Membrane Technology Thiab Vaccine Clarification (Ⅰ)

Cov tshuaj tiv thaiv los ntawm ntau qhov chaw, suav nrog cov ntaub so ntswg rho tawm, cov kab mob hauv cov kab mob, cov kab mob kis, cov proteins thiab nucleic acids uas tsim los ntawm mammalian recombinant hlwb, cov poov xab thiab kab.

Txoj kev siv tshuaj tiv thaiv ntau tshaj plaws yog ua raws li cov txheej txheem fermentation pib ua raws li kev ua kom huv. Kev tsim tshuaj tiv thaiv yog ib txheej txheem nyuaj uas muaj ntau yam sib txawv thiab cov txheej txheem. Xaiv txoj kev purification txoj cai yog qhov tseem ceeb rau kev ua tiav qhov xav tau qhov kawg khoom purity. Kev qhia meej ntawm cov tshuaj tiv thaiv yog ib kauj ruam tseem ceeb uas muaj kev cuam tshuam loj rau cov khoom lag luam rov qab thiab kev ua kom huv tom qab. Muaj ntau txoj kev siv tau los qhia meej txog cov tshuaj tiv thaiv. Kev xaiv ntawm kev sau thiab cov khoom siv yog nyob ntawm hom roj teeb, qhov xwm txheej ntawm cov khoom raug sau thiab cov txheej txheem ua kua. Cov txheej txheem no muaj xws li membrane filtration (microfiltration, tangential flow filtration), centrifugation, thiab sib sib zog nqus filtration (ib txwm txaus filtration). Los ntawm kev paub ntev, cov tshuaj tiv thaiv sau qhia meej feem ntau yog ua tiav los ntawm centrifugation tom qab pom kev sib sib zog nqus.

 

Nyob rau hauv xyoo tas los no, cov txheej txheem membrane-raws li tau txais kev pom zoo hauv kev qhia meej txog tshuaj tiv thaiv. Cov txheej txheem sab saud tau nce siv cov thev naus laus zis pov tseg, yog li cov tswv yim sau qoob loo yuav tsum hloov pauv. Kab lus no muab cov ntsiab lus dav dav ntawm cov txheej txheem sib txawv raws li cov thev naus laus zis thiab lawv cov kev siv hauv kev qhia meej txog tshuaj tiv thaiv.

 

01 Taw Qhia

Cov tshuaj tiv thaiv yog ib feem tseem ceeb ntawm peb kev tiv thaiv kab mob sib kis, uas tseem ua rau muaj kev poob siab. Nyob nruab nrab ntawm 2 thiab 3 lab tus tib neeg tau txais kev cawmdim los ntawm kab mob diphtheria, tetanus, hnoos hawb pob thiab qhua pias txhua xyoo ua tsaug rau kev txhaj tshuaj. Cov tshuaj tiv thaiv npog ntau yam, los ntawm cov proteins me me mus rau tag nrho cov kab mob thiab tag nrho cov kab mob. Lawv tuaj yeem tsim tau los ntawm cov tshuab sib txawv: qe, cov qe qe, cov kab mob, thiab lwm yam. Vim yog qhov nyuaj thiab ntau hom tshuaj tiv thaiv, tam sim no tsis muaj cov txheej txheem purification los yog qauv, txawm tias muaj kev txaus siab rau cov tshuaj tiv thaiv platform.

 

Feem ntau, cov txheej txheem tshuaj tiv thaiv tuaj yeem muab faib ua peb ntu: sab sauv (kev tsim khoom thiab kev qhia meej), kev kho cov dej ntws (purification suav nrog ultrafiltration, chromatography, thiab tshuaj kho mob), thiab kev tsim kho (kev ua haujlwm zaum kawg). Kev ywj pheej ntawm cov txheej txheem tsim khoom, kev qhia meej (thawj zaug tshem tawm cov khoom tsis xav tau) ua lub luag haujlwm tseem ceeb hauv kev txiav txim siab txog kev ua kom huv huv. Cov kauj ruam qhia kom meej yog tsuas yog tshem tawm tag nrho cov hlwb, cov khib nyiab ntawm tes, colloids, thiab cov khoom sib xyaw loj kom txo tau lub nra ntawm cov dej ntws. Qee zaum, kev qhia meej kuj tseem tuaj yeem txo cov impurities insoluble, host cell proteins (HCP), thiab host cell nucleic acids. Zoo li lwm yam kev ua kom huv huv, cov kauj ruam qhia meej yuav tsum tau ua kom zoo kom ua tiav cov khoom lag luam siab tshaj plaws thiab kev coj dawb huv thaum hloov mus rau qhov tshwj xeeb thiab kev txwv ntau lawm ntawm cov tshuaj tiv thaiv.

 

Vim yog hom tshuaj tiv thaiv heterogeneity, ntau yam txuj ci yog siv los qhia kom meej, nrog rau centrifugation lossis filtration (Table 1).

 

Ntau qhov kev ua haujlwm feem ntau yog xav tau kom ua tiav qhov xav tau kom meej. Thawj qhov kev ua haujlwm yog tsim los tshem tawm cov khoom loj dua (piv txwv li kev qhia meej) thiab kev ua haujlwm thib ob yog tsim los tshem tawm cov colloids thiab lwm cov sub-micron hais (qib qhia meej). Tsawg-ceev centrifugation ua hauj lwm raws li ib qho kev qhia meej ib zaug, tso cai rau cov hlwb thiab cov khib nyiab ntawm tes tshem tawm los ntawm nag lossis daus. Centrifugation tuaj yeem tuav cov khoom ntim siab thiab tau siv dav hauv batch lossis nruam hom thiab disc centrifuges. Nws xav tau kev nqis peev siab thiab kev saib xyuas cov nqi, thiab ntsib cov teeb meem hauv kev nce qib vim tsis muaj tus qauv txhim khu kev qha-down. Txawm li cas los xij, qee cov tuam txhab tshuaj tiv thaiv kev lag luam siv cov centrifuges hauv kev tsim khoom loj uas muaj cov khoom lag luam siab thiab ntau cov haujlwm ntau lawm.

 

Kev pom meej meej tuaj yeem ua tau los ntawm kev lim dej ib txwm muaj (NFF, tseem hu ua dead-end filtration) lossis tangential flow filtration (TFF, tseem hu ua cross-flow filtration). Kuj tseem muaj cov qauv lim tshwj xeeb (cov ntxaij lim dej sib sib zog nqus) uas muaj cov khoom siv tau zoo thiab lim AIDS uas txhim khu kev tuav cov khib nyiab ntawm tes, colloids, thiab cov khoom tsis zoo uas tsis xav tau.

 

Cov ntxaij lim dej membrane khaws cov khoom los ntawm qhov loj me me thiab tsis muaj lub peev xwm khaws cov av siab, yog li lawv tsim nyog rau cov kauj ruam qhia ntxiv. Ob qhov sib sib zog nqus lim thiab cov lim membrane yog ib qho yooj yim rau scale thiab siv (kev tsim qauv yooj yim). Tsis zoo li NFF, TFF feem ntau yog siv los ua kom pom tseeb (microfiltration). Membranes nrog kev txiav tawm ntau ntawm 0.1-0.65 μm (zoo dua nrog cov channel qhib) tau siv los khaws cov hlwb, cov khib nyiab ntawm tes, thiab lwm yam kab mob loj. Feem ntau cov khoom siv TFF yog linearly scalable thiab reusable tom qab ntxuav, uas txo tus nqi ntawm consumables rau cov kauj ruam.

 

Cov kauj ruam qhia meej yog nyob nruab nrab ntawm cov txheej txheem dej ntws thiab cov dej ntws thiab qee zaum raug saib tsis pom thaum lub sijhawm tsim cov tshuaj tiv thaiv vim tias lub sijhawm thiab cov peev txheej yog qhov tseem ceeb rau lwm cov kauj ruam ua kom huv, xws li chromatography lossis qhov ntom ntom gradient centrifugation.

 

Txawm tias patents rau cov txheej txheem tsim tshuaj tiv thaiv feem ntau tshem tawm cov kauj ruam qhia meej. Lub clarifying efficiency ncaj qha cuam tshuam rau kev ua tau zoo ntawm cov txheej txheem downstream. Cov kauj ruam qhia meej tsis zoo tuaj yeem cuam tshuam tsis zoo rau lub peev xwm ntawm cov lim dej tsis huv lossis tuaj yeem ua rau lub neej ua haujlwm luv luv ntawm chromatographic resin. Niaj hnub no, kev tswj hwm nruj dua qhov kev cia siab yuav ua rau cov tuam txhab tshuaj tiv thaiv tsim cov tshuaj tiv thaiv zoo dua, muaj tus cwj pwm zoo tab sis pheej yig. Hauv qhov no, txhua kauj ruam yuav tsum tau muab cov xim nws tsim nyog. Cov qauv tam sim no qhia tias cov txheej txheem dej ntws mus rau qhov "ntxhiab" qhia qhov system (xws li cov kab mob hauv cov ntshav tsis pub dawb hloov cov qe) nrog cov khoom tsim tau ntau dua thiab ntau dua ntawm tes.

 

Cov txheej txheem purification hauv qab yog ua kom yooj yim thiab ua kom yooj yim kom ua kom huv huv ntawm cov khoom kawg. Txhawm rau tswj cov kev hloov pauv no, cov kauj ruam qhia meej tsis tuaj yeem dhau los ua ib qho tsis muaj zog. Nyob rau hauv cov ntaub ntawv no, filtration technology raws li ib tug tshiab clarification kev sib tw, hais txog cov teeb meem ntawm cov txheej txheem ntau zog, muaj peev xwm siv ib leeg, thiab txo nqi peev.

 

02 Qhia meej txog cov tshuaj tiv thaiv kab mob

Ntau hom kev qhia meej, tsis yog ib leeg lossis ua ke, tau siv los ua kom paub meej txog kev sau qoob loo ntawm kev noj zaub mov kawg ntawm cov tshuaj tiv thaiv.

Pilot scale:1-20L, ntau lawm scale: ntau tshaj 20L

 

2.1 Kev ceev faj txog cov tshuaj tiv thaiv kab mob kom meej meej

Ntau cov tshuaj tiv thaiv muaj tag nrho lossis ib feem ntawm cov kab mob los tsim kev tiv thaiv kab mob. Lawv feem ntau poob rau hauv plaub pawg dav:

Cov tshuaj tiv thaiv nyob attenuated (LAV), uas yog raws li tus kab mob tsis muaj zog ntawm tus kab mob los txo nws txoj kev ua phem. Attenuated viruses tuaj yeem rov ua dua hauv lub cev tab sis tsis yog pathogenic.

- Cov tshuaj tiv thaiv kab mob inactivated virus (IV), uas muaj cov tshuaj tiv thaiv kab mob los yog ultraviolet inactivated virus kom tshem tawm cov kab mob. Cov kab mob tuaj yeem ua tiav, faib, lossis ua kom huv (antigenic proteins nkaus xwb).

- Cov tshuaj tiv thaiv kab mob viral vector (VV) yog ib yam kab mob uas tsis muaj kab mob uas nthuav tawm cov tshuaj tiv thaiv kab mob ntawm tus kab mob pathogenic. Cov qauv tsim tsis ntev los no kuj tau siv rau kev siv tshuaj kho noob.

Cov tshuaj tiv thaiv kab mob zoo li cov kab mob (VLPs), ib chav kawm tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob sib kis uas ua rau tag nrho cov qauv ntawm cov kab mob sib kis tab sis tsis muaj cov kab mob sib kis.

 

Feem ntau, cov kab mob kis tau zoo tag nrho thaum lub sij hawm qhia meej, txawm tias thaum lub sij hawm lysis ntawm cov tshuaj tiv thaiv kab mob (cleavage feem ntau yog nqa tawm hauv qab dej, nyob rau hauv ib qho chaw huv dua).

Qhov kev sib tw tseem ceeb ntawm kev nthuav qhia tus kab mob yog qhov rov qab los ntawm cov kab mob kis tau zoo thaum tshem tawm cov khib nyiab ntawm tes, cov khoom sib xyaw loj, thiab cov khoom tsis huv. Raws li tau piav qhia nyob rau hauv cov tshooj hauv qab no, muaj ntau yam uas tuaj yeem ua rau tus kab mob no degradation lossis poob. Tsis tas li ntawd, cov kab mob kis tau tuaj yeem nyuaj rau kev vam khom vim qhov kev hloov pauv siab ntawm cov kab mob sib kis ntau ntawm cov txheej txheem no, tshwj xeeb tshaj yog rau cov tshuaj tiv thaiv LAV thiab VV. Rau cov tshuaj tiv thaiv no, cov kab mob kis tau feem ntau raug ntsuas los ntawm kev kuaj kab mob xws li cov quav hniav lossis TCID 50 kuaj. Qhov tseeb tias qee qhov sib xyaw ua ke tuaj yeem cuam tshuam nrog lub peev xwm ntawm tus kab mob kom kis tau tus kab mob qhia cov hlwb ua rau muaj qhov sib txawv ntawm qhov kev ua kom muaj nuj nqis no.

 

2.2 Cov tshuaj tiv thaiv kab mob kom meej meej

Cov tshuaj tiv thaiv kab mob sib txawv heev hauv qhov loj, qauv, cov duab, thiab cov kab lus qhia (Table 3).

Yog li ntawd, feem ntau tsis muaj qauv rau nws cov txheej txheem qis qis, tshwj xeeb tshaj yog cov kauj ruam qhia meej. Nyob rau hauv txoj kev xav, tag nrho cov txheej txheem uas muaj (tsawg-ceev centrifugation, microfiltration TFF, NFF) tuaj yeem raug xaiv thiab muaj peev xwm ua ke kom paub meej tus kab mob. Qhov tseeb, kev ua tiav ntawm txoj kev qhia meej yog cuam tshuam los ntawm cov kab lus qhia thiab cov khoom siv lub cev ntawm cov kab mob cuam tshuam.

 

Recently, the high cell density process of viral vaccines is being explored. High cell density processes add to the challenge of clarification. Many treatment methods are by preconditioning, i.e. polymer-induced flocculation, precipitation, alternating TFF, etc. For example, Tomic et al. describe a method for high cell density collection (>{{0}} hlwb /ml) txoj kev qhia meej, siv cationic polymers, txo qhov tob pom qhov tob los ntawm 4 npaug piv rau cov txheej txheem ib txwm muaj. Cov txheej txheem no tso cai rau kev sau qoob loo ntawm lub peev xwm loj fermenters yam tsis tas siv cov centrifuges. Ib yam li ntawd, Riske et al. qhia tau hais tias kev kho chitosan ntawm 40 L cell kab lis kev cai (0.02%) muaj 1.4-2.6% cov khib nyiab coj mus rau 7-fold nce hauv lub peev xwm lim tom qab sib sib zog nqus pom.

 

Lawv kuj hais tias chitosan zoo li txhim kho kev ua kom pom tseeb los ntawm flocculating submicron hais, uas feem ntau nyob hauv centrifuges. Pretreatment thiab flocculation txoj kev zoo li yuav txuas ntxiv yog ib feem ntawm kev pom tseeb ntawm cov tshuaj tiv thaiv yav tom ntej. Penetrants, nrog rau cov suab thaj, glycols, thiab amino acids, nyiam flocculate viruses. Viral flocculation nrog osmotic kua ua raws li los ntawm 0.2µ microfiltration yuav siv tau raws li ib tug txheej txheem rau purification tus kab mob. Penetrants tuaj yeem txhawb nqa cov kab mob sib sau los ntawm kev txo cov hydration txheej nyob ib puag ncig cov khoom kom flocculate hydrophobic non-encapsulated virus particles thiab encapsulated virus hais. Txawm hais tias penetrants tau pom tias muaj kab mob flocculate, txoj kev muaj peev xwm los ua lub platform rau kev lim dej rau yav tom ntej, thiab nws txoj kev ua tau zoo hauv kev tsav tsheb lossis kev siv tshuaj tiv thaiv loj loj tsis tau muaj pov thawj. Txoj kev flocculation pretreatment, uas zoo li yuav txuas ntxiv yog ib feem ntawm cov tshuaj tiv thaiv kab mob yav tom ntej, yog ua nyob rau hauv 2L fermenter. Txhawm rau kom muaj peev xwm siv tau hauv kev tsim khoom loj, cov txheej txheem no yuav tsum tau ua kom raug cai ntawm kev sim thiab cov nplais ntau lawm.

 

2.2.1 Qhia qhov cuam tshuam qhov system

Txoj kev qhia meej feem ntau yog nyob ntawm cov txheej txheem ntawm cov dej ntws thiab cov txheej txheem qhia, uas txiav txim siab hom thiab theem ntawm cov kab mob kom tshem tawm. Cov tshuaj tiv thaiv kab mob feem ntau yog tsim nyob rau hauv qaib embryos los ntawm cov kab mob txuas ntxiv ntawm cov tsiaj txhu los yog noog lossis cov kab mob baculovirus / kab kab, uas yog ib qho kev qhia ntau dua. Qee hom VLPs kuj tseem tuaj yeem tsim los ntawm lwm cov kab mob heterologous (cov kab mob, cov poov xab, cov hlwb cog).

 

2.2.1.1 Tus kab mob tsim tawm hauv qaib embryo

Cov tshuaj tiv thaiv tau tsim nyob rau hauv cov qe rau ntau xyoo lawm. Qhov kev ua haujlwm no hnub rov qab mus rau xyoo 1931, thaum Woodruff thiab Pasture zoo tau siv cov chorio-allantoic membrane ntawm cov qe fertile ua substrate rau kev loj hlob ntawm tus kab mob. Niaj hnub no, ntau cov tshuaj tiv thaiv tib neeg thiab tsiaj txhu tseem tsim los siv cov txheej txheem qub no. Qhov paub zoo tshaj plaws yog qhov tshuaj tiv thaiv kab mob khaub thuas raws caij nyoog. Lub hauv paus ntsiab lus yog kom inoculate qaib qe nrog cov kab mob ntawm kev txaus siab thiab tom qab ntawd rov ua dua hauv chorio-allantoic membrane. Tom qab yug me nyuam, allantoic kua uas nplua nuj nyob rau hauv cov kab mob kis tau sau thiab purified.

 

Allantoic kua yog ib qho nyuaj clarifying pub. Nws cov ntxhia pob zeb thiab cov protein ntau (xws li ovalbumin) ua rau nws muaj viscosity siab. Allantoic kua kuj muaj cov ntaub so ntswg tseem ceeb ntawm cov nqaij qaib embryos, xws li plaub, beaks, cov hlab ntsha, lossis cov qe ntshav. Vim muaj cov ntsiab lus siab siab, qis-ceev centrifugation yog qhov kev xaiv zoo tshaj plaws rau kev qhia meej, feem ntau ua rau tus nqi rov qab li ntawm 70%. Txawm li cas los xij, thawj qhov kev qhia meej uas siv cov tswv yim pom zoo kuj ua tau. Rau NFF, polypropylene thiab cellulose-raws li cov ntxaij lim dej sib sib zog nqus muaj peev xwm sau cov kua dej allantoic zoo. Cov ntxaij lim dej saum npoo ua los ntawm polypropylene lossis cellulose-raws li cov ntxaij lim dej sib sib zog nqus tuaj yeem muaj peev xwm ntawm 150-210 L / m² thiab txo cov khoom noj turbidity txog li 3 zaug. Qhov qhib pub channel TFF cov cuab yeej tseem tsim nyog rau kev qhia cov kua dej allantoic, vim tias cov cuab yeej tsim nyog rau qhov tsawg kawg nkaus poob los ntawm cov khoom noj channel, yog li txo cov channel thaiv.

 

Cov kauj ruam qhia meej thib ob tuaj yeem ua tau yooj yim nrog NFF. Kev sib xyaw ua ke ntawm polypropylene, cellulose thiab iav fiber ntau cov ntaub ntawv feem ntau qhia tau hais tias muaj txiaj ntsig zoo, thiab lwm txoj hauv kev qhia meej thib ob yog siv TFF thiab ib qho {{0}}}.65μm lossis 0.45μm microfiltration membrane ntaus ntawv thiab ua haujlwm osmotic flux tswj. Ib qho qhib channel TFF cov cuab yeej nrog hydrophilic PVDF lossis hydrophilic PES membrane tuaj yeem siv rau hauv cov kauj ruam no.

Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias cov kab mob kis tau tuaj yeem cuam tshuam nrog cov khoom khib nyiab insoluble, uas tuaj yeem txo cov kab mob loj zuj zus thaum lub sij hawm qhia meej. Kev siv cov tshuaj ntsev tuaj yeem txo qhov kev sib koom ua ke ntawm cov kab mob khaub thuas thiab cov khoom tawg, ua rau muaj kev tsim tawm kwv yees li ob zaug yam tsis muaj kev cuam tshuam rau kev ncaj ncees ntawm cov kab mob.

 

2.2.1.2 Cov kab mob uas tsim nyob rau hauv cov kab mob mammalian thiab avian cell kab

Tsis ntev los no, qee cov tshuaj tiv thaiv kab mob tau txav mus deb ntawm cov txheej txheem qe-raws li kev pom zoo ntawm cov kab lis kev cai ntawm tes (tshwj xeeb tshaj yog rau cov tshuaj tiv thaiv kab mob khaub thuas). Qhov laj thawj tseem ceeb ntawm qhov kev hloov no yog los tiv thaiv cov teeb meem kev tsim tshuaj tiv thaiv uas cuam tshuam nrog embryonic qe (piv txwv li qhov tsis txaus ntawm cov qe uas yuav tshwm sim thaum muaj kab mob nqaij qaib). Raws li qhov tshwm sim, ntau hom tshuaj tiv thaiv thiab cov kab mob kis tau tam sim no tau tsim los siv cov kab txuas txuas ntxiv los ntawm cov tsiaj nyeg lossis noog, cov kab ntawm tes (xws li Vero, MDCK, lossis HEK293 cell kab), los yog cov kab ntawm tes.

 

Nyob ntawm cov kab lus qhia, tus kab mob tuaj yeem nyob hauv lub cell, xav tau cov kauj ruam ntawm tes, lossis nws yuav nyob sab nraud ntawm tes (lysis lossis budding). Cov khoom thauj khoom thiab cov ntsiab lus soluble tau los ntawm cov kab lis kev cai ntawm tes yog huv dua li cov kua allantoic theem. Raws li qhov tshwm sim, NFF thev naus laus zis tau yooj yim dua rau kev siv thiab lub peev xwm ntau dua. Txawm li cas los xij, lub peev xwm pom tau zoo nyob ntawm cov kab lis kev cai ntawm tes, xws li cell ceev lossis cell viability thaum sau. Cov kev txwv no cuam tshuam rau cov khib nyiab ntawm tes thiab macroaggregates uas tuaj yeem txhaws cov lim dej sib sib zog nqus thiab daim nyias nyias, ua rau muaj peev xwm txo qis.

 

Thomassen thiab al muab ib qho piv txwv zoo ntawm NFF qhia meej. Siv nyob rau hauv inactivated poliovirus (IPV) txheej txheem ntau lawm. Vero cells kab lis kev cai ntawm microcarriers tau siv rau kev loj hlob ntawm cov kab mob nrog lub cell ceev ntawm {{{0}}.78 x 106 cells /ml TOI. Lub vijtsam 75μm stainless hlau yog siv rau kev qhia ua ntej kom tshem tawm cov microcarriers los ntawm kev sau qoob loo. Qhov kev muab qhab nia ob txheej yog qhia meej siv lub lim dej sib sib zog nqus (0.2-2.0μm), ua raws li cov lim dej kom tsis muaj menyuam.

Cov chav tsev uas tau xaiv scalable pov tseg tau ua tiav siv rau kev npaj ntawm Sabin IPV cov khoom siv kho mob ntawm 350 L nplai. Nyob ntawm tus kab mob serotype, tus kab mob rov qab los ntawm 86 mus txog 96 feem pua ​​yog tau.

 

2.2.1.3 Baculovirus/virus-like particles uas tsim nyob rau hauv cov kab cell system

Kev txiav txim siab ntawm cov kab mob baculovirus / kab kab mob rau cov tshuaj tiv thaiv loj loj yog qhov tshiab, tab sis yog qhov txaus siab rau kev nthuav dav ntawm cov kab mob kab mob thiab VLPs. Qhov txiaj ntsig tseem ceeb ntawm cov kab ke no yog tias nws cuam tshuam nrog lub neej luv (tsis tas yuav tsim cov kab ntawm tes) thiab muaj kev nyab xeeb (tsis muaj kab mob DNA ua tiav). Kuj tseem muaj qee qhov tsis zoo, xws li kev xav tau kev tshem tawm baculovirus thiab cov khoom ruaj khov.

 

Cervarix, tshuaj tiv thaiv VLP tiv thaiv tib neeg papillomavirus kab mob uas tsim los ntawm GlaxoSmithKline, yog thawj tib neeg cov tshuaj tiv thaiv uas tau ua lag luam hauv cov kab kab. Ntau cov tshuaj tiv thaiv raws li VLPs thiab rAAV vectors tsim nyob rau hauv cov kab mob kab mob baculovirus tam sim no tab tom txhim kho. Kab cell kab muab los ntawm lub caij nplooj zeeg Armyworm Spodoptera frugiperda (Sf9 thiab Sf21) thiab cabbage bendworm Trichoplusia ni (BTI-TN5B1-4 hlwb) yog cov feem ntau siv vim lawv muaj peev xwm loj hlob nyob rau hauv ncua kev kawm ntawv, uas ua kom yooj yim amplification ntawm cov dej ntws. txheej txheem. Tom qab loj hlob mus rau qhov xav tau ceev ntawm cov cell nyob, cov hlwb no kis tau tus kab mob baculovirus nyob rau hauv lub sij hawm exponential cell loj hlob rau cov protein qhia. Qhov loj genome ntawm baculoviruses tso cai rau kev nthuav qhia ntawm tsib los yog ntau tshaj cov proteins sib txawv, uas phim cov complexity ntawm VLP thiab kab mob vectors.

 

Bernard et al. piav qhia txog cov txheej txheem hauv qab ntawm kab kab lis kev cai. Cov txheej txheem yog qhov sib txawv heev, qhia txog kev sib txawv ntawm cov proteins uas tsim nrog cov txheej txheem no. Thawj kauj ruam ntawm kev ua kom huv si yog hnyav heev los ntawm cov yam ntxwv ntim ntawm bioreactor (piv txwv li, cell density thiab viability), lossis los ntawm qhov xwm txheej ntawm cov khoom tso tawm (secreted los ntawm budding lossis cell lysis). Kab mob kab mob Baculovirus tuaj yeem ua rau cell lysis hauv 3-5 hnub. Kev puas tsuaj ntawm cov hlwb tuaj yeem ua rau muaj kev nce ntxiv hauv kev ua haujlwm proteolytic thiab lwm yam hauv ib puag ncig uas tuaj yeem ua rau degradation ntawm recombinant proteins.

Muaj kev sim los tsim cov kab mob baculoviruses nrog lub peev xwm qis dua los pib lub cell lysis. Tus kab mob baculovirus lyses tsawg dua 10 feem pua ​​​​ntawm cov kab mob.

 

Cell lysis tuaj yeem ua tau siv ntau txoj kev, xws li khov-thaw, detergents, homogenizers, lossis kev kho mob ultrasound. Kab kab tsis muaj cov phab ntsa ntawm tes thiab yog li yaj sai sai. Txawm hais tias kev kho mob ultrasonic tau raug tshaj tawm nyob rau hauv ntau lub rooj ntev zaum, nws tsis tshua siv los ntawm kev sim lossis kev lag luam. Txoj kev siv ntau tshaj plaws yog siv homogenizer los rhuav tshem cov hlwb nyob rau hauv lub xub ntiag ntawm cov tshuaj detergent tsawg (0.1% Triton X-100 lossis NP-40). Kev siv cov tshuaj detergents thiab microfluidization lossis osmotic cuam tshuam homogenization tau ua tiav hauv ntau qhov kev tsim khoom loj. Feem ntau, cov kab mob raug tshem tawm hauv lysis buffers (50 mM TRIS pH7.7, 300 mM NaCl, 5% glycerol, 0.2 mM PMSF, thiab protease inhibitors), thaum lub sijhawm Triton-X100 yog ntxiv rau qhov kawg concentration ntawm 0.1%, ua raws li me me ultrasound los yog microfluidization. Tom qab ntawd cov sib tov yog centrifuged kom tshem tawm insoluble hais.

 

Nyob rau theem no, cov lysate yuav tshwm sim huab heev thiab nyuaj rau lim siv lub lim dej 0.45μm. Qee zaum kev poob txog li 30% tuaj yeem pom thaum lub sij hawm piav qhia pyrolysis. Qhov sib ntxiv ntawm benzoenzymes ntawm theem cleavage pab daws qhov teeb meem pom. Kev ntxuav cov hlwb nrog phosphate buffered brine tom qab sau thiab ceev "freeze-thaw" nyob rau hauv cov ntsev siab (500mM NaCl) uas muaj cov cracking tsis pab tshem tawm cov aggregates.

 

Kev nthuav qhia ntawm VLPs thiab cov kab mob kis kab mob uas tsim los ntawm cov kab mob tshwm sim tom qab cell lysis (los ntawm kev kho tshuaj lom neeg lossis kev kho tshuab), uas tso tawm tsis yog cov kab mob kis nkaus xwb tab sis kuj muaj cov cellular nucleic acid ntau. Kab kab muaj peev xwm loj hlob ntawm lub cell ceev, los ntawm 1-9 10 6 cells/ml. Yog li ntawd, cov kauj ruam qhia meej yuav tsum daws nrog cov cell ceev, siab nucleic acid cov ntsiab lus thiab, yog tias ua tau, tshem tawm cov kab mob baculovirus. Txhawm rau ua qhov teeb meem nyuaj, VLPs lossis kab mob kab mob thiab baculoviruses tuaj yeem muaj qhov sib xws (baculoviruses yog 60-80 nanometers dav thiab 300-400 nanometers ntev). Vim yog lub cell ceev ceev, centrifugation yog cov txheej txheem nyiam rau kev qhia meej rau ntau xyoo lawm. Txawm li cas los xij, cov txheej txheem membrane zoo li yog lwm txoj hauv kev zoo nkauj heev vim tias scalability yooj yim los txhais.

 

Cov lim dej sib sib zog nqus tau siv tau zoo hauv cov txheej txheem qis ntawm peb txheej rotavirus zoo li hais. Hauv chav kuaj, CsCl density gradient ultracentrifugation method feem ntau yog siv los ntxuav cov khoom nyuaj no. Cov kws tshawb fawb tau soj ntsuam tsis yog tsuas yog cov kauj ruam qhia meej ntawm cov lim dej sib sib zog nqus, tab sis kuj tseem ua rau tag nrho cov txheej txheem qis qis (Triton X-100 cleavage thiab tob pom, ua raws li ultrafiltration thiab particle size exclusion). Cov txiaj ntsig tau pom tias cov txiaj ntsig ntawm CsCl density gradient tuaj yeem ncav cuag 37%.

Txoj kev ultra-centrifugal yog li 10%. Raws li lwm qhov piv txwv, 0.45μm hollow fibers thiab 500 kDa zoo txoj kab uas hla fibers tau siv los kho kom zoo thiab mloog cov kab mob HIV zoo li cov kab mob uas ua rau cov kab mob. Nyob rau hauv txoj kev tshawb no, lub zog shear ntawm hollow fibers tau optimized raws li kev ncaj ncees ntawm tes. Cov txiaj ntsig Cov txheej txheem qis qis raug tsim los hloov cov lus qab zib gradient ultracentrifugation.

 

Cov txheej txheem muaj peev xwm siv tau rau ntau lawm. Cov ntxaij lim dej sib sib zog nqus kuj tau siv tau zoo hauv kev tsim cov kab mob sib txuas nrog adeno. Cell lysis tau ua nrog ob-piston mechanical cell jammer, ua raws li kev kho nuclease. Hauv cov kauj ruam qhia meej, 1.2μm iav fiber ntau lim dej tau siv, ua raws li ob txheej ntawm 0.8μm hydrophilic PES thiab 0.2μm hydrophilic PES. Cov ntxaij lim dej loj npaum li cas yog siv rau cov txheej txheem batch los ntawm me me mus rau 200 L qhov ntau thiab tsawg. Cov ntxaij lim dej sib sib zog nqus tau siv tau zoo, thiab TFF kev ntsuas nrog cov yeeb yaj kiab tiaj tus lossis hollow fibers ntawm 0.2µm lossis 0.45µm kuj tau tshaj tawm tias ua tau zoo heev.

 

Txoj kev tshawb fawb los ntawm Tecnologica (IBET) ntawm Oeiras Lub Tsev Kawm Txuj Ci Kev Tshawb Fawb hauv Portugal tau siv NFF los ua kom paub meej txog kab mob siab C VLP uas qhia los ntawm baculoviruses yam tsis muaj centrifugation. Nominally rated polypropylene lim (10,5,0.6 thiab 0.3μm) lim yog siv los qhia meej VLP sau qoob. Tib cov lim dej nrog 0.6μm thiab 0.3μm pore ratings yog siv rau kev pom hauv VLP cov chaw sau qoob loo. Tag nrho cov filtrates tau kawm tau sim rau HCV-VLP cov nqi rov qab thiab cov kev pom zoo pom tau muab piv rau. Cov txiaj ntsig tau pom nyob rau hauv Table 4.

Cov txiaj ntsig tau pom tias 5μm-0.3μm lim tau muaj cov khoom lag luam zoo tshaj plaws (100%) rau cov qoob loo ncaj qha rau kab mob siab C VLP pub. Cov lim dej polypropylene 0.6μm muaj cov khoom lag luam zoo tshaj plaws (82%) rau cov khoom noj hauv nruab nrab, thiab DNA tshem tawm ntawm tus tswv tsev cell yog li 70%.

 

2.2.1.4 Cov kab mob zoo li cov kab mob ua rau cov kab mob los yog cov kab mob poov xab

Hom kev qhia meej meej rau cov tshuaj tiv thaiv VLP uas tau hais tawm hauv cov kab mob lossis cov kab mob poov xab nyob ntawm qhov tso tawm ntawm VLP rau cov neeg nruab nrab sab nraud. Yog tias VLPs tsis tuaj yeem zais tsis tau zoo, cell lysis lossis lwm yam kev rho tawm yuav tsum tau ua ua ntej cov kauj ruam qhia tseeb. Thaum qhov no yog tus qauv kub hauv kev lag luam protein kom meej, centrifugation (nruam los yog batch) qhia nyob rau hauv cov kab mob los yog cov kab mob poov xab, tsis ntev los no, cov txheej txheem membrane tau dhau los ua lwm txoj hauv kev zoo nkauj vim lawv yooj yim ntawm scalability thiab compatibility nrog ib leeg-siv. kev kho mob.

 

Richter thiab Topell piav qhia txog kev siv centrifugation, TFF, los yog kev sib xyaw ua ke ntawm ob qho tib si thaum npaj VLPs uas ua rau pom tseeb E. coli. Hauv lawv txoj haujlwm, 0.45m TFF daim nyias nyias tau siv los dilute thiab qhia meej txog homogenized E. coli homogenates ntawm qhov kub ntawm 5 degree . Lawv kuj tseem nco ntsoov tias daim nyias nyias raws li TFF yog qhov tsim nyog rau kev tuav cov viscosity siab, nyiam siv TFF units nrog qhib channel configurations.

Centrifugal clarification kuj tau raug soj ntsuam raws li lwm txoj hauv kev rau TFF. Hauv qhov no, qhov tshwm sim homogenate tsis yog diluted thiab centrifuged ntawm 4 degree rau 105 feeb, 10000g. Yog tsis hloov cov txheej mos, cov supernatant tau nchuav tawm ntawm cov khoom thiab rov centrifuged ntawm 4 degree thiab 10,000 g rau 60 min. Tom qab ntawd lub supernatant raug tshem tawm ntawm cov khoom tam sim no, diluted nrog EB tsis (43.89 mM Tris HCl, 6.11 mM Tris Base, 5.0 mM EDTA, 10% (v/v) Triton X-100) 1: 2, thiab lim. ntawm 0.22 m sterile lim ntaus ntawv. Kev ua haujlwm ntxiv. Cov txheej txheem scale-up rau kev tsim cov tshuaj tiv thaiv VLP no hauv E. coli ntawm 800L nplai kuj tau qhia meej los ntawm kev sib xyaw ntawm centrifugation thiab TFF.

 

Cov tshuaj tiv thaiv kab mob siab hepatitis E HEV 239 tau raug tso cai hauv Suav teb rau kev txhaj tshuaj rau cov neeg laus hnub nyoog 16 xyoo thiab laus dua. Cov tshuaj tiv thaiv antigens (VLP) tau qhia nyob rau hauv E. coli, thiab scale-up ntawm cov txheej txheem antigen ntau lawm tau pom tias nyob rau ntawm 50L nplai. Cov khoom tau muab rho tawm los ntawm kev tawg E. coli thiab lub cev suav nrog tau sib cais los ntawm cov khoom tawg ntawm tes los ntawm kev ntxuav hnyav nrog cov tsis muaj 2% Triton X-100 thiab tom qab ntawd yaj nrog 4 M urea homogenizer. TFF qhia meej txog tib neeg papillomavirus VLP uas tsim tawm hauv Saccharomyces cerevisiae (15L). Lub cell lysate kho nrog nuclease tau qhia meej los ntawm crossflow microfiltration hauv transfiltration hom siv 0.65 μm hollow fiber lim. Tib txheej txheem yog siv hauv kev tsim tshuaj tiv thaiv. Txawm hais tias tsuas yog ib qho puv tes ntawm cov tshuaj tiv thaiv VLP tau mus txog kev lag luam, ntau qhov tshuaj tiv thaiv VLP tau tsim kho, feem ntau tau qhia meej siv centrifugation lossis membrane technology.

 

Kev txwv los ntawm kev cuam tshuam ntawm qhov chaw, peb yuav qhia ib nrab ntawm cov ntsiab lus hauv tshooj tom ntej. Hauv tsab xov xwm tom ntej no, peb yuav qhia qee qhov xwm txheej ntawm kev qhia meej txog tshuaj tiv thaiv thiab kev siv cov txheej txheem membrane.

 

Raws li thawj lub tuam txhab hauv cheeb tsam hauv cheeb tsam, Guidling Technology tau sau cov kev paub txaus txaus hauv kev qhia meej txog tshuaj tiv thaiv. Guidling Technology yog ib lub tuam txhab kev loj hlob thiab kev tsim khoom tsom mus rau biopharmaceutical thiab cell kab lis kev cai, purification thiab kev sib cais. Cov khoom siv dav siv hauv kev kho mob biomedicine, kuaj mob, kev lim dej hauv kev lag luam, kev tshawb nrhiav, kev qhia meej, kev ua kom huv thiab cov txheej txheem concentration; Guidling tau ntse tsim ultrafiltration centrifuge raj, ultrafiltration / microfiltration membrane cassette, lim tshem tawm tus kab mob, tangential txaus lim ntaus ntawv, sib sib zog nqus membrane pawg, thiab lwm yam., uas muaj tag nrho raws li daim ntawv thov scenarios ntawm biopharmaceutical thiab cell kab lis kev cai.

 

Peb cov membranes thiab cov lim lim lim tau dav siv hauv kev sib xyaw, kev rho tawm thiab kev sib cais ua ntej pom, microfiltration, ultrafiltration thiab nanofiltration. Peb cov kab ntau ntawm cov khoom siv, los ntawm cov khoom siv me me ib leeg siv rau kev siv cov tshuab lim ntau lawm, kev kuaj tsis muaj menyuam, fermentation, kab lis kev cai ntawm tes thiab lwm yam, tuaj yeem ua tau raws li qhov xav tau ntawm kev sim thiab ntau lawm.