Daim Ntawv Qhia Rau Cov Qauv Ua Haujlwm Zoo Tshaj Plaws

Cov tshuaj tiv thaiv cov protein ntau nyob hauv cov tshuaj tiv thaiv kab mob, thiab muaj kev nyab xeeb thiab muaj kev nyab xeeb} Kuv muaj kev nyab xeeb} Kev nyab xeeb} Lawv cov khoom siv txaus ntshai heev (TFF), vim yog nws txoj kev ua tau zoo thiab Scalability, yog nce los ua ib qho cuab yeej tseem ceeb hauv cov protein purification workflows .

Cov kab lus no qhia cov kauj ruam tseem ceeb hauv kev ua haujlwm ntawm TFF kev tshawb fawb thiab txhim kho cov protein zoo.

 

Kuv {. Cov kauj ruam tseem ceeb hauv kev ua kom huv ntawm cov roj ntsha proteins

1.} cell sau thiab lysis

FENTRIFUGATION / DEJ NTIM NO: Tshem tawm ntawm tes khib nyiab thiab kev tsis sib haum xeeb; Haum rau cov kab mob, poov xab, thiab lwm yam .} .

Sonication / High-siab homogenization: rhuav cov hlwb los tso cov khoom muaj cov khoom muaj cov khoom siv tsis zoo; tej yam kev mob yuav tsum muaj kev txhim kho kom tiv thaiv tau protein denaturation .

Enzymatic lysis: e {. g .}}, lysozyme kev kho rau cov kab mob; kev mob siab tab sis tus nqi siab dua .

{{02. purification: ntes ntawm lub hom phiaj protein

Afficy chromatography (e {{{{0} g}}}}}}}}}, nws-tagged, siv pro / g): qhov tshwj xeeb khi; ua tiav siab purity nyob rau hauv ib kauj ruam .

Ion txauv cov chromatography (iex): cais cov protein raws li them qhov sib txawv; Haum rau thaum ntxov-rau-theem theem purification .

Hydrophobic sib cuam tshuam chrataphyraphy (hic): Siv cov kev sib txawv hauv cov protein saum hydrophobicity; siv tau rau qee yam nyuaj-rau-purify protein .

3. Polishing: Txhim Kho Purity

Qhov loj me-cais tawm cov chromatography (sec): tshem tawm cov kab ke thiab cov molecule me me; Txwv tsis pub thauj khoom muaj peev xwm .

Mullimodal chromatography (e {. g}}}}}}}}, ua ke ntau yam kev cuam tshuam rau kev daws teeb meem siab dua.

{{04.}}}} concent concent concent thiab Cov Tsis Txiaj Ntsig

Ultrafiltration centrifugal pab kiag li lawm: haum rau cov qauv me me; nquag rau protein poob .

Tangential pom kev lim (TFF): muaj txiaj ntsig, haum, zoo tagnrho rau kev lag luam kev lag luam (cov ncauj lus kom ntxaws ntxiv).

5.} Tsis muaj Kom Tsis Muaj me me thiab cia

0 {{{. 22 μm Kev Pom: Tshem tawm cov kab mob methurganism ua kom muaj kev tsis muaj menyuam.

Ntxiv rau ntawm stabilizers (e {{{. g}}, glycerol, tiv thaiv protein degradadation.

 

Ii {.} Cov ntawv thov tseem ceeb ntawm Tange Flow pom kev (Tff) hauv kev ua kom huv huv

Kev ua kom pom kev txiav tawm (Tff) txo tus membrane tsis muaj kev pom zoo, ua rau nws muaj kev sib haum xeeb (}}}

 

1. Cov txiaj ntsig ntawm TFF ICS

✔ Kom siab rov zoo: minimizes protein adsorption kev poob, tshwj xeeb tshaj yog qhov tseem ceeb rau cov qauv muaj txiaj ntsig .

✔ Tawm Scandability: Siv los ntawm Lab Teev (10 ML) rau ntau lawm (1000L +).

✔ Kev Yooj Yim: Ib qho system yuav ua tau zoo, lim ntshav (tsis sib pauv), thiab diafiltration .

 

{{02. TFF Cassette / Daim Ntawv Qhia Txog Cim Cim

Daim nyias nyias cov khoom	Cov yam ntxwv	Daim Ntawv Thov Kev Pab Cuam
Polyethersulfone (Pes)	Tsawg protein khi, chemically ruaj khov (part resistant), siab flux	Harsh buffer tej yam kev mob
Resenerated cellulose (RC)	Tsawg protein khi, siab flux, cov protein ntau	Niaj hnub cov protein / tshuaj tiv thaiv kev ua kom huv

Molecular hnyav txiav-tawm (MWCO) cov lus qhia xaiv:

1/3 txog 1/5 ntawm lub hom phiaj protein cov molecular hnyav (e {}} para} pa 1 kda protein).

Txhawm rau tshem tawm cov aggregates, xaiv ib qho me me pore loj (e .} Kuv siv 50 kda rau 100 kda protein).

 

{{0 {}}} kev ua haujlwm ntawm cov kev ua haujlwm tseem ceeb Tff ua haujlwm

Transmembrane siab (TMP): Feem ntau 3-15 psi; ntau dhau tmp txhawb fouling .

Tus Nqi Kub Txaus Ntshai: tswj kev kub ntxhov kom txo qis polarration; feem ntau 4-8 l / minen {{2 2}}

Kev Txhim Kho Kev Txhim Kho:

Siv 2-5 swb coldumes thaum diafiltration rau kev hloov pauv tiav .

Ua rov qab-yaug ntawm qhov kawg kom rov qab muaj protein protein .

 

4. Kev Kawm Kev Kawm: Monoclonal Antibody (Mab) Purification

Piav kom meej ntawm cov kab lis kev cai cell → Protein a ap

 

TFF lub luag haujlwm:

Ceev ceev cov dilute protein ib tug eluate mus rau lub hom phiaj concentration {}}

Sib pauv lub ntas yeej mus rau PBS lossis tsim ua qauv (e {{{} g . .

 

III {{. Cov teeb meem thiab cov kev daws teeb meem

❌ Qhov Teeb Meem 1: Cov txiaj ntsig muaj protein tsawg

Tau ua rau muaj: membrane adsorption; Nag lossis daus vim yog tshaj-siab .

Kev daws teeb meem: Hloov mus rau cov kab xev qis qis; Ntxiv surfactant (e {{{{1} g .}}}} 01% Tween 20).

Muaj teeb meem 2: Fai sai poob qis

Tau ua rau muaj: membrane fouling lossis concentration polarization .

Kev daws teeb meem: Ua kom zoo tangentic tus nqi ntws; siv cov ris tsho rov qab los tsis tu ncua; hloov mus rau ib qho qhib membrane ntau dua (e . g}!}}}) {{5} {5}

Pencs Teeb meem 3: Protein sib sau ua ke

Ua tau rau: ntau dhau shear zog; Tsis tsim nyog ua tsis tau zoo .

Kev daws teeb meem: Txo cov twj tso kua mis ceev; siv cov guffer (E {{{0} g .}}, muaj Sucrose lossis NacL) {2}.

 

Iv . cov ntsiab lus

Tau Txais Cov Khoom Siv Hluav Taws Xob Loj Tshaj Plaws Kev Sib Koom Zoo ntawm Kev Pom Zoo Ua Ntej, thiab Kev Ua Liaj Ua Haujlwm, Ua Liaj Ua Ke Thaum Ua Haujlwm Xaiv, Kev Ua Liaj Ua Ke Los Ntawm Qhov Kev Xaiv Cov Tshuaj Yooj Yim, thiab cov protein puricial thiab protein tau ua kom zoo dua, ntsib qhov kev xav tau ntawm ob qho kev tshawb fawb thiab kev ntsuas-ntsuas ntau lawm.