Protein Solution Viscosity Control in Ultrafiltration

Ukulawula i-viscosity yezisombululo zeproteni kubalulekile ekuphuculeni iinkqubo zoxinzelelo lwe-ultrafiltration kwimveliso ye-biopharmaceutical. I-viscosity ephakamileyo kwizisombululo zeproteni—ingakumbi kwi-protein concentrations ephezulu—ichaphazela ngokuthe ngqo ukusebenza kwe-membrane, ukusebenza kakuhle kwenkqubo, kunye noqoqosho kwizicelo zoxinzelelo lweproteni ye-ultrafiltration. I-viscosity yesisombululo iyanda kunye nomxholo weproteni ngenxa ye-anti-body clustering kunye nokusebenzisana kwe-electrostatic, okwandisa ukumelana nokuhamba kunye nokwehla koxinzelelo kwi-membrane ye-ultrafiltration. Oku kubangela ukuba i-permeate fluxes ephantsi kunye namaxesha amade okusebenza, ingakumbi kwiinkqubo ze-transverse flow filtration (TFF).

Uxinzelelo lwe-Transmembrane (TMP), amandla aqhubayo emva kokucoca i-ultramembrane, luqhagamshelwe ngokusondeleyo kwi-viscosity. Ukusebenza ngaphandle koluhlu oluqhelekileyo loxinzelelo lwe-transmembrane kukhawulezisa ukungcoliswa kwe-membrane kwaye kwandisa ukungcoliswa kwe-concentration-ukwakheka kweeproteni kufutshane ne-membrane okwandisa rhoqo i-viscosity yendawo. Zombini i-concentration polarization kunye ne-membrane fouling ziphumela ekusebenzeni okunciphileyo kwe-ultrafiltration membrane kwaye zinokunciphisa ubomi be-membrane ukuba azijongwanga. Umsebenzi wovavanyo ubonisa ukuba ukungcoliswa kwe-membrane kunye ne-concentration polarization kwi-ultrafiltration zibonakala ngakumbi kumaxabiso aphezulu e-TMP kwaye kunye nokutya okunamandla ngakumbi, okwenza ulawulo lwe-TMP lwexesha langempela lube lubalulekile ukuze kuphuculwe ukugqithiswa kunye nokunciphisa amaxesha okucoca.

Ukuphucula uxinaniso lwe-ultrafiltration kufuna amaqhinga adibeneyo:

Ukulinganiswa kwe-viscosity yesisombululo seprotheniUvavanyo oluqhelekileyo lwe-viscosity—ukusebenzisaii-viscometer ezikwi-intanethi—nceda ukuqikelela amazinga okuhluza kunye nokuqikelela imiqobo yenkqubo, ukuxhasa utshintsho olukhawulezileyo lwenkqubo.
Ukulungisa ukutya: Ukulungisa i-pH, amandla e-ionic, kunye nobushushu kunokunciphisa i-viscosity kwaye kunciphise ukungcoliswa. Umzekelo, ukongeza ii-ion ze-sodium kwandisa ukugxojwa kwamanzi phakathi kweeproteni, kunciphisa ukuhlangana kunye nokungcoliswa, ngelixa ii-ion ze-calcium zihlala zikhuthaza ukudibana kunye nokungcoliswa kweeproteni.
Ukusetyenziswa kwezinto ezincedisayo: Ukufaka izinto ezinciphisa i-viscosity kwizisombululo zeprotheyini ezixineneyo kuphucula ukuvuleka kwe-membrane kwaye kunciphisa uxinzelelo lwe-transmembrane kwi-ultrafiltration, okunyusa ukusebenza kakuhle ngokubanzi.
Iindlela zokuhamba kwamanzi eziphucukileyo: Ukunyusa isantya sokuhamba kwamanzi ahambayo, ukusebenzisa ukuhamba kwamanzi ahambayo ngokutshintshana, okanye ukusebenzisa i-air jet injection kuphazamisa iileya ezingcolisayo. Ezi ndlela zinceda ukugcina ukuhamba kwamanzi angena ngaphakathi kwaye zinciphise ukuphindaphinda kwe-membrane ngokunciphisa ukwakheka kwediphozithi.
Ukukhetha kunye nokucoca i-membrane: Ukukhetha ii-membrane ezikwaziyo ukumelana neekhemikhali (umz., i-SiC okanye ii-thermosalient hybrids) kunye nokwenza ngcono ukucocwa kwee-membrane rhoqo ngeeprotokholi ezifanelekileyo (umz., ukucocwa kwe-sodium hypochlorite) kubalulekile ekwandiseni ubomi be-membrane kunye nokunciphisa iindleko zokusebenza.

Lilonke, ulawulo olusebenzayo lwe-viscosity kunye nolawulo lwe-TMP zezona zinto zibalulekileyo ekusebenzeni ngempumelelo kwesigaba soxinzelelo lwe-ultrafiltration, ezichaphazela ngokuthe ngqo isivuno semveliso, ukuhlanjwa rhoqo kwe-membrane, kunye nexesha elide lezinto ze-membrane ezibizayo.

Ukuqonda i-Protein Solution Viscosity kwi-Ultrafiltration

1.1. Yintoni iViscosity yeProteni Solutions?

I-Viscosity ichaza ukumelana kolwelo nokugeleza kwalo; kwizisombululo zeproteni, ibonisa ukuba ingakanani i-molecular friction ethintela intshukumo. Iyunithi ye-SI ye-viscosity yi-Pascal-second (Pa·s), kodwa i-centipoise (cP) isetyenziswa rhoqo kwi-biological fluids. I-Viscosity ichaphazela ngokuthe ngqo indlela izisombululo zeproteni ezinokupompa okanye zihluzwe lula ngayo ngexesha lokwenziwa kwaye ichaphazela ukuhanjiswa kwamayeza, ngakumbi kwi-biotherapeutics enoxinzelelo oluphezulu.

Uxinaniso lweproteni yeyona nto iphambili echaphazela i-viscosity. Njengoko amanqanaba eproteni enyuka, ukusebenzisana phakathi kweemolekyuli kunye nokuxinana kuyanda, okubangela ukuba i-viscosity inyuke, rhoqo ngokungangqalanga. Ngaphezulu komda othile, ukusebenzisana kweproteni kunye neproteni kuyayicinezela ngakumbi i-dispersion ngaphakathi kwesisombululo. Umzekelo, izisombululo ze-antibody ze-monoclonal ezixineneyo ezisetyenziswa kumayeza zihlala zifikelela kumanqanaba e-viscosity afaka umngeni kwi-subcutaneous injection okanye athintele amazinga okucubungula.

Iimodeli eziqikelela i-viscosity kwizisombululo zeproteni ezixineneyo ngoku ziquka i-molecular geometry kunye ne-aggregation tendencies. I-protein morphology—nokuba inde, i-globular, okanye ithambekele ekuhlanganiseni—ichaphazela kakhulu i-viscosity kumanqanaba aphezulu. Inkqubela phambili yakutshanje kuvavanyo lwe-microfluidic ivumela ukulinganiswa okuchanekileyo kwe-viscosity ukusuka kwizambuku ezincinci zesampulu, okwenza kube lula ukuphononongwa ngokukhawuleza kwe-protein formulations ezintsha.

1.2. Indlela Ubungakanani Obutshintsha Ngayo Ngexesha Lokuhluza Nge-Ultra

Ngexesha lokuhluzwa kwe-ultrafiltration, i-concentration polarization iqokelela iiproteni ngokukhawuleza kwi-interface ye-membrane-solution. Oku kudala i-local concentration gradients ende kwaye kunyusa i-viscosity kufutshane ne-membrane. I-viscosity ephakamileyo kule ndawo ithintela ukudluliselwa kobunzima kwaye inciphise i-permeating flux.

Ukuphola koxinzelelo kwahlukile ekupholeni kwe-membrane. Ukuphola kuguquguqukayo kwaye kuyaguquka, kwenzeka kwimizuzu embalwa njengoko ukuhluza kuqhubeka. Xa kuthelekiswa, ukuphola kuyaqhubeka ngokuhamba kwexesha kwaye kudla ngokubandakanya ukubekwa okungaguqukiyo okanye ukuguqulwa kweekhemikhali kumphezulu we-membrane. Ukuxilongwa ngokuchanekileyo kuvumela ukulandelelwa kwexesha langempela kwe-concentration polarization layer, kutyhila uvakalelo lwayo kwisantya sokuhamba kunye noxinzelelo lwe-transmembrane. Umzekelo, ukwandisa isantya okanye ukunciphisa uxinzelelo lwe-transmembrane (TMP) kunceda ukuphazamisa umaleko womda oqinileyo, ukubuyisela ukugeleza.

Iiparameter zokusebenza zichaphazela ngokuthe ngqo indlela esebenza ngayo i-viscosity:

Uxinzelelo lwe-Transmembrane (TMP): I-TMP ephezulu iqinisa i-polarization, inyusa i-viscosity yendawo kwaye inciphise ukuhamba kwamanzi.
Isantya sokuhamba komnqamlezo: Isantya esiphuculweyo sithintela ukuqokelelana, silinganisela i-viscosity kufutshane ne-membrane.
Ukucocwa rhoqo kwe-membrane: Ukucoca rhoqo kunciphisa ukwakheka kwexesha elide kwaye kunciphisa ukulahleka kokusebenza okubangelwa kukuxinana.

Izigaba zoxinzelelo lwe-Ultrafiltration kufuneka ziphucule ezi parameters ukuze kuncitshiswe imiphumo emibi ye-viscosity kwaye kugcinwe umbane ophumayo.

1.3. Iimpawu zeSombululo seProtheni ezichaphazela ukuxinana

Ubunzima be-molecularkwayeukwakhekaikakhulu kumisela i-viscosity. Iiproteni ezinkulu nezintsonkothileyo okanye ii-aggregates zivelisa i-viscosity ephezulu ngenxa yokuphazamiseka kwentshukumo kunye namandla amakhulu aphakathi kweemolekyuli. Imilo yeeproteni ilawula ngakumbi ukuhamba-imixokelelwane emide okanye ethanda ukuhlanganiswa ibangela ukuxhathisa okungaphezulu kuneeproteni ezixineneyo ze-globular.

pHIchaphazela kakhulu itshaja yeproteni kunye nokunyibilika kwayo. Ukulungisa i-pH yesisombululo kufutshane nendawo ye-isoelectric yeproteni kunciphisa itshaja eseleyo, kunciphisa ukugxothwa kweproteni-protein, kwaye kunciphisa okwethutyana i-viscosity, okwenza kube lula ukuhluza. Umzekelo, ukusebenzisa i-ultrafiltration kufutshane nendawo ye-isoelectric ye-BSA okanye i-IgG kunokuphucula kakhulu i-permeate flux kunye nokukhetha ukwahlukana.

Amandla e-IonicIchaphazela i-viscosity ngokutshintsha umaleko ombane ophindwe kabini ojikeleze iiproteni. Amandla e-ionic anda ajonga ukusebenzisana kwe-electrostatic, ekhuthaza ukudluliselwa kweproteni ngee-membranes kodwa ikwanyusa umngcipheko wokudibana kunye nokunyuka kwe-viscosity ehambelanayo. Ukutshintshiselana phakathi kokusebenza kakuhle kokudluliselwa kunye nokukhetha kudla ngokuxhomekeke ekuhlengahlengiseni kakuhle amanqanaba etyuwa kunye nokwakheka kwe-buffer.

Izongezo ezincinci zemolekyuli—ezifana ne-arginine hydrochloride okanye i-guanidine—zingasetyenziswa ukunciphisa i-viscosity. Ezi arhente ziphazamisa ukutsala kwe-hydrophobic okanye i-electrostatic, zinciphisa ukuhlangana, kwaye ziphucula iipropati zokuhamba kwesisombululo. Ubushushu busebenza njengotshintsho olongezelelweyo lolawulo; amaqondo obushushu aphantsi anyusa i-viscosity, ngelixa ubushushu obongezelelweyo budla ngokuyinciphisa.

Ukulinganiswa kwe-viscosity yesisombululo seprotheni kufuneka kuqwalaselwe oku:

Ukusasazwa kobunzima be-molecular
Ukwakheka kwesisombululo (iityuwa, izinto ezincedisayo, izongezo)
Ukukhethwa kwe-pH kunye nenkqubo ye-buffer
Useto lwamandla e-Ionic

Ezi zinto zibalulekile ekwenzeni ngcono ukusebenza kwe-membrane ye-ultrafiltration kunye nokuqinisekisa ukuhambelana kuzo zonke izigaba zoxinzelelo kunye neenkqubo ze-TFF.

Iziseko zoQokelelo lweeProtheyini ze-Ultrafiltration

Imigaqo yeSigaba sokuQinisekisa i-Ultrafiltration

Uxinzelelo lweproteni ye-Ultrafiltration lusebenza ngokusebenzisa uxinzelelo lwe-transmembrane (TMP) kwi-membrane engakwazi ukungena kancinci, iqhuba i-solvent kunye ne-solutes ezincinci ngelixa igcina iiproteni kunye neemolekyuli ezinkulu. Le nkqubo isebenzisa ukungenelela okukhethiweyo ngokusekelwe kubukhulu beemolekyuli, kunye ne-membrane's molecular weight cut-off (MWCO) echaza ubungakanani obukhulu beemolekyuli ezidlulayo. Iiproteni ezidlula i-MWCO ziqokelelana kwicala elingasemva, zinyusa uxinano lwazo njengoko i-permeate irhoxiswa.

Isigaba soxinzelelo lwe-ultrafiltration sijolise ekunciphiseni umthamo kunye nokutyebisa isisombululo seproteni. Njengoko ukuhluza kuqhubeka, i-viscosity yesisombululo seproteni idla ngokunyuka, ichaphazela ukugeleza kunye neemfuno ze-TMP. Iiproteni ezigciniweyo zinokusebenzisana kunye kunye ne-membrane, okwenza inkqubo yokwenyani ibe nzima ngakumbi kunokukhupha ubungakanani obulula. Ukusebenzisana kwe-electrostatic, ukuhlanganiswa kweproteni, kunye neempawu zesisombululo ezifana ne-pH kunye namandla e-ionic zichaphazela iziphumo zokugcina kunye nokwahlukana. Kwezinye iimeko, ukuthuthwa kwe-advective kulawula ngaphezu kokusasazwa, ngakumbi kwii-membrane ezinee-pores ezinkulu, okwenza kube nzima ukulindela ngokusekelwe kuphela kukhetho lwe-MWCO [jonga isishwankathelo sophando].

Inkcazo ye-Transverse Flow Filtration (TFF)

I-transverse flow filtration, ekwabizwa ngokuba yi-tangential flow filtration (TFF), ihambisa isisombululo seprotheyini ngendlela ethe tyaba kumphezulu we-membrane. Le ndlela iyahluka kwi-dead-end filtration, apho ukuhamba kuthe tyaba kwi-membrane, kutyhala amasuntswana ngqo kwi-filter nakwi-filter.

Umahluko kunye nefuthe eliphambili:

Ulawulo lokungcolisa:I-TFF inciphisa ukwakheka kweproteni kunye neenxalenye ezincinci, ezaziwa ngokuba kukwakheka kwekhekhe, ngokuhlala isusa izinto ezinokubangela ukubola kwi-membrane. Oku kubangela ukuba i-permeate flux ihlale izinzile kwaye kube lula ukuyigcina.
Ukugcinwa kweeProteni:I-TFF ixhasa ulawulo olungcono lwe-concentration polarization—umaleko wee-molecules ezigciniweyo kufutshane ne-membrane—ezinokuthi, ukuba azilawulwa, zinciphise ukukhetha ukwahlukana kwaye zonyuse ukungcoliswa. Ukuhamba okuguquguqukayo kwi-TFF kunciphisa esi siphumo, kunceda ukugcina ukugcinwa okuphezulu kweproteni kunye nokusebenza kakuhle kokwahlukana.
Uzinzo lweFlux:I-TFF ivumela ixesha elide lokusebenza ngokuguquguquka rhoqo, inyusa ukusebenza kakuhle kwiinkqubo ezineproteni ephezulu okanye ukutya okutyebileyo. Ukucoca okungapheliyo, ngokuchaseneyo, kuphazamiseka ngokukhawuleza kukungcola, ukunciphisa ukuphuma kwamanzi kunye nokufuna amanyathelo okucoca rhoqo.

Iinguqulelo ze-TFF eziphambili, ezifana nokuhamba kwe-tangential alternating (ATF), ziphazamisa ngakumbi ukungcoliswa kunye nokwenziwa kwekhekhe ngokuguqula okanye ukutshintsha isantya se-tangential rhoqo, ukwandisa ubomi besihluzo kunye nokuphucula i-protein throughput [jonga isishwankathelo sophando]. Kuzo zombini iisetingi ze-TFF zakudala kunye neziphambili, useto lokusebenza—ezifana ne-TMP, isantya se-crossflow, kunye nobuninzi bokucoca—kufuneka zilungelelaniswe nenkqubo ethile yeproteni, uhlobo lwe-membrane, kunye noxinzelelo olujoliswe kulo ukuze kuphuculwe ukusebenza kunye nokunciphisa ukungcoliswa.

Uxinzelelo lwe-Transmembrane (TMP) kwi-Ultrafiltration

3.1. Yintoni uxinzelelo lwe-Transmembrane?

Uxinzelelo lwe-Transmembrane (TMP) ngumahluko woxinzelelo kwi-membrane yokucoca, eqhuba i-solvent ukusuka kwicala lokutya ukuya kwicala elingaphezulu. I-TMP yeyona nto iphambili kwinkqubo yokwahlula kwi-ultrafiltration, ivumela i-solvent ukuba idlule kwi-membrane ngelixa igcina iiproteni kunye nezinye ii-macromolecules.

Ifomula ye-TMP:

Umahluko olula: TMP = P_feed − P_permeate
Indlela yobunjineli: TMP = [(P_feed + P_retentate)/2] − P_permeate
Apha, i-P_feed luxinzelelo lokungena, i-P_retentate luxinzelelo lokukhupha kwicala elingasemva, kwaye i-P_permeate luxinzelelo lwecala elingaphandle. Ukubandakanya uxinzelelo olungasemva (okanye olugxininisiweyo) kubonelela ngexabiso elichanekileyo ngakumbi kumphezulu we-membrane, kubalwa i-gradients yoxinzelelo ebangelwa kukumelana nokuhamba kwamanzi kunye nokungcola.
Uxinzelelo lokutya kunye nesantya sokuhamba kwamanzi
Uxinzelelo oluhlala ixesha elide (xa lukhona)
Uxinzelelo olungena (oluhlala lunomoya)
Ukumelana ne-membrane
I-TMP iyahluka ngohlobo lwe-membrane, uyilo lwenkqubo, kunye neemeko zenkqubo.

Ukulawula iiNguqu:

3.2. I-TMP kunye neNkqubo yokuhluza nge-Ultrafiltration

I-TMP idlala indima ephambili ekuxinaniseni iiproteni nge-ultrafiltration, iqhuba izisombululo zeproteni kwi-membrane. Uxinzelelo kufuneka lube phezulu ngokwaneleyo ukuze loyise ukuxhathisa kwi-membrane nayo nayiphi na into eqokelelweyo kodwa lungadluli kakhulu kangangokuba lukhawulezise ukungcola.

Impembelelo yeSolution Viscosity kunye neProtein Concentration

Ukuqina kwezisombululo zeproteni:I-viscosity ephezulu inyusa ukumelana nokuhamba kwamanzi, nto leyo efuna i-TMP ephezulu ukuze kugcinwe i-permeating flux efanayo. Umzekelo, ukongeza i-glycerol kwi-feed okanye ukusebenzisa iiproteni ezixineneyo kunyusa i-viscosity kwaye ngaloo ndlela i-TMP esebenzayo iyafuneka.
Uxinzelelo lweeproteni:Njengoko uxinzelelo lusanda ngexesha lesigaba soxinzelelo lwe-ultrafiltration, i-viscosity yesisombululo iyanda, i-TMP iyanda, kwaye umngcipheko wokungcoliswa kwe-membrane okanye i-concentration polarization iyanda.
UMthetho kaDarcy:I-TMP, i-permeate flux (J), kunye ne-viscosity (μ) zinxulumene nge-TMP = J × μ × R_m (ukumelana ne-membrane). Kwizisombululo zeprotheyini ezixineneyo kakhulu, ukulungiswa kwe-TMP ngononophelo kubalulekile ukuze kuhluzwe kakuhle.

Imizekelo:

Ukuhluzwa kwe-ultrafiltration kwezisombululo ze-antibody ezixineneyo kufuna ulawulo olucokisekileyo lwe-TMP ukuze kuthintelwe ukunyuka kwe-viscosity.
I-PEGylation okanye ezinye iinguqulelo zeproteni zitshintsha indlela ezisebenzisana ngayo ne-membrane, nto leyo echaphazela i-TMP efunekayo ukuze kubekho ukugeleza okufunekayo.

3.3. Ukubeka iliso kunye nokuphucula i-TMP

Ukugcina i-TMP ngaphakathiuluhlu oluqhelekileyo loxinzelelo lwe-transmembraneKubalulekile ekusebenzeni kakuhle kwe-membrane ye-ultrafiltration kunye nomgangatho wemveliso. Njengoko i-ultrafiltration iqhubeka, i-concentration polarization kunye ne-fouling zinokubangela ukuba i-TMP inyuke, ngamanye amaxesha ngokukhawuleza.

Iindlela zokuBeka eSweni:

Ukubeka esweni ngexesha langempela:I-TMP ilandelwa ngokungena, ukuvala, kunye nokungena ngaphakathiabathumeli boxinzelelo.
I-Raman Spectroscopy:Isetyenziselwa ukujonga ngokungangenisi ubuninzi beeproteni kunye nee-excipient, iququzelela ulawulo lwe-TMP oluhambelanayo ngexesha lokuhluza nge-ultrafiltration kunye nokuhluza nge-diafiltration.
Ulawulo oluPhambili:Ii-Extended Kalman Filters (EKF) zinokucubungula idatha yesensa, zilungise i-TMP ngokuzenzekelayo ukuze kuthintelwe ukungcoliswa kakhulu.
Misela i-TMP yokuqala ngaphakathi koluhlu oluqhelekileyo:Ingabi phantsi kakhulu ukuze kuncitshiswe ukugeleza kwamanzi, ingabi phezulu kakhulu ukuze kuthintelwe ukungcoliswa ngokukhawuleza.
Lungisa i-TMP njengoko i-viscosity isanda:Ngexesha lesigaba soxinzelelo lwe-ultrafiltration, nyusa i-TMP kancinci kancinci kuphela xa kufuneka.
Ulawulo lokuhamba kokutya kunye ne-pH:Ukwandisa ukuhamba kokutya okanye ukunciphisa i-TMP kunciphisa ukuxinana koxinzelelo kunye nokungcola.
Ukucocwa nokutshintshwa kwe-membrane:Ii-TMP eziphezulu zinxulunyaniswa nokucocwa rhoqo kunye nokuncipha kobomi be-membrane.

Ukuphucula Amaqhinga:

Imizekelo:

Ukungcoliswa komhlwa kwimigca yokucubungula iiproteni kukhokelela ekwandeni kwe-TMP kunye nokuncipha kokuhamba kwamanzi, okufuna ukucocwa okanye ukutshintshwa kwe-membrane ukuze kubuyiselwe ukusebenza okuqhelekileyo.
Unyango lwangaphambi kwe-enzyme (umz., ukongezwa kwe-pectinase) lunokunciphisa i-TMP kwaye lwandise ubomi be-membrane ngexesha le-high-viscosity rapeseed protein ultrafiltration.

3.4. I-TMP kwiinkqubo ze-TFF

I-Tangential (transverse) flow filtration (TFF) isebenza ngokuhambisa isisombululo se-feed kwi-membrane endaweni yokusidlula ngqo kuyo, nto leyo echaphazela kakhulu i-TMP dynamics.

Ulawulo kunye neBalance ye-TMP

Uxinzelelo lwe-transmembrane ye-TFF (i-TFF TMP):Ilawulwa ngokulawula isantya sokuhamba kokutya kunye noxinzelelo lwepompo ukuze kuthintelwe i-TMP egqithisileyo ngelixa kusetyenziswa kakhulu i-permeating flux.
Ukuphucula iiparameter:Ukwanda kokuhamba kokutya kunciphisa ukubekwa kweeproteni kwindawo ethile, kuzinzise i-TMP, kwaye kunciphisa ukungcola kwe-membrane.
Umodeli wekhompyutha:Iimodeli zeCFD ziqikelela kwaye ziphucula i-TFF TMP ukuze imveliso ibuyiselwe, icoceke, kwaye ivelise imveliso—ingakumbi ebaluleke kakhulu kwiinkqubo ezifana ne-mRNA okanye i-extracellular vesicle isolation.

Imizekelo:

Kwi-bioprocessing, i-TFF TMP efanelekileyo ivelisa ukubuyiswa kwe-mRNA engaphezulu kwama-70% ngaphandle kokubola, nto leyo ephumelela kakhulu kuneendlela ze-ultracentrifugation.
Ulawulo lwe-TMP oluhambelanayo, olusekelwe kwiimodeli zezibalo kunye nempendulo yenzwa, lunciphisa ukuphindaphinda kokutshintshwa kwe-membrane kwaye luphucula ubomi be-membrane ngokunciphisa i-fouling.

Izinto ezibalulekileyo ekufuneka uziqwalasele:

Uxinzelelo lwe-TMP transmembrane kufuneka lulawulwe ngokukhutheleyo kwi-TFF ukuze kugcinwe ukusebenza kakuhle kwenkqubo, ukuguquguquka, kunye nempilo ye-membrane.
Ukulungiswa kwe-TMP okucwangcisiweyo kunciphisa iindleko zokusebenza, kuxhasa ukubuyiswa kwemveliso ecocekileyo kakhulu, kwaye kwandisa ubomi be-membrane kwi-protein ultrafiltration kunye neenkqubo ezinxulumene nayo.

Jonga kwaye Ulinganise Uxinzelelo lweeProtheyini Eziphezulu

Iindlela Zokungcolisa Ubudlelwane Bazo Nokuxinana

Iindlela Eziphambili Zokungcolisa Kwi-Protein Ultrafiltration

Ukucoca iiproteni kakhulu kuchaphazeleka ziindlela ezahlukeneyo zokungcolisa:

Ungcoliseko loMhlwa:Kwenzeka xa iimveliso zokugqwala—ngokuqhelekileyo ii-iron oxides—ziqokelelana phezu kwe-membrane. Ezi zinciphisa ukugeleza kwaye kunzima ukuzisusa ngee-arhente zokucoca iikhemikhali eziqhelekileyo. Ukungcoliswa komhlwa kukhokelela ekulahlekelweni rhoqo kokusebenza kwe-membrane kwaye kwandisa amaxesha okutshintshwa kwe-membrane ngokuhamba kwexesha. Impembelelo yayo inzima ngakumbi kwi-PVDF kunye ne-PES membranes ezisetyenziswa kunyango lwamanzi kunye nokusetyenziswa kweproteni.

Ukungcola kwezinto eziphilayo:Ibangelwa kakhulu ziiproteni ezifana ne-bovine serum albumin (BSA), kwaye inokwandiswa xa kukho ezinye izinto eziphilayo ezifana neepolysaccharides (umz., i-sodium alginate). Iindlela ziquka ukufunxwa kwi-membrane pores, ukuvaleka kwe-pore, kunye nokwakheka komaleko wekhekhe. Iziphumo ze-synergistic zenzeka xa kukho izinto ezininzi eziphilayo, kunye neenkqubo ezixutyiweyo ezifumana ukungcola okukhulu kunezo zeproteni enye.

Ukwahlulahlula koxinzelelo:Njengoko i-ultrafiltration iqhubeka, iiproteni ezigciniweyo ziqokelelana kufutshane nomphezulu we-membrane, nto leyo enyusa uxinano lwendawo kunye ne-viscosity. Oku kudala umaleko we-polarisation ophucula ukuthambekela kokungcolisa kwaye unciphise ukugeleza. Le nkqubo ikhawuleza njengoko isigaba soxinano lwe-ultrafiltration siqhubeka, sichatshazelwa ngokuthe ngqo luxinzelelo lwe-transmembrane kunye ne-flow dynamics.

Ukungcola kweColloidal kunye neMixed-Foulant:Izinto ezibizwa ngokuba yiColloidal matter (umz., isilika, iiminerali ezingaphiliyo) zinokusebenzisana neeproteni, zenze iileya ezintsonkothileyo ezidibanisa izinto ezibangela ukuba i-membrane ingcole. Ukubakho kwe-colloidal silica, umzekelo, kunciphisa kakhulu amazinga okuhamba kwamanzi, ingakumbi xa zidityaniswe nezinto eziphilayo okanye phantsi kweemeko ze-pH ezingagqibelelanga.

Impembelelo yoBungqingqwa beSombululo kuPhuhliso lweNgcoliso

I-viscosity yezisombululo zeproteni ichaphazela kakhulu i-fouling kinetics kunye ne-membrane compaction:

Ukungcola okukhawulezileyo:I-viscosity ephezulu yesisombululo seproteni inyusa ukumelana nokuhanjiswa kwee-solutes ezigciniweyo, nto leyo enceda ukwakheka kongqimba lwekhekhe ngokukhawuleza. Oku kwandisa uxinzelelo lwe-transmembrane (TMP), kukhawulezise ukuxinana kwe-membrane kunye nokungcoliswa kwayo.

Iziphumo zoBume beSisombululo:Uhlobo lweproteni lutshintsha i-viscosity; iiproteni ze-globular (umz., i-BSA) kunye neeproteni ezinde ziziphatha ngendlela eyahlukileyo malunga nokuhamba kunye ne-polarization. Ukongeza iikhompawundi ezifana nee-polysaccharides okanye i-glycerol kuphakamisa kakhulu i-viscosity, okukhuthaza ukungcoliswa. Izongezo kunye nokuhlanganiswa kweproteni kumanqanaba aphezulu kuqinisa ngakumbi isantya apho ii-membranes zivala khona, kunciphisa ngokuthe ngqo ukuhamba kunye nobomi be-membrane.

Iziphumo Zokusebenza:Ukuxinana okuphezulu kufuna i-TMP eyongeziweyo ukuze kugcinwe amazinga okuhluza kwiinkqubo zokuhluza ezihamba ngokunqamlezayo. Ukuvezwa ixesha elide kwi-TMP ephezulu kunyusa ukungcola okungenakuguqulwa, okuhlala kufuna ukucocwa rhoqo kwe-membrane okanye ukutshintshwa kwe-membrane kwangoko.

Indima yeempawu zesondlo

Iimpawu zokutya—ezifana neempawu zeproteni kunye nekhemikhali yamanzi—zichonga ubukhali bokungcola:

Ubungakanani kunye nokuSasazwa kweProtheyini:Iiproteni ezinkulu okanye ezidityanisiweyo zinotyekelo olukhulu lokubangela ukuvalwa kweembobo kunye nokwakheka kweekhekhe, nto leyo ephakamisa ukuxinana kunye nokuxinana ngexesha loxinzelelo lweeproteni ezifakwe kwi-ultrafiltration.

i-pH:I-pH ephezulu inyusa ukugxojwa kwe-electrostatic, ithintela iiproteni ukuba zingahlangani kufutshane ne-membrane, ngaloo ndlela inciphisa ukugxojwa. Ngokwahlukileyo koko, iimeko ze-asidi zinciphisa ukugxojwa, ingakumbi kwi-colloidal silica, nto leyo ebangela ukugxojwa kwe-membrane kube kubi kakhulu kwaye inciphise amazinga okugeleza.

Ubushushu:Amaqondo obushushu aphantsi enkqubo adla ngokunciphisa amandla e-kinetic, nto leyo enokunciphisa amazinga okungcolisa kodwa ikwanyusa nokuqina kwesisombululo. Amaqondo obushushu aphezulu akhawulezisa ukungcolisa kodwa anokuphucula nokusebenza kakuhle kokucoca.

Izinto ezingaphiliyo/ezingekho kwindalo:Ukubakho kwe-colloidal silica okanye iimetali kwandisa ukungcola, ingakumbi phantsi kweemeko ze-asidi. Iiparticles ze-silica zonyusa i-viscosity yesisombululo iyonke kwaye zivale ii-pores ngokwasemzimbeni, nto leyo eyenza ukuba uxinzelelo lwe-ultrafiltration lungasebenzi kakuhle kwaye lunciphise ubomi be-membrane iyonke kunye nokusebenza kwayo.

Ukwakhiwa kwe-Ionic:Ukongeza iintlobo ezithile ze-ionic (Na⁺, Zn²⁺, K⁺) kunokunciphisa ukungcoliswa ngokuguqula amandla e-electrostatic kunye nokufuma phakathi kweeproteni kunye nee-membranes. Nangona kunjalo, ii-ion ezifana neCa²⁺ zihlala zikhuthaza ukuhlangana kwaye zonyusa amandla okungcoliswa.

Imizekelo:

Ngexesha lokuhluza ukuhamba okuhambayo, ukutya okutyebileyo kwiiproteni ezinobunzima beemolekyuli eziphezulu kunye ne-viscosity ephezulu kuya kwehla ngokukhawuleza kokuhamba kwamanzi, ukunyuka kokucoca kunye nokutshintshwa kweenkqubo.
Xa amanzi okutya equlethe i-colloidal silica kwaye e-asidi, ukuhlanganiswa kwe-silica kunye nokufakwa kwayo kuyaqina, okunyusa kakhulu amazinga okungcolisa kunye nokunciphisa ukusebenza kwe-membrane.

Ngamafutshane, ukuqonda unxibelelwano phakathi kwe-viscosity yesisombululo, iintlobo ze-fouling, kunye neempawu zokutya kubalulekile ekuphuculeni uxinano lwe-ultrafiltration, ukunciphisa i-membrane fouling, kunye nokwandisa ubomi be-membrane.

Ukuqhekeka koxinzelelo kunye noLawulo lwayo

Yintoni i-Concentration Polarization?

I-Concentration polarization kukuqokelelwa kwendawo ye-retained solute—njengeeproteni—kwi-membrane/solution interface ngexesha lokuhluzwa kwe-ultrafiltration. Kwimeko yezisombululo zeproteni, njengoko ulwelo luhamba ngokuchasene ne-semi-permeable membrane, iiproteni ezingavunyelwanga yi-membrane zihlala ziqokelelana kumaleko omncinci okufutshane nomphezulu. Oku kwakheka kubangela i-gradient ye-concentration ephezulu: i-protein concentration ephezulu kanye kwi-membrane, iphantsi kakhulu kwisisombululo esikhulu. Le nto iyakwazi ukubuyiselwa kwaye ilawulwa yi-hydrodynamic forces. Iyahluka kwi-membrane fouling, equka ukufakwa okungapheliyo okanye ukufunxwa ngaphakathi okanye kwi-membrane.

Indlela i-Concentration Polarization eyonyusa ngayo i-Viscosity kunye ne-Fouling

Kumphezulu we-membrane, ukuqokelelana okuqhubekayo kweeproteni kwenza umaleko womda owandisa uxinzelelo lwe-solute lwendawo. Oku kunemiphumo emibini ebalulekileyo:

Ukwanda kobungqindilili bendawo:Njengoko uxinzelelo lweproteni lunyuka kufutshane ne-membrane, uxinano lwesisombululo seprotheni kule ndawo incinci nalo luyanda. Uxinano oluphezulu luthintela ukuhanjiswa kwe-solute ukusuka kwi-membrane, nto leyo eqinisa ngakumbi i-concentration gradient kwaye idale i-feedback loop yokumelana nokuhamba kwamanzi. Oku kubangela ukuncipha kokujikeleza kwamanzi kunye nesidingo samandla esiphezulu sokuqhubeka nokuhluza.

Ukuncedisa ukungcoliswa kwe-membrane:Ubuninzi beeproteni kufutshane ne-membrane bunyusa amathuba okuhlangana kweeproteni, kwaye kwezinye iinkqubo, ukwakheka kwe-gel layer. Le layer ithintela ii-membrane pores kwaye yandisa ukumelana nokuhamba kwamanzi. Ezi meko zilungele ukuqala kokungcola okungenakulungiseka, apho iiproteni zihlangana kwaye ukungcola kunamathela ngokwasemzimbeni okanye ngokweekhemikhali kwi-membrane matrix.

Uvavanyo lokufota (umz., i-electron microscopy) luqinisekisa ukuhlanganiswa ngokukhawuleza kwamaqela eeprotheyini ezincinci kwi-membrane, ezinokukhula zibe yi-deposits ebalulekileyo ukuba izicwangciso zokusebenza azilawulwa ngokufanelekileyo.

Amaqhinga okunciphisa ukuHlanganisa koXinzelelo

Ukulawula i-concentration polarization kwi-ultrafiltration protein concentration okanye i-transverse flow filtration kufuna indlela ephindwe kabini: ukulungisa i-hydrodynamics kunye nokulungisa iiparameter zokusebenza.

Ukuphucula isantya sokuhamba kombane:
Ukunyusa isantya sokuhamba kwe-cross-flow kwandisa ukuhamba kwe-tangential kwi-membrane, kukhuthaza ukucheba kunye nokunciphisa umaleko womda woxinzelelo. Ukucheba okunamandla ngakumbi kususa iiproteni eziqokelelweyo kumphezulu we-membrane, kunciphisa ukubola kunye nomngcipheko wokungcoliswa. Umzekelo, ukusebenzisa ii-static mixers okanye ukungenisa i-gas sparging kuphazamisa umaleko onyibilikayo, ngokukodwa ukuphucula ukuhamba kwe-permeate kunye nokusebenza kakuhle kwinkqubo yokucoca ukuhamba kwe-transverse.

Ukutshintsha iiParameters zokusebenza:

Uxinzelelo lwe-Transmembrane (TMP):I-TMP ngumahluko woxinzelelo kwi-membrane kunye namandla aqhubayo e-ultrafiltration. Nangona kunjalo, ukutyhala i-TMP phezulu ukuze kukhawuleziswe ukuhluzwa kunokubuyisela umva ngokuqinisa i-concentration polarization. Ukunamathela kuluhlu oluqhelekileyo loxinzelelo lwe-transmembrane—olungadluli kwimida ebekiweyo ye-protein ultrafiltration—kunceda ukuthintela ukwakheka kwe-solute egqithisileyo kunye nokwanda okunxulumene noko kwi-viscosity yendawo.

Izinga lokucheba:Isantya sokucheba, umsebenzi wesantya sokuhamba kwamanzi kunye noyilo lwetshaneli, idlala indima ephambili kwi-solute transport dynamics. I-high shear igcina umaleko we-polarization uncinci kwaye uhamba, okuvumela ukuhlaziywa rhoqo kwendawo eyomileyo kufutshane ne-membrane. Ukwanda kwesantya sokucheba kunciphisa ixesha ekufuneka iiproteni ziqokelele kwaye kunciphisa ukunyuka kwe-viscosity kwindawo yokujonga.

Iipropati Zokutya:Ukulungisa iipropati zesisombululo seprotheyini esingenayo—njengokunciphisa i-viscosity yesisombululo seprotheyini, ukunciphisa umxholo opheleleyo, okanye ukulawula i-pH kunye namandla e-ionic—kunokunceda ukunciphisa ubungakanani kunye nempembelelo ye-concentration polarization. Unyango lwangaphambi kokutyisa kunye notshintsho lwefomyula lunokuphucula ukusebenza kwe-membrane ye-ultrafiltration kwaye lwandise ubomi be-membrane ngokunciphisa i-frequency yokucoca i-membrane.

Umzekelo weSicelo:
Isityalo esisebenzisa i-tangential flow filtration (TFF) ukugxininisa ii-antibodies ze-monoclonal sisebenzisa isantya esilungelelanisiweyo se-cross-flow kwaye sigcina i-TMP ngaphakathi kwefestile engqongqo. Ngokwenza njalo, abaqhubi banciphisa i-concentration polarization kunye ne-membrane fouling, benciphisa zombini i-membrane replacement frequency kunye ne-cleaning cycles—inciphisa ngokuthe ngqo iindleko zokusebenza kunye nokuphucula isivuno semveliso.

Ukulungiswa ngokufanelekileyo kunye nokubekwa esweni kwezi zinto ziguquguqukayo—kuquka umlinganiselo we-viscosity yesisombululo seprotheyini ngexesha langempela—zibalulekile ekuphuculeni ukusebenza koxinzelelo lwe-ultrafiltration kunye nokunciphisa iziphumo ezimbi ezinxulumene ne-concentration polarization ekucutshungulweni kweprotheyini.

Ukuphucula i-Ultrafiltration kwiZisombululo zeProtein eziNamaqondo aphezulu

6.1. Iindlela ezilungileyo zokusebenza

Ukugcina ukusebenza kakuhle kwe-ultrafiltration ngezisombululo zeprotheyini eziphezulu kufuna ibhalansi ethambileyo phakathi koxinzelelo lwe-transmembrane (TMP), uxinzelelo lweprotheyini, kunye ne-viscosity yesisombululo. I-TMP—umahluko kuxinzelelo kulo lonke i-membrane—ichaphazela ngokuthe ngqo izinga loxinzelelo lweprotheyini ye-ultrafiltration kunye nenqanaba lokungcoliswa kwe-membrane. Xa kusetyenzwa izisombululo ezingcolisayo ezifana nee-antibodies ze-monoclonal okanye iiprotheyini ze-serum ezingcolisayo eziphezulu, naluphi na ukwanda okugqithisileyo kwe-TMP kunokunyusa ukugeleza ekuqaleni, kodwa kukwakhawulezisa ngokukhawuleza ukungcoliswa kunye nokuqokelelwa kweprotheyini kumphezulu we-membrane. Oku kukhokelela kwinkqubo yokucoca ebuthathaka nengazinzanga, eqinisekiswe zizifundo zemifanekiso ezibonisa iileya zeprotheyini ezixineneyo ezenza kwi-TMP ephezulu kunye noxinzelelo lweprotheyini ngaphezulu kwe-200 mg/mL.

Indlela efanelekileyo ibandakanya ukuqhuba inkqubo kufutshane, kodwa ingadluli, i-TMP ebalulekileyo. Okwangoku, imveliso iyanda kodwa umngcipheko wokungcoliswa okungenakuguqulwa uhlala umncinci. Kwi-viscosities ephezulu kakhulu, iziphumo zakutshanje zibonisa ukunciphisa i-TMP kunye nokunyusa ukuhamba kokutya ngaxeshanye (i-transverse flow filtration) ukunceda ukunciphisa i-concentration polarization kunye ne-protein deposition. Umzekelo, izifundo kwi-Fc-fusion protein concentration zibonisa ukuba useto oluphantsi lwe-TMP lunceda ukugcina i-flux ezinzileyo ngelixa kunciphisa ukulahleka kwemveliso.

Ukwanda kancinci kancinci nangokucwangcisiweyo koxinzelelo lweproteni ngexesha lokuhluzwa kwe-ultrafiltration kubalulekile. Amanyathelo okuhluzwa ngequbuliso anokunyanzela isisombululo ukuba singene kwinkqubo ye-viscosity ephezulu ngokukhawuleza, okwandisa umngcipheko wokuhlanganiswa kunye nobunzima bokungcola. Endaweni yoko, ukunyusa amanqanaba eprotheni ngokuthe ngcembe kuvumela ukuba iiparameter zenkqubo ezifana ne-TMP, isantya sokuhamba kwe-cross-flow, kunye ne-pH zilungiswe ngaxeshanye, zinceda ukugcina uzinzo lwenkqubo. Izifundo zetyala le-enzyme ultrafiltration ziqinisekisa ukuba ukugcina uxinzelelo oluphantsi lokusebenza ngeli xesha kuqinisekisa ukwanda okulawulwayo koxinzelelo, kunciphisa ukwehla kokuguquguquka ngelixa kukhuselwa ukuthembeka kwemveliso.

6.2. Ukuphindaphinda kunye nokugcinwa kweMembrane

Ukuphindaphinda kokutshintshwa kwe-membrane kwi-ultrafiltration kunxulunyaniswa kakhulu nezibonakaliso zokungcoliswa kunye nokuncipha kokuhamba kwamanzi. Endaweni yokuxhomekeka kuphela ekunciphiseni kokuhamba kwamanzi njengesalathisi sokuphela kobomi, ukujonga ukumelana nokungcoliswa okuthile—umlinganiselo wobungakanani omele ukumelana okubangelwa zizinto eziqokelelweyo—kuye kwabonakala kuthembekile ngakumbi, ngakumbi kwi-mixed-protein okanye i-protein-polysaccharide feeds, apho ukungcoliswa kunokwenzeka ngokukhawuleza nangokuqatha.

Ukubeka esweni ezinye izalathisi zokungcolisa kubalulekile. Iimpawu ezibonakalayo zokuma komphezulu, ukuhamba kwamanzi okungenakulinganiswa, okanye ukunyuka okuqhubekayo kwe-TMP (nangona kucocwa) zonke ziimpawu zesilumkiso zokungcolisa okuphambili okuphambi kokusilela kwe-membrane. Iindlela ezifana nokulandelela i-modified fouling index (MFI-UF) kunye nokuyidibanisa nokusebenza kwe-membrane kuvumela ukucwangciswa kwangaphambili kokutshintshwa endaweni yokutshintsha okusebenzayo, ngaloo ndlela kunciphisa ixesha lokungasebenzi kunye nokulawula iindleko zokulungisa.

Ukuthembeka kwe-membrane akuphazamisi nje kuphela ngenxa yokwakheka kwe-organic foluant kodwa kwanangenxa yokugqwala, ingakumbi kwiinkqubo ezisebenza kwi-pH ephezulu okanye ezinomlinganiselo ophezulu wetyuwa. Ukuhlolwa rhoqo kunye neenkqubo zokucoca iikhemikhali kufuneka zenziwe ukulawula ukugqwala kunye nokufakwa kwe-foluant. Xa kujongwa ukungcoliswa okunxulumene nokugqwala, amaxesha okucoca i-membrane kunye namaxesha okutshintshwa kufuneka ahlengahlengiswe ukuqinisekisa ukuba i-membrane ihlala ixesha elide kunye nokusebenza kwe-membrane okuhlala ixesha elide. Ukugcinwa okupheleleyo nokucwangcisiweyo kubalulekile ekunciphiseni impembelelo yale miba kunye nokwandisa ukusebenza ngempumelelo.

6.3. Ulawulo lweNkqubo kunye noLinganiselo lwe-Inline Viscosity

Ukulinganisa ngokuchanekileyo nangexesha langempela kwe-viscosity yesisombululo seprotheyini kubalulekile kulawulo lwenkqubo kwi-ultrafiltration, ngakumbi njengoko uxinaniso kunye ne-viscosities zisanda. Iinkqubo zokulinganisa i-viscosity ezikwi-inline zibonelela ngokubekwa esweni okuqhubekayo, zivumela impendulo kwangoko kwaye zivumela uhlengahlengiso oluguquguqukayo kwiiparameter zenkqubo.

Iitekhnoloji ezintsha zitshintshe indlela yokulinganisa i-viscosity yesisombululo seprotheni:

I-Raman Spectroscopy kunye neKalman Filtering: Uhlalutyo lweRaman lwexesha langempela, oluxhaswa zizihluzi zeKalman ezandisiweyo, luvumela ukulandelelwa okuqinileyo koxinzelelo lweproteni kunye nokwakheka kwe-buffer. Le ndlela yonyusa uvakalelo kunye nokuchaneka, ixhasa ukwenziwa ngokuzenzekelayo kwenkqubo yoxinzelelo lwe-ultrafiltration kunye ne-diafiltration.

I-Automated Kinematic Capillary Viscometry: Isebenzisa umbono wekhompyutha, le teknoloji ilinganisa ngokuzenzekelayo i-viscosity yesisombululo, yoyisa iimpazamo ezenziwe ngesandla kwaye inikezela ngokubekwa esweni okuphindaphindwayo, okuphindaphindwayo kwimijelo yenkqubo emininzi. Iqinisekisiwe kuzo zombini iifomyula zeproteni eziqhelekileyo nezintsonkothileyo kwaye inciphisa ukungenelela ngexesha lesigaba soxinzelelo lwe-ultrafiltration.

Izixhobo zeMicrofluidic Rheology: Iinkqubo zeMicrofluidic zibonelela ngeeprofayili ze-rheological ezineenkcukacha, eziqhubekayo, kwanakwizisombululo zeproteni ezingezizo ezeNewtonian, ezine-viscosity ephezulu. Ezi zibaluleke kakhulu ekuvelisweni kwamayeza, ukuxhasa amaqhinga eteknoloji yohlalutyo lwenkqubo (i-PAT) kunye nokudibanisa ne-feedback loops.

Ulawulo lwenkqubo olusebenzisa ezi zixhobo luvumela ukusetyenziswa kwee-feedback loops zokulungisa i-TMP ngexesha langempela, isantya sokutya, okanye isantya sokuhamba kwe-crossflow ukuphendula utshintsho lwe-viscosity. Umzekelo, ukuba i-inline sensing ibona ukunyuka ngequbuliso kwe-viscosity (ngenxa yokwanda kwe-concentration okanye ukuhlanganiswa), i-TMP inokunciphisa ngokuzenzekelayo okanye isantya sokuhamba kwe-crossflow sinyuswe ukunciphisa ukuqala kwe-concentration polarization kwi-ultrafiltration. Le ndlela ayigcini nje ngokwandisa ubomi be-membrane kodwa ikwaxhasa umgangatho wemveliso ohambelanayo ngokulawula izinto ezichaphazela i-viscosity yezisombululo zeprotheni ngokuguquguqukayo.

Ukukhethwa kwetekhnoloji yokujonga i-viscosity efanelekileyo kuxhomekeke kwiimfuno ezithile zesicelo se-ultrafiltration, kubandakanya uluhlu lwe-viscosity olulindelekileyo, ubunzima bokwenziwa kweproteni, iimfuno zokudibanisa, kunye neendleko. Olu phuculo ekujongeni ngexesha langempela kunye nolawulo lwenkqubo oluguqukayo luphucule kakhulu amandla okwenza ngcono i-ultrafiltration kwizisombululo zeprotheni ezine-viscosity ephezulu, ukuqinisekisa uzinzo lokusebenza kunye nemveliso ephezulu.

Ukusombulula iingxaki kunye neengxaki eziqhelekileyo kwi-Protein Ultrafiltration

7.1. Iimpawu, Oonobangela, kunye namayeza

Uxinzelelo lwe-Transmembrane olunyukileyo

Ukunyuka koxinzelelo lwe-transmembrane (TMP) ngexesha lokuhluzwa kwe-ultra-filtration kubonisa ukuxhathisa okukhulayo kwi-membrane. Iziphumo zoxinzelelo lwe-transmembrane kwi-ultra-filtration zithe ngqo: uluhlu oluqhelekileyo loxinzelelo lwe-transmembrane luhlala luxhomekeke kwinkqubo, kodwa ukunyuka okuqhubekayo kufanele kuphandwe. Zimbini izizathu eziqhelekileyo ezibalaseleyo:

Isisombululo seproteni esinoburhabaxa obuphezulu:Njengoko i-viscosity yezisombululo zeproteni isanda—ngokuqhelekileyo xa i-ultrafiltration concentration ephezulu yeproteni—uxinzelelo olufunekayo ukuze kubekho ukugeleza luyanda. Oku kuvakala kumanqanaba okugqibela okuxinezeleka kunye nokuphuma kwamanzi apho izisombululo zixineneyo kakhulu.
Ukungcola kwe-membrane:Izinto ezibangela iifouli ezifana neeprotein aggregates okanye imixube yepolysaccharide-protein zinokunamathela okanye zivale iipores zemembrane, nto leyo ebangela ukunyuka kwe-TMP ngokukhawuleza.

Amayeza:

Yehlisa i-TMP kwaye wonyuse ukuhamba kokutya: Ukunciphisa i-TMP ngelixa kunyusa isantya sokutya kunciphisa i-concentration polarization kunye nokwakheka komaleko wejeli, okukhuthaza ukujiya okuzinzileyo.
Ukucoca rhoqo i-membrane: Misela ixesha elifanelekileyo lokucoca i-membrane ukuze kususwe izinto ezingafunekiyo eziqokelelweyo. Jonga ukusebenza kakuhle ngokulinganisa i-viscosity yesisombululo seproteni emva kokucoca.
Buyisela ii-membranes ezigugayo: Ukwanda kokuphindaphinda kokutshintshwa kwe-membrane kungafuneka ukuba ukucoca akwanelanga okanye ixesha lokuphelelwa yi-membrane lifikelelwe.

Ukwehla kweFlux Rate: Umthi wokuxilonga

Ukwehla rhoqo kokuguquguquka ngexesha lesigaba soxinzelelo lwe-ultrafiltration kubonisa ukuba kukho iingxaki zokuvelisa. Landela le ndlela yokuxilonga:

Jonga i-TMP kunye ne-viscosity:Ukuba zombini zinyukile, jonga ukuba azikho na izinto ezingcolisayo okanye kukho umaleko wejeli.
Hlola ukwakheka kokutya kunye ne-pH:Utshintsho apha lunokutshintsha i-viscosity yezisombululo zeprotheyini kwaye lukhuthaze ukungcoliswa.
Vavanya ukusebenza kwe-membrane:Ukuncipha kokuhamba kwamanzi nangona kucocwa imiqondiso yokuba kunokwenzeka ukuba kubekho umonakalo kwi-membrane okanye ukungcola okungenakuguqulwa.

Izisombululo:

Lungiselela ubushushu, i-pH, kunye namandla e-ionic ekutyeni ukuze kuncitshiswe ukungcoliswa kunye nokuqhekeka koxinzelelo kwi-ultrafiltration.
Sebenzisa iimodyuli ze-membrane eziguqulwe ngumphezulu okanye ezijikelezayo ukuze uphazamise iileya zejeli kwaye ubuyisele ukuhamba kwamanzi.
Yenza umlinganiselo wesiqhelo we-viscosity yesisombululo seprotheni ukuze uqikelele utshintsho oluchaphazela ukuhamba kwamanzi.

Ukwenziwa kweLayer yeGel okanye iRapid Fouling

Ukwakheka ngokukhawuleza komaleko wejeli kubangelwa kukuqhekeka okukhulu koxinzelelo kumphezulu we-membrane. Uxinzelelo lwe-transmembrane ye-transverse flow filtration (TFF) luchaphazeleka ngakumbi phantsi kweemeko zokutya ezine-viscosity ephezulu okanye ezine-protein ephezulu.

Amaqhinga okunciphisa le meko:

Faka imiphezulu ye-membrane ethanda ukunyibilika emanzini, etshajelwe kakubi (umz., ii-membrane ze-Polyvinylidene fluoride [PVDF]) ukuze unciphise ukubopha kunye nokunamathela kweproteni.
Yitya ngaphambi kokuba uyinyange usebenzisa i-coagulation okanye i-electrocoagulation ukuze ususe izinto ezingcolisa kakhulu ngaphambi kokuba i-ultrafiltration iqale.
Hlanganisa izixhobo zoomatshini ezifana neemodyuli ezijikelezayo kwinkqubo yokucoca ukuhamba okunqamlezileyo ukunciphisa ubukhulu beleya yekhekhe kunye nokulibazisa ukwakheka kwebeleya yejeli.

7.2. Ukulungelelanisa Ukuguquguquka Kwesondlo

Iinkqubo zokucoca iiproteni ultrafiltration kufuneka zilungelelanise nokuguquguquka kweempawu okanye ukwakheka kweeproteni zokutya. Izinto ezichaphazela ukuxinana kwezisombululo zeeproteni—ezifana nokwakheka kwe-buffer, uxinano lweeproteni, kunye nokuthambekela kokuhlanganiswa—zinokutshintsha indlela esebenza ngayo inkqubo.

Amaqhinga okuphendula

Ukubeka esweni i-viscosity kunye nokwakheka kwayo ngexesha langempela:Sebenzisa izinzwa zohlalutyo ezikwi-intanethi (i-Raman spectroscopy + i-Kalman filtering) ukuze kufunyanwe ngokukhawuleza utshintsho lokutya, iindlela ze-UV okanye ze-IR ezisebenza ngcono kunezinye.
Ulawulo lwenkqubo oluhambelanayo:Lungisa useto lweparameter (umgangatho wohambo, TMP, ukukhethwa kwe-membrane) ukuphendula utshintsho olufunyenweyo. Umzekelo, ukwanda kwe-viscosity yesisombululo seprotheni kunokufuna i-TMP ephantsi kunye namazinga aphezulu okucheba.
Ukukhetha i-membrane:Sebenzisa ii-membrane ezinobukhulu beembobo kunye ne-chemistry yomphezulu elungiselelwe iipropati zokutya zangoku, ukulinganisela ukugcinwa kweproteni kunye nokuguquguquka.
Unyango lwangaphambi kokutyisa:Ukuba utshintsho olukhawulezileyo kwindlela ukutya okutyiwa ngayo lubangela ukungcola, faka amanyathelo okujiya okanye okucoca phezulu kwe-ultrafiltration.

Imizekelo:

Kwi-bioprocessing, ii-buffer switches okanye utshintsho kwi-antibody aggregates kufuneka lubangele uhlengahlengiso lwe-TMP kunye nokuhamba kwegazi ngenkqubo yolawulo.
Kwi-ultrafiltration edityaniswe ne-chromatography, ii-adaptive mixing-integer optimization algorithms zinokunciphisa ukuguquguquka kwaye zinciphise iindleko zokusebenza ngelixa zigcina ukusebenza kwe-ultrafiltration membrane.

Ukulandelela rhoqo ukulinganiswa kwe-viscosity yesisombululo seprotheni kunye nokulungiswa kwangoko kweemeko zenkqubo kunceda ukuphucula uxinaniso lwe-ultrafiltration, ukugcina ukuphuma kwayo, kunye nokunciphisa ukungcola kwe-membrane kunye ne-concentration polarization.

Imibuzo ebuzwa qho

8.1. Luluphi udidi oluqhelekileyo loxinzelelo lwe-transmembrane kwisisombululo se-ultrafiltration seprotheyini?

Uluhlu oluqhelekileyo loxinzelelo lwe-transmembrane (TMP) kwiinkqubo zoxinzelelo lwe-ultrafiltration protein luxhomekeke kuhlobo lwe-membrane, uyilo lwemodyuli, kunye neempawu zokutya. Kwiinkqubo ezininzi ze-protein ultrafiltration, i-TMP idla ngokugcinwa phakathi kwe-1 ukuya kwi-3 bar (15–45 psi). Amaxabiso e-TMP angaphezu kwe-0.2 MPa (malunga ne-29 psi) anokubeka emngciphekweni umonakalo we-membrane, ukungcola okukhawulezileyo, kunye nobomi obufutshane be-membrane. Kwizicelo ze-biomedical kunye ne-bioprocessing, i-TMP ecetyiswayo ngokubanzi akufuneki idlule kwi-0.8 bar (~12 psi) ukuthintela ukuqhekeka kwe-membrane. Kwiinkqubo ezifana nokuhluzwa kwe-transverse flow, ukuhlala ngaphakathi kolu luhlu lwe-TMP kukhusela zombini isivuno kunye nokuthembeka kweproteni.

8.2. I-viscosity yezisombululo zeprotheni iyichaphazela njani ukusebenza kwe-ultrafiltration?

I-viscosity yesisombululo seprotheyini ichaphazela ngokuthe ngqo ukusebenza koxinzelelo lwe-ultrafiltration. I-viscosity ephezulu inyusa ukumelana nokuhamba kwamanzi kwaye iphakamisa i-TMP, nto leyo ebangela ukuncipha kokujikeleza kwamanzi kunye nokungcoliswa kwe-membrane ngokukhawuleza. Esi siphumo sibonakala nge-monoclonal antibodies okanye ii-Fc-fusion proteins kwi-concentration ephezulu, apho i-viscosity iyanda ngenxa yokusebenzisana kweprotheyini kunye neprotheyini kunye nemiphumo yokutshaja. Ukulawula kunye nokwenza ngcono i-viscosity ngee-excipients okanye unyango lwe-enzymes kuphucula i-flux, kunciphisa i-foul, kwaye kuvumela uxinano oluphezulu olunokwenzeka ngexesha lesigaba soxinano lwe-ultrafiltration. Ukubeka esweni ukulinganiswa kwe-viscosity yesisombululo seprotheyini kubalulekile ekugcineni ukucutshungulwa okusebenzayo.

8.3. Yintoni i-concentration polarization kwaye kutheni ibalulekile kwi-TFF?

Ukuhluzwa koxinzelelo kwi-ultrafiltration kukuqokelelana kweeproteni kumphezulu we-membrane, okubangela i-gradient phakathi kwesisombululo esikhulu kunye ne-interface ye-membrane. Kwi-transverse flow filtration, oku kukhokelela ekwandeni kwe-viscosity yendawo kunye nokwehla kwe-flux okunokuguqulwa. Ukuba ayilawulwa, inokukhuthaza ukungcoliswa kwe-membrane kwaye inciphise ukusebenza kakuhle kwenkqubo. Ukujongana ne-concentration polarization kwi-ultrafiltration kubandakanya ukuphucula amazinga okuhamba kwe-cross-flow, i-TMP, kunye nokukhetha i-membrane ukugcina umaleko omncinci we-polarization. Ulawulo oluchanekileyo lugcina i-throughput iphezulu kwaye umngcipheko we-foil uphantsi.

8.4. Ndingasigqiba njani ukuba ndiza kuyitshintsha nini i-membrane yam ye-ultrafiltration?

Buyisela i-membrane ye-ultrafiltration xa ubona ukwehla okuphawulekayo kokuphuma kwayo (i-flux), ukunyuka okuqhubekayo kwe-TMP okungenakusombululwa kukucoca okuqhelekileyo, okanye ukungcola okubonakalayo okushiyekileyo emva kokucoca. Ezinye izalathiso ziquka ukulahleka kokukhetha (ukungaphumeleli ukwala iiproteni ezijoliswe kuzo njengoko kulindelekile) kunye nokungakwazi ukufikelela kwiinkcukacha zokusebenza. Ukubeka esweni imvamisa yokutshintshwa kwe-membrane ngokujikeleza rhoqo kunye novavanyo lokukhetha sisiseko sokwandisa ubomi be-membrane kwiinkqubo zoxinzelelo lwe-protein solution ultrafiltration.

8.5. Zeziphi iiparameter zokusebenza endinokuzilungisa ukuze ndinciphise ukungcoliswa kweeproteni kwi-TFF?

Iiparamitha eziphambili zokusebenza ukunciphisa ukungcola kweproteni kwi-transverse flow filtration ziquka:

Gcina isantya sokuhamba kwamanzi esifanelekileyo ukuze unciphise ukwakheka kweeproteni kwindawo ethile kwaye ulawule ukuxinana koxinzelelo.
Sebenza ngaphakathi koluhlu lwe-TMP olucetyiswayo, ngesiqhelo i-3–5 psi (0.2–0.35 bar), ukuthintela ukuvuza okugqithisileyo kwemveliso kunye nomonakalo we-membrane.
Sebenzisa iindlela zokucoca i-membrane rhoqo ukuze unciphise ukungcola okungalungisekiyo.
Jonga kwaye, ukuba kuyimfuneko, lungisa isisombululo sokutya kwangaphambili ukuze ulawule i-viscosity (umzekelo, usebenzisa unyango lwe-enzymes olufana ne-pectinase).
Khetha izinto ze-membrane kunye nobukhulu beembobo (MWCO) ezifanelekileyo kubungakanani beprotheyini ekujoliswe kuyo kunye neenjongo zenkqubo.

Ukudibanisa i-hydrocyclone prefiltration okanye i-enzymatic pretreatment kunokuphucula ukusebenza kwenkqubo, ingakumbi kwii-feeds ezine-viscosity ephezulu. Landelela ngokusondeleyo ukwakheka kwe-feed kwaye ulungise useto ngendlela eguquguqukayo ukuze unciphise ukungcola kwe-membrane kwaye ulungiselele isigaba soxinzelelo lwe-ultrafiltration.