• Nghien CIJU - Ky thuat
NI Chien lirffc moi trong viec bieu hien erythropoietin (EPO) tren dong tk bao
CHO-DG44 bang vector tricistronic
Nguyen Anh Xuan', Lao Due Thuan^ Doan Ng^c Hai^ D 8 Minh ST'-^' ' Khoa Sinh iipc, Truang Bgi hoc Kfioa hoc Tu nhien - Dgi hoc Quoc gia Tp. Ho Chi Minh
^ Cong ty TNHH CNSHDu^c NANOGEN
^ Khoa Cong ngh^ sink hpc, Tnrdng Dgi hgc Mcr Tp. Ho Clii Minh ' Vi^n Sue khoe nghe nghidp va Moi truang
'E-mail: i
Summary
Erythropoietin (EPO)gene was cloned Into the expression vector pDIGF-EPO, which carries other two expression cassettes (GFP and DHFR). The dihydrofolate reductase deficiency (DHFR-) CHO (DG44) cell line was transfected by this vector using lipofectin method. The GFP-based system was used to shorten the cloning duration and enhance the opportunity to select super clones. The DHFR-based system was used for gene amplification. The transfected cells were selected and processed through 2 cycles of FACS followed by gene amplification and single cloning by limited dilution method. GFP gene from the single cell clone was removed by recombination. An optimized fed-batch was developed to culture the EPO expressing cells. The cells could survive for 14 days; the protein titer was 902.6 ± 19.96 pg/mL (comparable to the previously reported values for EPO production In pilot scale). The purified recombinant erythropoietin from the fed-batch had the molecular weight within the range of 30-40 l<Da as checked by SDS-PAGE. Us isoform pattern by isoelectric focusing (lEF) electrophoresis satisfied the requirement ofisoforms issued by the European pharmacopoeia 7.0 (EP 7.0) with ratios in appropriate ranges. Peptide mapping and sialic acid content analysis strongly passed the requirement stated in EP 7.0. Biological activity of the obtained EPO-alfa versus the 3rd WHO International Standard for Erythropoietin for bioassay was 112.545 IU/mg protein.
Keyw/ords; Recombinant human erythropoietin (rHuEpo), CHO-dhfr<-' cell, GFP, single clone, gene amplification, recombination.
O a t v ^ n (Je EPO t y nhien hoan todn giong nhau v l trinh t y
^ aeid amin va co chuoi oligosaccharid giong nhau Erythropoietin (EPO), mOt nhdn to tang tn^ang ^^^^^ ^ ^ ^^^^ ^^^^^^ ^^^^^ ^ p ^ ^^^ ^. ^^^ ^ kich thich tao h i n g eau, giup cdi thien dang ke hoat tinh sinh hpe t u a n g t y nhu EPO npi sinh "^-^i.
ehat l u y n g cupc song cua bpnh nhan mac chung ^ , . . . . . , . . . . _ , , _ ,
^ , . . Z Nhung te bao dpng vgt n h u CHO d u y c lua suy thSn hoac chu'ng thieu mau t r i m trong. EPO ^ ^ ^, ^^ ^ ^ , , ^^ p^^,^,^ ^ . „ ^ ^ ^ ^ ^ „ ; , ^ adng vai tro quan tnjng trong s<f tang sinh vS biet ^ p g ^^^^ ^^ ^ , - „ ^ j . ^ ^ ^ ^.^^ ^ . ^ . ^ , . ^^ ^^^
h6a cCia cac t^ bdo tien than h6ng cau trong tuy ^^,p „ ^ i ^^ , ^ ; , protein cua ngifoi v^ mat chi/c x y o n g ra. Phan I6n EPO (Juvc tao ra chu yeu bffi ^ j ^ g ^^^ nhif du-ffc dOng hpc "">'. Viec sir dung t h | n d ^ d i p Lpng vdi tinh trang thieu oxy trong ,^ j j j g C H O cho nudi cay s i n xuat protein v « mS, mot phan nh6 (10%-14%) do gan tong hop quy mo Ion ngay cang pho bi^n bU cac dong t^
(gan id ca quan chfnh s i n xuat ra EPO d b i o bao nay co the thich i>ng voi viec phat trien trong thai) ra. Trong nhu-ng nam gan dSy. viec O'ng dyng moi tru^ong huyen phii Ithong chtra huyet thanh.
cua EPO tai t6 htyp trong d i l u trj b#nh ngay cang Ngoai ra. cac dong te bao nay du'gc cong nhan la ph6 biSn tr^n toan the gi6i. EPO tai to h o p va cac te bao an toan cho viec s i n xuat protein tai t6
T.M'CHf DU'OC Hpc - 11/2015 (S6 475 NAM 55)
Nghien CIPU - Ky thuat
hyp ^. Tuy nhien, d ^ lya etipn d u y c dong t^ bao dpng vpt th^ hidn cac protein mpc tieu (bi6n doi sau dieh ma) vol hipu sudt cao, mOt s6 van db can d u y c giai quyet nhu he thong bieu hien kem hipu qua, thai gian chuyen gen vd tao dong, miFe dp bieu hipn protein thap a quy mo lan. Day la nhung kho khan g | p phai trong hdu het eac dong te bao hien nay ^•^. Tnang nghien cuu trudc day. Meng va cpng s y da phat trien he thong dong bieu hidn gen mue tieu vai gen chpn lpc DHFR (ma hda cho enzym dihydrofolat reductase) vd gen GFP (ma hoa cho protein phat huynh quang xanh) de lya ehpn dong te tiao bieu hipn protein tdi to hyp muc tidu vai hidu suSt cao nhat f^^. Dong t^ bao t6t nhdt d u y e ehpn lpe d y a tren hp thong ehpn lpe DHFR va hi th6ng phdn tach te bdo dya tren tin hidu huynh quang cua GFP (FACS).
Trong nghien CCPU nay, ehung t5l dua ra mpt chien l u y c md'i cho vide tao ddng te bao bieu hipn 6n djnh protein EPO tai to hyp. Hp th6ng vector tricistronic g6m gen mye tidu, gen GFP (tao thu$n lyi cho viee rut ngdn thai gian tpo dong vd tang kha nang chpn lpc d6ng te bdo tot nhat) va gen DHFR (dung chpn lpe tren dong te bdo khuyet duang DHFR CHO-DG44). Buac tiep theo eua nghidn cuu la loai bd gen GFP bdng phuang phdp tdi to hyp sau khi chpn d u y c te bdo dan dong bieu hien protein mgc tieu tdt nhat.
Qua trinh ndy se giup gidm hay Iopi bo s y bi^u hidn cua GFP.
Vat li^u, phircng phap nghien ciru
Thiet ke vector
Trong nghien ciru nay, plasmld mang gen ma hoa EPO tdi t6 hyp nguai (EPO), gen GFP (md hoa protein GFP), gen khdng Zeocin vd gen ehpn lpc DHFR (md h6a dihydrofolat reductase). Trinh t y Hu-EPO d u y c thiet ke va tong hyp bai hang Genscript (Piscataway, My). Gen eGFP d u y e phan taeh t u plasmld pEGFP-NI (Clonteeh, My).
Gen DHFR du<?c phan tdch ti> plasmld pSV2-dhfr (ATCC, My). Promoter CMV d u y e s u dung de dieu hoa bieu hl§n EPO tai to hyp ngu6i. Trinh t y intron cua gen beta-globin tren tho (GenBank:
V00882.1) vd vung CpG cua gen HNRPA2B1 nguai ma h6a cho 2 Iopi protein hnRNP A2 va B l (GenBank: D28877.1) d u y c bo sung de
tang c u a n g kha nang bieu hipn gen mgc tidu.
Ket thue qua trinh biSu hien GFP bdng phuang phdp tai to hyp. Hai vj trf tdi to h y p tuang dong FRT d u y e them vdo trude promoter vd sau trinh t y gen GFP. Dk dam bao eho GFP bilu hidn thip han gen ma hoa EPO, trinh t y IRES d u a c chen vao t r u a c promoter eua GFP. Vung promoter (SV40) d u y c s u dgng dk dieu hoa bieu hipn DHFR. Plasmid iJipac dong hoa vao te bao vi khuan £. coli ToplOF theo huang din cua nhd san xuat (Invitrogen, My). Plasmid bilu hidn d u a c tinh spch bdng bp kit taeh chiet DNA (Qiagen, My).
Quy t r i n h c h u y e n gen
T l bdo CHO-DG44 (ngan hang cGMP, Invitrogen, My), dong te bao mang dot bien gen reductase dihyrofolat (DHFR) nam trdn nhiem sdc t h i s6 2, d u y c s u dung rdng rai trong san xuat protein tai to h y p thong qua phuang phap chpn lpc khuyet d u a n g DHFR. Te bdo CHO-DG44 dugc nuoi cay trong m6i truang PowerCH02 (Lonza, My) co bo sung hypoxanthin va thymldin (HT, Sigma-Aldrich, My) cf 37°C, 5% CO^.
Dk chuyen plasmid vdo te bdo CHO- DG44, nghien euu s u dgng b6 kit chuyin gen Lipofectamine LTX with Plus Reagent kit (Invitrogen, My). Quy trinh thye hipn d u y c tiln hanh theo huang ddn eua nhd sdn x u i t . Nghien cuu tien hanh c h u y i n vector trietstronie vdo t l bdo CHO-DG44. Sau 48 gia ehuyen gen, te bao ehuyin gen bieu hidn GFP d u y c quan sdt b i n g kinh hien vi huynh quang va s y bleu hidn protein EPO trong djch nuoi eay te bao d u y c djnh luyng bdng bp kit human EPO ELISA (Stem Cell technology, Canada).
Chpn lpc va tpo d d n g te bao CHO-DG44 b i l u hipn EPO tai t o hp'p
Qua ti-inh chon lpc d u y c tien hanh bang cddi nudi te bao da ehuyen gen trong m6i truang chpn lpe bo sung 400 pg/ml zeocin, 50 nM methroxat (MTX) va khuyet d u a n g HT (Sigma-Aldrich, My).
Sau chpn lpc, te bao mang plasmid d u y c nudi cay tang smh trong moi truang duy tri c6 b l sung 50 pg/ml zeocin. Te bao ehuyen gen b i l u hipn manh GFP d u y e ehpn tpe 2 lan trin hp thing mdy FACS Calibur (BD Bioseiences, My) vd si>
T ^ P CHI DlTOfC HQC - 11/2015 (SO 475 NAM 55)
Nghien ciru - Ky thuat
dgng eho nhung nghidn c u u tiep theo.
Te bdo sau khi lya ehpn bang GFP tilp tgc d u y e tiln hanh chpn lpc thdng qua k h u l e h dpi gen. Te bdo d u y c cdm i>ng trong moi trud'ng nuoi c i y bo sung MTX vai n6ng dp tang d i n t u 50nM den 500 nM. Sau do, t l bao d u y e phuc hoi vd t i l p tgc nuoi tdng sinh. Dong te bao dan (single clone) bieu hien on djnh protein mge tieu t i l p tgc d u y c tpo ra bang phuang phap pha loang tai han. Sang lpc nhu'ng dong t l bdo dan c6 b i l u hipn GFP (quan sdt d u a l h i l n vi hu^nh quang) va EPO (djnh l u y n g b i n g phuang phap ELISA) cao nhat. C u l i cung, s y bieu hipn GFP d u y c Iopi bo bdng cdch chuyen plasmid pOG44 mang gen md hoa reeombinase (Invitrogen, My) vdo d6ng te bdo lya chpn. Hi$u s u i t tdi to h y p d u y c ddnh gid bdng hi thong may FACS Calibur.
Nudi cay tdng sinh te bao san x u i t EPO tdi to hp'p trdn quy mo thu> nghidm
D6ng dan te bao sau khi chpn lpe theo quy trinh neu trdn (bieu hipn mpnh n h i t EPO tai to hyp) d u y e s u dgng d l nuoi cay tang sinh bang moi trud'ng PowerCH02 (Lonza). Trud'c tien, te bao d u y e nudi c i y trong ong nudi eay l i e 50 ml (BD Biosicenee, My), sau dd d u y c nudi cay tang sinh Idn t h i tich cao ban (600ml) trong hd t h i n g nudi cay Tube spin flask (TPP, Thuy 37) ket hyp vdi tu nudi eay l i e (Kuhner, Thyy ST). C u l l cung, qud trinh nudi cay te bao san xuat EPO tai td h y p d u y c thyc hidn trong binh nudi cay ldc 5 L (Thompson, My). Te bao d u y c nudi c i y vdi m^t dd ban dau 0,5 x 10^ te bao/mL trong 2 L mdi trudng sdn x u i t d 37°C, 5% CO^, toe dp lae 100 vdng/phOt. Sau 3 ngdy nudi c i y , 500 mLdung djch feed (bao gom Feed 3 ti> Exeellgene, Galactose, Manose, Insulin.Transferrin va hda chat khae de tang qud trinh g i n d u d n g cua EPO t u Sigma) d u y c b l sung hdng ngdy. Qud trinh nudi c i y t l bdo sdn x u i t EPO k i t thue khi ti lp phan tram t l bdo song d u d i 70% (khoang 12-14 ngay). Djch nudi e l y t l bdo sau dd d u y e thu n h | n va tinh c h l bang hp t h i n g sac kf Akta - Mabselect Sure (GE Healthcare, Anh) theo hudng d i n cOa nhd sdn x u i t . Protein sau tinh spch d u y e kiem tra, ddnh gid cdc cht tieu n h u cau tnJc hda hpe, dp tinh spch, n i n g dO protein, hopt tinh sinh hpc d y a trdn cdc quy trinh da d u y e cdng bo [Jelkmann,
Lee, Breidbach].
Ket qua va ban luan Vector bieu hien EPO tai t l hyp
Hinh 1 : Cau true vector pDlGF-EPO C h i l n l u y c b i l u hidn protein EPO trdn ddng t l bao CHO-DG44 trong nghidn c u u nay mang Ipl hieu qud cao khi xem xet tren khia cpnh thdi gian tao ddng vd luyng protein d u y c tao ra.
Vung bieu hipn rHu-EPO bao g i m gen mye tidu (Hu-EPO) d u y c dieu hda bdi promoter/enhancer CMV. Dya tren k i t qud cdng bo eua patent W O 2004067701A2, vung CpG cua gen HNRPA2B1 ma hda cho protein hnRNPA2 vd B l d u y c sit dyng nhu Id yeu to tang cudng bieu hipn gen (enhancer) '^i. VCing CpG d u y c chung minh nhu la yeu to tang cudng b i l u hien gen ngoai lai va cd nhieu thupn lyi khi t h i l t k l vector cho vide tpo ddng bleu hien i"i. Nghien euu eua Buchman vd cdng s y dd ehung minh s y hipn dien eua vung intron thupc gen beta-globin d thd eo kha nang tang cudng s y b i l u hi^n gen cao gap 2 den 500 lan ^. Ket h y p cdc y l u to tang cudng tren, s y bieu hipn cua GOI tdng len ddng ke (du lipu khdng trinh bay).
Trinh t y IRES (internal ribosome entry site) Id trinh t y d u y c tim thay trong virus ed kha nang huy dpng ribosome d l b i l u hipn protein mdt cdch dpc lpp va ddng vai trd quan trpng trong qud trinh phdt trien cac vector polycistronie. VCing IRES thu nh|ln tCp vims Eneephalomyocarditis (EMCV) hipn nay d u y c sD" dung chu yeu trong h i u het eae vector.
Tuy nhidn, luyng protein b i l u hipn dudi s y dieu hda eua EMCV IRES thudng khdng du d l dat d u y c cae yeu cau thye nghidm hay ehu'a tri. Nghidn CLPU
TAP CHI Dirpc HQC - 11/2015 (S6 475 NAM 551
• Nghien CLFU - Ky thuatj
eua Lieursi va cdng sy cho ttiay vipc siJ dyi^Hung IRES cua nhan to tang tnfdng nOi mach^S^aular endothelial growth factor) va protein cam T>ng collagen Iopi 1 (type 1 coltagen-indudble protein) tdng cudng khd nang bilu hien gen cao h a n ^ vdi eac vector chCra EMCV IRES'"«. Trong nghidn cuu nay, chung tdi sir dung trinh ty ndy d l dilu hd^ sy bilu hidn eua GFP (mariter chpn lpc). C a ^ ^ M cua vector pDIGF-EPO duyc md ta trong h i n h l ^
Tao ddng te bao CH0-DG44 bieu hien EPO tai td hyp
Sau 48 gid chuyen gen, s u bieu hien EPO va GFP tam thdi d u y c k i l m tra Idn luyt bing phuang phap ELISA va phuang phap quan sat mau d u d i kinh hien vi huynh quang. Du lieu ti>
s y bieu hien GFP cho thay hieu suat chuyen gen dat 50 - 70% va te bao bieu hien EPO d miFc 2,3 pg protein/te bao/ngay (bang 1).
Bang 1 : S y bleu hiin EPO va GFP cua te bao CHO-DG44
Thing s6 Bieu hien Sau 1 lin sorting
Ham lirgng EPO (pg/t4 tjaofngay)
T71dbiluhi^nGFP(%) " ^ Q , 0 2 " "
Sau 2 lan sorting
4,78 + 3,27 32,57 ±6,33 78,22 ±8,96 94,66 + 9,13 Sau 2 ngdy ehuyen gen, te bao duycqhpn
lpe bdng mdi trudng nudi ciy khuyit dudng HT-, b l sung khdng sinh va MTX nhdm chpn ra cac te bdo chuyen gen on ^nh. Protein G F R g p y e ehung minh Id huu ich tnsng h$ thong ^ H lpc t>di protein GFP eho phep tpo ra m p ^ ^ n ' n g thue chpn lpe nhanh. dan gidn vd ^ ^ ^ q u a tren eac t l bdo dd duyc chuyen gen thdng qua phuang phdp dong ehuyen. Dya trdn ky ( i u a t phdn taeh va thu nhdn te bao cd chpn I p e l d n g qua tin hi$u huynh quang (FACS), nghien cuu SU" dyng chiln luyc nay de phat hidn cac t l bao
bieu hien manh protein muc tieu. Cac dong te bao cd tin hieu GFP cao nhdt trong mdi trudng d u y c cho la cd kha nang b i l u hien protein muc tieu EPO vdi hieu s u i t tuong ty. Trong nghien cuu nay, sau hai lan phan tach va thu nhan te bao bdng ky thuat FACS, khoang 94% te bao hieu hien GFP cho tin hieu vdi cudng dp khac nhau (hinh 2). Ket qua nay cho phep nghien cii'u loai bd cac te bao bieu hien thSp protein muc tieu EPO Ngoai ra, cac te bao chuyen gen on djnh cho thay s y tang len dang ke luyng EPO dupc tao ra.
Hinh 2: Subieuhi$n GFP eua te bao CHO-DG44 truac va sau khi chuyin gen.
Tlii te bio phit huynh quang tm-dc va sau khi chuyen gen. phan tach ti bao duac xac dinh b^ng ky thuit FACS. Tin hieu phit huynh quang^a ti bao duac kiim tra dual kinh hiin vi huynh quang.
Cdc te bao sau khi lya chpn theo q u y i - i n h neu tren tilp tge nudi c i y tang sinh va tao dong t l bdo dan bing phuang phap pha lodng toi hpn nhim chpn lpc ddng t l bdo b i l u , , ^ ^ on djnh nhat. Dich huyen phu Kr dTa 96 giengmpyc thu 12 ngay sau khi chuyen te bao vdo dTanuoi d y . 48 ddng te bao tot nhat duye nudi tren dTa
24 gieng bdng each lya chpn cac t l bao t u dia 96 gieng chuyen qua dTa 24 g i l n g . Sau 5 ngay nuoi, djch huyen phu t u cac gieng cua dTa nudi cdy 24 g i l n g d u a c phan tich. Sau dd, 12 dong te bao tot nhdt d u y c chpn ra va chuyen vao dTa nudi cay 6 g i l n g de tiep tuc nudi cdy tang sinh. Dya tren k i t qua phan tich tCf dieh nudi cdy t l bao, 6 ddng
T A P C H i DL OC HQC -11/2015 (SO 475 NAM 55)
• Nghien CCPU - Ky thuat
t l bao bleu hien tot n h i t (EPO 1 - 6, bdng 2) d u y e lya chpn vd nudi e l y tdng sinh trong 6 ngay sir dgng hi thdng lac on nhipt (Kuhner). Dya ti-en k i t qua thu nhdn d u y c tCr nudi eay lac (mpt dp t l bdo ban dau 5 x l O M i bdo/mL, the tich 100 mL mdi trudng nudi c i y ) , ddng t l bao b i l u hidn tot
nhdt (EPO 5) d u y c luu tri> trong ngdn hang giong vd ky hipu EPO0414. K i t qua ddnh gia hipu s u i t san xuat protein mgc tieu va dudng eong tang trudng cho eae ddng t l bao tot nhdt d u y c trinh bay trong bang 2.
Bang 2: M e u suit cOa cic ddng ti bio dan bleu hien EPO
Ddng te bio EP01 EPO 2 EPO 3 EPO 4 EP0 5*
EPO 6
l1Vt6bito/ml) 2,23 ±0,25 2,21 ± 0,39 2,37 ±0,45 3,09 ±0,51 3,13 ±0,62 2,61 ±0,47
Ngay 3 Ham lm?ng protein
(iig/ml) 135,2 ±6,33 122,1 ±7,49 136,3 ±5,01 141,7±8,23 198,6 ±7,22 153,7 ±9,16
Lm?ng piotein/
t4b^o/ngiy 20,2 ±0,97 18,4±1,23 19,2 ±0,85 15,3 ±0,98 21,2 ±1,51 19,6 ±0,98
Thai gian nhin (36i (glo) 33,38 ±0,13 33,58 ±0,25 32,07 ±0,36 27,40 ±0,24 27,21 ±0,19 30,20 ±0,27
Ngiy 6 Mata^t^bao (irftibioM)
4,24 ±0,31 4,37 ±0,27 4,62 ±0,39 4,91 ±0,52 5,23 ±0,68 4,31 ±0,29
Ham iut?ng [initein (lig/ml) 365,8 ±10,25 371,2 ±11,36 312,9 ±15,26 344,8 ± 14,33 405,6 ±17,66 311,2±12,38 (') Ddng te bao dem tfuyc /ya chon
Hien nay, eac enzym tai to h y p tpi cac vj trf chuyen bi0t (site specific recombinases) d u y e phdt t r i l n thanh nhQ'ng cdng cy s u dung trong ky thupt thao tac tren gen vi chung cd t h i xuc tae chtnh xdc cho eac phan dng cat, chen, dao dopn h o l e e h u y i n vi tri cua DNA thdng qua cdc trinh t y nhdn b i l t dpc hipu '^l Nghien cuu s u dyng hi thong Flp-ln (Invitrogen) chua vj tri tai t l hyp Flp (Flp Recombination Target site - FRT) de
di tao ngin h^ng ti b^o. EPO0514
Iopi bd gen GFP. S y bleu hipn GFP da giam dang ke khi dua he thong ndy (vector pOG44) vao t l bao CHO-DG44 b i l u hien GFP. Theo hinh 3, k i t qud cho thay t l bao EPO0414 sau khi Iopi bd GFP vdn bleu hipn EPO vdi hdm luyng tuang t y . Trong khi dd, mCrc dp bieu hipn GFP giam dang k l (8,98%) so vdl trudc khi e h u y i n vector pOG44 (93,64%). T l bdo sau khi loai bd GFP d u y e luu t r u vd ky hipu EPO0514.
Doi chi'nig Tivo^c kill tai lo hop S»ii klii t»i to Ii<^
Hinh 3: Si/ biiu hiin GFP cOa ti bio CHO-DG44 truac va sau khi tii tS h(?p.
Tlii ti bio phit huynh quang trudc va sau khi tii to hgp dugc xic dinh bing ky thuit FACS.
Tin hiiu phat huynh quang cua te bao dugc kiim tra dual kinh hien vl huynh quang Nuoi c i y te bao va s a n xuat EPO tai t o hp'p
Hidu s u i t cua EPO0514 dufgc danh gid khi nudi d y theo phuang phap nudi eay fed- batch (quy md 5 L) vdi mdt trudng d u y c t i i u u (mdi trudng d u y e bo sung theo thdi gian.
t h i tich c u l i 2500 mL) s d dung binh nudi d y 5 L (Thomson) va hd thong tu d y lac (Kuhner).
L u y n g protein d u y c xdc djnh bdng phuang phap ELISA s u dyng bp kit human Erythropoietin (EPO) ELISA (Stemcell Technologies). K i t qua
T A P C H i DUQC HQC-11/2015 ( S 6 4 7 5 NAM 55)
• Nghien CIPU - Ky thuat
dat 902,6 ±19,96 ng/mL, t u a n g duang vdi cdc edng bd trudc dd v l sdn xuat EPO.
d u y e trinh bdy trong bang 3. Vdi phuang phap nudi d y fed-batch d u y c t i i uu hda, EPOG514cd t h i duyti-i trong vdng 14 ngdy. Ham luyng protein
Bang 3: Kit qua nuoi ciy ddng te bio EPO0514 theo phucmg phip nudi cay Fed-Batch Ngay
NgayO Ngay 1 Ngay 2 N9Sy3 Ngdy 5 Ngdy 6 Ngdy?
Ngay 8 NgayO Ngay 12 Ngay 13*
M#rdo (llflibao/ml)
0,55 0,66 ±0,09 1,43±0,11 2,32 ±0,25 2,84 ± 0 3 3,65 ±0,37 3,53 ±0,26 3,52 ±0,38 4,36 ±0,52 2,76 ±0,63 2,62 ±0,61
TT/elebao s6ng(%) 99,21 ±0,25 98,51 ±0,16 98,62 ±0,22 95,87 ±0,31 97,26 ±0,25 94,19±0,47 93,07 ±0,51 95,65 ±1,33 92,37 ±3,51 90,6414,73 73,44 ±4,33
Hamlu^gEPO „.. ^^^^
(liglml)
221,3±12,11
425,1 ± 13,24
668,7 ±21,12
902,6 ±19,96
2000 mL MOI tnjdng Power CH02 40 mL Feed 3 IJL,^ bo sung mSj ngdy
+ Galactose + Mannose + Non essental ammo aad + Undin + L-Glutamin+ Insulin + Transferrin
• NEMtUridin
+ Insulin + Transfemn N.glucosamin + Gaiactr)se + Mannose
+ Uridin + L-Cystin, + NEAA + N^giucosamin + Pluronic + Insulin + Treinsfenin
+ NEAA+ N-glucx>samin + Undin
C) thu ho$ch Phdn t i c h EPO tai t l hp^p
Protein tdi to hyp EPO tinh sach tCr ddng te bdo EPO0514 nudi d y fed-bateh ed trpng luyng phdn t u khoang tCr 30 - 40 kDa khi d u y e kiem tra
vao ngay 14
bdng phuang phap SDS-PAGE (hinh 4). K i t qud nay phu h y p vdi tieu ehuan d u y c dua ra trong D u y e dien Chau Au (European pharmacopoeia 7 . 0 - E P 7.0).
Hinti 4: Ket qui phin tich mau protein EPO.
(A) Ket qua f^^in fie/) pepM cua mau EPO t)ing phuong phip pepUd mapf^ng.Miu EPO (trin) vi miu (^uin quoc te EPO jEDQM, dudi). (B) Kk qua (Mn ti<^ Isoform oia EPO bing phumg f^ap dien di diim dang den (lEF).
Giing 1: Mau EPO. ^eng 2: TTiang chuin pi, giing 3: Miu EPO chuan quoc ti. (C) Ket qua phin tidi tmng /ugng phan (u-EPO t)dng phuong phap SDS-PAGE. Giing 1: Miu EPO; giing 2: Miu EPO chuan quoc ti, geng 3: TTiang <^uin protein.
K i t qua khao sat ti lp isoform bdng phuang phdp dipn di diem dang dien (lEF) cho thay EPO tpo ra dam bao cdc yeu cau v l isoform vdi eae ty lp nam trong khoang gidi hpn. Cg the, isoform 1 khdng dugc phdt hipn thay, trong khi dd isoform 6 c h i l m p h i n Idn luyng isoform (bang 4). Ket qua pho s i c ky peptide mapping cho thdy EPO cd p h i peptid tuang t y vdi EPO chuan q u i c te.
Ham l u y n g acid sialic cua protein sau tinh sach la 14,90 ± 0,30 moi sialic acid/mol EPO (dya tren phuang phap cua EP 7.0). K i t qud ndy eung thda man cae yeu d u eua EP 7.0 (a 10 moi sialic acid/mol EPO).
Hopt tinh sinh hpc eua EPO tpo ra dupc ddnh gia trdn md hinh chupt cai khde mgnh
TAP CHi DlTQC HQC - 11/2015 (S6 475 NAM 55)
i i i
• Nghien ci>u - Ky thuat
(20 - 22 g) va so sdnh vdi EPO e b u i n quoe t l theo tidu chuan cCia WHO danh cho EPO (3""
WHO International Standard for Erythropoietin- ma NIBSC: 11/170). Hopt tinh sinh hpe eua EPO d u y c tinh todn d y a tren ehi s l h i n g eau l u d i cda chupt khi tiem EPO. Ket qua cho thay EPO tao ra cd hoat tinh 112.545,00 ± 6,32 lU/mg protein. K i t qua ndy thda man yeu d u cua EP 7.0 (2 100.000 lU/mg protein).
Bang 4: 77/p isoforms cua EPO Insoform
Insoform 1 Insofomi 2 Insoform 3 Insoform 4 Insofomi 5 Insoform 6 Insoform 7 Insoform 8
Tile phan tram chuan (%) 0-15 0-15 1-20 10-35 15-40 10-35 5-25 0-15
Kit qua (%) 0 0 4.9 12 7
23 29 5 23.9 4.9 Nghidn ciru sir dgng hp thong chpn lpe mdi giCip gidm ddng k l thdi gian tao ddng (so luyng the hi eua ddng d u y c chpn lpc Id 30) va tdng eudng kha n§ng thu nhpn cae ddng t l bao bieu hipn mpnh protein EPO. Tu" dd, eae ddng te bao cd the d u y c t h i l t ldp vd san xuat protein tri lipu vdi muc dO t u a n g d u a n g so vdi cdc he t h i n g chpn lpc hi$n nay. Dpc bi^t vdi nganh cdng nghidp d n t i c dd tpo ra cae nguyen lipu eho eae nghien eCru ldm sdng giai dopn I la h i t sue quan trpng, hp t h i n g chpn lpe d u y e de xuat trong nghien euu ndy hi vpng tpo ra d u y c s y khac bipt n h i t djnh.
K e t l u a n
Da nghien euu vd phdt t r i l n thdnh cdng ddng t l bao CHO-DG44 b i l u hi^n I n djnh protein EPO tdi to h y p vdi hidu s u i t dat 902,6 ±19,96 mg/mL.
Cde k i t qua eua nghidn c d u ndy cho thay EPO tdi t l h y p d u y c tao ra cd trpng luyng phan tu', t!
I§ isoform, d u tmc peptid t u a n g t y EPO ehuan q u i c t l theo D u y e d i l n Chdu Au (EP 7.0). Ngoai
ra. hoat tinh sinh hpe eua EPO dat 112.545 lU/mg, t u a n g d u a n g vdi EPO c h u i n q u i c te WHO (3"*
WHO Intemational Standard for Erythropoietin).
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(Ngiy nhin bai: 13/06/2015 - Ngiy duyet ding: 02/11/2015)
r\PCHi DlTQC HQC-11/2015(50475 NAM 55)