1. Mikropartikel-Vorbereitung <ol><li> Label Antikörper mit Biotin NHS Chemie </li><li> Bestimmung des Grades der Biotinylierung </li><li> Konjugat Streptavidin Beads mit biotinylierter Antikörper <ol><li> Resuspendieren der Perlen in der Original-Durchstechflasche durch Vortexen für 1-2 Minuten </li><li> Aliquot die gewünschte Lautstärke aus Perlen in ein Mikrozentrifugenröhrchen </li><li> Waschen Sie die Kugeln 3-mal mit PBST: <ol><li> Das Röhrchen mit den Perlen auf den Magneten für 2 Minuten </li><li> Entfernen Sie den Überstand durch Absaugen mit dem Schlauch auf den Magneten. Achten Sie darauf, um die Perlen zu stören </li><li> Entfernen Sie das Röhrchen aus dem Magneten und 1 ml PBST </li><li> Vortex für 20 Sekunden, um die Perlen zu resuspendieren </li><li> Zentrifugieren Sie für 5 Sekunden, um die Perlen aus der Kappe entfernen </li><li> Wiederholen Sie 2.3.1-2.3.5 noch zweimal </li></ol></li><li> Nach dem dritten Waschen statt der Röhre auf dem Magneten und saugen Sie den PBST </li><li> Add biotinylierten Antikörper an die Beads und resuspendieren </li><li> Bei Bedarf bringen die Lautstärke des biotinylierten Antikörpers bis zu 300 ul mit PBS. Wenn der Antikörper Volumen ist bereits mehr als 300 ul fügen Sie keine PBS </li><li> Inkubieren Sie die Perle / Antikörper-Suspension für 30 Minuten bei RT auf dem Vortex-Schüttler oder Tube Rotator </li><li> 3 x waschen mit PBST wie in Schritt 3. </li><li> Block Perlen mit biotinylierten BSA <ol><li> Nach dem dritten Waschen add 10 ug / mg biotinylierten BSA, die Lautstärke auf 300 ul mit PBS, wenn nötig, und inkubieren unter Rühren für 30 Minuten </li></ol></li><li> 3 x waschen mit PBST wie in Schritt 3 </li><li> Resuspendieren der Beads in 100 ul PBS/0.05% BSA. Natriumazid kann für die langfristige Lagerung hinzugefügt werden </li></ol></li><li> Mit Standard-NHS Fluorescein-Protokolle, fluoresceinate der Detektor-Antikörper </li><li> Bestimmen den Grad der Fluorescination </li></ol> 2. HCP - Entwicklung <ol><li> Bereiten Sie funktionierende Lösungen: <ol><li> Fluorescein-markierten Antikörper (Fl-Ab): Die meisten CHO-HCP-Assays verwenden Fl-Ab bei einer Endkonzentration von 400 ng / mL. Die Fl-Ab als ¼ der endgültigen Assayvolumen hinzugefügt wird, damit die Konzentration der Arbeitslösung beträgt 1,6 pg / mL. Diese Lösung durch Verdünnen Flab Lager mit BioScale Bead / Flab Diluent. </li><li> Perlen: Die meisten CHO-HCP-Assays verwenden Perlen an einer Endkonzentration von 2x10 5 Perlen / mL. Die Bead-Reagenz bildet ¼ der endgültigen Assayvolumen, daher die Konzentration der Arbeitslösung ist 8x10 5 Perlen / mL. Diese Lösung durch Verdünnen bead Lager mit BioScale Bead / Flab Diluent. Vortex die Perle Lager auch um sicherzustellen, dass alle Perlen sind in Suspension vor dem Entfernen eines aliquoten. </li><li> Laufpuffer: Die Zusammensetzung der Laufpuffer Möglicherweise müssen Sie für die Antikörper verwendet, um den Test zu entwickeln optimiert werden jedoch mehrere CHO-HCP-Assays entwickelt worden sind, mit 10 mM HEPES-Puffer, 1% Tween, pH 7,5. BioScale hat ein Additiv für den Laufpuffer (im Lieferumfang des Development Kit), die in den Puffer zu einer Konzentration von 1% vor Gebrauch hinzugefügt wird entwickelt. </li></ol></li><li> Verdünnen Sie die Kalibratoren und Proben mit BioScale Probendiluent. </li></ol> 3. ViBE System Set Up <ol><li> Drehen Sie den ViBE Workstation auf </li><li> Schließen Sie das System-und-entsorgung </li><li> Prime das System </li><li> Legen Sie eine ViBE Cartridge </li><li> Verlegen Sie die Rohre </li><li> Bereiten Assay Vorlage, indem Sie das Arbeitsblatt mit der Bezeichnung &quot;Sample Setup&quot;. BioScale bietet die Möglichkeit, einen Test Vorlage für jeden ViBE Workstation-Test haben. Dies ermöglicht es dem Benutzer, bearbeiten und speichern Sie bestimmte Noten in den &quot;Notes&quot; Blatt der Vorlage für die verschiedenen Tests, die eine vollständige Aufzeichnung des Experiments. </li><li> Erste Test-Template eingerichtet: Beginnen Sie mit dem Ausfüllen der Tabelle (oben links) der Konzentrationsbereich der Standards in den Test in der &quot;Sample Setup&quot; Blatt. </li><li> Als nächstes geben Sie zusätzliche Informationen in der Tabelle auf der rechten Seite des Standards (Simple Spalte Read Setup, ein Reagenz hinzufügen und Zwei-Reagenz Add-Setups), um die ViBE Workstation, wie lange Proben inkubieren müssen anweisen, wie viel Volumen von Reagenz zu übertragenden , wie lange die Probe Akkumulation zu sein braucht, und ob der Sensor ausgestattet und Dekontamination von Sonden und Patrone sind notwendig, etc </li><li> Füllen Sie die Platte Layout. </li><li> Laden Probe / Reagenzien in Platten. Der Vibe wird die erforderlichen Volumina für das Reagens Platte. Probenvolumen kann variieren, aber in der Hälfte der insgesamt Assay Volumen von 120 ul ist ein guter Ausgangspunkt. </li><li> Bewerben Platte abdeckt. Laden Platten auf Workstation-Deck. </li><li> Führen Sie den Test starten Assistenten durch Assistenten und starten Assay. </li></ol> 4. Repräsentative Ergebnisse <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Abbildung 1" /> Abbildung 1: AMMP Sandwich-Immunoassay </p&gt; Auf einer ViBE Workstation, misst ein AMMP Assay Antigen-Konzentration. Signal wird gemessen, wenn der Wulst-Antigen-Hapten-Antikörper auf den Sensor bindet durch das Hapten / Anti-Hapten-Wechselwirkung. Regeneration bringt der Sensor wieder an den Anfang Staat durch die Beseitigung der Immunkomplexe. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Abbildung 2" /> Abbildung 2: CHO-HCP AMMP Assay Kalibrierkurve Abbildung 2 oben ist ein Beispiel für eine CHO HCP AMMP Assay Kalibrierkurve. Die Analyse kann in Proben aus der ganzen Reinigung Strom einschließlich der Reaktor mit hoher Präzision und Reproduzierbarkeit durchgeführt werden. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Protein A AMMP Assay Kalibrierkurve Abbildung 3 ist eine Kalibrierkurve für einen Rest-Protein A AMMP Assay, mit einer VIBE Residual Protein A-Assay-Kit durchgeführt. Das gleiche Kit kann für eine schnellere Assay mit einer 30-minütigen Inkubationszeit, oder für empfindlichere Ergebnisse und einen größeren Dynamikbereich mit einer längeren Inkubationszeit ohne Verlust an Präzision und Genauigkeit, die beide in der Regel besser sind als gleichwertig ELISA ist in Benutzern verwendet werden &#39; Hände <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Abbildung 4" /> Abbildung 4: Produkt-Titer AMMP Assay Der Vibe Workstation kann auch für ein Produkt Titer-Test, Ergebnisse in Abbildung 4 dargestellt verwendet werden. Anders als andere AMMP Assays durchgeführt, hat dieser Test keine Probe einer anderen Behandlung als einfaches Verdünnen oder Perlen, und die Quantifizierung hat in &quot;no spin&quot; Proben mit mehr als 10 Millionen Zellen über einen weiten Bereich von Bewohnbarkeit überprüft worden. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Abbildung 5" /> Abbildung 5: AMMP Assay Standardkurve für GST-Gadd34 Biomarker Assay In Abbildung 5 ist ein Standard-Kurve des Signals von der Stimmung BioAnalyzer (Panel A) oder AlphaScreen Technologie unter Verwendung von rekombinanten GST-Gadd34 erhalten. Ergebnisse auf der ViBE Plattform wurden rund ein Protokoll empfindlicher mit dem gleichen Antikörper. AMMP nutzt die Messung von weniger größeren Partikeln dennoch Vorteile aus der Lösung Phase Erfassung und Erkennung der Komplexbildung. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Abbildung 6" /> Abbildung 6: AMMP Assay Standardkurve für GST-Gadd34 in Tumorprobe In Abbildung 6 wurde die Induktion von Gadd34 aus CWR22 Xenotransplantate in Anwesenheit oder Abwesenheit eines Boronat Proteasom-Inhibitor, wie entweder durch die ViBE BioAnalyzer (Panel A) oder AlphaScreen (Panel B) analysiert getestet. Der Vibe-Plattform war in der Lage, sensible und reproduzierbare Ergebnisse in der in vivo Tumorxenotransplantaten während die AlphaScreen Signal wurde mit einem erhöhten ug Protein abgeschreckt zu produzieren. Dieses Beispiel zeigt die Macht der AMMP Technologie in komplexen Proben. Aufgrund der empfindlichen Nachweis-Chip und einen hohen Wirkungsgrad des Magneten in der Nähe des Chips kann geringe Anzahl von magnetischen Mikropartikeln effizient und reproduzierbar für niedrige Fülle Ziel mitten in der Komplexität der lysierten Zellen (einschließlich Lyse-Reagenzien) und Blut Schmutz abzufangen.

Die Autoren dieses Artikels sind Bioscale, Inc, dass einige der Instrumente und Reagenzien in diesem Artikel verwendet produziert beschäftigt.

Die vorstehenden Beispiele zeigen, AMMP hochempfindliche quantitative Ergebnisse in komplexen Proben. Diese Leistung, wenn sie mit der Leichtigkeit und Kontrolle der Formatierung Reagenzien in das System geliefert wird, für eine Vielzahl von Anwendungen ermöglicht. Diese Anträge können spezifisch für ein Ziel, wie in Gadd34 in Tumor, oder allgemein für ein Spektrum von Zielen, wie sie in Wirtszellprotein Detektion und Quantifizierung in Proben, die die Reinigung Strom zu überqueren. Der geringe Overhead ViBE Plattform mit hochflexiblen AMMP kombiniert ein leistungsstarkes Tool für die Bereitstellung von kritischen Tests.

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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

Benutzerdefinierte Assays für einzigartige Proteine ​​werden oft zeitraubende manuelle Erfassung und Quantifizierung Techniken wie ELISA oder Western Blots auf Grund der Komplexität der Entwicklung auf alternative Plattformen beschränkt. BioScale proprietären Acoustic Membrane Mikropartikel (AMMP)-Technologie ermöglicht Sandwich-Immunoassays zu leicht für die Verwendung auf dem ViBE Plattform entwickelt werden, was eine bessere Empfindlichkeit, Reproduzierbarkeit und automatisierten Betrieb. Vorausgesetzt, als Beispiel, werden in diesem Protokoll das Verfahren für die Entwicklung eines benutzerdefinierten Chinesischen Hamsters-Host Cell Protein (CHO-HCP)-Assay. Die allgemeinen Grundsätze hier skizzierten kann für die Entwicklung einer breiten Palette von Immunoassays verfolgt werden. Ein AMMP Assay misst Antigen-Konzentration durch die Messung von Veränderungen in Schwingfrequenz durch die Bindung von Mikropartikeln verursacht, die Sensorfläche zu berechnen. Es besteht aus vier Hauptkomponenten: (1) eine Patrone, die eine funktionalisierte acht Sensor-Chip enthält (2) Antikörper markiert magnetische Mikropartikel, (3) Hapten markierten Antikörper an die Oberfläche der funktionalisierten Chip (4) Proben, die das Antigen bindet Interesse. BioScale der Biosensor ist ein Resonanz-Gerät, das acht einzelnen Membranen mit separatem fluidische Pfade enthält. Die Membranen ändern Schwingungsfrequenz in Reaktion auf Masse ansammeln auf der Oberfläche und diese Frequenz ändern wird verwendet, um die Menge des zugegebenen Masse zu quantifizieren. Für die Verwendung in einer Vielzahl von Immunoassays der Sensor mit einem Anti-Hapten-Antikörper funktionalisierten erleichtern. Assay spezifische Antikörper werden durch die kovalente Konjugation von Hapten-Tag, um einen Antikörper und Biotin auf die andere geändert. Die Biotin-Markierung verwendet wird, um den Antikörper binden an gekoppelten magnetischen Beads, die in Kombination mit dem Hapten-markierten Antikörper, verwendet werden, um den Analyten in einer Sandwich-Abscheidung Streptavidin. Der Komplex bindet an den Chip durch die anti-hapten/hapten Interaktion. Am Ende eines jeden Testlauf werden die Sensoren mit einer verdünnten Säure ermöglicht die sequentielle Analyse von Spalten aus einer 96-Well-Platte gereinigt. Hier präsentieren wir die Methode zur Entwicklung eines benutzerdefinierten CHO-HCP AMMP Assay für Bioprozess-Entwicklung. Die Entwicklung AMMP Assays oder die Änderung bestehender Assays in AMMP Assays können eine bessere Leistung (Reproduzierbarkeit, Empfindlichkeit) in komplexen Proben und reduzierte Arbeitszeit. Das Protokoll zeigt die Schritte für die Entwicklung und die Diskussion der Bewertungen repräsentative Ergebnisse. Für eine ausführliche Erläuterung der Assay-Optimierung und Anpassung Parameter Kontakt BioScale. Dieses Kit bietet generische Bioprozessentwicklung Assays wie Residual Protein A, Produkt-Titer und 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

Entwicklung von kundenspezifischen Assays auf der ViBE Plattform für empfindlichere, reproduzierbare, automatisierte Ergebnisse in komplexen Matrices beschrieben ist. Die Universal-Kassette ermöglicht Assays zu leicht für die Verwendung mit benutzerdefinierten Tests angepasst werden. Diese Vielseitigkeit ermöglicht die schnelle Entwicklung und Validierung neuer Tests oder automatisierte Versionen bestehender manueller Tests, in diesem Video veranschaulicht.

Entwickeln von benutzerdefinierten Chinesischen Hamsters-Wirtszellprotein Assays mit Akustikvlies Mikropartikeltechnologie

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 Aufrufe.  BioScale, Inc.. Entwicklung von kundenspezifischen Assays auf der ViBE Plattform für empfindlichere, reproduzierbare, automatisierte Ergebnisse in komplexen Matrices beschrieben ist. Die Universal-Kassette ermöglicht Assays zu leicht für die Verwendung mit benutzerdefinierten Tests angepasst werden. Diese Vielseitigkeit ermöglicht die schnelle Entwicklung und Validierung neuer Tests oder automatisierte Versionen bestehender manueller Tests, in diesem Video veranschaulicht.

Serie: 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.

Steigende cDNA Erträge aus Einzel-Zell-Mengen von mRNA in Standard Laboratory Reverse-Transkriptase-Reaktionen mit 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 ...

Forschung

Biotechnik

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 und Charakterisierung von 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 ...

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.

Generischen Protokoll zur Optimierung der heterologen Proteinproduktion mit automatisierten Microbioreactor Technologie

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.

Reinigung und Analyse eines monoklonalen Antikörpers aus chinesischen Hamster-Ovarialzellen mit einem automatisierten Mikrobioreaktorsystem

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.

Prozessoptimierung mit automatisierten Mikro-Bioreaktoren mit hohem Durchsatz in der chinesischen Hamster-Ovarialzell-Kultivierung

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.

Herstellung von Proteinproduktion synthetischer Zellen mit zellfreien bakteriellen Extrakten, Liposomen und Emulsionstransfer

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.

Primärklärung der CHO geernteten Zellkulturflüssigkeit mittels eines akustischen Separators

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.

Formulierung und akustische Modulation von optisch verdampften Perfluorcarbon-Nanotröpfchen

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.

Schnelles Antikörper-Glykoengineering in chinesischen Hamster-Eierstockzellen

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 Spaltungstests mit gereinigten rekombinanten Drosophila-Caspasen für das Substrat-Screening

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