Laboratorieskala Produktion og oprensning af et terapeutisk antistof
Summary
Denne protokol beskriver fremstillingen af et terapeutisk antistof i en mammal ekspressionssystem. De beskrevne fremgangsmåder omfatter fremstilling af vektor-DNA, stabil transfektion og serumfrit tilpasning af en human embryonal nyre 293 cellelinie, der er nedsat af skala kulturer og oprensning store ved hjælp af affinitetskromatografi.
Abstract
Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.
Introduction
Succesen af terapeutiske antistoffer fortsætter med at drive betydelig investering i antistof udvikling som en bølge af næste generation af lægemidler begynder. Forventes Antistoffet marked, der skal omformes ved antistoffragmenter 1, antistof-drug konjugater 2, bispecifikke antistoffer 3 og manipuleret antistoffer med gunstige egenskaber 4. En anden klasse vinder farmaceutisk interesse er biosimilars. Biosimilære antistoffer er "meget ens« replikere produkter af en terapeutisk antistof, der allerede har modtaget myndighedernes godkendelse. Et forslag biosimilært skal være sammenlignelige med ophavsmanden antistof med hensyn til dets struktur, funktion, dyr toksicitet, kliniske sikkerhed og effektivitet, menneskelige farmakokinetik (PK), farmakodynamik (PD) og immunogenicitet 5, 6.
Godkendelsen rater af biosimilære antistoffer har været langsomme på grund af de strenge begrænsninger på den endelige kvalitet af produktet. De nøjagtige fremstillingsprocesser såsom specifikke cellelinjer og dyrkningsbetingelser igennem til de endelige procestrin kan forblive proprietær. Hvad mere er, fremstillingen af antistoffer i sagens natur indebærer en grad af variabilitet, som kan føje til den udfordring at producere et meget lignende produkt. En omfattende fysisk-kemiske og biofysisk karakterisering og sammenligning er ganske vanskeligt, men en række undersøgelser, der viser karakteristika biosimilære antistoffer dukker i litteraturen 7, 8, 9.
Generering af en terapeutisk antistof begynder med transfektion af pattedyrsværtsceller med en vektor, der bærer generne for det respektive antistof. Vector design, cellelinje og dyrkningsbetingelser er centrale overvejelser for opsætning af expression system.
DNA sekvenser af antistoffer kan hentes fra Drug Bank (www.drugbank.ca), IMGT (www.igmt.org) eller forskningspublikationer herunder patenter. For eksempel sekvensen af trastuzumab er tilgængelig via Drug Bank (DB ID: DB00072). Aminosyresekvensen af de variable regioner kan undergå gen design og optimering til syntese i de ønskede værtsart. Det er vigtigt for et biosimilært antistof der ingen ændring sker på aminosyresekvensen. Når syntetiseret, kan antistofgener subklones i den passende valgte vektor.
Humane IgG-antistoffer består af to identiske tunge kæder og to identiske lette kæder. Tæt reguleret ekspression af begge kæder er afgørende for optimal produktion af heterologt IgG protein i pattedyrceller 10. Intra- såvel som inter-kæde disulfidbindinger skal dannes, og en række post-translationelle modifikationer skal være iintroduceret under proteinbiosyntese. En række vektorer er til rådighed, der er blevet designet specifikt til at udtrykke antistofgener (se tabel of Materials). Disse antistof-specifikke vektorer udtrykker normalt de konstante regioner for både tunge og lette kæder så kun de variable regioner i hver kæde kræver kloning.
Transfektion af celler med to uafhængige konstruktioner (co-transfektion) er den mest almindelige metode til afgivelse tunge og lette kæde-gener. Det vil sige, er hvert gen drevet af sin egen promotor og transskriberes som separate antistofkæder inden de samles i det endoplasmatiske reticulum. På den anden side, multi-cistroniske vektorer har interne ribosomindgangssted (IRES) elementer inkorporeret som tillader ekspression af multiple gener som en enkelt mRNA-transkript med oversættelse tilladt fra interne regioner af mRNA 11. I dette tilfælde er de tunge og lette kæde gener er koblet i en Arrangement at opnå co-ekspression af begge antistofkæder 10, 12.
Mens forbigående transficerede celler gav tilstrækkelig protein til at udføre et begrænset antal eksperimenter, kan stabilt transficerede cellelinjer, der har undergået selektion for genom integration levere højere udbytter. Højere proteinmængder mulighed for assay udvikling vedrørende in vitro karakterisering og kan give en indikation af antistof kvalitet i betragtning for downstream-applikationer såsom klonal cellelinie og bly kandidat udvælgelse.
Målet med denne artikel er at beskrive den stabile ekspression og oprensning af et terapeutisk antistof produceret i en mammal ekspressionssystem. Faktisk kan denne fremgangsmåde anvendes til ekspressionen af et biosimilært antistof. Fremgangsmåden kan anvendes til den første karakterisering af antistoffer før man går videre til det kritiske, omend tidskrævende steps identificere en ønskelig klon for større produktionsskala. Desuden kan denne metode anvendes til at udtrykke andre proteiner og ikke kun antistoffer.
Følgende detaljeret protokol beskriver udtryk for terapeutiske antistoffer trastuzumab. Denne består af fremstilling af vektor-DNA, efterfulgt af stabil transfektion i HEK-293-cellelinien og oprensning af antistof-protein ved en automatiseret kromatografisk metode.
Protocol
Representative Results
Discussion
Denne protokol detaljer transfektionen stabil ekspression og oprensning af et terapeutisk antistof i HEK-293-celler. Stabil udtryk for antistofgener er det første skridt i at generere et antistof-producerende cellelinje til udvikling og fremstilling af et terapeutisk antistof. Mens kinesisk hamster ovarie (CHO) celler forbliver ekspressionen foretrukne platform for terapeutiske proteiner, er HEK-293-cellelinje få fremtrædende med erkendelsen af, at proteiner produceret i disse celler er en tættere match med naturlig…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/κ was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/κ.
Materials
| pFUSE vector series | N/A | InvivoGen | Heavy and light antibody genes expressed in separate vectors that require co-transfection. |
| mAbXpress vector series | N/A | ACYTE Biotech Pty Ltd. | Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010). |
| pVITRO1 vector | N/A | N/A | Heavy and light antibody genes are each driven by a separate promoter in a single vector. Refer to: Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014). |
| GS vector series | N/A | Lonza | Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. |
| Multi-cistronic vector series 1 | N/A | N/A | Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. et al. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007). |
| Multi-cistronic vector series 2 | N/A | N/A | Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012). |
| pVITRO1-Trastuzumab-IgG1/κ | 61883 | Addgene | Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/κ was a gift from Andrew Beavil. |
| Fast-Media Hygro Agar | fas-hg-s | Jomar Life Research | Used to prepare low salt LB agar containing 75 µg/ml hygromycin. |
| Fast-Media Hygro TB | fas-hg-l | Jomar Life Research | Used to prepare low salt TB broth containing 75 µg/ml hygromycin. |
| Glycerol, BioXtra, ≥99% | G6279 | Sigma-Aldrich | Prepare to 80% with water and autoclave. Store at room temperature. |
| Jestar 2.0/LFU Plasmid Maxi Kit | G221020 | Astral Scientific | Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5). |
| FreeStyle 293-F Cells | R790-07 | Life Technologies | HEK-293 cell line adapted to suspension culture in serum-free media. |
| FreeStyle 293 Expression Medium | 12338-018 | Life Technologies | Serum-free media specially formulated for maintaing 293-F cell line and high protein expression. |
| Kolliphor P188 | K4894 | Sigma-Aldrich | Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 μm filter. Store at 4oC. |
| DMEM, high glucose | 11995-065 | Life Technologies | |
| Heat-Inactivated Foetal Bovine Serum | 10082-147 | Life Technologies | |
| Polyethylenimine, Linear, MW 25,000 | 23966 | Polysciences, Inc. | Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 μm filter. Store at -80oC until use. |
| OptiPro SFM | 12309-050 | Life Technologies | Transfection formulated serum-free media |
| Hygromycin B Solution | ant-hg-1 | Jomar Life Research | |
| Dimethylsulphoxide (DMSO) | AJA2225 | Thermo Fisher Scientific | |
| Tryptone (casein peptone) | LP0042B | Thermo Fisher Scientific | Prepare to 20% in PBS and filter-sterilize using 0.22 μm filter. Store at 4oC. |
| Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml | 09-2051-100 | Astral Scientific | |
| HiTrap Protein A High Performance, 1 x 5 ml column | GE17-0403-01 | Sigma-Aldrich | |
| AKTApurifier 100 | 28406266 | GE Healthcare | Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector. |
| Glycine-HCl | G2879 | Sigma-Aldrich | |
| Citric Acid, monohydrate | BIOC2123 | Astral Scientific | |
| Sodium Citrate, trisodium salt dihydrate | BIOCB0035 | Astral Scientific | |
| 1 M Tris-HCl solution pH 9.0 | BIOSD8146 | Astral Scientific | |
| Amicon Ultra Centrifugal Filters (30 MWCO) | UFC803008/UFC903008 | Merck Millipore | Used to buffer exchange and concentrate purified protein. |
| Pierce Bicinchoninic Acid (BCA) Assay Kit | 23227 | Thermo Fisher Scientific | |
| BLItz System | 45-5000 | fortéBIO | Instrument used for bio-layer interferometry (BLI) measurements. |
| Protein A biosensors | 18-5010 | fortéBIO | |
| Acrylamide/Bisacrylamide (37.5:1), 40% solution | 786-502 | Astral Scientific | |
| Ammonium Persulfate (APS) | AM0486 | Astral Scientific | |
| TEMED | AM0761 | Astral Scientific | |
| Coomassie Brilliant Blue R-250 | 786-498 | Astral Scientific | |
| Precision Plus Dual-Color Protein Standard | 1610374 | Bio-Rad |
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