Kawm Txog Tus Kab Mob Tshem Tawm Muaj Peev Xwm Ntawm Monoclonal Antibody Downstream txheej txheem
Cov khoom siv biotechnology muab tau los ntawm cov kab ntawm tes yuav muaj kev pheej hmoo ntawm cov kab mob sib kis - qhov twg yog nyob ntawm ntau yam, suav nrog qhov chaw ntawm kev npaj, cov khoom siv raw siv, cov txheej txheem tsim khoom, purification reagents thiab excipients. Kev kawm txog kev tshem tawm lossis tsis ua haujlwm ntawm cov kab mob hauv cov txheej txheem tsim khoom yog ib kauj ruam tseem ceeb hauv kev txo cov peev txheej ntawm iatrogenic kis ntawm cov kab mob pathogenic thiab yog li txo qhov kev pheej hmoo, uas yog qhov tseem ceeb rau kev nyab xeeb ntawm cov khoom. Ua ntej cov khoom siv roj ntsha tuaj yeem nkag mus rau kev sim tshuaj thiab ua lag luam, lawv cov txheej txheem tsim khoom yuav tsum tau ua kom pom tias muaj peev xwm tshem tawm cov kab mob uas paub thiab xav tau.
01 Monoclonal antibody khoom kuaj kab mob kev nyab xeeb
Kev tshawb fawb txog kev tshem tawm tus kab mob muaj peev xwm feem ntau yog los ntawm kev ntxiv cov kab mob titer paub txog rau cov khoom nruab nrab ntawm cov theem ntau lawm, tom qab ntawd txiav txim siab tus kab mob titer seem hauv cov qauv kho los ntawm cov txheej txheem tshwj xeeb, thiab thaum kawg ntsuas tus nqi txo qis (LRV) ntawm tus kab mob titer. Cov kauj ruam rau LRV Ntau dua lossis sib npaug rau 4log10 feem ntau suav tias yog cov kauj ruam tshem tawm cov kab mob zoo. Cov txheej txheem nrog LRV<41og10 can be considered as helpful to increase the total virus clearance in the production process. However, due to the limitations of virus verification, the process with LRV≤11og10 has little significance for virus removal, and its LRV value is not included in the total amount of the production process.
Thaum siv cov txheej txheem simulated ntau lawm (txo cov txheej txheem), nws yog ib qho tsim nyog los tsim cov kab mob tsim nyog tshem tawm / inactivation kev tshawb fawb thiab kev sim cov phiaj xwm cuam tshuam txog cov txheej txheem tsim khoom tiag tiag kom deb li deb tau, tshwj xeeb tshaj yog cov txheej txheem tsim khoom tau zoo. Cov txheej txheem simulated yuav tsum nruj me ntsis raws li cov txheej txheem tiag tiag hauv cov ntsiab lus ntawm cov ntsuas ntsuas thiab kev tswj xyuas.
02 Feem ntau qhia cov kab mob thiab cov txheej txheem pov thawj
Cov kab mob qhia tau pom muaj nyob hauv Table 1.
Ntawm qhov kev tshawb nrhiav tshuaj tshiab (IND) theem, cov khoom lag luam biotech xav tau kev tshawb fawb txog kev kis kab mob tsawg kawg 2 tus qauv kab mob, feem ntau xaiv ib tus kab mob retrovirus (X-MuLV) thiab ib qho parvovirus (MMV). Thaum lub sijhawm thov daim ntawv tso cai biologics (BLA), cov kev tshawb fawb tshem tawm tus kab mob feem ntau yog ua los ntawm kev siv 3-5 cov kab mob tshwj xeeb / tsis tshwj xeeb thiab 3-5 cov txheej txheem cov txheej txheem raug ntsuas los ua pov thawj tias cov khoom muaj kev nyab xeeb txaus los ntawm ib tus kab mob kev nyab xeeb foundations. Vim muaj cov txheej txheem sib txawv hauv tsev thiab txawv teb chaws, muaj qee qhov sib txawv ntawm cov txheej txheem kuaj xyuas tus kab mob uas siv rau kev tshaj tawm. Feem ntau siv cov kab mob tshem tawm pov thawj cov txheej txheem hauv mAb cov txheej txheem tsim tawm tau qhia hauv Table 2.
03 Tus kab mob tshem tawm muaj peev xwm ntawm cov txheej txheem qis
3.1 Lub peev xwm ntawm cov txheej txheem hauv qab los tshem tawm cov kab mob sib txawv
Raws li xyoo 2009, cov khoom lag luam monoclonal antibody xa mus rau FDA rau IND thiab BLA, Protein A affinity chromatography, Low pH, anion exchange chromatography AEX (flow penetration mode), cation exchange chromatography CEX (binding - elution mode), and virus clearance validation excluding. virus filtration yog cov feem ntau siv qhov taw qhia cov tsev neeg muaj kab mob thiab qhov nruab nrab thiab ntau yam ntawm lawv cov txiaj ntsig tshem tawm tau pom hauv daim duab 1.
Rau retroviruses, tshwj tsis yog CEX, uas muaj LRV txog 3log10 (raws li qhia hauv A-bar daim duab hauv daim duab 1), lwm cov txheej txheem tau muaj zog (LRV Ntau dua lossis sib npaug rau 4log10). Herpesviruses thiab retroviruses muaj qhov cuam tshuam zoo sib xws thiab LRV> 4log10 (raws li qhia hauv B-bar chart hauv daim duab 1). Cov kab mob me me, xws li parvovirus, yog qhov nyuaj rau tshem tawm tau zoo, tiv taus tshuaj kho mob, thiab tsis yooj yim los ntawm cov lim dej (pom hauv C-bar daim duab hauv daim duab 1). Tsuas yog AEX thiab cov lim dej me me tau siv tau los tshem tawm parvoviridae (qhov nruab nrab LRV> 4log10).
Cov txheej txheem qhia hauv daim duab 1 yog ProteinA, AEX (flow penetration mode), CEX (concomitation-elution mode), Low pH inactivation ("tsis tiav" piv" ua tiav "tshem tawm), thiab loj thiab me aperture devirus pom.
3.2 Muaj feem cuam tshuam rau cov txheej txheem hauv qab ntawm cov peev txheej tshem tawm tus kab mob
Kev faib tawm ntawm LRV qhov tseem ceeb rau txhua tus txheej txheem yog pom hauv daim duab 2. Cov kev tshawb fawb nrog 0 ~ 21og10 tau muab faib ua pawg LRV qis, thiab cov uas muaj ntau tshaj 6log10 tau muab faib ua pawg LRV siab rau kev sib piv.
3.2.1 ProteinA affinity chromatography
Tsis muaj kev sib raug zoo ntawm LRV tus nqi ntawm Protein A txheej txheem thiab qhov sib npaug / ntxuav tsis pub, muab tub lim, eluent muaj pes tsawg leeg thiab elution pH tus nqi. Qhov feem ntau tus lej ntawm pawg LRV qis yog tias cov khoom noj khoom haus yog qhov nyuaj heev, thiab nws yuav yog qhov kev sib cuam tshuam ntawm cov khoom noj khoom haus thiab cov muab tub lim tsis zoo cuam tshuam rau lub peev xwm ntawm chromatographic kem kom tshem tawm cov kab mob.
3.2.2 AEX (Flow Through Mode)
Strauss et al. pom tias pH thiab conductivity ntawm AEX cov qauv tuaj yeem cuam tshuam rau LRV. Txawm li cas los xij, cov txiaj ntsig ntawm Miesegaes thiab al qhia tau hais tias, zoo ib yam li ProteinA, LRV ntawm AEX (flow nkag mus) tsis muaj kev sib raug zoo nrog cov sib txawv buffers, fillers, pH thiab conductivity, uas tej zaum yuav yog vim lub optimization ntawm pH thiab conductivity. nyob rau hauv cov ntaub ntawv txheej txheem txheej txheem.
3.2.3 CEX (Kev khi - Elution hom)
Kev tshem tawm kab mob los ntawm cov txheej txheem CEX tsis muaj zog txaus. Tsis muaj qhov sib cuam tshuam tseem ceeb ntawm LRV hauv pawg LRV siab thiab qhov tsis sib xws, qib kev paub, lossis ib qho khoom siv chromatographic kem, xws li kem qhov siab thiab hom resin. Txawm li cas los xij, qhov sib npaug / thauj khoom thiab elution pH qhov tseem ceeb siv hauv kev tshawb fawb LRV siab yog ob qho tib si qis dua, ntawm 5.3 ±0.2; Hauv pawg LRV qis, pH yog 6.0±0.2. Lwm qhov tseem ceeb yog tias ntsev concentration hauv qhov tsis tuaj yeem ua rau LRV tus nqi sib txawv. Qhov tsis siv nyob rau hauv txoj kev tshawb fawb LRV qis muaj cov ntsev ntau dua, nrog qhov nruab nrab ntawm 290 ± 120 mM hauv kev thauj khoom / ntsuas kev daws teeb meem thiab 340 ± 70 mM hauv eluent, thaum cov concentrations ntawm NaCl hauv kev tshawb fawb LRV siab yog 58. ± 27 mM thiab 183 ± 31 mM feem.
3.2.4 Tsawg pH inactivation
Hauv cov txheej txheem pH qis, pH yog cov txheej txheem tseem ceeb, thiab pH ntawm 3.8 yog txaus los ua kom tsis txhob muaj tus kab mob retrovirus los ntawm qib. Cov pab pawg LRV qis muaj pH ntawm 3.9, thaum txoj kev tshawb fawb LRV siab muaj pH ntawm 3.4.
3.2.5 Tshem tawm cov kab mob lim
Rau ob qho tib si me thiab loj aperture lim, LRV tus nqi ntawm MuLV tshem tawm tsis muaj tsawg dua 2log10, thiab tsuas yog 1% ntawm cov kev tshawb fawb tau qis dua 3log10 (pom hauv kab h thiab kuv hauv daim duab 2). Tag nrho kev tshem tawm ntawm cov kab mob retroviruses los ntawm cov kab mob loj aperture lim tau qis dua li qhov me me aperture virus lim (raws li qhia hauv C-bar daim duab hauv daim duab 1). Qhov laj thawj tseem ceeb cuam tshuam qhov tshwm sim ntawm kev tshem tawm parvovirus yog cov lim dej sib txawv. Feem ntau, anti-virus filtering tuaj yeem txhim kho cov kab mob.
Cov txheej txheem yog Protein A(a), AEX penetration mode (b), CEX binding - elution mode (c), AEX binding Ⅰ elution mode (d), Low pH inactivation ("tsis tiav" thiab "ua tiav" clearance) (e ~ g), thiab qhov pore loj (h) thiab qhov pore me me (i ~ j) devirus filtration (qhov twg, a ~ i: Retrovirus; j: Parvovirus).
04 Kev tsim kho tshiab thiab cov teeb meem hauv kev ntsuas kev nyab xeeb ntawm tus kab mob
Kev txhim kho tas mus li thiab kev tsim kho tshiab hauv thaj chaw ntawm cov txheej txheem biopharmaceutical downstream yuav ua rau muaj kev hloov pauv tshiab hauv kev ntsuam xyuas tus kab mob tshem tawm. Kev nce qib hauv cov txheej txheem dej ntws thiab qis qis-xws li cov kwj dej txuas ntxiv bioprocesses-tau ua rau nws tsim nyog los ntsuas thiab tsim cov txheej txheem tshem tawm cov kab mob zoo thiab muaj zog. Thaum lub sij hawm tsis tu ncua, cov kauj ruam xws li kev tsis ua haujlwm ntawm tus kab mob thiab kev lim cov kab mob tseem yuav tsum tau ua hauv batch hom. Txawm hais tias tus kab mob tshem tawm muaj peev xwm ntawm cov txheej txheem chromatographic nyob rau hauv kev nruam ntws yuav tsum tsis txhob sib txawv ntawm cov txheej txheem batch, nws yuav tsum tau txais kev txhawb nqa los ntawm cov ntaub ntawv.
05 Outlook
Los ntawm kev pom kev nyab xeeb ntawm tus kab mob, kev lag luam biopharmaceutical kev nyab xeeb zoo heev tuaj yeem raug ntaus nqi rau kev lag luam kev ua raws li peb lub tswv yim tseem ceeb rau kev nyab xeeb ntawm tus kab mob: tsim kom txaus thiab kuaj cov khoom siv thiab cov chaw txhab nyiaj ntawm tes, sau cov ntaub ntawv kuaj kab mob (kab mob validation kev tshawb fawb), thiab nyob rau hauv-txoj kev kuaj kab mob. Cov yam ntxwv ntawm cov tshuaj biologic uas tsim los ntawm cov tshiab cell substrates yuav hloov nrog cov kev pheej hmoo sib txawv, thiab cov tswv yim tswj kev pheej hmoo zoo yuav tsum tau ua kom muaj kev nyab xeeb ntawm cov tshuaj biologic. Guidling Technology tau ntsib cov kws tshaj lij kev tshaj lij, npog cov txheej txheem pom los ntawm kev sim me me, kev sim sim, mus rau qhov loj-loj ntau lawm, peb cov membrane thiab membrane lim tau dav siv hauv pre-filtration, microfiltration, ultrafiltration, nanofiltration concentration, extraction thiab sib cais; Peb cov khoom lag luam ntau, los ntawm kev siv ib zaug siv me me mus rau kev tsim khoom-oriented filtration systems, sterility kuaj, fermentation, cell kab lis kev cai thiab ntau dua, ua kom cov khoom tsim tau nce ntxiv.
Jiuling los xyuas kom meej cov khoom zoo raws li thawj qhov taw qhia, hauv kev nrhiav "txo cov nqi, nce kev ua tau zoo" ntawm txoj kev tshawb nrhiav, tos ntsoov ua haujlwm nrog koj yav tom ntej kom ntsib cov teeb meem ntawm kev lag luam thiab nrhiav kev txhim kho zoo dua.