Sn xut protein ti t

hp c dc tnh sinh hc (phn II)

Kim tra cc im mnh ca quy trnh Cc t bo l cc h thng phn ng sinh hc nh sn xut hiu qu nhiu hay t cc protein ti t hp da vo cc yu t bit hay cha

bit. Sn phm qu trnh ln men cha nhiu loi protein, mc d quy trnh c thit k tt trong giai on ny. iu ny ph thuc rt nhiu vo cc bc tinh sch. Trong mt quy trnh hiu qu, cc cht nn ct c th gn nhiu protein (10-20% nhiu hn hay t hn s lng mong i). Tc dng chy ct v s lng mu c kh nng cha c th c mt nhiu trong sut qu trnh chy ct v sch ca qu trnh phn tch. Cc bc kim tra cn c thc hin tnh ton khong thi gian hiu qu ca ct v sn phm c tinh sch xc nh cc iu kin m protein vn c kh nng hot

ng. Cui cng, tnh hiu qu ca quy trnh ph thuc mnh vo cc thng s nh pH, dn in v tnh phn cc. Nhn chung, mt ct tinh sch linh ng th tnh hiu qu ca quy trnh cng tng thm. Tnh ton quy m nh Mt khi tnh hiu qu ca quy trnh c kim tra, quy trnh ny cn lp li t ba n nm ln xc nh cc thng s, bt u t t bo nui cy n sn xut quy m ln. Mc ch l xc nh lp li v tnh hiu qu ca mi bc lin quan n cht lng v c tnh ca cc cht trung gian v cc cht thuc nng ln. Sau khi vn hnh thnh cng quy

m phng th nghim, quy trnh c nng cp t t thnh quy m pilot ln hn gp 10 ln quy m phng th nghim v sau l sn xut quy m ln. Tt c cc thng s ca cc bc n r c kim tra tt c ngay c khi quy trnh vn hnh quy m ln thnh cng, quy trnh cn c kim tra quy m nh mt ln na tnh ton t hp cc thng s k thut trong mt h thng y . Lc ny, khi quy trnh nh c thm nh li. qu trnh lm sch virus phi c thc hin. Loi b virus Mt thnh phn cn thit ca cc nghin cu thm nh quy trnh ca

cc sn phm thu nhn t t bo eukaryotic l hiu c quy trnh tinh sch loi b cc cc thnh phn virus bt hot b nhim. Cc virus nhim vo quy trnh sinh hc c chia thnh loi:

Cc virus ni sinh tn ti nh mt phn ca t bo c s dng biu hin sn phm protein (retrovirus chut). Cc virus khng thuc loi virus ni sinh l cc virus t cc ngun bn ngoi hin din trong cc ngn hng t bo cao cp (nh Epstein Barr virus). Cc virus ngoi sinh thng nhim vo sn phm thng qua trnh sn xut cng nh nhim t nguyn liu th hay do tp nhim

(parvovirus). Ngy hm nay cc sn phm cng ngh sinh hc thu nhn t cc dng t bo bt buc khng c cha bt k thnh phn virus no. Nhng trong cc quy trnh sn xut hin nay, do thin nhin c th bnhim t cc ngun virus ngoi sinh. Do , chc chn c sn phm an ton, gim nh cc bc lm sch virus phi tr thnh mt phn ca qu trnh sn xut. Loi nhim virus Cc bc lm virus phi c gim nh trc giai on I ca sn phm v c thc hin bi chuyn gia. Phng php chung cho cc nh sn xut l s dng cc dng t bo

chut lang (rodent cell lines) gim st cc bc tinh sch virus loi b hay bt hot cc virus gy ung th mu chut (murine leukemia virus), virus ny ng vai tr nh l mt m hnh chuyn bit i vi cc tiu phn ging retrovirus c sn xut bi cc h thng biu hin trong dng t bo chut v thm vo v gim st bc tinh sch parvo virus trong qu trnh sn xut sn phm giai on I. S bt hot virus c th c thc hin bng nhit , pH, cht ty ra, phng x v cc dung mi s loi b virus c th c thc hin bng phng php lc v chy sc

k. Trong mt nghin cu gim st virus , c thc hin truc khi sn xut cc vt liu cho giai on III c sn phm, cc nh xut s chn mt quy trnh bao gm t hai ti bn bc kim tra b sung. S la chn virus ph thuc vo dng t bo v ngun gc ca mi trng bao gm v hai loi virus m hnh chuyn bit v khng chuyn bit. Cc dng virus c la chn bt hot v loi b nhiu loi virus trong qu trnh sn xut m vn suy tr hot tnh v cht lng sn phm. Mt virus ng quan tm l cng nh virus c xc nh cng nh

cc chng loi virus c bit cng nh nhim vo vt liu trong qu trnh sn xut. Mt m hnh virus chuyn bit cn c chnh xc nht cc chng loi virus (cng chng hay h), c cc c tnh ho hc v vt l tng t nhau. Cui cng, mt m hnh virus chuyn bit c s dng c tnh ho kh nng loi b hay bt hot virus tng s cho thy tnh hiu qu ca quy trnh tinh sch. Quan trng l, virus trong hn mc l s la chn u tin. Nu s dng bit chnh xc l iu khng th. Cc nh sn xut chn mt m hnh virus chuyn bit nht lm mt m hnh virus cn kho

st. kho st s sch ca virus, cc nghin cu h tr tnh ton s sch ca virus ca mt nhm virus khng chuyn bit no . Nhng virus ny c kt hp cac m hnh chuyn bit v tng ng to ra mt quy trnh kim tra s hin din ca virus t cc b gene khc nhau (DNA v RNA), kch thc v cc c tnh b mt (virus c v hay khng) Nghin cu v s b sung virus gim st Trong nghin cu v s b sung virus gim st, cc bc ny nm trong mt qu trnh sn xut vi tnh hiu qu cao nht loi b hay bt hot cc thnh phn

virus l mt thch thc khi gp phi s lng virus cao. S lng virus cao cho php cc yu t kh virus ln hn v cho php gp phi nhng kh khn khi lm sch virus trong mt quy trnh. Vi l do , cc vn bn hng dn cho php lng virus c th b nhim vo trong vt liu ban u vo bc sn xut phi c gim st cng nhiu cng tt. Tuy nhin khn phi thnh phn ban u no qu trnh sn xut cng c chp nhn. Th tch cho vo phi c gi mc thp, bnh thng phi thp hn 10%. Trong khi nghin cu lng cn c thm vo, m hnh virus c

b sung trc tip vo cc bc thch hp quy m nh c tnh ton trc. Nhng nghin cu ny cho php nh sn xut tin lng c kh nng loi b ton b virus ca quy trnh sn xut v xc nh quy trnh sn xut no c kh nng cung cp mt mc thch hp trong quy trnh lm sch virus chc chn rng cc sn phm thuc c an ton. Yu t kh Cn thit bit c bao nhiu virus c trong mu khng qua ch bin bi v qua trnh phi c thit k c kh nng loi b ton b virus. Bnh thng virus trong mu khng qua ch bin c nh

lng bng knh hin vi in t truyn qua theo mt nng m c hay bng cc thc nghim cho nhim (bioassays). Yu t kh c xc nh theo log10 theo t l gia tng s virus cho vo trc khi lm sch v tng s virus cho vo c o lng trong sn phm sau khi lm sch. Nhn chung cc i hi v yu t kh c thit k chuyn bit cho cc virus bit nhim vo quy trnh sn xut. Cc tiu phn ging nh retrovirus c sn xut bi cc h thng biu hin protein trong cc t bo chut l cc mu ph bin. Vi mc tiu ca yu t kh chuyn

bit theo quy trnh c thit k sn, cc nh sn xut b sung cc bc lm sch virus vo quy trnh v thc hin cc bc c tnh gim st theo di tnh hiu qu ca qu trnh lm sch virus khi vn hnh mt vi h thng. iu c bn ca yu t kh v tnh hiu qu, tnh hiu qu c t ln hng u. Mt s vn hnh c th c tnh ton theo tnh hiu qu, khng hiu qu hay hiu qu tng i. Cc bc hiu qu cung cp mt yu t kh ca t nht 4 yu t v khng nh hng bi bi sai s trong mt quy trnh nhiu yu t. Cc bc khng hiu qu cung cp mt yu t kh ca mt hay t hn

v cc bc hiu qa mc tng i ri vo mt trong hai yu t c tnh quyt nh. Thit k quy trnh vn cn l mt thch thc trong vic pht trin quy trnh mc nh v cc yu t c tnh phn tch bi v quy trnh phi thch hp vi s sn xut cc protein c hot tnh trong mt tiu chun nghim gt theo cc quy nh an ton dc phm.