1. Preparação de micropartículas <ol><li> Anticorpos etiqueta com Biotina NHS Química </li><li> Determinação do Grau de Biotinilação </li><li> Beads conjugado estreptavidina com anticorpo biotinilado <ol><li> Volte a suspender as contas no frasco original em vortex por 1-2 minutos </li><li> Alíquota o volume desejado de contas em um tubo de microcentrífuga </li><li> Lave as contas 3 vezes com PBST: <ol><li> Coloque o tubo que contém as contas sobre o ímã por 2 minutos </li><li> Retire o sobrenadante por aspiração com o tubo sobre o ímã. Tenha cuidado para não atrapalhar as contas </li><li> Retire o tubo do ímã e adicionar 1 mL PBST </li><li> Vortex por 20 segundos para voltar a suspender as contas </li><li> Centrifugar durante 5 segundos para remover as contas da tampa </li><li> Repita mais duas vezes 2.3.1-2.3.5 </li></ol></li><li> Após a terceira lavagem lugar do tubo sobre o ímã e aspirar o PBST </li><li> Adicionar anticorpo biotinilado para as contas e ressuspender </li><li> Se for necessário reduzir o volume do anticorpo biotinilado até 300 mL com PBS. Se o volume de anticorpos é já superior a 300 mL não adicionar qualquer PBS </li><li> Incubar a suspensão de esferas / anticorpo por 30 minutos em temperatura ambiente no agitador vortex ou tubo rotador </li><li> Lavar 3 vezes com PBST como no passo 3. </li><li> Bloco de contas com BSA biotinilado <ol><li> Após a terceira lavagem adicionar 10 mg / mg biotinilado BSA, ajustar o volume para 300 mL com PBS, se necessário, e incubar com agitação por 30 minutos </li></ol></li><li> Lavar 3 vezes com PBST como no passo 3 </li><li> Volte a suspender as contas em 100 mL de BSA PBS/0.05%. Azida de sódio pode ser adicionado para armazenamento a longo prazo </li></ol></li><li> Utilizando protocolos padrão NHS fluoresceína, o anticorpo fluoresceinate detector </li><li> Determinar o grau de Fluorescination </li></ol> 2. HCP - Desenvolvimento <ol><li> Prepare soluções de trabalho: <ol><li> Anticorpo marcado com fluoresceína (Fl-Ab): A maioria dos CHO-HCP ensaios use Fl-Ab na concentração final de 400 ng / mL. O Fl-Ab é adicionado como ¼ do volume final do ensaio, por isso a concentração da solução de trabalho é de 1,6 mcg / mL. Preparar esta solução diluindo estoque Flab com BioScale Bead / Diluente flacidez. </li><li> Contas: a maioria dos CHO-HCP ensaios use bolinhas em uma concentração final de 2x10 5 gotas / mL. O reagente talão constitui ¼ do volume final do ensaio, por isso a concentração da solução de trabalho é 8x10 5 gotas / mL. Preparar esta solução diluindo estoque talão com BioScale Bead / Diluente flacidez. Vortex do estoque talão bem para garantir que todas as contas estão em suspensão antes de remover uma alíquota. </li><li> Execução Buffer: A composição do tampão de corrida pode ter que ser otimizado para os anticorpos utilizados para desenvolver o ensaio, no entanto várias CHO-HCP ensaios foram desenvolvidos utilizando tampão HEPES 10 mM, Tween 1%, pH 7.5. BioScale desenvolveu um aditivo para o tampão de corrida (fornecido com o kit de desenvolvimento), que é adicionado ao buffer para uma concentração de 1% antes do uso. </li></ol></li><li> Diluir os calibradores e amostras com Diluente de Amostra BioScale. </li></ol> 3. ViBE Sistema de Set Up <ol><li> Vire o Workstation ViBE em </li><li> Ligue a fonte de sistema e resíduos </li><li> Primeiro o sistema </li><li> Insira um cartucho ViBE </li><li> Rota dos tubos </li><li> Prepare modelo de ensaio, preencha a folha chamada &quot;Setup Amostra&quot;. BioScale fornece a capacidade de ter um modelo de análise para cada ensaio Workstation ViBE. Isto permite ao usuário editar e salvar notas específicas nas &quot;Notas&quot; da folha do modelo para diferentes testes de manter um registro completo do experimento. </li><li> Modelo de teste de configuração inicial: Comece por preencher a tabela (canto superior esquerdo) da faixa de concentração dos padrões no ensaio da &quot;Sample Setup&quot; folha. </li><li> Em seguida, insira informações adicionais na tabela à direita dos padrões (Setup Coluna Simples Read, Uma Adicionar reagente e dois setups Reagente Add) para instruir o Workstation ViBE como amostras longa necessidade de incubar, o quanto de volume de reagente deve ser transferida , quanto tempo a acumulação amostra precisa ser, e se condicionado sensores e descontaminação de sondas e cartucho são necessários, etc </li><li> Preencha o layout da placa. </li><li> Carga de amostra / reagentes em placas. O ViBE irá fornecer os volumes necessários para a placa de reagente. Volume de amostra pode variar, mas na metade de um volume de ensaio total de 120 mL é um bom lugar de partida. </li><li> Aplicar cobre da placa. Placas de carga no convés em estação de trabalho. </li><li> Executar o ensaio de arranque assistente, passar por assistente e iniciar ensaio. </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Figura 1" /> Figura 1: AMMP Imunoensaio Sandwich </p&gt; Numa estação de trabalho ViBE, um ensaio AMMP mede a concentração de antígeno. Sinal é medido quando o anticorpo-antígeno hapteno bead-liga-se ao sensor através do hapteno / anti-hapteno interação. Regeneração traz o sensor de volta ao estado inicial, removendo os complexos imunes. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Figura 2" /> Figura 2: CHO-HCP AMMP curva de calibração do ensaio Figura 2 acima é um exemplo de uma AMMP HCP CHO curva de calibração do ensaio. Análise pode ser realizada em amostras de todo o fluxo de purificação, incluindo o reator com alta precisão e reprodutibilidade. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Figura 3" /> Figura 3: Proteína A curva de calibração AMMP Assay Figura 3 é uma curva de calibração para uma proteína residual Um Ensaio AMMP, realizada com uma proteína VIBE Residual Um Kit de Ensaio. O mesmo kit pode ser usado para um rápido ensaio com um período de incubação de 30 minutos, ou para resultados mais sensível e maior gama dinâmica, com um período mais longo de incubação, sem perda de precisão ou precisão, sendo que ambos são normalmente melhor do que equivalentes ELISA em usuários mãos <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Figura 4" /> Figura 4: Produto testes sorológicos AMMP Assay O Workstation ViBE também pode ser usado para um ensaio de Produto testes sorológicos, os resultados mostrados na figura 4. Realizada de forma diferente Ensaios AMMP, este ensaio não necessita de tratamento da amostra que a diluição simples, ou grânulos, e quantificação foi verificado em &quot;nenhum spin&quot; amostras contendo mais de 10 milhões de células em uma ampla faixa de viabilidades. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Figura 5" /> Figura 5: AMMP curva padrão do ensaio para o ensaio GST-Gadd34 biomarcador Mostrado na Figura 5 é uma curva padrão de sinal obtido pela Bioanalyzer ViBE (Painel A) ou Tecnologia AlphaScreen usando recombinante GST-Gadd34. Resultados na plataforma ViBE foram de aproximadamente um log mais sensível utilizando os mesmos anticorpos. AMMP utiliza a medição de menos partículas maiores benefícios ainda assim, desde a captura solução fase e formação do complexo de detecção. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Figura 6" /> Figura 6: Ensaio AMMP curva padrão para GST-Gadd34 em amostra de tumor Na Figura 6, a indução de Gadd34 de CWR22 xenoenxertos foi testada na presença ou ausência de um inibidor de proteassoma boronato como analisado tanto pela Bioanalyzer ViBE (Painel A) ou AlphaScreen (Painel B). A plataforma ViBE foi capaz de produzir sensíveis, resultados reprodutíveis na in vivo xenoenxertos tumor enquanto o sinal AlphaScreen se apagou com o aumento mg de proteína. Este exemplo mostra o poder da tecnologia AMMP em amostras altamente complexo. Devido ao chip de detecção sensível e de alta eficiência do ímã perto do chips, baixo número de micropartículas magnéticas podem de forma eficiente e reproduzível para limpar alvo baixa abundância em meio à complexidade de lisados ​​celulares (incluindo os reagentes de lise) e os restos de sangue.

Os autores deste artigo são empregados por Bioscale, Inc, que produz alguns dos instrumentos e reagentes utilizados neste artigo.

Os exemplos anteriores demonstram altamente sensível AMMP de resultados quantitativos em amostras complexas. Esse desempenho, quando entregues com a facilidade e controle de formatação reagentes para o sistema é propício para uma variedade de aplicações. Os pedidos podem ser específicos para um alvo, como em Gadd34 no tumor, ou genérico para um espectro de alvos, como na série de detecção e quantificação de proteínas de células em amostras que cruzam o fluxo de purificação. A plataforma ViBE baixo overhead combinado com AMMP altamente flexível fornece uma ferramenta poderosa para a entrega de ensaios críticos.

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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

Ensaios personalizado para proteínas específicas são muitas vezes limitadas ao tempo de detecção manual de consumir e as técnicas de quantificação, tais como ELISA ou Western blot, devido à complexidade do desenvolvimento em plataformas alternativas. Proprietários BioScale de Membrana acústica micropartículas tecnologia (AMMP) permite imunoensaios sanduíche para ser facilmente desenvolvidos para uso na plataforma ViBE, proporcionando uma melhor sensibilidade, reprodutibilidade e operação automatizada. Fornecido como um exemplo, este protocolo descreve o procedimento para o desenvolvimento de um costume chinês Hamster Proteína de Célula Ovário-Host (CHO-HCP) ensaio. Os princípios gerais aqui delineados podem ser seguidas para o desenvolvimento de uma ampla variedade de imunoensaios. AMMP um ensaio concentração do antígeno medidas medindo mudanças na freqüência de oscilação causada pela ligação de micropartículas à superfície do sensor de calcular. É composto de quatro componentes principais: (1) um cartucho que contém um chip sensor funcionalizados oito (2) anticorpo marcado micropartículas magnéticas, (3) anticorpo hapteno marcado que se liga à superfície do chip funcionalizados (4) amostras contendo o antígeno de interesse. BioScale biosensor é um dispositivo ressonante que contém oito individuais com membranas separadas caminhos fluídico. As membranas alterar a freqüência de oscilação em resposta a massa acumulando na superfície e essa mudança de freqüência é usada para quantificar a quantidade de massa adicionada. Para facilitar o uso em uma ampla variedade de imunoensaios o sensor é funcionalizada com um anticorpo anti-hapteno. Anticorpos específicos ensaio são modificados por meio da conjugação covalente de um tag hapteno a um anticorpo e biotina para o outro. O rótulo biotina é usada para vincular o anticorpo para estreptavidina acoplado esferas magnéticas que, em combinação com o anticorpo hapteno-tagged, são usados ​​para capturar o analito em um sanduíche. O complexo se liga ao chip através da interação anti-hapten/hapten. No final de cada ensaio os sensores são limpas com um ácido diluído permitindo a análise seqüencial de colunas de uma placa de 96 poços. Aqui, apresentamos o método para o desenvolvimento de um costume CHO-HCP ensaio AMMP para o desenvolvimento de bioprocessos. Desenvolvimento de ensaios AMMP ou modificar ensaios existentes em ensaios AMMP pode proporcionar um melhor desempenho (sensibilidade, reprodutibilidade) em amostras complexas e tempo do operador. O protocolo mostra os passos para o desenvolvimento ea seção de discussão opiniões resultados representativos. Para uma explicação mais aprofundada da otimização do ensaio e os parâmetros de personalização contato BioScale. Este kit oferece genéricos ensaios de desenvolvimento de bioprocessos, como proteína residual Um título do produto, e CHO-HCP.

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

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

Desenvolvimento de ensaios personalizado na plataforma ViBE para mais sensíveis, reprodutíveis, os resultados automatizados em matrizes complexas é descrito. O cartucho universal permite ensaios para ser facilmente adaptadas para uso com ensaios personalizado. Esta versatilidade permite o rápido desenvolvimento e validação de ensaios de romance ou versões automatizada de ensaios existentes manual, exemplificado neste vídeo.

Desenvolvimento personalizado Ovário de Hamster Chinês hospedar-Cell ensaios de proteína utilizando a Tecnologia micropartícula Acústico Membrana

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

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

Research

JoVE Journal

Bioengineering

13.1K visualizações.  BioScale, Inc.. Desenvolvimento de ensaios personalizado na plataforma ViBE para mais sensíveis, reprodutíveis, os resultados automatizados em matrizes complexas é descrito. O cartucho universal permite ensaios para ser facilmente adaptadas para uso com ensaios personalizado. Esta versatilidade permite o rápido desenvolvimento e validação de ensaios de romance ou versões automatizada de ensaios existentes manual, exemplificado neste vídeo.

Vídeo: 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 os rendimentos cDNA Single-cell quantidades de mRNA em Laboratório Padrão Reações transcriptase reversa usando Microstreaming Acústico

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 ...

Bioengenharia

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

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

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

Isolamento e caracterização de proteínas quimricas que expressam fc humanos usando adsorbers de membrana de proteína e um fluxo de trabalho simplificado

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 otimização da produção de proteína heteróloga usando tecnologia automatizada de Microbioreactor

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

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

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

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

Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

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.

Otimização de processos usando micro-bioreatores automatizados de alto throughput no cultivo de células de ovário de hamster chinês

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.

Preparando proteínas que produzem células sintéticas usando extratos bacterianos livres de células, liposomos e transferência de emulsão

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.

Esclarecimento Primário do Fluido de Cultura Celular Colhida cho usando um 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.

Formulação e modulação acústica de nanogotículas de perfluorocarbono opticamente vaporizadas

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.

Glicoengenia de anticorpos rápidos em células de ovário de hamster chinês

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 Ensaios de Clivagem usando Caspases de Drosophila Recombinante Purificada para Triagem de Substrato

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