1. Preparación de micropartículas <ol><li> Los anticuerpos con biotina etiqueta NHS Química </li><li> Determinación del grado de biotinilación </li><li> Conjugado estreptavidina bolas con anticuerpos con biotina <ol><li> Volver a suspender las bolas en el vial original por agitación durante 1-2 minutos </li><li> Alícuota del volumen deseado de granos en un tubo de microcentrífuga </li><li> Lavar las perlas de tres veces con PBST: <ol><li> Coloque el tubo que contiene las cuentas en el imán durante 2 minutos </li><li> Eliminar el sobrenadante por aspiración con el tubo en el imán. Tenga cuidado de no alterar las cuentas </li><li> Retire el tubo del imán y añadir 1 ml de PBST </li><li> Vortex durante 20 segundos para volver a suspender las cuentas </li><li> Centrifugar durante 5 segundos para eliminar las cuentas de la tapa </li><li> Repetir dos veces más 2.3.1-2.3.5 </li></ol></li><li> Después del tercer lavado lugar el tubo en el imán y aspirar el PBST </li><li> Añadir anticuerpo biotinilado a las perlas y resuspender </li><li> Si es necesario llevar el volumen de los anticuerpos con biotina hasta 300 ml con PBS. Si el volumen de anticuerpos ya es mayor de 300 l no añadir PBS </li><li> Incubar la suspensión de bolas / anticuerpo durante 30 minutos a temperatura ambiente en un agitador vortex o rotor del tubo </li><li> Lavar 3 veces con PBST como en el paso 3. </li><li> Bloquear cuentas con biotina BSA <ol><li> Después del tercer lavado añadir 10 mg / mg biotina BSA, ajustar el volumen a 300 ml con PBS, si es necesario, e incubar con agitación durante 30 minutos </li></ol></li><li> Lavar 3 veces con PBST como en el paso 3 </li><li> Volver a suspender las bolas en 100 L de PBS/0.05% de BSA. Azida de sodio puede ser agregado para almacenamiento a largo plazo </li></ol></li><li> Utilizando protocolos estándar NHS con fluoresceína, fluoresceinato el anticuerpo detector </li><li> Determinar el grado de Fluorescination </li></ol> 2. HCP - Desarrollo <ol><li> Prepare las soluciones de trabajo: <ol><li> Anticuerpos fluoresceína (FL-Ab): La mayoría de los ensayos de CHO-HCP uso Fl-Ab a una concentración final de 400 ng / mL. El FL-Ab se agrega como ¼ del volumen final del ensayo, por lo tanto, la concentración de la solución de trabajo es de 1,6 mg / ml. Preparar esta solución diluyendo acciones gordura con BioScale bolas / diluyente de flacidez. </li><li> Cuentas: La mayoría de los ensayos de CHO-HCP utilizar cuentas a una concentración final de 2x10 5 cuentas / ml. El reactivo de cuentas constituye una cuarta parte de el volumen final del ensayo, por lo tanto la concentración de la solución de trabajo es de 8x10 5 cuentas / ml. Preparar esta solución diluyendo acciones de bolas con bolas BioScale / diluyente de flacidez. Vortex el stock de cuentas y para asegurarse de que todas las cuentas están en suspensión antes de extraer una alícuota. </li><li> Running Buffer: La composición del tampón de que tenga que ser optimizado para los anticuerpos utilizados para desarrollar el ensayo, sin embargo, varios CHO-HCP ensayos se han desarrollado utilizando tampón HEPES 10 mM, 1% de Tween, pH 7,5. BioScale ha desarrollado un aditivo para el funcionamiento de amortiguación (suministrado con el kit de desarrollo) que se añade al tampón a una concentración del 1% antes de su uso. </li></ol></li><li> Diluir los calibradores y las muestras con diluyente de la muestra BioScale. </li></ol> 3. VIBE de configuración del sistema <ol><li> A su vez la estación de trabajo Vibe </li><li> Conecte el sistema de suministro y de los residuos </li><li> Cebe el sistema de </li><li> Introducir los cartuchos de ViBE </li><li> Ruta de los tubos </li><li> Preparar plantilla de ensayo rellenando la hoja de la etiqueta &quot;Configuración de la muestra&quot;. BioScale ofrece la posibilidad de tener una plantilla de ensayo para cada ensayo de estación de trabajo VIBE. Esto permite al usuario editar y guardar notas específicas en las &quot;Notas&quot; hoja de la plantilla para los ensayos de diferentes manteniendo un registro completo de la experiencia. </li><li> Plantilla de ensayo de la configuración inicial: Comience por llenar la mesa (en la esquina superior izquierda) del rango de concentración de los estándares en el ensayo en la &quot;configuración de muestra&quot; de hoja. </li><li> A continuación, introducir información adicional en la tabla a la derecha de las normas (configuración de columna simple lectura, One Agregar reactivo y dos configuraciones de reactivo Añadir) para instruir a la estación de trabajo ViBE el número de muestras a largo necesario para la incubación, la cantidad de volumen de reactivo se va a transferir , ¿cuánto tiempo la acumulación de la muestra tiene que ser, y si de acondicionamiento del sensor y la descontaminación de las sondas y el cartucho son necesarios, etc </li><li> Rellene el diseño de la placa. </li><li> Carga de la muestra / reactivos en las placas. El VIBE proporcionará los volúmenes necesarios para el plato de reactivos. Volumen de la muestra puede variar, pero en medio de un volumen de ensayo total de 120 l es un buen punto de partida. </li><li> Aplicar cubiertas de la placa. Placas de carga sobre la cubierta de estación de trabajo. </li><li> Realizar el ensayo en marcha el asistente, vaya a través de asistente e iniciar la prueba. </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Figura 1" /> Figura 1: Como parte del proyecto de Inmunoensayo Sandwich </p&gt; En una estación de trabajo VIBE, un ensayo parte del proyecto se mide la concentración de antígeno. La señal se mide cuando el anticuerpo-antígeno-cordón hapteno se une al sensor a través del hapteno / interacción anti-hapteno. Regeneración lleva el sensor de nuevo al estado comenzando por la eliminación de los complejos inmunes. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Figura 2" /> Figura 2: CHO-HCP Ensayo Como parte del proyecto curva de calibración La figura 2 es un ejemplo de una curva de calibración CHO HCP Como parte del proyecto de ensayo. El análisis puede llevarse a cabo en muestras de toda la corriente de purificación incluyendo el reactor de alta precisión y reproducibilidad. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Figura 3" /> Figura 3: Proteína Una curva de calibración del ensayo parte del proyecto La figura 3 es una curva de calibración para una proteína residual Un ensayo Como parte del proyecto, realizado con una proteína residual VIBE un kit de ensayo. El mismo kit puede ser utilizado para un ensayo más rápido con un período de incubación de 30 minutos, o para obtener resultados más sensible y más amplio rango dinámico, con un período de incubación más largo, sin pérdida de precisión o exactitud, los cuales suelen ser mejores que sus equivalentes ELISA en los usuarios manos <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Figura 4" /> Figura 4: El producto Titer Como parte del proyecto de ensayo La estación de trabajo ViBE también se puede utilizar para un ensayo Titer producto, resultados que se muestran en la Figura 4. Realiza de forma diferente que los otros ensayos Como parte del proyecto, este ensayo no requiere tratamiento de la muestra que no sea la simple dilución, o perlas, y la cuantificación se ha verificado en &quot;sin efecto&quot; las muestras que contienen más de 10 millones de células en un amplio rango de viabilidad. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Figura 5" /> Figura 5: Ensayo Como parte del proyecto curva estándar para GST-Gadd34 biomarcador ensayo Muestra en la Figura 5 es una curva de nivel de señal que se obtiene por el Bioanalyzer Vibe (Grupo A) o Tecnología AlphaScreen utilizando recombinante GST-Gadd34. Resultados en la plataforma ViBE fueron de aproximadamente un registro más sensible con los mismos anticuerpos. Como parte del proyecto aprovecha la medición de partículas de mayor tamaño menos y aún así los beneficios de la captura en fase de solución y la formación de complejos de detección. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Figura 6" /> Figura 6: Curva Como parte del proyecto de ensayo estándar para la GST-Gadd34 en una muestra de tumor En la figura 6, la inducción de Gadd34 de CWR22 xenoinjertos fue probado en la presencia o ausencia de un inhibidor del proteasoma boronato como se ha analizado tanto por el Bioanalyzer Vibe (Grupo A) o AlphaScreen (Grupo B). La plataforma ambiente era capaz de producir resultados sensibles y reproducibles en los xenoinjertos de tumores in vivo, mientras que la señal se interrumpió con AlphaScreen mg cada vez mayor de proteínas. Este ejemplo muestra el poder de la tecnología Como parte del proyecto en las muestras de gran complejidad. Debido a que el chip de detección sensibles y de alto rendimiento del imán cerca de los chips, bajo número de micropartículas magnéticas de manera eficiente y reproducible para la meta recoger baja abundancia en medio de la complejidad de la lisis celular (incluidos los reactivos de lisis) y restos de sangre.

Los autores de este artículo son empleados por Bioscale, Inc, que produce algunos de los instrumentos y reactivos utilizados en este artículo.

Los ejemplos anteriores muestran muy sensibles los resultados cuantitativos Como parte del proyecto en muestras complejas. Esta actuación, cuando se entrega con la facilidad y el control de formato de reactivos en el sistema es propicio para una variedad de aplicaciones. Estas aplicaciones pueden ser específicos para un objetivo, como en Gadd34 en el tumor, o genérico para un espectro de objetivos, como en la detección de proteínas de la célula huésped y la cuantificación de las muestras que cruzar el arroyo de purificación. La plataforma de arriba ViBE bajas combinadas con AMMP altamente flexible proporciona una herramienta poderosa para la entrega de los ensayos de crítica.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Los ensayos de encargo de proteínas únicas a menudo se limitan a la detección manual de tiempo y técnicas de cuantificación, como ELISA o Western blot, debido a la complejidad del desarrollo de plataformas alternativas. Propiedad BioScale de membrana acústica de micropartículas (AMMP) permite inmunoensayos sándwich para ser fácilmente desarrollado para su uso en la plataforma de VIBE, proporcionando una mejor sensibilidad, reproducibilidad y funcionamiento automatizado. Siempre como un ejemplo, este protocolo se describe el procedimiento para el desarrollo de una costumbre de ovario de hámster chino-Host proteína celular (CHO-HCP) de ensayo. Los principios generales enunciados aquí se puede seguir para el desarrollo de una amplia variedad de inmunoensayos. Un ensayo de las medidas de parte del proyecto de concentración del antígeno mediante la medición de los cambios en la frecuencia de oscilación causada por la unión de las micropartículas de la superficie del sensor para calcular. Consta de cuatro componentes principales: (1) un cartucho que contiene un chip sensor funcionalizados ocho (2) anticuerpo marcado micropartículas magnéticas, (3) anticuerpos hapteno marcado que se une a la superficie del chip funcionalizadas (4) las muestras que contienen el antígeno de de interés. Biosensor BioScale es un dispositivo de resonancia que contiene ocho membranas individuales con distintos caminos fluidos. Las membranas de cambio de frecuencia de oscilación en respuesta a la acumulación de masa en la superficie y el cambio de frecuencia se utiliza para cuantificar la cantidad de masa añadida. Para facilitar su uso en una amplia variedad de inmunoensayos se funcionaliza el sensor con un anticuerpo anti-hapteno. Anticuerpos específicos de ensayo se han modificado a través de la conjugación covalente de una etiqueta a un anticuerpo hapteno y biotina a la otra. La etiqueta de biotina se utiliza para enlazar el anticuerpo unido a la estreptavidina partículas magnéticas que, en combinación con el anticuerpo hapteno-etiquetados, se utilizan para capturar a analizar en un sándwich. El complejo se une al chip a través de la interacción anti-hapten/hapten. Al final de cada ensayo de ejecución de los sensores se limpian con un ácido diluido que permite el análisis secuencial de las columnas de una placa de 96 pocillos. A continuación, presentamos el método para el desarrollo de una costumbre CHO-HCP ensayo Como parte del proyecto para el desarrollo de bioprocesos. El desarrollo de ensayos Como parte del proyecto o la modificación de los ensayos existentes en los ensayos de parte del proyecto se puede proporcionar un mejor rendimiento (reproducibilidad, sensibilidad) en muestras complejas y el tiempo del operador. El protocolo se muestran los pasos para el desarrollo y la sección de discusión revisa los resultados representativos. Para una explicación más detallada de la optimización del ensayo y parámetros de personalización de contacto BioScale. Este kit ofrece genérico ensayos de desarrollo de bioprocesos, tales como proteína residual Un título del producto, y HCP-CHO.

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

Desarrollo de ensayos personalizados en la plataforma de Vibe para más sensibles, reproducibles, automatizado en matrices complejas se describe. El cartucho universal permite ensayos para ser adaptado fácilmente para su uso con los ensayos de costumbre. Esta versatilidad permite un rápido desarrollo y validación de nuevos ensayos o versiones automatizadas de los actuales ensayos manuales, ejemplificado en este video.

Desarrollar ovario de hámster chino anfitrión-ensayos con células de proteínas utilizando la tecnología de membrana acústica de micropartículas

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 vistas.  BioScale, Inc.. Desarrollo de ensayos personalizados en la plataforma de Vibe para más sensibles, reproducibles, automatizado en matrices complejas se describe. El cartucho universal permite ensayos para ser adaptado fácilmente para su uso con los ensayos de costumbre. Esta versatilidad permite un rápido desarrollo y validación de nuevos ensayos o versiones automatizadas de los actuales ensayos manuales, ejemplificado en este 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.

Aumentar el rendimiento de cDNA a partir de una sola célula cantidades de ARNm en las reacciones de laboratorio estándar de la transcriptasa inversa con microcorrientes acústica

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 ...

Investigación

Bioingeniería

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.

Aislamiento Y Caracterización De Proteínas Quiméricas Humanas Que Expresan Fc Usando Adsorbentes De Membrana De Proteína A Y Un Flujo de Trabajo Optimizado

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.

Protocolo genérico para la optimización de la producción de proteína heteróloga utilizando tecnología Microbioreactor automatizada

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.

Purificación y análisis de un anticuerpo monoclonal de células de ovario de hámster chino utilizando un sistema automatizado de microbiorreactores

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.

Optimización de procesos mediante microbiorreactores automatizados de alto rendimiento en el cultivo de células de ovario de hámster chino

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.

Preparación de células sintéticas productoras de proteínas mediante extractos bacterianos libres de células, liposomas y transferencia de emulsión

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.

Clarificación primaria del fluido de cultivo celular cosechado con CHO utilizando un separador acústico

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.

Formulación y modulación acústica de nanogotas de perfluorocarbono vaporizadas ópticamente

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.

Glicoingeniería rápida de anticuerpos en células de ovario de hámster chino

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 Ensayos de escisión utilizando Caspasas de Drosophila recombinantes purificadas para cribado de sustrato

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