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Nanoparticle Removal and Brownian Diffusion by Virus Removal Filters: Theoretical and Experimental Study

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Nanoparticle Removal and Brownian Diffusion by Virus Removal Filters: Theoretical and Experimental Study UPTEC K17 017 Examensarbete 30 hp November 2017 Nanoparticle Removal and Brownian Diffusion by Virus Removal Filters: Theoretical and Experimental Study Olof Gustafsson Abstract Nanoparticle Removal and Brownian Diffusion by Virus Removal Filters: Theoretical and Experimental Study Olof Gustafsson Teknisk- naturvetenskaplig fakultet UTH-enheten This study aims to examine the throughput of nanoparticles through a Cladophora cellulose based virus removal filter. The effect of Besöksadress: Brownian motion and flow velocity on the retention of 5 nm gold Ångströmlaboratoriet Lägerhyddsvägen 1 nanoparticles, 12.8 nm dextran nanoparticles and 28 nm phiX174 Hus 4, Plan 0 bacteriophages was examined through MATLAB simulations and filtration experiments. Modeling of Brownian motion at different flow velocities Postadress: was performed in MATLAB by solving the Langevin equation for particle Box 536 751 21 Uppsala position and velocity for all three types of particles. The motion of all three particle types was shown to be constrained at local flow Telefon: velocities of 1e-2 m/s or greater. The constraint was greatest for 018 – 471 30 03 phiX174 bacteriophages, followed by dextran particles and then gold Telefax: particles as a result of particle diameter. To verify the effect 018 – 471 30 00 experimentally, virus removal filters were prepared with a peak pore width of 23 nm. Filtration experiments were performed at different Hemsida: flux values where gold and dextran particles did not exhibit any http://www.teknat.uu.se/student difference in retention between fluxes. However, a significant amount of gold and dextran particles were removed by the filter despite being smaller than the measured pore size. A decrease in retention with filtrated volume was observed for both particle types. Filtration of phiX174 bacteriophages exhibited a difference in retention at different fluxes, where all bacteriophages where removed at a higher flux. The results from both simulations and experiments suggest that the retentive mechanism in filtering is more complex than what can be described only by size exclusion sieving, Brownian diffusion and hydrodynamic constraint of particles. Handledare: Simon Gustafsson & Albert Mihranyan Ämnesgranskare: Kristofer Gamstedt Examinator: Peter Broqvist ISSN: 1650-8297, UPTEC K17 017 Populärvetenskaplig Sammanfattning Filtrering är ett viktigt sätt att avlägsna virus och ett avgörande steg vid framställningen av terapeutiska proteiner samt potentiellt en metod för rening av dricksvatten. Virus är en typ av mikroorganismer som är orsaken till en rad olika sjukdomar såsom hepatit, lunginflammation och vinterkräksjuka för att nämna några. Virus är väldigt små, där de minsta varianterna kan vara 18–20 nanometer i diameter, vilket är ungefär 3000 gånger mindre än tjockleken av ett hårstrå. Terapeutiska proteiner är en typ av läkemedel som används vid behandling av sjukdomar som orsakas av felaktigheter, brister eller frånvaro av proteiner som annars förekommer naturligt i kroppen. Dessa proteiner kan vara livsviktiga för vissa funktioner i kroppen och orsaken till dessa sjukdomar kan ibland vara genetisk. Ett vanligt exempel på ett terapeutiskt protein är insulin som personer drabbade av diabetes inte kan producera själva och därför måste få som ett externt tillskott. Ett annat exempel är så kallade monoklonala antikroppar som används i kliniska studier för behandling av till exempel cancer och sjukdomar som påverkar immunförsvaret. Framställningsprocessen av terapeutiska proteiner gör att de alltid löper en viss risk att innehålla virus, vilket gör avlägsnandet av virus en viktig del av framställningen för att undvika spridning av virussjukdomar. Avlägsnandet av virus sker i många olika steg där filtrering ofta är det sista och oftast också det mest kostsamma steget. Förekomsten av virus i dricksvatten är ett globalt hälsoproblem och den främsta källan till denna förekomst är förorening av vattnet via avföring. Tillgången till dricksvatten som genomgått en reningsprocess är en viktig del i att minska spridandet av virusrelaterade sjukdomar och Världshälsoorganisationen (WHO) rapporterade år 2015 att uppskattningsvis 663 miljoner människor fick sitt dricksvatten från obehandlade källor. Behovet av storskalig och billig virusfiltrering ter sig därmed angeläget, men begränsas av den höga kostnaden hos de flesta kommersiellt tillgängliga filter på marknaden idag. Det filter som presenteras i denna studie är baserat på cellulosafiber från alger av typen Cladophora och framställs genom en enkel metod som påminner om den metod som används för att tillverka vanligt papper. Cellulosafibrerna dispergeras först i vatten och bildar en slags massa som sedan torkas till ett tunt ark. Det resulterande materialet är poröst med porer (hålrum) i storleksordningen 5–40 nanometer, där den största volymsandelen utgörs av porer med en diameter på 23 nanometer. Dessa porer är därmed tillräckligt små för att inte släppa igenom de flesta typer av virus. Användandet av alger som råmaterial gör denna typ av filter intressant ur både ett ekonomisk och hållbart perspektiv. Tillvaratagandet av alger skulle kunna påverka ekosystem i hav och sjöar positivt samtidigt som det är en källa till förnyelsebart material. Blomningen av alger är ett problem som uppstår i både hav och sjöar, där mänsklig övergödning är en bidragande faktor. Blomningen skapar problem i ekosystemen genom att hindra solljus från att nå ner till arter på djupet samt genom att skapa syrebrist då algerna bryts ner. Kraven på virusfilter är stora eftersom förekomsten av även små mängder virus kan skapa problem då virus kan replikera sig vid kontakt med exempelvis mänskliga celler eller bakterier. USA:s livsmedels- och läkemedelsmyndighet, FDA, har tillsammans med EU:s läkemedelsmyndighet, EMA, satt en rekommendation för virusfilter där den rekommenderade effektiviteten bör vara högre än 99,99%. Detta innebär att minst 99,99% av alla virus måste tas bort vid filtrering. Dessa krav gör det viktigt att kunna kontrollera tillverkade filter för att se att de kan möta dessa krav. Att använda sig av virus för att kvalitetssäkra filter är dock väldigt kostsamt och krångligt. Istället för att använda virus är det därför vanligt att använda sig av väldigt små partiklar, så kallade nanopartiklar, som är lika stora som de virus filtren ska kvalitetstestas mot. Två vanliga typer av nanopartiklar som används är antingen guldpartiklar eller dextranpartiklar, där dextran är en typ av socker. i Tidigare studier har dock visat att både virus och nanopartiklar inte alltid beter sig som förväntat vid filtrering och att det finns fler faktorer än bara storlek som påverkar om virus och partiklar fastnar i filtret eller inte. Bland annat har hastigheten av flödet genom filtret visat sig haft en inverkan på hur virus och partiklar fastnar i ett filter vid filtrering. Syftet med denna studie har varit att undersöka hur nanopartiklar, i form av guld och dextran, beter sig i det pappersbaserade virusfiltret och om dessa kan användas för att kvalitetssäkra filtren. Resultaten från denna studie innehåller både datorsimuleringar samt experiment. Datorsimuleringarna visade att flödet genom filtret har en effekt på hur virus och partiklar kan röra sig, vilket har visat sig vara en faktor som kan påverka effektiviteten vid filtrering. Om virus och partiklar kan röra sig genom filtret så ökar möjligheten att de hittar vägar genom filtret där de kan smita igenom. Simuleringarna visade att rörelsen minskade desto högre flödet var och desto större virus eller partiklar var. Vid experiment där filtrering av 28 nanometer stora virus (ΦX174 bakteriofager) genomfördes vid olika flöden så syntes en tydlig skillnad i effektivitet. Vid ett högre flöde fångades samtliga virus upp av filtret medan virus smet igenom filtret vid lägre flöden. Vid filtrering av 5 nanometer stora guldpartiklar och 12,8 nanometer stora dextranpartiklar syntes ingen skillnad i effektivitet vid olika flöden. Vad som var intressant var att en stor del av partiklarna fastnade i filtret trots att en större del av dessa förväntades passera filtret på grund av sin storlek. 60– 75% av guldpartiklarna fastnade i filtret i början av filtreringen och motsvarande siffra var 98–99% för dextranpartiklarna. Detta tyder på att mekanismen vid filtrering är mer komplex än vad som enbart kan förklaras med storleken på porerna i ett filter samt storleken på partiklarna. Resultaten uppmuntrar till ytterligare studier kring mekanismerna vid filtrering och manar till försiktighet vid användning av nanopartiklar som metod för att kvalitetssäkra virusfilter. ii Contents 1. Introduction ...................................................................................................................................... 3 1.1 Virus Removal in Therapeutic Protein Manufacturing ............................................................ 3 1.2 Virus Removal in Drinking Water ............................................................................................ 3 1.3 Virus Removal Filters ............................................................................................................... 3 1.4 Virus Removal Filter Paper ......................................................................................................4 1.5 Aim ...........................................................................................................................................4 2. Theory...............................................................................................................................................5
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