1。微粒子の調製<ol><li>ビオチンNHS化学を使用してラベルの抗体</li><li>ビオチン化の程度の決定</li><li>ビオチン化抗体とコンジュゲートストレプトアビジンビーズ<ol><li> 1〜2分間ボルテックスして、元のバイアルでビーズを再懸濁します</li><li>マイクロ遠心チューブに分注しビーズの目的のボリュームを</li><li> PBSTでビーズを3回洗浄。 <ol><li> 2分間磁石でビーズを含むチューブを置きます</li><li>磁石上にチューブで吸引して上清を取り除きます。ビーズを妨害しないように注意してください</li><li>磁石からチューブを外し、1 mLのPBSTを追加</li><li>ビーズを再懸濁するために20秒間ボルテックス</li><li>キャップからビーズを除去するために5秒間遠心分離します。 </li><li>さらに2回2.3.1-2.3.5を繰り返します</li></ol></li><li>第三洗浄は、磁石にチューブを配置し、PBSTを吸引除去した後</li><li>ビーズにビオチン化抗体を追加し、再懸濁</li><li>必要な場合は、PBSで300μLにビオチン化抗体の量を持ち出す。抗体の量は任意のPBSを追加しない既に超えて300μLである場合</li><li>ボルテックスシェーカーまたはチューブローテーターで室温で30分間ビーズ/抗体サスペンションをインキュベート</li><li>ステップ3のようにPBSTで3回洗浄する。 </li><li>ビオチン化BSAでビーズをブロックする<ol><li>第三洗浄には10μg/ mgのビオチン化BSAを追加した後、PBS必要に応じて300μLに音量を調節し、30分間撹拌しながらインキュベート</li></ol></li><li>ステップ3のようにPBSTで3回洗浄する</li><li> PBS/0.05％のBSAの100μLでビーズを再懸濁します。アジ化ナトリウムは、長期保管のために追加することができます。 </li></ol></li><li>標準NHSフルオレセインのプロトコルを使用して、検出抗体をfluoresceinate </li><li> Fluorescinationの程度を決定</li></ol> 2。 HCP - 開発<ol><li>ワーキング溶液の調製法<ol><li>フルオレセイン標識抗体（FL - AB）：ほとんどのCHO - HCPアッセイは、400 ng / mLの最終濃度でFL - Abを使用してください。 FL - ABは、ワーキング溶液の濃度が1.6μg/ mLとなるため、最終アッセイ体積の¼として追加されます。 BioScaleビーズ/ぜい肉希釈液でぜい肉の株式を希釈することにより、この溶液を調製します。 </li><li>ビーズ：ほとんどのCHO - HCPアッセイは、2 × 10 5ビーズ/ mlの最終濃度でビーズを使用してください。ビーズ試薬は、最終アッセイ体積の¼を構成しているのでワーキング溶液の濃度は、5ビーズ/ mLの8 × 10です。 BioScaleビーズ/ぜい肉希釈液でビーズ株式を希釈することにより、この溶液を調製します。渦ビーズ株式はよく確認するために、すべてのビーズは、アリコートを除去する前に、懸濁液です。 </li><li>ランニングバッファー：ランニングバッファーの組成は、アッセイを開発するために使用される抗体のために最適化されなければならないかもしれない、しかし、いくつかのCHO - HCPアッセイは、10mMのHEPES緩衝液を使用して開発された、1％のTween、pH7.5の。 BioScaleは使用前に1％の濃度にバッファに追加されるランニングバッファー（開発キットに付属）のための添加剤を開発しました。 </li></ol></li><li> BioScaleサンプル希釈液で校正し、試料を希釈する。 </li></ol> 3。 ViBEプロダクトシステムのセットアップ<ol><li>グラスバレーのワークステーションの電源をONに</li><li>システムの供給と廃棄物を接続する</li><li>プライムシステム</li><li>グラスバレーカートリッジを挿入</li><li>チューブを配線します。 </li><li> &quot;サンプルの設定&quot;というラベルシートに記入することによってアッセイのテンプレートを準備します。 BioScaleは、それぞれのグラスバレーのワークステーションのアッセイ用のアッセイのテンプレートを持っている機能を提供します。これにより、ユーザーは実験の完全な記録を保持する別のアッセイのためのテンプレートの&quot;注意事項&quot;シート内の特定のノートを編集して保存することができます。 </li><li>初期アッセイのテンプレートは、設定する：&quot;サンプルの設定&quot;シートのアッセイの標準の濃度範囲の表（左上隅）を記入することから始めます。 </li><li>次に、試薬の量を転送する場合どの程度、インキュベートする必要があるどのくらいのサンプルがグラスバレーのワークステーションを指示する（単純な列は、一つの試薬の追加と2つの試薬の追加セットアップをセットアップを読む）規格の右側のテーブルに追加情報を入力してください、サンプルの蓄積がする必要があるどのくらいの時間、およびセンサーの調整とプローブとカートリッジの汚染除去が必要かどうか、など</li><li>プレートのレイアウトを記入してください。 </li><li>プレートに試料/試薬をロードします。雰囲気は試薬のプレートのために必要なボリュームを提供します。サンプル量が異なりますが、120μLの全アッセイ体積の半分でよい出発点であることができる。 </li><li>プレートカバーを適用します。ワークステーションのデッキの上にプレートをロードします。 </li><li>ウィザードを起動アッセイを実行、ウィザードの指示に従って移動し、アッセイを開始します。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/2493/2493fig1.jpg" alt="図1" /> 図1：AMMPサンドイッチ免疫測定法 </p&gt; グラスバレーのワークステーションで、AMMPアッセイは、抗原の濃度を測定する。信号はビーズ - 抗原 - ハプテン抗体がハプテン/抗ハプテン相互作用を介してセンサーに結合するときに測定されます。再生には、免疫複合体を除去することによって最初の状態に戻ってセンサーをもたらします。 <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="図2" /> 図2：CHO - HCP AMMPアッセイの検量線 上記の図2は、CHO HCP AMMPアッセイの検量線の例です。分析は高精度と再現性を持つ原子炉を含む精製ストリーム全体からのサンプルで実行することができます。 <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="図3" /> 図3：プロテインAMMPアッセイの検量線 図3は、VIBE残留タンパク質アッセイキットを使用して実行によりタンパクの残渣AMMPアッセイのための検量線です。同キットは、30分間の潜伏期間を持つ高速なアッセイのための、またはより高感度な結果のために使用され、ユーザーは通常、同等のELISAのより良いが、どちらも精度や正確さを失うことなく、長い潜伏期間を持つ広いダイナミックレンジ、することができます&quot;手<img src="/files/ftp_upload/2493/2493fig4.jpg" alt="図4" /> 図4：製品タイターAMMPアッセイ グラスバレーのワークステーションは、結果が図4に示すように、製品の力価検定にも使用できます。他のAMMPアッセイとは異なる方法で実行される、このアッセイは、単純な希釈、またはビーズよりサンプル処理が他の必要としない、と定量は、生存率の広い範囲にわたって1000万人以上の細胞を含む&quot;無スピン&quot;のサンプルで確認されています。 <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="図5" /> 図5：GST - Gadd34バイオマーカーアッセイのためのAMMPアッセイ標準曲線 図5に示すには、グラスバレーバイオアナライザ（パネルA）または組換えGST - Gadd34を使用してAlphaScreenテクノロジーによって得られた信号の標準曲線である。グラスバレーのプラットフォーム上での結果はほぼ同じ抗体を使用して、より敏感なログでした。 AMMPはまだ少数の大きい粒子の液相捕捉および検出複合体形成から、まだ利益の測定を利用しています。 <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="図6" /> 図6：腫瘍サンプル中のGST - Gadd34用AMMPアッセイ標準曲線 図6では、CWR22異種移植片からGadd34の誘導は、グラスバレーバイオアナライザ（パネルA）またはAlphaScreen（パネルB）のいずれかの方法で分析したボロネートプロテアソーム阻害剤の存在下または非存在下で試験された。 ViBEプロダクトプラットフォームはAlphaScreenシグナルはタンパク質の増加μgのでクエンチしたのに対し、in vivoでの腫瘍異種移植片での敏感な、再現性のある結果を生み出すことができた。 この例では、非常に複雑なサンプル中のAMMP技術の力を示しています。チップの近くに磁石の高感度な検出チップや高効率のために、磁性微粒子の低い数字は、効率的かつ再現性よく溶解した携帯電話（溶解試薬を含む）と血液の残骸の複雑さの中に低濃度の目標のために清掃できます。

この記事の著者は、この記事で使用する機器や試薬の一部を生成するBioscale、株式会社によって雇用されている。

上記の例では、複雑なサンプルにAMMPの高感度定量結果を示す。システムに試薬をフォーマットの使いやすさとコントロールで提供されるこのパフォーマンスは、さまざまなアプリケーションのために有効になっている。それらのアプリケーションは、精製の流れを横切るサンプル中の宿主細胞タンパク質の検出と定量のように、ターゲットのスペクトルのための一般的な腫瘍のGadd34のように、ターゲットに特異的である、または可能性があります。柔軟性の高いAMMPと組み合わせ、低オーバーヘッドの雰囲気のプラットフォームは、重要なアッセイを提供するための強力なツールが用意されています。

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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

ユニークなタンパク質のカスタムアッセイは、しばしば時間のかかる手動の検出とそのような代替プラットフォーム上で開発の複雑さのためにELISAやウェスタンブロットなどの定量手法に限定されています。 BioScale独自のアコースティック膜の微粒子（AMMP）技術は、より良い感度、再現性、そして自動化された操作を提供し、サンドイッチイムノアッセイを簡単にViBEプロダクトプラットフォーム上で使用するために開発することができます。一例として提供されて、このプロトコルは、カスタムチャイニーズハムスター卵巣-宿主細胞タンパク質（CHO - HCP）アッセイを開発するための手順を概説します。ここで概説した一般原則は、イムノアッセイの多種多様な開発のために追跡することができる。 計算するためにセンサーの表面に微粒子の結合による発振周波数の変化を測定することにより、AMMP検定対策の抗原濃度。それは、4つの主要コンポーネントで構成されています。官能化されたeightセンサーチップが含まれている（1）カートリッジ（2）抗体を標識した磁気微粒子を、官能化されたチップの表面（4）の抗原を含むサンプルに結合する（3）ハプテンタグ抗体関心。 BioScaleのバイオセンサーは、別々の流体のパスを持つ8個の膜を含む共振デバイスです。膜は、質量が表面に蓄積し、この周波数の変化は添加量の量を定量するために使用されるに応答して発振周波数を変更してください。 センサは、抗ハプテン抗体と官能化されたイムノアッセイの多種多様な使用を促進する。アッセイ特異的抗体は、1つの抗体と他のビオチンへのハプテンタグの共有結合を介して変更されます。ビオチン標識は、ハプテン-タグ抗体と組み合わせて、サンドイッチで分析物を捕捉するために使用される、結合磁気ビーズをストレプトアビジンに抗体を結合するために使用されます。複合体はanti-hapten/hapten相互作用を介してチップに結合する。各アッセイの最後にセンサーを実行して96ウェルプレートからの列の連続分析を可能に希酸で洗浄されています。 ここで、我々は、バイオプロセス開発のためのカスタムCHO - HCP AMMPアッセイを開発するための手法を提案する。 AMMPアッセイを開発したり、AMMPアッセイに既存のアッセイを変更すると、複雑なサンプルと減少演算子の時間でより良い性能（再現性、感度）を提供することができます。プロトコルは開発のための手順を示し、議論のセクションでは、代表的な結果をレビュー。アッセイの最適化とカスタマイズパラメータのより詳細な説明についてはBioScaleに連絡してください。このキットによりタンパクの残渣、製品の力価、およびCHO - HCPのような一般的なバイオプロセス開発のアッセイを提供しています。

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

複雑なマトリックスで、より敏感な、再現性、自動化された結果のViBEプロダクトプラットフォーム上でカスタムアッセイの開発が説明されています。ユニバーサルカートリッジはアッセイが容易にカスタムアッセイで使用するために適応させることができます。この汎用性は、新規アッセイまたはこのビデオに例示される既存のマニュアルアッセイの自動化されたバージョン、の迅速な開発および検証を行います。

アコースティック膜の微粒子技術を使用してカスタムチャイニーズハムスター卵巣-宿主細胞タンパク質のアッセイを開発する

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 ビュー.  BioScale, Inc.. 複雑なマトリックスで、より敏感な、再現性、自動化された結果のViBEプロダクトプラットフォーム上でカスタムアッセイの開発が説明されています。ユニバーサルカートリッジはアッセイが容易にカスタムアッセイで使用するために適応させることができます。この汎用性は、新規アッセイまたはこのビデオに例示される既存のマニュアルアッセイの自?...

ビデオ: 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.

アコースティックMicrostreamingを使用して標準的な実験系逆転写反応でmRNAの単セルの物理量からcDNA収量を増加させる

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 ...

生物工学

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.

タンパク質A膜吸着体と合理化されたワークフローを用いたキメラヒトFc発現タンパク質の単離と特性評価

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.

自動 Microbioreactor 技術を用いた異種タンパク質生産の最適化のための汎用プロトコル

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.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

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.

中国ハムスター卵巣細胞培養におけるハイスループット自動マイクロバイオリアクターを用いたプロセス最適化

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.

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

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.

音響セパレータを用いたCHO採取細胞培養液の一次解明

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.

光気化したパーフルオロカーボンナノ液滴の定式化と音響変調

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.

チャイニーズハムスター卵巣細胞における迅速な抗体糖工学

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.

インビトロ 基質スクリーニングのための精製組換え ショウジョウバエ カスパーゼを用いた切断アッセイ

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