1. Préparation de microparticules <ol><li> Anticorps étiquette utilisant la biotine NHS Chimie </li><li> Détermination du degré de biotinylation </li><li> Conjugué avec des billes de streptavidine anticorps biotinylé <ol><li> Resuspendre les perles dans le flacon d&#39;origine par vortex pendant 1-2 minutes </li><li> Aliquoter le volume souhaité de perles dans un tube à centrifuger </li><li> Laver les billes 3 fois avec PBST: <ol><li> Placer le tube contenant les billes sur l&#39;aimant pendant 2 minutes </li><li> Retirer le surnageant par aspiration avec le tube sur l&#39;aimant. Soyez prudent de ne pas perturber les perles </li><li> Retirer le tube de l&#39;aimant et ajouter 1 ml de PBST </li><li> Vortex pendant 20 secondes pour remettre en suspension les perles </li><li> Centrifuger pendant 5 secondes pour enlever les perles de la PAC </li><li> Répéter deux fois plus 2.3.1-2.3.5 </li></ol></li><li> Après le troisième lavage lieu le tube sur l&#39;aimant et aspirer le PBST </li><li> Ajouter anticorps biotinylé aux billes et resuspendre </li><li> Si nécessaire porter le volume de l&#39;anticorps biotinylé jusqu&#39;à 300 uL de PBS. Si le volume d&#39;anticorps est déjà supérieure à 300 ul ne pas ajouter de PBS </li><li> Incuber la suspension de billes / anticorps pendant 30 minutes à température ambiante sur l&#39;agitateur vortex ou le tube des rotateurs </li><li> Laver 3 fois avec PBST comme dans l&#39;étape 3. </li><li> Bloc perles avec biotinylé BSA <ol><li> Après le troisième lavage ajouter 10 pg / mg de BSA biotinylé, ajuster le volume à 300 ul avec du PBS, si nécessaire, et incuber sous agitation pendant 30 minutes </li></ol></li><li> Laver 3 fois avec PBST comme dans l&#39;étape 3 </li><li> Resuspendre les perles dans 100 uL de BSA PBS/0.05%. L&#39;azide de sodium peut être ajouté pour le stockage à long terme </li></ol></li><li> Utilisation de la norme du NHS protocoles fluorescéine, fluoresceinate l&#39;anticorps détecteur </li><li> Déterminer le degré de Fluorescination </li></ol> 2. HCP - Développement <ol><li> Préparer des solutions de travail: <ol><li> Anticorps marqué à la fluorescéine (FL-Ab): La plupart des cellules CHO-HCP dosages utilisent Fl-Ab à une concentration finale de 400 ng / mL. Le FL-Ab est ajouté en tant que ¼ du volume d&#39;essai final, donc la concentration de la solution de travail est de 1,6 mg / ml. Préparer cette solution en diluant avec de stock Flab BioScale billes / Flab diluant. </li><li> Perles: La plupart des cellules CHO-HCP dosages utilisent des billes à une concentration finale de 2x10 5 billes / mL. Le réactif perles constitue ¼ du volume d&#39;essai final, donc la concentration de la solution de travail est 8x10 5 billes / mL. Préparer cette solution en diluant stock de billes avec billes BioScale / Flab diluant. Vortex du stock de perles et de s&#39;assurer que toutes les perles sont en suspension avant de retirer une partie aliquote. </li><li> Tampon: La composition du tampon de migration peut être optimisé pour les anticorps utilisés pour développer le test, mais plusieurs CHO-HCP tests ont été développés en utilisant 10 mM de tampon HEPES, 1% de Tween, pH 7,5. BioScale a développé un additif pour le tampon courant (fournie avec le kit de développement) qui est ajouté au tampon à une concentration de 1% avant de l&#39;utiliser. </li></ol></li><li> Diluer étalons et d&#39;échantillons avec le diluant échantillon BioScale. </li></ol> 3. ViBE Système Set Up <ol><li> Tournez le Workstation Vibe </li><li> Branchez l&#39;alimentation du système et des déchets </li><li> Premier système </li><li> Insérez une cartouche ViBE </li><li> Route des tubes </li><li> Préparer modèle de test en remplissant la feuille intitulée «Exemple de configuration&quot;. BioScale offre la possibilité d&#39;avoir un modèle de test pour chaque dosage Workstation ViBE. Cela permet à l&#39;utilisateur de modifier et enregistrer des notes spécifiques dans les &quot;Notes&quot; feuille de modèle pour des dosages différents conserver un dossier complet de l&#39;expérience. </li><li> Modèle de test initial mis en place: Commencez par remplir le tableau (en haut à gauche) de la gamme de concentration de ces normes dans le test dans le &quot;Setup Sample&quot; feuille. </li><li> Ensuite, entrer des informations supplémentaires dans le tableau à droite de l&#39;normes (Configuration simple colonne Lire, Ajouter un réactif et réactif Deux configurations Ajouter) pour charger le Workstation ViBE combien de longs échantillons doivent incuber, quel volume de réactif doit être transféré , combien de temps l&#39;accumulation de l&#39;échantillon doit être, et si conditionné capteur et la décontamination des sondes et des cartouches sont nécessaires, etc </li><li> Remplissez le disposition de la plaque. </li><li> Charger l&#39;échantillon / réactifs en plaques. La Vibe fournira les volumes nécessaires à la plaque de réactif. Le volume d&#39;échantillon peut varier, mais dans la moitié d&#39;un volume de dosage total de 120 uL est un bon point de départ. </li><li> Appliquer couvre la plaque. Charge plaques sur le pont poste de travail. </li><li> Exécutez le test de démarrage Assistant, passez par l&#39;assistant et commencer dosage. </li></ol> 4. Les résultats représentatifs <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Figure 1" /> Figure 1: immunoessai sandwich AMMP </p&gt; Sur un poste ViBE, un test mesure la concentration d&#39;antigène AMMP. Le signal est mesuré lorsque l&#39;anticorps-antigène-billes haptène se lie à la sonde à travers l&#39;haptène / anti-haptène interaction. Régénération apporte le capteur revient à l&#39;état commence par enlever les complexes immuns. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Figure 2" /> Figure 2: CHO-HCP courbe de calibration du dosage du PMMA Figure 2 ci-dessus est un exemple d&#39;une courbe CHO HCP AMMP calibration du dosage. L&#39;analyse peut être effectuée dans les échantillons de l&#39;ensemble du flux d&#39;épuration, y compris le réacteur avec une grande précision et la reproductibilité. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Figure 3" /> Figure 3: Protéines Une courbe de calibration Assay AMMP La figure 3 est une courbe de calibration pour une protéine résiduelle Une analyse du PMMA, réalisée avec une protéine résiduelle VIBE une trousse de dosage. Le même kit peut être utilisé pour un rapide essai avec une période d&#39;incubation de 30 minutes, ou pour des résultats plus sensibles et plus large gamme dynamique, avec une période d&#39;incubation plus longue, sans perte de précision ou de précision, qui tous deux sont généralement mieux que leurs équivalents en ELISA dans des usagers les mains <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Figure 4" /> Figure 4: Produit Titer AMMP Assay La station de travail ViBE peut également être utilisé pour un dosage Titer produit, résultats présentés dans la figure 4. Réalisée différemment que d&#39;autres dosages PMMA, ce test ne nécessite pas de traitement de l&#39;échantillon autre que la dilution simple, ou de perles, et la quantification a été vérifié dans &quot;sans essorage&quot; échantillons contenant plus de 10 millions de cellules sur une large gamme de viabilités. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Figure 5" /> Figure 5: courbe standard de dosage AMMP pour le dosage de la TPS-GADD34 biomarqueur Montre la figure 5 est une courbe standard de signal obtenu par le BioAnalyzer ViBE (Groupe A) ou la technologie en utilisant AlphaScreen recombinant GST-GADD34. Résultats sur la plate-forme ViBE ont été d&#39;environ un journal plus sensibles en utilisant les mêmes anticorps. AMMP s&#39;appuie sur la mesure de moins de particules plus grandes mais encore les avantages de la capture en phase solution et la formation de complexes de détection. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Figure 6" /> Figure 6: Courbe AMMP dosage standard pour la TPS-GADD34 dans l&#39;échantillon de la tumeur Dans la figure 6, l&#39;induction de GADD34 partir CWR22 xénogreffes a été testé dans la présence ou l&#39;absence d&#39;un inhibiteur du protéasome boronate que analysées soit par le BioAnalyzer ViBE (Groupe A) ou AlphaScreen (Groupe B). La plate-forme ViBE était capable de produire sensibles, des résultats reproductibles dans le in vivo xénogreffes tumorales tandis que le signal a été stoppée avec AlphaScreen mg de protéine accrue. Cet exemple montre la puissance de la technologie dans des échantillons de PMMA très complexe. Grâce à la puce de détection sensible et une grande efficacité de l&#39;aimant à proximité de la chips, un faible nombre de microparticules magnétiques peuvent efficacement et de façon reproductible récupérer des cibles de faible abondance au milieu de la complexité de lysées cellulaire (y compris les réactifs de lyse) et les débris de sang.

Les auteurs de cet article sont employés par Bioscale, Inc qui produit certains des instruments et des réactifs utilisés dans cet article.

Les exemples qui précèdent montrent PMMA est très sensible des résultats quantitatifs dans des échantillons complexes. Cette performance, lorsqu&#39;ils sont livrés avec la facilité et le contrôle de la mise en forme des réactifs dans le système est propice à une variété d&#39;applications. Ces applications peuvent être spécifiques d&#39;une cible, comme dans GADD34 dans la tumeur, ou générique pour un large éventail de cibles, comme dans la détection des protéines de la cellule hôte et la quantification dans des échantillons qui traverse le ruisseau de purification. La plate-forme ViBE faible surcharge combinée avec PMMA extrêmement souple offre un outil puissant pour offrir des tests critiques.

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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

Dosages personnalisés pour les protéines uniques sont souvent limitées à la détection manuelle du temps et des techniques de quantification telles que ELISA ou Western blot en raison de la complexité du développement sur les plates-formes de substitution. BioScale de membrane exclusive microparticules acoustique (PMMA) technologie permet immunodosages sandwich pour être facilement développé pour une utilisation sur la plate-forme ViBE, offrant une meilleure sensibilité, la reproductibilité et un fonctionnement automatisé. À titre d&#39;exemple, ce protocole définit la procédure d&#39;élaboration d&#39;une coutume chinoise protéine cellulaire d&#39;ovaire de hamster-Host (CHO-HCP) dosage. Les principes généraux énoncés ici peuvent être suivies pour le développement d&#39;une grande variété de dosages immunologiques. Une concentration de dosage d&#39;antigène AMMP mesures en mesurant les changements dans la fréquence d&#39;oscillation provoquée par la liaison de microparticules à la surface du capteur pour calculer. Il se compose de quatre éléments principaux: (1) une cartouche qui contient une puce de capteur fonctionnalisés huit (2) anticorps marqué microparticules magnétiques, (3) d&#39;anticorps haptène marqués qui se lie à la surface de la puce fonctionnalisés (4) échantillons contenant l&#39;antigène de intérêt. Biocapteur BioScale est un appareil de résonance qui contient huit membranes individuels avec des chemins séparés fluidiques. Les membranes changement de fréquence d&#39;oscillation en réponse à la masse qui s&#39;accumule sur la surface et ce changement de fréquence est utilisée pour quantifier la quantité de masse ajoutée. Pour faciliter l&#39;utilisation dans une grande variété de tests immunologiques de la sonde est fonctionnalisé avec un anticorps anti-haptène. Anticorps spécifiques de dosage ont été modifiés par la conjugaison covalente d&#39;un tag haptène à un anticorps et de la biotine à l&#39;autre. L&#39;étiquette biotine est utilisé pour lier l&#39;anticorps à la streptavidine couplée billes magnétiques qui, en combinaison avec l&#39;anticorps haptène marqué, sont utilisés pour capturer l&#39;analyte dans un sandwich. Le complexe se lie à la puce grâce à l&#39;interaction anti-hapten/hapten. A la fin de chaque essai exécuté les capteurs sont nettoyés avec un acide dilué permettant l&#39;analyse séquentielle des colonnes d&#39;une plaque de 96 puits. Ici, nous présentons la méthode pour développer une coutume CHO-HCP test AMMP pour le développement de bioprocédés. Développer des dosages PMMA ou en modifiant des dosages existants dans des dosages PMMA peut offrir de meilleures performances (reproductibilité, sensibilité) dans des échantillons complexes et le temps de l&#39;opérateur réduit. Le protocole décrit les étapes de développement et de la section de discussion critiques des résultats représentatifs. Pour une explication plus approfondie de l&#39;optimisation des essais et des paramètres de personnalisation de contact BioScale. Ce kit offre générique de tests de développement de bioprocédés comme les protéines résiduelles Un titre produit, et 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

Développement de tests personnalisés sur la plate-forme ViBE pour les plus sensibles, reproductibles, des résultats automatisé dans des matrices complexes est décrite. La cartouche universelle permet des dosages pour être facilement adapté pour une utilisation avec des dosages personnalisés. Cette polyvalence permet un développement rapide et la validation de nouveaux tests ou de versions automatisées de tests existants manuel, illustré dans cette vidéo.

Développement de ovaire de hamster chinois-hôte dosages de protéines cellulaires utilisant la technologie acoustique microparticules membranaires

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

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JoVE Journal

Bioengineering

13.1K vues.  BioScale, Inc.. Développement de tests personnalisés sur la plate-forme ViBE pour les plus sensibles, reproductibles, des résultats automatisé dans des matrices complexes est décrite. La cartouche universelle permet des dosages pour être facilement adapté pour une utilisation avec des dosages personnalisés. Cette polyvalence permet un développement rapide et la validation de nouveaux tests ou de versions automatisées de tests existants manuel, illustré dans cette vidéo.

Vidéo: 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.

L&#39;augmentation des rendements d&#39;ADNc de cellule unique quantités d&#39;ARNm dans les réactions de laboratoire standard de la transcriptase inverse en utilisant Microstreaming acoustique

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 ...

Recherche

Bio-ingénierie

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.

Isolation et caractérisation de protéines humaines chimériques exprimant fc à l’aide d’adsorbeurs membranaires de protéine a et d’un flux de travail rationalisé

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.

Protocole générique pour l’optimisation de la Production de protéines hétérologues à l’aide de la technologie automatisée de MICROBIOREACTEUR

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.

Purification et analyse d'un anticorps monoclonal à partir de cellules ovaires de hamster chinois à l'aide d'un système automatisé de microbioréacteur

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.

Optimisation des processus à l’aide de micro-bioréacteurs automatisés à haut débit dans la culture chinoise de cellules d’ovaires de Hamster

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.

Préparation des cellules synthétiques produisant des protéines à l’aide d’extraits bactériens exempts de cellules, de liposomes et de transfert d’émulsion

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.

Clarification primaire du fluide de culture cellulaire récolté par CHO à l’aide d’un séparateur acoustique

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.

Formulation et modulation acoustique de nanogouttelettes de perfluorocarbone vaporisées optiquement

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.

Glycoingénierie rapide des anticorps dans les cellules ovaires du hamster chinois

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 Essais de clivage utilisant des caspases de drosophile recombinantes purifiées pour le criblage de substrats

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