Name: laboratory scale production and purification of a therapeutic antibody
Uploaded: 2017-01-24T13:00:00
Description: Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

MERK: En egnet pattedyr-ekspresjonsvektor som må brukes for denne protokollen. Her blir en enkel konstruksjon inneholdende to ekspresjonskassetter brukt (dvs. tung og lettkjede-ekspresjon drives av separate promotere). Trastuzumab tunge og lette kjeder ble tidligere klonet inn i vektoren. Denne vektoren var en gave fra Andrew Beavil, innhentet gjennom en ikke-for-profit plasmid depot 13. 1. Recovery og oppskalering av Vector DNA MERK: Vektor DNA ble mottatt som en myk agar...

<ol>
	<li>Nelson, A. L., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+therapeutic+antibody+fragments.">Development trends for therapeutic antibody fragments.</a> Nat Biotechnol. 27 (4), 331-337 (2009).</li><li>Drake, P. M., Rabuka, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+playbook+for+antibody-drug+conjugates:+lessons+from+the+laboratory+and+clinic+suggest+a+strategy+for+improving+efficacy+and+safety.">An emerging playbook for antibody-drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety.</a> Curr Opin Chem Biol. 28, 174-180 (2015).</li><li>Kontermann, R. E., Brinkmann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies.">Bispecific antibodies.</a> Drug Discov Today. 20 (7), 838-847 (2015).</li><li>Beck, A., Wurch, T., Bailly, C., Corvaia, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+challenges+for+the+next+generation+of+therapeutic+antibodies.">Strategies and challenges for the next generation of therapeutic antibodies.</a> Nat Rev Immunol. 10 (5), 345-352 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food and Drug Administration. 22, (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> European Medicines Agency. 13, (2014).</li><li>Lopez-Morales, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+and+biological+characterization+of+a+biosimilar+trastuzumab.">Physicochemical and biological characterization of a biosimilar trastuzumab.</a> Biomed Res Int. 2015, 427235 (2015).</li><li>Jung, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+of+Remsima.">Physicochemical characterization of Remsima.</a> MAbs. 6 (5), 1163-1177 (2014).</li><li>Visser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+and+functional+comparability+between+the+proposed+biosimilar+rituximab+GP2013+and+originator+rituximab.">Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.</a> BioDrugs. 27 (5), 495-507 (2013).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+different+vector+designs+for+the+mammalian+expression+of+recombinant+IgG+antibodies.">A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies.</a> J Immunol Methods. 318 (1-2), 113-124 (2007).</li><li>Fitzgerald, K. D., Semler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+IRES+elements+in+mRNAs+to+the+eukaryotic+translation+apparatus.">Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.</a> Biochim Biophys Acta. 1789 (9-10), 518-528 (2009).</li><li>Ho, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRES-mediated+Tricistronic+vectors+for+enhancing+generation+of+high+monoclonal+antibody+expressing+CHO+cell+lines.">IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.</a> J Biotechnol. 157 (1), 130-139 (2012).</li><li>Dodev, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tool+kit+for+rapid+cloning+and+expression+of+recombinant+antibodies.">A tool kit for rapid cloning and expression of recombinant antibodies.</a> Sci Rep. 4, 5885 (2014).</li><li>Walsh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+protein+biopharmaceuticals.">Post-translational modifications of protein biopharmaceuticals.</a> Drug Discov Today. 15 (17-18), 773-780 (2010).</li><li>Backliwal, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+vector+design+and+multi-pathway+modulation+of+HEK+293E+cells+yield+recombinant+antibody+titers+exceeding+1+g/l+by+transient+transfection+under+serum-free+conditions.">Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.</a> Nucleic Acids Res. 36 (15), 96 (2008).</li><li>Bebbington, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+expression+of+a+recombinant+antibody+from+myeloma+cells+using+a+glutamine+synthetase+gene+as+an+amplifiable+selectable+marker.">High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.</a> Biotechnology (N Y). 10 (2), 169-175 (1992).</li><li>Kaufman, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coamplification+and+coexpression+of+human+tissue-type+plasminogen+activator+and+murine+dihydrofolate+reductase+sequences+in+Chinese+hamster+ovary+cells.">Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.</a> Mol Cell Biol. 5 (7), 1750-1759 (1985).</li><li>Dalton, A. C., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+secreted+proteins+from+mammalian+cell+lines.">Over-expression of secreted proteins from mammalian cell lines.</a> Protein Sci. 23 (5), 517-525 (2014).</li><li>Pham, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol Bioeng. 90 (3), 332-344 (2005).</li><li>Pham, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+transient+transfection+of+serum-free+suspension-growing+HEK293+EBNA1+cells:+peptone+additives+improve+cell+growth+and+transfection+efficiency.">Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.</a> Biotechnol Bioeng. 84 (3), 332-342 (2003).</li><li>Yang, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+valproic+acid+to+CHO+cell+fed-batch+cultures+improves+monoclonal+antibody+titers.">Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.</a> Molecular Biotechnology. 56 (5), 421-428 (2014).</li><li>Backliwal, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid:+a+viable+alternative+to+sodium+butyrate+for+enhancing+protein+expression+in+mammalian+cell+cultures.">Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.</a> Biotechnol Bioeng. 101 (1), 182-189 (2008).</li><li>Jiang, Z., Sharfstein, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulates+monoclonal+antibody+over-expression+in+CHO+cells+by+improving+gene+accessibility.">Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility.</a> Biotechnol Bioeng. 100 (1), 189-194 (2008).</li><li>Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+adherent+and+suspended+cells+by+calcium+phosphate.">Transfection of adherent and suspended cells by calcium phosphate.</a> Methods. 33 (2), 136-143 (2004).</li></ol>

Denne protokollen beskriver transfeksjon stabil ekspresjon og rensing av et terapeutisk antistoff i HEK-293-celler. Stabil ekspresjon av antistoffgener er det første trinnet i å generere en antistoffproduserende cellelinje for utvikling og fremstilling av et terapeutisk antistoff. Mens kinesiske hamsterovarie (CHO) celler forblir uttrykket plattformen for terapeutiske proteiner, er HEK-293 cellelinje få prominence med realisering at proteiner som produseres i disse cellene er en tettere match til naturlig forekommend...

Suksessen av terapeutiske antistoffer fortsetter å drive betydelig investering i antistoffutvikling som en bølge av neste generasjons therapeutics begynner. Antistoffet markedet forventes å bli omformet av antistoff-fragmenter 1, antistoff-stoffet konjugater 2, bispesifikke antistoffer 3 og utviklet antistoffer med gunstige egenskaper 4. En annen klasse få farmasøytisk interesse er biosimilars. Biosimilar antistoffer er &quot;svært lik &#39;replikere produkter av en terapeutisk antistoff som allerede har fått regulatorisk godkjenning. En foreslått biosimilar må være sammenlignbare med opphavs antistoff med hensyn til sin struktur, funksjon, dyr toksisitet, kliniske sikkerheten og effektiviteten, humane farmakokinetikk (PK), farmakodynamikken (PD) og immunogenisitet 5, 6. Godkjenningen rAtes av biosimilar antistoffer har vært treg på grunn av de strenge begrensninger på den endelige kvaliteten på produktet. De nøyaktige produksjonsprosesser som bestemt cellelinjer og dyrkingsforhold gjennom til den endelige behandlingstrinn kan forbli fortrolig. Hva mer er, produksjon av antistoffer i seg selv innebærer en grad av variasjon som kan legge til utfordringen med å produsere en svært lignende produkt. En omfattende fysio og biofysiske karakterisering og sammenligningen er ganske vanskelig, men en rekke studier som viser egenskapene til biosimilar antistoffer dukker opp i litteraturen 7, 8, 9. Generering av et terapeutisk antistoff begynner med transfeksjon av mammalske vertsceller med en vektor som bærer gener for de respektive antistoff. Vektor design, cellelinje og kultur forholdene er viktige hensyn for å sette opp expression system. DNA-sekvenser av antistoffer kan være hentet fra Drug Bank (www.drugbank.ca), IMGT (www.igmt.org) eller forskningspublikasjoner inkludert patenter. For eksempel sekvensen av trastuzumab er tilgjengelig gjennom Drug Bank (DB ID: DB00072). Aminosyresekvensen til de variable regionene kan gjennomgå genet utforming og optimalisering for syntese i de ønskede vertsart. Det er viktig for en biosimilar antistoff som ingen modifikasjon er laget for å aminosyresekvensen. Når syntetisert, kan antistoffgener blir subklonet inn i passende vektor for valg. Humane IgG antistoffer består av to identiske tungkjeder og to identiske lette kjeder. Strengt regulert ekspresjon av begge kjedene er avgjørende for optimal produksjon av heterologe IgG-protein i pattedyrceller 10. Intra- og inter-kjede-disulfidbindinger måtte bli dannet og en rekke post-translasjonelle modifikasjoner må være iinnført i løpet av proteinsyntese. En rekke vektorer er tilgjengelige som har blitt designet spesielt for å uttrykke antistoffgener (se tabell for material). Disse antistoff-spesifikke vektorer som vanligvis uttrykker de konstante områder for både tunge og lette kjeder slik at bare de variable områder i hver kjede krever kloning. Transfeksjon av celler med to uavhengige konstrukter (co-transfeksjon) er den mest vanlige metode for tilførsel av tung og lett kjede-kodende gener. Det er, er hvert gen drives av sin egen promoter og transkribert som separate antistoffkjeder før de blir satt sammen i det endoplasmatiske retikulum. På den annen side, multi-cistronisk vektorer har interne ribosom innføringsstedet (IRES) elementer som inngår som tillater ekspresjon av multiple gener som et enkelt mRNA-transkript med oversettelse tillates fra interne regioner av mRNA 11. I dette tilfelle blir de tunge og lette kjede-kodende gener koblet i en Arrangeement å oppnå co-uttrykk for begge antistoff kjedene 10, 12. Mens forbigående transfekterte celler, hvilket ga tilstrekkelig protein for å utføre et begrenset antall forsøk, kan stabilt transfekterte cellelinjer som har gjennomgått seleksjon for integrasjon genomet gir høyere utbytter. Høyere mengder protein tillate assay utvikling i forbindelse med in vitro karakterisering og kan gi en indikasjon av antistoff kvalitet i betraktning for nedstrøms applikasjoner som klonal cellelinje og bly kandidat valg. Målet med denne artikkelen er å beskrive den stabile uttrykk og rensing av et terapeutisk antistoff produsert i en pattedyr uttrykk system. Faktisk kan denne metoden anvendes for ekspresjon av et biosimilar antistoff. Fremgangsmåten kan anvendes for den innledende karakterisering av antistoffer før fortsetter videre til den kritiske, enskjønt tidkrevende steps for å identifisere en ønskelig klone for større skala produksjon. Videre kan denne fremgangsmåte anvendes for å uttrykke andre proteiner og ikke bare antistoffer. Den følgende detaljerte protokollen beskriver ekspresjon av terapeutiske antistoff trastuzumab. Denne består av fremstilling av vektor-DNA, etterfulgt av stabil transfeksjon i HEK-293 cellelinje og rensing av antistoff-protein ved en automatisert kromatografisk metode.

<table border="0" cellpadding="0" cellspacing="0" width="1730">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">pFUSE vector series</td>
			<td style="width:189px;">N/A</td>
			<td style="width:191px;">InvivoGen</td>
			<td style="width:1003px;">Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAbXpress vector series</td>
			<td>N/A</td>
			<td>ACYTE Biotech Pty Ltd.</td>
			<td>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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1 vector</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&#160; 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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GS vector series</td>
			<td>N/A</td>
			<td>Lonza</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1-Trastuzumab-IgG1/&#954;</td>
			<td>61883</td>
			<td>Addgene</td>
			<td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro Agar</td>
			<td>fas-hg-s</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt LB agar containing 75 &#181;g/ml hygromycin.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro TB</td>
			<td>fas-hg-l</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt TB broth containing 75 &#181;g/ml hygromycin.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol, BioXtra, &#8805;99%</td>
			<td>G6279</td>
			<td>Sigma-Aldrich</td>
			<td>Prepare to 80% with water and autoclave. Store at room temperature.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jestar 2.0/LFU Plasmid Maxi Kit</td>
			<td>G221020</td>
			<td>Astral Scientific</td>
			<td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293-F Cells</td>
			<td>R790-07</td>
			<td>Life Technologies</td>
			<td>HEK-293 cell line adapted to suspension culture in serum-free media.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293 Expression Medium</td>
			<td>12338-018</td>
			<td>Life Technologies</td>
			<td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Kolliphor P188</td>
			<td>K4894</td>
			<td>Sigma-Aldrich</td>
			<td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose&#160;</td>
			<td>11995-065</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat-Inactivated Foetal Bovine Serum</td>
			<td>10082-147</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Polyethylenimine, Linear, MW 25,000&#160;</td>
			<td>23966</td>
			<td>Polysciences, Inc.</td>
			<td>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 &#956;m filter. Store at -80oC until use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiPro SFM</td>
			<td>12309-050</td>
			<td>Life Technologies</td>
			<td>Transfection formulated serum-free media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B Solution</td>
			<td>ant-hg-1</td>
			<td>Jomar Life Research</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethylsulphoxide (DMSO)</td>
			<td>AJA2225</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tryptone (casein peptone)</td>
			<td>LP0042B</td>
			<td>Thermo Fisher Scientific</td>
			<td>Prepare to 20% in PBS and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td>
			<td>09-2051-100</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiTrap Protein A High Performance, 1 x 5 ml column</td>
			<td>GE17-0403-01</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AKTApurifier 100</td>
			<td>28406266</td>
			<td>GE Healthcare</td>
			<td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine-HCl</td>
			<td>G2879</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric Acid, monohydrate</td>
			<td>BIOC2123</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Citrate, trisodium salt dihydrate&#160;</td>
			<td>BIOCB0035</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M Tris-HCl solution pH 9.0</td>
			<td>BIOSD8146</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal Filters (30 MWCO)</td>
			<td>UFC803008/UFC903008</td>
			<td>Merck Millipore</td>
			<td>Used to buffer exchange and concentrate purified protein.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Bicinchoninic Acid (BCA) Assay Kit</td>
			<td>23227</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BLItz System</td>
			<td>45-5000</td>
			<td>fort&#233;BIO</td>
			<td>Instrument used for bio-layer interferometry (BLI) measurements.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein A biosensors</td>
			<td>18-5010</td>
			<td>fort&#233;BIO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide/Bisacrylamide (37.5:1), 40% solution</td>
			<td>786-502</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Persulfate (APS)</td>
			<td>AM0486</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>AM0761</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Brilliant Blue R-250</td>
			<td>786-498</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Precision Plus Dual-Color Protein Standard</td>
			<td>1610374</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory scale production and purification of a therapeutic antibody

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.

Stabil produksjon av trastuzumab ved transfekterte HEK-293 celler ble bekreftet ved hjelp av bio-lag interferometri (BLI) som presentert i figur 1. En IgG standardkurve ble generert ved å måle bindingshastigheten mellom et IgG-antistoff standard og Protein A biosensor (figur 1A). Det urene supernatant Prøven ble på lignende måte målt, og dens konsentrasjon interpoleres fra standardkurven (figur 1B). Supernatanten konsentrasjonen bl...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

Denne protokollen beskriver fremstillingen av et terapeutisk antistoff i et pattedyr ekspresjonssystem. Metodene som beskrives omfatter fremstilling av vektor-DNA, stabil transfeksjon og serumfritt tilpasning av en human embryonisk nyrecellelinje 293, setter opp av store kulturer og rensing ved hjelp av affinitetskromatografi.

Laboratorieskala Produksjon og rensing av et terapeutisk antistoff

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Regissert Protein Emballasje innen ytre membran Blemmer fra<em&gt; Escherichia coli</em&gt;: Design, produksjon og rensing

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Produksjon, krystallisering og struktur Bestemmelse av<em&gt; C. difficile</em&gt; PPEP-en via Microseeding og sink-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Expression, Purification, and Antimicrobial Activity of S100A12

Ekspresjon, rensing og antimikrobiell aktivitet av S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

En Guide til produksjon, krystallisering og proteinstrukturer av menneskelig IKK1/α

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Metode for effektiv Refolding og rensing av Chemoreceptor Ligand bindende domene

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Rensing og rekonstituering av TRPV1 for Spectroscopic analyse

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Forbigående uttrykk i Nicotiana Benthamiana forlater Triterpene produksjon i skytevåpen skala

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Storskalaproduksjon av rekombinant RNAs på en sirkulær stillaset med en Viroid-avledet System i Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Uttrykk og rensing av Nuklease-gratis Oxygen renovasjons Protocatechuate 3, 4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

En tilpasningsvennlig tilnærming for enzymatisk produksjon og rensing av Diterpenoid naturlige produkter

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...