdm dap ung ydu clu v l hdm lugng nfii

ddc t i cho phdp dii vih sdn phim dgng tidm md vin dam bdo chit lupng sdn pham. . VAT LI^U VA PHl/ONG PHAP NGHIEN CCTU

V$t llfu

Mlu 8i> dung d l djnh lupng vd ttitf nghidm phucmg phdp logi ndi ddc td Id IL-2 ngudi tdi t l hpp dupc tao bang k? thujt DNA tdi td hpp vd bilu hidn ttong t l bdo chu Id E. coli BL21, tgi phdng Ky ttiugt Di tiuyin vidn Cdng nghd sinh hgcgim cddjch IL-2 ttifdc tinh chl, djch IL-2 sau khi t i n tinh chl (tin tai d ttil djch trong Guanidine 7M); djch IL-2 h o ^ nguydn sau khi tinh chd vd ddng khd. Bd Kit xdc djnh hdm lupng ndi dpc t l LAL QCL 1000 ciia hdng Lonza Mdng sidu lgc ultracel 30 kDa cua hdng Millipore

Phuong phdp nghien cOu t h ^ nghifm s!

Tlnh chi IL'2 bing mdy sac kjr ldng hiiu ndng cao ^ Mlu IL-2 tiln tnh che dupc hda tan ttong Guadinine 7M, pH 7,5 Djch IL-2 tiln tinh chl sau 66 dirge bi simg

Tnfluoroacetic acid CTFA) d nj^g d l cudi ciing Id 15 mM d l dua pH v l 3,5 -4. Sau do djch dupc dua ldn cOt bdnSSu

? P L c T ^ i ; ^ S ; f N i i t R^"n^ n ^ ^ ^ ^ ^ ^ ^ . " ^ ^^' ' * '°1^ ^ ° ^P ^f^LC tnh Chl bing sic' k* d S ^ h a sCr dyng h? thfiw HPLC (Shimazu Nhgt Bdn). D|ch tinh chl IL-2 dupc kilm tta bing phuong phdp d i n di SDS-PAGE d gel .^ o l n g *

XCrliymSuv&iCaCa d IL-2 ngudi tdi t l hpp d cac dgng khdc nhau dupc b l sung CaCI; d cdc ning dd khac nhau (0,5 M, 0.25 M, 0,1 M) tproc

; CONG NGHE SINH HUU I UAN UUOU 2013

khi t i n hanh lpc

tog/ ch^( ngi d^ t6 bing mang sieu Igc Millipore

5mL cac m l u IL-2 ngudi tdi t i hpp dupc chia vao cac ong falcoi 15 mL, sau do dupc bo sung cdc dung djch c i n ttiilt t^i«5rc khi tiln hdnh lpc. ThCr tu vd thdnh phin phdn img mlu dupc md td trong Bang 1.

Quy trinh xdc djnh hdm lupng nfi dpc t i trong mlu dupc tiln hanh nhu sau: 40 ^1 t h i tich mau hogc chit chuan hodc nude {dll chung am) dupc cho vdo cdc gilng trdn dTa ELISA 96 gilng. B l sung 40 p| LAL lysate vdo cdc gieng ttieo trlnh tu nhlt djnh. Trpn d i u vd 0 d 37^ C tt^ng 10 phiit B l sung 80 ul ca chit vdo moi gilng. Trpn diu va Ci d 37" C trong 6 phiit. Dirng phdn img bdng dung djch axit acetic 30%. Do dp hap ttiy d birdc sdng 405 hodc 410 nm.

Bfing 1 Thanh phin mau trwo« khi Igc Thfinh phSn

Miu s6 1 HjO xir ly nfii dfic t i bfing nhi$t MSu so 2 1L.2 sail tinh chl + HjO (11) Mdu s6 3 IL-2 sau tinti chS Miu s6 4 IL-2 dfing kho + HjO Miu s6 5 IL-2 dfing kho

Miu s6 6 IL-2 chira tnh chl + 0,5 M CaCb Mau s i 7 IL-2 chua tinh chl + 0,25 M CaCli Mau s i 8 IL-2 chira Hnh chl + 0,01 M CaCIa Phuvng phap Westem Blot

Djch protein dug^: di#n di bdng phuong phdp SDS-PAGE (12% bis acr^amide) sau dd protein dupc chuyin sang mdng PVDF. M.^n9 sau khi hip thy protein dupc phii stfa tdch ba 5%, sau dd dupc phO vdl khdng t h i 1 (khdng ttil khdng vdi IL-2), vd khdng t h i 2 (khdng thd IgG) khdng vdl khdng t h i 1. Phdn iing hign mdu nhd ttiuic nhudm peroxidase TMB (3, 3', 5, 5'-tetramethylbenzidene).

K£T QUA VA THAO LUAN

Tinh c h l IL-2 ngu^l tdi t l hp'p bang h# thing sac ky ldng hidu ndng cao HPLC

Djch lL-2 ngudi tdi t l hpp bllu hidn trong t l bao E. coli BL21 dupc xir ly tiln tlnh c h l qua cdc budc lam sgch vd dupc hda tan trong Guanidine 7 M, pH 7,5. Djch IL-2 tiln tinh d i l sau do dupc bo sung p-mecaptoettianol d ning 3d cull ciing Id 15 mM d l dua pH cua dung djch v l 3,5 - 4. Djch IL-2 trong Guanidine 7 M dupc dua Idn cpt ban dieu c h l Bio- Vflrepore 05 vd tinh c h l trdn hd thing sic ky ldng cao dp HPLC (Shimadzu).Dich tinh c h l iL-2 dupc kilm tra bing phuang phdp di$n di SDS-PAGE, kit hpp vdi phuang phdp miin djch Western Blots.Kit qud dupc thi hidn d Hlnh 1.

116,0 66,2

Hinh 1- Kit qua kiem tra djch tinh che IL-2 ngu*l tfil to t\<rc bang phuwng phap dien dl SDS-PAGE .nhufim bac (A) va Western blot (B). Dudng chay 1: lL-2: Djch tmh chl IL-2 ngirdi tfii t l hqrp Dudng chgy 2: IL-2 flii chirng cOa Tmng Quoc Dirdng chgy M: Thang protein chuin (Fermentas - #SM0341).

6 dudng chgy 1 va 2 ttdn Hinh IA xuat hi#n 1 bang protein duy nhat cd kich ttiudc khodng 15 kDa. Kit qua Western blot (Hlnh 1B) cho thiy xuit hidn 1 bang cd kich thudc tuong u-ng vdi 15 kDa chirng td IL-2 da dupc bleu hi§n trong E.

coli. Dich tinh c h l ndy sau dd dupc dem di kiem tra ham iupng ndi ddc t i bdng LAL kit.

Xay dyng phuang phap logi bo c h i t ndi ddc to

lntGrieukin-2 (IL-2) id mdt polypeptide cd 133 glc axit amin vdi khii luang phan tip lupng khodng 15 kOa (Granucci e( ai.

2004) Vi khii lupng phan tip cCia iL-2 nam trong khoang khii lupng phdn ti> cua ndi ddc to endotoxin dgng monomer (10-20 kDa) vl vdy vide phan tdch IL-2 vd nii dpc to theo kich thudc khd kho khan.

Dich IL-2 ngudi tai to hpp tnrdc khi tiln hdnh lpc dupc xu ly vdi dung djch 60%jsDpro|3anoi nhim phdn tdch cdc phdn tli lipopolysaccharide lien kit vdi cdc phdn tu protein tt-ong dung djCh. Nil ddc to cd the tin tgi d 3 dgng khdc nhau Id dano dan (Khii lupng phdn td khoang ttr 10 din 20 kDa tiiy thupc vao dp ddi chuii oligosaccharides, dgng micelles /khii lupng tir 300 -1000 kDa) vd dgng vesicles (khoi iupng Idn hon 1000 kDa). Vide ton tgi d dang nao Id tiiy thupc vdo mdi trudng vl dg nhu sy cd m^t cua cde cation hda trj 2 nhu Mg^* vd Ca^* Dua vdo dac tinh tgo sidu clu tnic md chung tdi sir dyng CaCiz d ning dd khdc nhau nhdm Idm cde ndi ddc t i kit lgi vdi nhau, tgo ra clu triic c6 khoi lupng cao (>

H O I N G H I K H O A H Q C C O N I J N u n c o i i i

3 0 0 k D a ) h o n t i J n s o v d i IL-2 ( M w - 15k Da).T(t 66 qu4 Irinh s i c ky h o # c s i d g n g m a n g s B u Igc trer nfin hl$u qua lioi, D u n g d i c h di«7C n r l y b 6 s u n g t h e m d u n g dich C a C b l o i n6ng d o 0,1 M ; 0 2 5 ra 0,5 M d e H n g d o t i c h t y c u a C4c phin li> n i l d o c 14 n h a m tern B n g k i d i l h i / 6 c phan li> cua kh6i n6l doc l 6 I v d o , t a o diSu kien t h u ^ n l o i c h o vi#c loc q u a mang sifiu lgc Ultracel Millipore 3 0 k D a .

X a c dinh him lu^ng n6i tf$c to bing LAL kit

C f i c mSu djch IL-2 S i n tinh c h ^ , s a u tlnh c h « vi djch IL-2 h o a n n g u y f i n s a u d d n j k h o difcrc m H J b 6 s u n g cfic dung djUi cSn M e t n h u m o l i t i o n g p h i n p h i K m g phfip. Cac mSu nfiy s a u d o d u o c tien hfinh 1 ^ q u a m f i n g sieu lgc Milipo, Ultracel 3 0 kDa d 4 Ihu thfinh cfic p h f i n d o a n c f i the tich 1 m L trong c f i c ong e p p e n d o r f . Kfit q u f i xfic dinh h a m luong n^l dOc to trong mau d u o c mfi tfi trong H l n h 2 .

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2,00 1,50 1.00 0.50

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Hlnh 2 : K i t qua xAc ^ n h hdm l u p n g n$l d$c l l cOa cdc Hinh 3: Anh h u d n g ciia n i n g d 9 CaCli Idn qud b i n h I09I b6 nfl d $ c t 6 . M i u sd 6: IL-2 chua ttnh c ^ + 0,5 M CaCli, MSU s i 7: IL-2 chua Unh che + 0,25 M CaCli, Mau s i 8 : IL-2 chua tinh c h l « 0,1 M m i u sau khi 19c qua mdng MllliporB bang k i t LAL. M l u :

HjO xir ly bSng nhidt. m i u s i 2: IL-2 sau t n h c h l + H^O (1:1), mSu s i 3: IL-2 sau tinh chS, mSu s i 4: IL-2 dflng khfl, m l u s i 5: IL-2 dflng kh&+ H,0, m l u s i 6: IL-2 chua t n h chd + 0,5M C a a , .

K i t q u d d H l n h 2 cho t h i y r i n g : vd'i m l u s i 1 Id nuirc dirge xir ly nhidt d l b i t h o g t n i l d | c to, thi h d m l u p n g endotoxin Id 0,13 E U / m L . O cdc m a u s 6 2 v d s i 3 t u o n g i>ng v d i djch IL-2 s a u tinh c h l v d IL-2 s a u tinh c h e d u p c b l sung thSm n u * c k h d n g chira ndi d d c t i v d i ty Id 1:1 c 6 hdm iupTig npi d | c t i t u a n g d i i t h i p ( n o n g d d ndi d i e t i t u o n g img U 0,014 E U / m L v d 0,061 EU/mL), ttilp h a n so v d i m§u n u d e doi chipng c h l b i t hogt n^i d d c t l b i n g nhidt d d eao. d i k nfly cd t h i gidi t h i d i d u p c d o h d m lu-png c h a t ndi d i e to d m l u IL-2 s a u tinh c h l Id k h d n g d d n g k l , v! v | y v u p t ngu&ng gi6l h g n c u a p h u a n g phdp. C d c m i u 4 v d 5 Id cdc m a u cd nguon g l c tir djch IL-2 h o d n n g u y d n s a u d i n g k h i , hdm lupng n i i d i e t i trong m l u 4 vd 5 ( l l n l u p t Id 0,70 EU/mL vd 1,70 E U / m L ) eao h a n s o vd'i m a u 2 v d 3. Nguydn nhdn cua k^

q u d ndy c d till gidi thich tjiri trong q u d trinh tgo cdng t h i r c c h o IL-2 n g u d i tdi t i h p p , c h i i n g t i i s i r d p n g m p t s i cdc thdnli p h i n d d n g vai t r i n h u td c h i t gidp tdng kha ndng hda tan vd mure d p on i^nh c u a IL-2.Cdc t h d n h p h i n ndy chua dirpc k i l m sodt chdt c h l v l chat l u g n g d i u v d o . do d d ed t h i da bj nhiem ndi d i e to trong q u d t r l n h s d n x u i t M l u 6 la iL-2 t i l n tinh c h l (trong dung m i i Guanidine 7 M c d b i sung CaCi2 0,5M) cd h d m i u p n g ndi dpc t i cao n h l t Id 2,08 EU/mL, b d i ndi d i e t i c h u a d u p c logi b d b i n g h i t h i n g s i c k^ Ifing cao d p H P L C .

V d i m o n g m u i n t l m mOt p h u a n g phdp r l tien v d tang hidu q u d logi b d n i l d d c t i ra khoi san p h i m tdi t i h p p chiing W t i l n hdnh thi> nghldm p h u a n g phdp lpc bang mdng sidu Ipe Ultracell Millipore 3 0 k D a , k i t h p p v d i si> dgng hda chSt nhtf C a C W l c h u y i n n i l dpc tiS v d d g n g micelles vd vesicles c 6 k h i i l u p n g p h d n t u c a o h a n khoi l u o n g cOa IL-2, giiip v i ^ Ipc b i ndi d d c t i hidu q u d h a n .

D d n h g i d d n h h u d ' n g c i l a n i n g d p C a C U l d n q u d t r l n h l o g i b i n p i d $ c t i

Vd'i cdc m l u 6,7,8 Id djch IL-2 t e n tinh c h l (trong d u n g moi G n d 7 M ) , d u p c b o s u n g C a C b v d i n i n g d p t u o n g dng 1*

0,5 M, 0,25 M vd 0 , 1 M. K i t q u a cho ttily m l u s i 8 Id djch IL-2 t i i n tinh c h l c d b l s u n g 0,1 M CaCi2 co ham lugng nOi ddc t i t h i p n h l t s o v i i 2 m l u c d n Igi. D i l u ndy cho t h i y n i n g d i 0,1 M C a C b Id n i n g d d phCi h p p d l cdc phdn tir nji d i e t ! k i t t y thdnh dgng c d k h i i l u p n g phdn t u cao h o n khoi l u p n g p h d n h> cOa IL-2 ( 1 5 kDa) t g o dieu k i d n r f » 1 " * ttinh Ipc logi b i ndi d i e tt) b d n g m d n g Millipore 30 k D a . D o do chOng tdi l u a c h p n n i n g d p 0 1 M C a C b d l x u I* ( T ^ cho cdc thi nghidm t i l p ttieo.

D d n h g l d h l ^ u s u i t Ipc e n d o t o x i n

Hidu s u i t Ipc ciia mdng lpc Millipore 3 0 k D a d u p c xac djnh b a n g t J 1§ p h i n trdm eda h a m l u o n g endottwin dupcgi&l^i s a u khi PC. Djch IL-2 hodn nguyen s a u d d n g k h d vd d|ch IL-2 sau Unh c h l d u p e sir d u n g d l lpc b i n g mdng Millipore 30 kDa v d k i l m tra hidu s u a L K l t q u a lpc endob)xin d u p c t h i hidn trong H i n h 4 .

K i t q u ^ k i ^ m tra hidu s u i t lpc c u a m d n g Ipc Millipore 30 kDa a H i n h 4 cho ttiay: h d m l u p n g n i i ddc t l bj gil> M I* • * * c a o > 9 0 % d i l v d . cde m l u IL-2 h o d n nguyen sau d i n g k h d vd IL-2 h o d n nguyen s a u d i n g k h i c d b i sung nude vditJIO 1 0 ^ ^ 2 K >,A I I ' ^ H A I i i " ^ " T ^ ^ ' * ^ P * ^ ^ ^ ° ' ' ^ ^ ' ^ " ^ y '^ ^^^ 9*^' *'!"='' ^"Vc d o h a m l u p n g c h i t npi d i e t i f l " * IL-2 s a u hnh c h l Id khdng ddng k e , v l v d y v u p t n g u d n g g i d i h g n cCia p h u a n g p h d p

; CONG NGH? SINH HOC TOAN Q U 6 C 2013

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^ UI

0,00 0.02 0,02 0 . 0 2 ^ 1 0.*

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^'.AM« *'ii!JI',^'* ^ ' * " *'"* ''*' " ^ " '^^^ "-'^ "^ng m ^ g sieu I9C MiHipore 30 Mta.l: Dich IL-2 sau tinh che bvtic vi sau loc (hi$u suit I S 94 %)' '^'' °^^ M • H20 (1 ;1) i,«i,c v i sau Kic (hieu suat kjc 93 %(. 3: D|ch IL-2 dOng kho tnroc ii sau loc (hl», BSnh gia phirong phap io?l U n^l d6c to bang vl|c Hit hop sft dung hoa chat va ming Igc Millipore

hl,'''S,°' '^-"'' • °''° ""^^ " ' * " """^ * ' ' * ^ ""^ xft I* voi 0,1 M CaCli sau khi loc c6 ham luong chit nOI aOc to thap h i . -• 2 S L " f ' " ' I ^ Pli^ong Ph4p loc bang mSng Millipore 30 kDa v i Bin »> 1} viii CaClj se loai bo duoc hau net nqi aoc 16 c6 trSn mSu hSm lugrng nffl dOc 16 cbn lai chi la 0,04 EU/mL (thap hon rat nhieu lan so v « h4m luffng n6i dpc tb cho phep trong 1 san ph4m ti§m 0,25 EU/mL)

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Hlnh 5 Ham lupng n$i d$c t i trong mlu dupc xu 1;^ vd khfing difpc xir Hlnh 8: Ket qud didn di djch pratein truvc vi sau ly vdi CaCli.Miu s i 1: HiO xi> l<? ndi d$c td bing nhidt dfy. miu s i 2: IL-2 khi di qua mdng loc bang phuang phdp SIIS-PAGE tinh chl dd xi> ly. tnj'dc v6J CaCI^ O.IM sau khi IQC, mlu s i 3: IL-2 tinh chl sau dd hifn mdu bing phuOTig phdp nhu$m bgc daxi>iy tni^cvdiCaCbO.IMtrudckhilqc, mSusi4:IL-2tinhchlsaukhi Duong chgy M: Thang protein chuin (Femientas lpc SM0341), dudmg chgy 1: Dich tinh chd IL-2 sau khi Ipc.

dujmg chgy 2: Djch tinh chl IL-2 tnrdc khi lpc.

Dinh gli hl$u suit xuyen mang cOa IL-2 qua mdng sieu Igc millipore 30 kDa

Sau khi danh gid hi#u qua lpc logi bd nQI ddc to khdi djch tinh chl IL-2 ngudi tdi t i hpp, chung tii ttln hdnh ddnh gld hdm lupng IL-2 c i n igi sau khi tpc qua mdng millipore 30 kDa. V l bdn chit, protein cOa chung ta c i kidi thuoc khodng 15 kDa, vi v§y chOng tdi lua chpn mdng Ipc ed kich thudc 30 kDa vdri hy vpng giCr igi ede phan tu npi die t i tich ty, mgt khdc v l n ddm bdo lupng IL-2 bj giir Igi trdn mdng Id it nhlt.

Kit qud di#n dl kiem tra protein truoc Ipc (duang chay 2) vd sau Ipc (du*ng chgy 1) o' Hlnh 6cho thiy, lupng protein qua mdng la tuang duang nhau, khdc nhau khong dang k l . Nhu vdy, mdng Ipc millipore kich thud'c 30 kDa Id phu hpp cho viic su dyng d l iogi bd ndi die to frong phuang phdp cua chung tdi.

Thdo l u | n

Ban ehit cOa IL-2 Id cdc pnatein tdi to hpp dupc su dung Idm thuoe dgn^ tidm cho ngudi, vr v$y vide su dgng cdc phuong phdp vgt ly vd hda hpc d l logi bd hogc bit hogt cdc nil dpc t i Id het sue khd khdn, do Idm gidm hogt tlnh stnh hpc vd tdng nguy ca tilm tdng do cac thdnh phdn hda hpe gay ndn. Di/a vdo bdn chit dudi polysaccharide thud'ng tich di^n dm vd dupc gan Uidm cdc goc Phosphate, mdt s6 cing trinh nghiin cuu da cdng b i kit qua su dyng cot lpc trao dii anion d l bat giO" cdc phdn tu lipopolysaccharide trdn cplTuy nhien bdn ehat cdc phan ti> protein dang tich di#n dm, hode duang tuy thuOc vdo trinh tu cdc axit amin, hay mdi tnrd'ng, bin vgy c i khd ndng dnh hudng din hidu suit eua qud trinh trao d i i ion tt^n cdt, phuang phdp ndy co nhupc diem id ciing vdn vide npi die t i bj mit di thi mit phan khong nhi cua protein cung bj mit ttieo. Vdi protein cd khii lupng lan nhu cdc immunoglobulin (M, - 150 000 Da) thudng khdng su dyng phuang phdp sidu loc. Oii vdi sdn phim c i kich thudc nho (Mr ~ 18 000 Da), phuang phdp hO'u dyng nhlt Id diing mdng sidu Ipc logi b i ndi dpc ti.Mpt van de cin phdi d l y trong qud ttinh xdy dyng phuang phap logi bo ndi dpe t i khdi djch protein tdi to hpp Id trgng ttidi tdn tgi cua cdc phdn tu lipopo[ysaccharide trong dung djch. Thing thudng ehung cd the t i n tgi d dgng tu do trong dung djeh, tuy nhidn chung cd ttie tin tgi d dgng lidn kit vdi cdc phan tu protein thing qua cde lidn k i t kieu tTnh ildn, hogc tuang tdc di lue (Magalhaes e( al. 2007). Didu ndy gdy khd khdn eho qud ttinh logi bd nd ddc t i khii dung djch chua protein. Cdc ky thugt trdn ttidnh cing hay khdng phy thuic nhieu vdo

HOl NGH! KHOA HQC C O N G NGHE SINM MVU i UAP

llnh chit cua r»6i die t i cflng nhu protem ttong sdn phlm.Dya vao cac nghien cuu cua Williams (2007) chiing tdi nh«r S i v Dhan ti> lipopolysaccharide c6 ttil dupc tdch khii phdn tt> protein bdng cdc dung dich nhu iso-propanol,.., (WUHams 2007) Trong djch tinh che IL-2 ngudi tdi t l hpp, chiing tdi su dgng h$ dung mdi Id 60 % Iso-propanol frong:

axit dfrfc 0 5 % H20 39,5 %, vl v ^ phil h ^ eho viec tdch cdc phdn tu n ^ flic tfi khii IL-2 frong djch tinh chl.Dpa vdo bdn chit cia dii fcfpng d n nghien ciiu Id protein tdi t l hop cua nguai, kit hpp vat t^c ddc Unh vdt ly vd hoa hpc c ^ Phan tir Upopolysaccharkte (nil dpc to), cflng nhu dnh hudng cua mpt s i ton (Ca , Mg ....)(Lp and Rg 1999) nfln chuna tdi 6d lua chpn phuang phap Ipc vdi mdng loc Millipore 30 kDa k i t hop vdi vi§c bdn xu ly bdng dung djch CaClj d ning dd khdc nhau d l tang cudng Ndu qud ipc. Tir cdc kit qud nghidn cdu dd dupc frlnh bdy. chiing ldi nhdn ttijy phuong phdp kit hpp gida lpc toai bo npi dpc t i bang mdng loc Millipore 30 kDa vd xu ly tnfdc lpc bdng dung djch CaClj 0 1 M dd cho hidu qua lpc t i t phu hpp cho vigc dp dyng phuong phdp a qui m i san xuit. Trong nghidn ciru cGa chiing tdi phuang phdp lpc dd dupc dp dgng ngay ti> khdu xir ly IL-2 tten ttnh che trong dung djch Guanidine 7 M vd c4 sau tinh chl. Kit qud ttiu dupc deu cho tti^ hidu qud toe t i t cua phuang phdp. Tuy nhien dp dyng phuang phdp lpc ngay ti> khdu xd ly tien tinh chl se ddm bdo cho qud ttinh ttnh sgch san phim trdn hg ttidng HPLC dgt higu qua cao, franh qud trinh tich hj tt*n cOt ldm anh hudng din qua ttlnh tinh c h l tilp ttieo.

K ^ L U J B M

Da ddnh gid, so sdnh duoc lupng ndi dOc t i cOa cdc mdu djdi IL-2 ngudi tdi t i tvyp khdc nhau vd so sanh vdi quy chuin an tt)dn till gidi bing bd kit LAL QCL 1000 dupc to chdc FDA My cdng nhdn.

Phuang phdp s i c ky ddo pha su dyng hd th&ig sac ky long cao dp HPLC dd ehung td Id phuang phdp hidu qud tong vide logi bd ndi die to ra khdi djch IL-2 tdi t l hop.

Sdn phim sau khi xu ly bdng CaClz vd Ipc bing mdng lpc Millipore 30 kDa cho k i t qud vdi hdm lupng chit ndi die tfi r i thip Id 0,04 EU/mL.

TAI LI$U THAM KHAO

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admlnistratkm of lntedeukin-2. Annafs of tfie/^ewVorfc Academy of Sciences 11&2' 14-27.

GortMt, Maud B. and h/6chad V (2005). Endotoxin: The uninvited guesL Somaterials 26 (34): 6811-6817.

Granucci F, Ivan Z, Norman P, Seiani L, Chnstopher E, Filippo B, Manapta A, Paola R (2004). A Contribution of mouse dendnlic cell- derived IL-2 for NK c«ll activation. The Joumd d Experimental MedKine 200 (3): 287-2S5.

U L, Luo R (1999). Quantitative determination of Ca^ effects on endotoxin removal and protein yield in a two-stage ultraHltialion piocass. Separadon Sdence and Technology 34 (9)- 1729-1741.

Magalhaes PO, Andrd M, PrlscBa G, Cartota R, Thereza C, Adalberto P (2007). Mettiods of endotoxin removal from bnlosical

preparatkms: a review. Joumd d Phwmacy 8. Pharmaceutical Sdances: a Publication d the Canadian Sodety for PhamtaceuHcal Sciences. Soci4td t^anadienne Des Sciences Phamjaceudques 10 (Zy 388-404.

MoiTison D, Lei M, Kirikae T, Chen T (1993). Endotoxin receptors on mammalian cells. Immunobiology MT (3-5): 212-226.

Rietschel E, RitSiard A LemerB, Berry L (1984) Handtiook of Endotoxin. Elsevier

Williams, Kevin L (2007). Endotoxins: pyrogens, LAL testing and depyrogenation, 3"" ed. Taytor & Francis

ENDOTOXIN REMOVAL FROM HUMAN RECOMBINANT INTERLEUKIN-2 PRODUCT BY ULTRAFILTRATION AND REVERSE PHASE

CHROMATOGRAPHY (RP-HPLC)

Le Phuong Hoang Anh, Dao Trong Khoa, Nguyen Hong Thanh, Phung Thu Nguyet, Truong Nam Hai Institute of Biotechndogy, Vietnam Academy of Sdence and Technology

SUMMARY

Recombinant proteins produced from Gram negative bactena (E coli) is often contaminated widi endotoxin during extracti(««S proiem punfication. Due to dieir high toxicity in vivo and in vitro, endotoxin removal &om injection products is essential. In 4"

study, we developed a method to remove endotoxin in recombinant IL-2 products using reverse phase chromatograihy RP-tSIf combmmg with ultrafiltration widi nulipore membrane of 30 kDa treated with 0.1 M CaClj. Endotoxin concentration was detemuwd by Limulus Amcl)ocyteLysate (LAL) kit measured at 405-410 nm in spectrophotometry. Results showed that >94% endoloxinwis, removed from IL-2 products usmg ultiafilier treated withO.l M CaClj. Usmg diis mettiod togettier widi RP-HPLC endotoxin«

reduced to final concentration of 0.04 EU/mL, lower than limitted dircshold of FDA for injection dnjg products of 0.2S EU/mL Key word: Endotoxin, HPLC, Intcrieukin-2, ultrafilter.