1. Microparticelle di preparazione <ol><li> Anticorpi etichette con Biotina NHS Chimica </li><li> Determinazione del grado di biotinilazione </li><li> Perle streptavidina coniugato con anticorpo biotinilato <ol><li> Risospendere le sfere nel flacone originale con il vortex per 1-2 minuti </li><li> Aliquota il volume desiderato di perline in una provetta </li><li> Lavare le perle 3 volte con PBST: <ol><li> Collocare la provetta contenente le perline sul magnete per 2 minuti </li><li> Eliminare il surnatante tramite aspirazione con il tubo sul magnete. Fare attenzione a non disturbare le perline </li><li> Togliere la provetta dal magnete e aggiungere 1 ml PBST </li><li> Vortex per 20 secondi per risospendere le sfere </li><li> Centrifugare per 5 secondi per rimuovere le perle dal tappo </li><li> Ripetere altre due volte 2.3.1-2.3.5 </li></ol></li><li> Dopo il terzo lavaggio posto il tubo sul magnete e aspirare il PBST </li><li> Aggiungi anticorpo biotinilato ai talloni, risospendere </li><li> Se necessario, portare il volume delle anticorpo biotinilato fino a 300 ml con PBS. Se il volume anticorpo è già superiore a 300 ml non aggiungere alcun PBS </li><li> Incubare la sospensione tallone / anticorpo per 30 minuti a temperatura ambiente mediante l&#39;agitatore vortex o rotatori tubo </li><li> Lavare 3 volte con PBST come nel punto 3. </li><li> Blocco con perline biotinilato BSA <ol><li> Dopo il terzo lavaggio aggiungere 10 mg / mg biotinilato BSA, regolare il volume a 300 ml con PBS, se necessario, e incubare con agitazione per 30 minuti </li></ol></li><li> Lavare 3 volte con PBST come nel punto 3 </li><li> Risospendere le sfere in 100 ml di PBS/0.05% BSA. Azoturo di sodio possono essere aggiunti per la conservazione a lungo termine </li></ol></li><li> Utilizzando protocolli standard di NHS fluorescina, fluoresceinate l&#39;anticorpo rivelatore </li><li> Determinare il grado di Fluorescination </li></ol> 2. HCP - Sviluppo <ol><li> Preparare le soluzioni di lavoro: <ol><li> Anticorpo marcato con fluoresceina (Fl-Ab): La maggior parte CHO-HCP saggi uso Fl-Ab ad una concentrazione finale di 400 ng / ml. Il Fl-Ab è aggiunto come ¼ del volume saggio finale, quindi la concentrazione della soluzione di lavoro è di 1,6 mg / ml. Preparare questa soluzione diluendo magazzino Flab con BioScale Bead / flab diluente. </li><li> Perle: la maggior parte CHO-HCP saggi uso perline ad una concentrazione finale di 2x10 5 pedine / ml. Il reagente tallone costituisce ¼ del volume saggio finale, quindi la concentrazione della soluzione di lavoro è 8x10 5 pedine / ml. Preparare questa soluzione diluendo magazzino tallone con BioScale Bead / flab diluente. Vortex lo stock tallone bene per assicurarsi che tutte le perle sono in sospensione prima di rimuovere una frazione. </li><li> Esecuzione di buffer: La composizione del tampone di corsa può essere ottimizzata per gli anticorpi utilizzati per sviluppare il test, tuttavia diversi CHO-HCP test sono stati sviluppati utilizzando 10 mM tampone HEPES, 1% di Tween, pH 7.5. BioScale ha sviluppato un additivo per il tampone di corsa (in dotazione con il kit di sviluppo) che si aggiunge al buffer ad una concentrazione di 1% prima dell&#39;uso. </li></ol></li><li> Diluire calibratori e campioni con diluente campione BioScale. </li></ol> 3. ViBE di configurazione del sistema <ol><li> Girare la workstation ViBE su </li><li> Collegare l&#39;alimentazione del sistema e dei rifiuti </li><li> Il primo sistema </li><li> Inserire una cartuccia ViBE </li><li> Far passare il tubo </li><li> Preparare modello di analisi compilando il foglio denominato &quot;Setup campione&quot;. BioScale offre la possibilità di avere un modello di analisi per ogni dosaggio Workstation ViBE. Questo permette all&#39;utente di modificare e salvare note specifiche nella scheda &quot;Note&quot; del modello di saggi diversi tenere un registro completo dell&#39;esperimento. </li><li> Modello di analisi configurazione iniziale: Inizia compilando il tavolo (angolo in alto a sinistra) del range di concentrazione degli standard nel saggio in &quot;Setup campione&quot; foglio. </li><li> Successivamente, inserire ulteriori informazioni nella tabella a destra degli standard (impostazione della colonna semplici Per saperne, One Aggiungi reagenti e due configurazioni reagente Aggiungi) per indicare la workstation ViBE come campioni lunghi bisogno di incubare, come è il volume di reagente deve essere trasferita , per quanto tempo l&#39;accumulo campione deve essere, e se condizionamento del sensore e la decontaminazione di sonde e cartucce sono necessari, ecc </li><li> Compila il layout del pannello. </li><li> Carico del campione / i reagenti in piastre. Il ViBE fornirà i volumi necessari per la piastra reagente. Volume del campione può variare, ma a metà di un volume dosaggio totale di 120 microlitri è un buon punto di partenza. </li><li> Applicare copertine piatto. Caricare le piastre sul ponte workstation. </li><li> Eseguire il test di avvio procedura guidata, passare attraverso procedura guidata e avviare test. </li></ol> 4. Rappresentante Risultati <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Figura 1" /> Figura 1: immunodosaggio a sandwich AMMP </p&gt; Su una workstation ViBE, un saggio AMMP misura la concentrazione di antigene. Il segnale è misurato quando il tallone-antigene-anticorpo aptene si lega al sensore tramite l&#39;aptene / anti-aptene interazione. Rigenerazione porta il sensore torna allo stato all&#39;inizio rimuovendo il immunocomplessi. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Figura 2" /> Figura 2: CHO-HCP curva di calibrazione Assay AMMP Figura 2 è un esempio di una curva di calibrazione CHO HCP saggio AMMP. Analisi può essere effettuata in campioni provenienti da tutto il flusso di purificazione compreso il reattore con alta precisione e riproducibilità. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Figura 3" /> Figura 3: Proteina A AMMP curva di calibrazione di analisi Figura 3 è una curva di calibrazione per una proteina residua un test AMMP, eseguita con una proteina VIBE residua un kit di analisi. Lo stesso kit può essere utilizzato per un test veloce con un periodo di incubazione di 30 minuti, o per risultati più sensibili e più ampia gamma dinamica, con un periodo di incubazione più lungo, senza perdita di precisione o accuratezza, entrambi i quali sono in genere migliori di equivalente ELISA nel utenti &#39; mani <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Figura 4" /> Figura 4: Prodotto titolo AMMP Assay La stazione di lavoro ViBE può essere utilizzato anche per un saggio titolo del prodotto, i risultati mostrati in figura 4. Eseguita in modo diverso rispetto saggi AMMP, questo test non richiede trattamento del campione diverso da semplice diluizione o perline, e quantificazione è stata verificata in &quot;no rotazione&quot; campioni contenenti oltre 10 milioni di cellule in un ampio intervallo di Viabilità. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Figura 5" /> Figura 5: Curva Assay AMMP standard per la GST-Gadd34 biomarker test Mostrata in Figura 5 è una curva standard di segnale ottenuto dal Bioanalyzer ViBE (Pannello A) o utilizzando la tecnologia AlphaScreen ricombinante GST-Gadd34. Risultati su piattaforma ViBE sono stati circa un registro più sensibili utilizzando gli stessi anticorpi. AMMP sfrutta la misura di un minor numero di particelle più grandi ma ancora beneficia soluzione di acquisizione e complessa fase di formazione di rilevamento. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Figura 6" /> Figura 6: Curva Assay AMMP standard per la GST-Gadd34 a campione del tumore Nella Figura 6, l&#39;induzione di Gadd34 da CWR22 xenotrapianti è stato testato in presenza o in assenza di un inibitore del proteasoma boronate come analizzato sia dal ViBE Bioanalyzer (Pannello A) o AlphaScreen (Pannello B). La piattaforma ViBE è stato in grado di produrre sensibili, risultati riproducibili in xenotrapianti tumorali in vivo, mentre il segnale è stato spento con AlphaScreen mcg maggiore di proteine. Questo esempio mostra la potenza della tecnologia AMMP in campioni molto complessi. Grazie al chip di rilevamento sensibili e ad alta efficienza del magnete vicino al chip, basso numero di microparticelle magnetiche possono pulire in modo efficace e riproducibile per target bassa abbondanza tra la complessità del lisato cellulare (compresi i reagenti lisi) e detriti sangue.

Gli autori di questo articolo sono impiegati da Bioscale, Inc che produce alcuni degli strumenti e dei reagenti utilizzati in questo articolo.

Gli esempi precedenti dimostrano AMMP I risultati altamente sensibile quantitativa in campioni complessi. Questa performance, al momento della consegna con la facilità e il controllo della formattazione dei reagenti nel sistema è abilitante per una varietà di applicazioni. Tali domande possono essere specifici per un target, come in Gadd34 nel tumore, o generico per uno spettro di obiettivi, come nella rilevazione delle proteine ​​della cellula ospite e la quantificazione in campioni che attraversano il torrente purificazione. La bassa piattaforma ViBE testa combinata con AMMP altamente flessibile fornisce un potente strumento per la distribuzione di saggi critici.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NHS-Chromogenic-biotin or NHS-Chromogenic Biotin Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Chromogenic-biotin is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS) or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer able to measure 354 and 280 nm &#x2013; for measuring protein and biotin concentrations</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated antibody (2-5 biotins/Ab)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin magnetic beads</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>(10 mg/mL) ~108 beads/mg</td>
</tr>
<tr>
<td>Magnet for microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>New England BioLabs</td>
<td>cat # S1510S</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated BSA for blocking</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS 0.1% Tween 20</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge holder for Vortex or tube rotator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Antibody or protein to be labeled</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NHS-Fluorescein or NHS-Fluorescein Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Fluorescein is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 mM borate, pH 8.5 or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Measurement of protein and fluorescein concentrations requires a spectrophotometer able to measure 493 and 280 nm.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute beads and FlAb</td>
</tr>
<tr>
<td>Sample Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute samples and calibrators</td>
</tr>
<tr>
<td>Running Buffer Additive</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

developing custom chinese hamster ovary-host cell protein assays using acoustic membrane microparticle technology

Test personalizzati per specifiche proteine ​​sono spesso limitati a rilevamento manuale che richiede tempo e le tecniche di quantificazione, quali ELISA o Western blot a causa della complessità dello sviluppo su piattaforme alternative. BioScale proprietaria membrana acustica microparticelle (AMMP), la tecnologia permette immunologici panino di essere facilmente sviluppati per l&#39;uso sulla piattaforma ViBE, fornendo una migliore sensibilità, la riproducibilità e il funzionamento automatico. Fornito come esempio, questo protocollo delinea le procedure per lo sviluppo di una consuetudine ovariche di criceto cinese-Host proteina cellulare (CHO-HCP) test. I principi generali descritti qui possono essere seguiti per lo sviluppo di una grande varietà di saggi immunologici. Un AMMP antigene concentrazione saggio misura misurando i cambiamenti nella frequenza di oscillazione causata dal legame di microparticelle alla superficie del sensore da calcolare. È composto da quattro componenti principali: (1) una cartuccia che contiene un funzionalizzato otto chip del sensore (2) anticorpo marcato microparticelle magnetiche, (3) anticorpo aptene tag che si lega alla superficie del chip funzionalizzati (4) campioni contenenti l&#39;antigene di interesse. BioScale biosensore è un dispositivo di risonanza che contiene otto membrane individuale con percorsi separati fluidica. Le membrane cambiare frequenza di oscillazione in risposta ad accumulare massa in superficie e questo cambiamento di frequenza è utilizzato per quantificare la quantità di massa aggiunto. Per facilitare l&#39;uso in una vasta gamma di immunodosaggi è funzionalizzati il ​​sensore con un anticorpo anti-aptene. Anticorpi specifici test vengono modificati attraverso la coniugazione covalente di un tag aptene di un anticorpo e biotina all&#39;altro. L&#39;etichetta biotina viene utilizzato per associare l&#39;anticorpo di streptavidina accoppiata sfere magnetiche che, in combinazione con l&#39;aptene-anticorpo tag, vengono utilizzati per catturare l&#39;analita in un panino. Il complesso si lega al chip attraverso l&#39;interazione anti-hapten/hapten. Alla fine di ogni test eseguire i sensori sono pulite con un acido diluito che permette l&#39;analisi sequenziale di colonne da una piastra da 96 pozzetti. Qui vi presentiamo il metodo per lo sviluppo di una consuetudine CHO-HCP test AMMP per lo sviluppo dei bioprocessi. Lo sviluppo di test AMMP o modificare i test esistenti in saggi AMMP in grado di fornire prestazioni migliori (riproducibilità, sensibilità) in campioni complessi e tempi operatore ridotto. Il protocollo viene illustrata la procedura per lo sviluppo e la sezione di discussione su risultati rappresentativi. Per una spiegazione più approfondita analisi e di ottimizzazione dei parametri di personalizzazione contatto BioScale. Questo kit offre generico test di sviluppo bioprocessi come proteina residua A, titolo del prodotto, e CHO-HCP.

Watch this Scientific Journal Video about Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology at JoVE.com

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Sviluppo di saggi di costume sulla piattaforma ViBE per più sensibili, riproducibili, i risultati automatizzati in matrici complesse è descritto. La cartuccia universale consente di test per essere facilmente adattati per essere utilizzati con test personalizzati. Questa versatilità consente un rapido sviluppo e validazione di test romanzo o versioni automatizzate di test manuali esistenti, esemplificata in questo video.

Sviluppo di ovaio di criceto cinese-dosaggi delle proteine ​​della cellula ospite con la tecnologia Acoustic microparticelle di membrana

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

developing-custom-chinese-hamster-ovary-host-cell-protein-assays

Research

JoVE Journal

Bioengineering

13.1K Views.  BioScale, Inc.. Sviluppo di saggi di costume sulla piattaforma ViBE per più sensibili, riproducibili, i risultati automatizzati in matrici complesse è descritto. La cartuccia universale consente di test per essere facilmente adattati per essere utilizzati con test personalizzati. Questa versatilità consente un rapido sviluppo e validazione di test romanzo o versioni automatizzate di test manuali esistenti, esemplificata in questo video.

Video: Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Aumentare le rese cDNA da singola cellula quantità di mRNA nelle reazioni standard di laboratorio della trascrittasi inversa utilizzando Microstreaming acustico

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Ricerca

Bioingegneria

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Isolamento e caratterizzazione delle proteine chimeriche che esprimono fc umano utilizzando proteine A adsorbenti di membrana e un flusso di lavoro semplificato

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Protocollo generico per l'ottimizzazione della produzione di proteine eterologhe utilizzando tecnologia automatizzata di Microbioreactor

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purificazione e analisi di un anticorpo monoclonale da cellule ovaio Cesster cinese utilizzando un sistema microbioreante automatizzato

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Ottimizzazione dei processi mediante microreattori automatizzati ad alta velocità effettiva nella coltivazione delle cellule ovaio dell'ammasso cinese

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Preparazione di proteine che producono cellule sintetiche utilizzando estratti batterici cell free, liposomi e trasferimento di emulsione

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Chiarificazione primaria del fluido di coltura cellulare raccolto da CHO utilizzando un separatore acustico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Formulazione e modulazione acustica di nanogoccioline di perfluorocarburi vaporizzate otticamente

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Glicoingegneria anticorpale rapida nelle cellule ovariche di criceto cinese

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Here we present a protocol to express and purify recombinant Drosophila caspases Dronc and Drice, and their use in in vitro cleavage assays.

In vitro Saggi di cleavage utilizzando Drosophila caspasi ricombinanti purificate per lo screening del substrato

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...

Sviluppo di ovaio di criceto cinese-dosaggi delle proteine ​​della cellula ospite con la tecnologia Acoustic microparticelle di membrana

Sviluppo di ovaio di criceto cinese-dosaggi delle proteine della cellula ospite con la tecnologia Acoustic microparticelle di membrana