Downstream Ultrafiltration / Diafiltration Concentration txheej txheem: Yuav ua li cas tiv thaiv Target Protein Permeation Thiab Poob

Downstream ultrafiltration / diafiltration concentration txheej txheem: Yuav ua li cas tiv thaiv lub hom phiaj protein permeation thiab poob

Hauv cov txheej txheem purification downstream rau recombinant proteins, ntshav khoom, thiab lwm yam biologics, ultrafiltration/diafiltration (UF/DF) yog siv rau feedstock concentration, tsis pauv, thiab tshem tawm cov ntsev thiab me me -molecule impurities. Kev sib cais yog ua tiav raws li daim nyias nyias: cov molecules loj xws li cov tshuaj tiv thaiv kab mob monoclonal (mAbs) khaws cia los ntawm daim nyias nyias, thaum cov ntsev, endotoxins, dawb host cell proteins (HCPs), thiab lwm yam me me -molecule impurities dhau los ntawm daim nyias nyias thiab raug tshem tawm hauv cov permeate. Thaum lub sij hawm txheej txheem teev- nce, lub hom phiaj cov protein poob los ntawm daim nyias nyias tuaj yeem tshwm sim, ncaj qha txo cov txiaj ntsig thiab nce nqi batch. Hauv qee qhov kev ua haujlwm, cov protein yuav raug kuaj pom hauv cov permeate, xav tau kev txiav txim siab sai sai ntawm seb cov khoom tawg puas yog lub hom phiaj mAb, degraded / fragmented aggregates, los yog host cell proteins. Kab lus no tau muab faib ua ob qhov tseem ceeb: nws tshuaj xyuas cov txheej txheem ib txwm siv- tswj kev ua kom zoo thiab, ua ke nrog Tsim Kev Tshawb Fawb (DOE), tsim kom muaj lub qhov rais txheej txheem muaj zog, txhawb nqa los ntawm ntau txoj kev tshuaj ntsuam xyuas los txiav txim seb tus kheej ntawm cov proteins pom nyob rau hauv permeate.

 

 

I. Cov Uas Tseem Ceeb Ntawm Lub Hom Phiaj Protein Permeation Los ntawm Membrane Thaum UF/DF

1. Ntau dhau transmembrane siab (TMP)
Thaum qhov sib txawv ntawm qhov sib txawv ntawm daim nyias nyias yog siab dhau lawm, hnyav hnyav yog txhim kho, yuam qee cov protein monomeric rau hauv cov permeate. Thaum lub sij hawm teev- nce, lub tank pub muaj cov khoom ntim ntau dua thiab cov kev hloov pauv siab hauv cov kav dej tau nthuav tawm ntau dua, ua rau lub sijhawm muaj kev kub ntxhov ntau dua li ntawm qhov ntsuas ntsuas.

2. Tsis tsim nyog tangential flow rate
Yog tias tus nqi ntws qis dhau lawm, qhov ntom ntom ntom ntom - cov txheej txheem polarization ntawm daim nyias nyias, thiab cov protein ntau hauv zos tuaj yeem dhau ntawm daim nyias nyias qhov kev ua siab ntev. Yog tias tus nqi ntws siab dhau lawm, kev ntxhov siab shear tuaj yeem ua rau cov txheej txheem tawg ntawm mAb, tsim cov khoom me me uas tuaj yeem dhau los ntawm cov membrane pores.

3. Muaj protein ntau ntau hauv cov khoom noj
Thaum cov protein concentration nyob rau hauv pub yog siab dhau lawm, protein ntau accumulates ntawm daim nyias nyias, thiab ib co monomeric protein yuav nqa mus rau hauv lub permeate. Qhov no yog ib qho teeb meem tshwm sim hauv loj - teev ntau lawm.

4. pH lossis conductivity sab nraum cov txheej txheem
Cov kev hloov pauv ntawm cov protein cov yam ntxwv tuaj yeem txo nws cov hydration vojvoog thiab pom qhov loj me me, ua rau muaj qhov txawv txav ntawm daim nyias nyias permeation.

5. Membrane cassette aging, siab diffusive flux, los yog tsis txaus ua ntej -conditioning
Lub cev tsis xws luag hauv daim nyias nyias tuaj yeem ua rau muaj protein ntau thiab sawv cev rau cov khoom siv -txog kev pheej hmoo. Rov ua dua CIP nrog cov kev daws teeb meem alkaline maj mam ua rau cov khoom siv polyethersulfone (PES) puas.

6. Ntau dhau diafiltration ntim
Thaum lub sij hawm ntev diafiltration, cov kuab tshuaj tas mus li yuav maj mam nqa tawm me me ntawm cov protein monomeric.

 

II. Ib txwm siv tswv yim ntsuas los tiv thaiv Target Protein Permeation thiab poob

(A) Cov txheej txheem Parameter Control

1. Tswj transmembrane siab (TMP)
Txhais lub siab ceeb toom txwv kev txwv thiab tsim kom muaj kev cuam tshuam lub tswb kom tiv thaiv kev hloov pauv siab - muaj kev cuam tshuam rau cov txheej membrane. Tom qab txiav txim siab qhov ntsuas qhov nruab nrab ntawm qhov ntsuas me me, txo qhov kev nyab xeeb ntawm kev ua haujlwm siab thaum lub sijhawm teev - nce ntau lawm thiab khaws cov txheej txheem tsim nyog kam rau ua.

2. Optimize tus crossflow tus nqi
Tsim kom muaj ib tug ciam teb ruaj khov txheej thiab txo concentration polarization ntawm daim nyias nyias nto los ntawm crossflow, yog li txo cov protein tsub zuj zuj ntawm daim nyias nyias ntawm qhov chaw.

3. Txwv qhov kawg concentration tom qab concentration
Txhais qhov kev txwv tsis pub muaj kev txwv raws li cov khoom siv physicochemical ntawm cov protein thiab zam kev pub noj ntau dhau. Rau mAbs, qhov kev niaj hnub zaum kawg concentration feem ntau tsis tshaj 50 mg / mL; rau qee qhov sib sau ua ke -mAbs yooj yim, qhov concentration yuav tsum raug txo ntxiv.

4. Tswj pub pH thiab conductivity
Ua kom ruaj khov hydrated particle loj ntawm cov protein thiab zam kev sib cog lus molecular los ntawm cov kua qaub / puag lossis ionic- muaj zog cuam tshuam.

(B) Cov khoom siv thiab kev tswj xyuas kev ua haujlwm

1. Ua kev ntsuam xyuas kev ruaj ntseg ntawm daim nyias nyias ua ntej txhua batch
Ua npuas -point xeem ua ntej tsim tawm ntawm txhua batch los txheeb xyuas cov membrane puas lawm. Kev puas tsuaj ntawm daim nyias nyias yuav tsum tau muab pov tseg tam sim ntawd txhawm rau tiv thaiv kom tsis txhob muaj protein ntau.

2. Ua raws li cov txheej txheem ntxuav CIP
Tswj cov NaOH concentration thiab lub sijhawm tiv tauj. Tom qab muaj zog alkaline tu, tag nrho equilibrate cov ntaub ntawv membrane kom txo tau qhov kev pheej hmoo ntawm daim nyias nyias pore deformation.

3. Siv staged diafiltration
Siv qhov loj - ntim tsis hloov pauv thaum lub sijhawm ntxov kom tshem tawm cov ntsev, thiab ua kom cov kua dej ntws qeeb qeeb raws li qhov kawg ntawm qhov kawg kom txo tau cov protein ntau thiab poob.

 

III. Tsim Tus Txheej Txheem Tsim Chaw Los Ntawm DOE (QbD Txheej Txheem Tus Qauv; Muaj feem xyuam rau IND Xa)

1. Kev tshuaj ntsuam xyuas
Tshuaj ntsuam tsib qhov tseem ceeb UF / DF txheej txheem tsis (P): transmembrane siab, pub txaus tus nqi, thawj zaug protein concentration, pub pH, thiab kev khiav hauj lwm kub.
Teb variables: khaws cia yield, protein cov ntsiab lus nyob rau hauv lub permeate, membrane flux, thiab feem pua ​​ntawm siab -molecular- hnyav aggregates.

Los ntawm kev txheeb xyuas qhov tseem ceeb, txheeb xyuas cov ntsiab lus tseem ceeb uas ua rau muaj protein ntau.

2. Teb nto qauv qauv
Xaiv plaub qhov tsis muaj qhov tseem ceeb tshaj plaws los tsim cov lus teb thiab txheeb xyuas kev sib cuam tshuam ntawm cov tsis. Nyob rau hauv ntau qhov kev khiav hauj lwm, ib qho kev ntsuas yuav nyob hauv nws qhov kev tsim nyog, thaum kev sib xyaw ntawm ob qhov tsis tuaj yeem ua rau muaj protein ntau permeation. Cov kev cuam tshuam zoo li no tsis tuaj yeem raug txheeb xyuas los ntawm ib txwm muaj ib qho -factor- ntawm - ib - lub sijhawm sim. Raws li tus qauv, txhais qhov chaw ua haujlwm tsim tau. Ntev npaum li cov txheej txheem tsis nyob hauv qhov ntau, cov protein tuav tau zoo tuaj yeem ua kom ntseeg tau thaum cov impurities raug tshem tawm los ntawm diafiltration.

3. Qhov phem tshaj - rooj plaub kev sib tw (yuav tsum tau ua rau tus cwj pwm ntawm tus txheej txheem)
Xaiv cov txheej txheem tsis zoo ntawm qhov kawg ntawm kev ua haujlwm ciam teb kom simulate kev tsim khoom sib txawv: ntau tshaj TMP, qhov tsawg kawg nkaus crossflow tus nqi, thiab sab sauv - txwv cov protein ntau. Xyuas kom tseeb tias txawm tias nyob rau hauv kev ua haujlwm hnyav, loj - nplai lub hom phiaj cov protein poob tsis tshwm sim, yog li ua tau raws li CMC tshuaj xyuas cov cai.

 

IV. Muaj ntau txoj hauv kev los txheeb xyuas cov Proteins pom nyob rau hauv Permeate thiab Distinguish Target mAb, Degradation Fragments, thiab HCP

Raws li kev txheeb xyuas kev ua tau zoo, tus nqi, thiab kev tswj hwm cov xwm txheej, cov txheej txheem tuaj yeem muab faib ua ob pawg: kev tshuaj ntsuam sai thiab kev kuaj xyuas. Cov txheej txheem nrawm yuav tsum tau ua qhov tseem ceeb rau kev saib xyuas cov txheej txheem niaj hnub; thaum muaj qhov txawv txav tshwm sim, cov txheej txheem qhia meej meej yuav tsum tau siv.

1. UV spectrophotometry (kev tshuaj ntsuam online ceev ceev; nyiam rau cov khw niaj hnub- saib xyuas hauv pem teb)
Sau cov permeate thiab ntsuas nws A280 absorbance. Qhov no tsuas tuaj yeem txiav txim siab seb puas muaj protein nyob hauv cov permeate thiab tsis tuaj yeem paub qhov txawv ntawm hom protein. Nws yog tsuas yog siv rau cov txheej txheem niaj hnub saib xyuas. Ib qho kev nce ntxiv ntawm A280 tus nqi qhia tau tias muaj protein ntau ntxiv thiab yuav tsum tau ncua tam sim ntawm kev lim ntshav thiab tshawb xyuas cov txheej txheem.

2. SDS-PAGE electrophoresis (feem ntau yog siv txoj kev txheeb xyuas qhov zoo)
Siv txo thiab tsis yog -txo electrophoresis thiab sib piv cov haujlwm band nrog cov hom phiaj mAb hauv cov khoom noj:
1) Yog hais tias lub band txoj hauj lwm yog tag nrho raws li lub hom phiaj mAb, zoo mAb tau permeated daim nyias nyias.
2) Yog tias qis dua -molecular- qhov hnyav bands tshwm, cov protein tau dhau los ntawm hydrolytic cleavage, tsim mAb fragments.
3) Yog hais tias cov bands tawg nrog ntau lwm yam bands, feem ntau yog host cell proteins (HCPs) los ntawm E. coli/CHO host cells es tsis yog cov khoom lag luam.

3. High-kev ua tau zoo loj-exclusion chromatography (HPSEC)
Cais cov khoom raws li molecular loj thiab sib piv lawv lub sij hawm elution nrog cov feedstock chromatogram:
- Lub ncov tseem ceeb nrog tib lub sijhawm khaws cia raws li mAb qhia txog cov protein tsis zoo.
- Lub ncov eluting ua ntej qhia tias siab -molecular- hnyav aggregates/fragments.
- Lub ncov eluting tom qab qhia tias qis -molecular- hnyav degradation fragments.
- Qhov tsis muaj tus yam ntxwv tseem ceeb ncov qhia tias HCP cov protein tsis huv.
Txoj kev no tuaj yeem muab cov ntaub ntawv ntau thiab tuaj yeem suav nrog hauv cov txheej txheem kev tshawb nrhiav kev sib txawv; nws muaj kev lees paub siab hauv cov ntaub ntawv xa mus kuaj mob.

4. HCP host-cell protein ELISA cov khoom kuaj
Yog tias pom muaj protein ntau hauv cov permeate, ELISA cov txiaj ntsig tau nce siab, thiab HPSEC tsis pom cov yam ntxwv ntawm mAb ncov, cov khoom xau tuaj yeem txiav txim siab tias yog CHO tus tswv tsev - cell proteins. Qhov no qhia tias tsis tiav impurity tshem tawm thaum lub sij hawm purification ntws; cov impurities raug ntxuav tawm thaum lub sij hawm UF / DF theem thiab tsis sawv cev rau cov khoom poob.

5. Affinity chromatography pov thawj (Protein A chromatography)
Protein A tshwj xeeb khi rau thaj tsam Fc ntawm mAbs. Thauj cov qauv permeate mus rau hauv kem: qhov tsos ntawm tus yam ntxwv elution ncov qhia tias muaj cov phiaj xwm tsis zoo; Qhov tsis muaj elution ncov qhia tau hais tias cov proteins nyob rau hauv cov permeate yog tag nrho cov impurity proteins.

6. Mass spectrometry (LC-MS) (nyob rau hauv -qhov tob traceability; siv rau kev tshawb xyuas ntawm cov txheej txheem loj sib txawv)
LC-MS tuaj yeem txheeb xyuas cov kab mob amino acid ncaj qha thiab txiav txim siab qhov keeb kwm ntawm cov protein. Nws yog tsuas yog siv rau kev tshawb nrhiav ntawm qhov nyuaj lossis txawv txawv txawv thiab tsis yog siv raws li niaj hnub hauv - kev ntsuas kev tswj xyuas.

V. Kev Kho Kom Zoo Ua Raws Li Kev Tshawb Fawb Sib Txawv

1. Intact target mAb kuaj pom nyob rau hauv permeate
Txo cov transmembrane siab thiab qhov kawg concentration tom qab concentration; nqaim cov txheej txheem khiav hauj lwm raws li DOE cov txiaj ntsig; thiab tshawb xyuas cov membrane cassette kev ncaj ncees.

2. Antibody degradation fragments kuaj pom nyob rau hauv permeate
Tshawb xyuas kev ntxhov siab thoob plaws hauv cov txheej txheem thiab pH siab; ua kom zoo tshaj cov kab mob kis mus rau theem qis kom txo tau cov protein cleavage.

3. Tsuas yog HCP impurity proteins pom nyob rau hauv permeate
Tsis muaj kev hloov kho ntawm UF / DF tsis yog yuav tsum tau. Taug qab qhov teeb meem rov qab mus rau cov kauj ruam ua ntej chromatography thiab txhim kho kev tshem tawm impurity thaum muaj kev sib raug zoo thiab ion- pauv chromatography.

 

VI. Cov ntsiab lus

Txhawm rau tiv thaiv kom tsis txhob muaj protein ntau thaum lub sijhawm UF / DF, cov txheej txheem kev tswj hwm ib txwm siv los ntawm kev paub keeb kwm los txhais cov ntsiab lus ntawm tus kheej, uas tuaj yeem ua kom txaus nyob ruaj khov me me - teev ntau lawm. Txhawm rau ua kom muaj kev ruaj ntseg thaum lub sij hawm txheej txheem txheej txheem theem- nce, DOE- raws li tus txheej txheem tus yam ntxwv yuav tsum tau siv los tsim kom muaj qhov chaw tsim txheej txheem nrog kev ua siab ntev txaus. Interval-raws li kev tswj tsis tau tuaj yeem them nyiaj rau kev tsim khoom sib txawv. Thaum kuaj pom cov protein nyob rau hauv cov permeate, ib qho yooj yim UV nrhiav kom tau qhov kev ceeb toom thawj zaug, ua raws li electrophoresis, HPSEC, thiab ELISA rau traceability thiab txheeb xyuas. Qhov no tso cai rau qhov sib txawv ntawm lub hom phiaj cov khoom lag luam thiab tus tswv tsev -cell impurities, ua rau lub hom phiaj hauv paus- ua rau kev tshawb nrhiav thiab kev kho kom raug. Qhov no kuj yog ib qho kev tshawb fawb tus qauv tam sim no muaj nyob rau hauv CMC cov ntaub ntawv rau mAb tej yaam num.