Name: comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts
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We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. Coltivare le piante di tabacco <ol><li> Lavare ogni blocco lana minerale con 1 a 2 L di acqua deionizzata e quindi con 1 L di 0,1% [w / v] Soluzione di concime. Mettere un seme di tabacco in ogni blocco lana minerale e delicatamente a filo con 0,25 L di soluzione fertilizzante senza lavare via il seme 16. </li><li> Coltivare le piante di tabacco per 7 settimane in una serra con il 70% di umidità relativa, 16 hr fotoperiodo (180 mmol sec - 1 m - 2; λ = 400 - 7...

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I tre metodi per la precipitazione di calore sopra descritti possono rimuovere efficacemente operatori sanitari tabacco prima di ogni fase di purificazione cromatografica 16,17. Esse completano altre strategie che mirano ad aumentare la purezza iniziale del prodotto, ad esempio, guttazione 29, rhizosecretion 30 o centrifuga estrazione 31,32, che sono tutti limitati alle proteine ​​secrete. Tuttavia, i metodi di calore a base possono essere usati solo in modo signi...

I moderni sistemi di assistenza sanitaria dipendono sempre più sulle proteine ​​biofarmaceutiche 1. Produrre queste proteine ​​nelle piante è vantaggioso a causa del basso carico patogeno e maggiore scalabilità rispetto ai sistemi di espressione convenzionali 2-4. Tuttavia, la lavorazione a valle (DSP) di farmaci di origine vegetale può essere difficile perché le procedure di estrazione dirompenti si traducono in un onere elevato di particelle, con torbidità superiore a 5.000 unità nefelometrica torbidità (NTU), e di proteine ​​della cellula ospite (HCP) concentrazioni spesso superiore a 95 % [m / m] 5,6. Procedure di chiarificazione elaborati sono tenuti a rimuovere particelle disperse 7-9, ma l&#39;apparecchiatura cromatografia è meno costoso per operare in modalità bind-and-eluire durante il recupero iniziale del prodotto se c&#39;è un passaggio precedente per la rimozione efficiente degli operatori sanitari 10,11. Ciò può essere ottenuto sia da precipitare la proteina bersaglio utilizzando flocculformiche 12 o basso pH 13,14, così come provocando la operatori sanitari di aggregare. L&#39;aggregazione selettiva di ribulosio-1,5-bisfosfato carbossilasi / ossigenasi (Rubisco), l&#39;operatore sanitario più abbondante nelle piante verdi come il tabacco (Nicotiana tabacum), può essere promossa aggiungendo polietilene glicole 15, ma questo è costoso e incompatibile con grande la produzione -scale. Il trattamento termico è stato dimostrato per denaturare e precipitare più del 95% degli operatori sanitari tabacco, mentre vaccini candidati proteina malaria come Vax8 rimangono stabili in soluzione 16-18. Tre diversi approcci sono stati usati per ottenere la precipitazione indotta dal calore degli operatori sanitari tabacco: (i) la scottatura, cioè, l&#39;immersione delle foglie intatte in liquido caldo, (ii) una temperatura controllata agitata nave, e (iii) uno scambiatore di calore ( Figura 1) 16. Per foglie intatte, scottatura raggiunto la precipitazione rapida ed efficace degli operatori sanitari ed era anche facilea scalare e compatibile con i processi di produzione su larga scala esistenti che includono un primo passo per lavare la biomassa vegetale 19. Al contrario, navi temperatura controllata sono già disponibili in alcuni processi e possono essere utilizzati per il trattamento termico di estratti vegetali 20, ma la loro scalabilità e velocità di trasferimento di energia è limitato perché il rapporto superficie-volume dei serbatoi è progressivamente ridotta e diventa inadatto a scala processo. Uno scambiatore di calore è un&#39;alternativa tecnicamente ben definito per riscaldata navi agitate ma richiede un abbondante approvvigionamento di riscaldamento e mezzi di raffreddamento, ad esempio, vapore e acqua fredda, nonché una portata volumetrica strettamente controllato atto a geometria scambiatore di calore e proprietà dei media, ad esempio., la capacità termica specifica. Questo articolo mostra come tutti i tre metodi possono essere usati per la precipitazione indotta dal calore degli operatori sanitari tabacco e HCPs vegetali in genere. L&#39;istituzione e il funzionamento di EACMetodo h in un ambiente di laboratorio può essere utilizzato per valutare la loro idoneità per processi su larga scala. La sfida principale è quello di individuare modelli in scala-down adeguati e condizioni di funzionamento per ogni operazione che assomigliano i dispositivi e le condizioni utilizzate durante la produzione processo di scala. I dati qui presentati si riferiscono ad esperimenti condotti con piante di tabacco transgeniche che esprimono la malaria candidato vaccino Vax8 e proteina fluorescente DsRed 16, ma il metodo è stato applicato con successo anche a N. piante benthamiana esprimere transitoriamente altre proteine ​​biofarmaceutiche 21. Un design-di-esperimenti (DOE) si avvicinano 22 può facilitare lo sviluppo di processo, e flocculanti 23 può anche essere utile in questo contesto, come descritto in precedenza 8. La differenza principale tra la scottatura, serbatoi riscaldati e scambiatori di calore è che sbiancamento viene applicata alle foglie intatte nelle prime fasi del processo mentre l&#39;otlei sono applicati a estratti vegetali (Figura 1). <img alt="Figura 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> Figura 1:. Processo Schema di flusso che illustra la realizzazione di tre diversi metodi per il tabacco HCP calore Precipitazioni Il materiale vegetale è lavato ed omogeneizzato prima chiarificazione e purificazione. L&#39;attrezzatura per il passo scottatura (rosso) può essere facilmente aggiunto al macchinario esistente. Al contrario, utilizzando un recipiente agitato (arancione) e, soprattutto, uno scambiatore di calore (blu) richiede uno o più dispositivi e tubi aggiuntivi. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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			<td height="21" style="height:21px;width:360px;">2100P Portable Turbidimeter</td>
			<td style="width:195px;">Hach</td>
			<td style="width:119px;">4650000</td>
			<td style="width:352px;">Turbidimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Amine Coupling Kit</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100050&#160;</td>
			<td>SPR chip coupling kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Autoclaving basket</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:119px;">6917-0230</td>
			<td>Basket for leaf blanching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9750-01</td>
			<td>SPR device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bio Cell Analyser BCA 003 R&#38;D with 3D ORM</td>
			<td style="width:195px;">Sequip</td>
			<td style="width:119px;">n.a.</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Blender</td>
			<td style="width:195px;">Waring</td>
			<td style="width:119px;">800EG</td>
			<td>Blender</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">BP-410</td>
			<td style="width:195px;">Furh</td>
			<td style="width:119px;">2632410001</td>
			<td>Bag filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 15 mL</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">1110504</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CM5 chip</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100012&#160;</td>
			<td>Chip for SPR measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cuvette 10x10x45</td>
			<td style="width:195px;">Sarsted</td>
			<td style="width:119px;">67.754</td>
			<td>Cuvette for Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Twentey-loop heat exchanger (4.8 m length)</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Heat exchanger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K200P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302303</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">L/S 24</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">SN-06508-24</td>
			<td>Tubing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">mAb 5.2</td>
			<td style="width:195px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-9148</td>
			<td>Vax8 specific antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Masterflex L/S</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium disulfit</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8554.1</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Container for heat precipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence and spectrometric plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">ZEN3600</td>
			<td>DLS particle size distribution measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Software v7.11</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">n.a.</td>
			<td>Software to operate the Zetasizer Nano ZS device</td>
		</tr>
	</tbody>
</table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

Le piante non solo forniscono alimenti, mangimi e materie prime per l&#39;uomo, ma sono stati sviluppati come un sistema di produzione economica per le proteine ​​biofarmaceutiche, come gli anticorpi, vaccini candidati ed enzimi. Questi devono essere purificati dalla biomassa vegetale, ma passaggi di cromatografia sono ostacolati dalle alte concentrazioni di proteine ​​della cellula ospite (operatori sanitari) in estratti di piante. Tuttavia, la maggior parte HCPs irreversibilmente aggregano a temperature superiori a 60 ° C agevolare successiva purificazione della proteina bersaglio. Qui, tre metodi vengono presentati per ottenere la precipitazione di calore degli operatori sanitari del tabacco in foglie sia intatte o estratti. Il pallore di foglie intatte può essere facilmente incorporato in processi esistenti, ma può avere un impatto negativo sulle successive fasi di filtrazione. L&#39;opposto è vero per la precipitazione di calore di estratti di foglie in un recipiente agitato, che può migliorare le prestazioni delle operazioni a valle anche se con grandi cambiamenti nella progettazione apparecchiature di processo, qualiGeometria omogeneizzatore. Infine, una configurazione scambiatore di calore è ben caratterizzato in termini di condizioni di trasferimento di calore e facile in scala, ma pulizia può essere difficile e potrebbe esserci un impatto negativo sulla capacità del filtro. L&#39;approccio progettuale-di-esperimenti può essere utilizzato per identificare i parametri di processo più importanti che influenzano la rimozione HCP e recupero del prodotto. Questo facilita l&#39;applicazione di ciascun metodo in altre piattaforme di espressione e l&#39;identificazione del metodo più adatto per una data strategia di purificazione.

La precipitazione di calore delle proteine ​​della cellula ospite tabacco da scottatura La procedura di sbiancamento descritto nella sezione 2. è stato utilizzato con successo per far precipitare operatori sanitari da foglie di tabacco con 70 ° C, la riduzione del TSP del 96 ± 1% (n = 3), mentre il recupero fino al 51% della proteina bersaglio Vax8, aumentando così la sua purezza dal 0,1% al 1,2% prima della separazione cromatografica 16. Era anche poss...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Tre calore metodi di precipitazione sono presentati che rimuove efficacemente oltre il 90% delle proteine ​​della cellula ospite (HCP) da tabacco estrae prima di qualsiasi altra fase di purificazione. L&#39;impianto di operatori sanitari in modo irreversibile aggregare a temperature superiori a 60 ° C.

Confronto di tabacco metodi Host cellulare della proteina di rimozione da Blanching Piante intatte o mediante trattamento termico di estratti

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Preparazione e frazionamento di Xenopus laevis Egg Estratti

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Ricerca

Biologia

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Rilevazione di modificazioni post-traduzionali in nativo nucleosomi intatto da ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

The MultiBac Protein Complex Production Platform at the EMBL

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Interazioni proteina-proteina visualizzati tramite bimolecular complementazione fluorescenza in protoplasti di tabacco e foglie

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

DamID-seq: Genome-wide Mappatura delle interazioni proteina-DNA da High Throughput Sequencing di Adenina-metilato frammenti di DNA

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Procedura per valutare l&#39;efficienza di flocculanti per la rimozione di particelle disperse di estratti di piante

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Confronto di tre diversi metodi per la determinazione proliferazione cellulare in Breast Cancer linee cellulari

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...