Name: laboratory scale production and purification of a therapeutic antibody
Uploaded: 2017-01-24T13:00:00
Description: Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

注：適切な哺乳動物発現ベクターは、このプロトコルを使用する必要があります。ここでは、2つの発現カセットを含有する単一の構築物が使用される（ すなわち、重鎖および軽鎖の発現は別個のプロモーターによって駆動されます）。トラスツズマブ重鎖および軽鎖は、以前にベクターにクローニングしました。このベクターは、非営利のプラスミドリポジトリ13を経て得られたア...

<ol>
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このプロトコルは、HEK-293細胞にトランスフェクションは、安定な発現および治療用抗体の精製を詳細に説明します。抗体遺伝子の安定な発現は、治療用抗体の開発および製造のための抗体産生細胞株を生成するための最初のステップです。チャイニーズハムスター卵巣（CHO）細胞は、治療用タンパク質のための最適な発現プラットフォームまま、HEK-293細胞株は、これらの細胞中で産生され?...

治療用抗体の成功は、次世代の治療薬の波が開始されるように、抗体の開発に多額の投資を駆動し続けます。抗体市場は良好な特性4を有する抗体断片1、抗体-薬剤コンジュゲート2、二重特異性抗体3と操作された抗体により再形成されることが期待されます。医薬品の関心を集めて別のクラスは、バイオシミラーです。バイオシミラーの抗体は、すでに規制当局の承認を受けている治療用抗体の製品を複製する「高度に類似」しています。提案されたバイオシミラーは、その構造、機能、動物の毒性、臨床安全性と有効性、人間の薬物動態（PK）、薬力学（PD）および免疫原性5、6に関して創始抗体と同等でなければなりません。 承認rをバイオシミラーの抗体のATEは、製品の最終品質に厳しい制約のために遅れています。このような最終処理工程に至る細胞株及び培養条件の特定等の正確な製造プロセスは、独自のままであり得ます。詳細は何ですか、抗体の製造は、本質的に非常に類似した製品を製造するの挑戦に追加できる変動率を伴います。総合的な物理化学的および生物物理学的特徴との比較は非常に困難である、まだバイオシミラー抗体の特性を実証する多くの研究が文献7、8、9に浮上しています。 治療用抗体の生成は、それぞれの抗体のための遺伝子を保持するベクターを哺乳動物宿主細胞のトランスフェクションから始まります。ベクター設計、細胞株および培養条件は、元の設定に重要な考慮事項でありますPRESSIONシステム。 抗体のDNA配列は、特許を含む医薬品銀行（www.drugbank.ca）、IMGT（www.igmt.org）や研究出版物から供給することができます。例えば、トラスツズマブのシーケンスは、医薬品銀行（：DB00072 DB ID）を介して利用可能です。可変領域のアミノ酸配列は、所望の宿主種における合成のための遺伝子の設計及び最適化を行うことができます。それは修飾アミノ酸配列に対して行われていないバイオシミラー抗体のために重要です。合成された後、抗体遺伝子を選択した適当なベクターにサブクローニングすることができます。 ヒトIgG抗体は、2つの同一の重鎖と2つの同一の軽鎖からなります。両鎖の密調節された発現は、哺乳動物細胞10における異種IgGタンパク質の最適な生産に不可欠です。鎖間ジスルフィド結合を形成する必要があり、翻訳後修飾の数であることを持っているだけでなく、イントラタンパク質生合成の間にtroduced。ベクトルの数は、抗体遺伝子を（材料の表を参照）を発現するように設計されていますが利用可能です。これらの抗体に特異的ベクターは、通常、それぞれの鎖のように可変領域のみがクローニングを必要とする重鎖及び軽鎖の双方のための定常領域を発現します。 二つの独立した構築物（コトランスフェクション）を用いた細胞のトランスフェクションは、重鎖および軽鎖をコードする遺伝子を送達するための最も一般的なアプローチです。すなわち、各遺伝子は、それ自身のプロモーターにより駆動され、小胞体に組み立てられる前に、別個の抗体鎖として転写されます。一方、多シストロン性ベクターは、mRNA 11の内部領域から許可翻訳有する単一のmRNA転写物として複数の遺伝子の発現を可能に組み込まれた内部リボソーム侵入部位（IRES）要素を有します。この例では、重鎖および軽鎖をコードする遺伝子はARRANGに接続されています両方の抗体鎖10、12の同時発現を達成するement。 一過性にトランスフェクトされた細胞は、実験の限られた数を実行するのに十分なタンパク質を得ながら、ゲノム組込みのための選択を受けた安定にトランスフェクトされた細胞株は、高い収率を提供できます。より高いタンパク質の量は、in vitroでの特徴付けに関連するアッセイ開発を可能にし、そのようなクローン細胞株とリード候補の選択などの下流の用途を考慮して、抗体の質の指標を提供することができます。 この記事の目的は、哺乳動物発現系において産生さ治療抗体の安定な発現および精製を記載することです。実際、この方法は、バイオシミラーの抗体の発現に適用することができます。この方法は、時間がかかりSTEいえ、重要へ進む前に、抗体の最初の特徴付けのために使用することができますより大規模な製造のための望ましいクローンを同定するPS。また、この方法は、他のタンパク質だけでなく、抗体を発現するために使用することができます。 以下の詳細なプロトコルは、治療用抗体トラスツズマブの発現を記載します。これは、自動化されたクロマトグラフィー法によってHEK-293細胞株と抗体タンパク質の精製における安定なトランスフェクションに続いてベクターDNAの調製から成ります。

<table border="0" cellpadding="0" cellspacing="0" width="1730">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">pFUSE vector series</td>
			<td style="width:189px;">N/A</td>
			<td style="width:191px;">InvivoGen</td>
			<td style="width:1003px;">Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAbXpress vector series</td>
			<td>N/A</td>
			<td>ACYTE Biotech Pty Ltd.</td>
			<td>Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1 vector</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&#160; Refer to: Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GS vector series</td>
			<td>N/A</td>
			<td>Lonza</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. et al. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1-Trastuzumab-IgG1/&#954;</td>
			<td>61883</td>
			<td>Addgene</td>
			<td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro Agar</td>
			<td>fas-hg-s</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt LB agar containing 75 &#181;g/ml hygromycin.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro TB</td>
			<td>fas-hg-l</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt TB broth containing 75 &#181;g/ml hygromycin.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol, BioXtra, &#8805;99%</td>
			<td>G6279</td>
			<td>Sigma-Aldrich</td>
			<td>Prepare to 80% with water and autoclave. Store at room temperature.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jestar 2.0/LFU Plasmid Maxi Kit</td>
			<td>G221020</td>
			<td>Astral Scientific</td>
			<td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293-F Cells</td>
			<td>R790-07</td>
			<td>Life Technologies</td>
			<td>HEK-293 cell line adapted to suspension culture in serum-free media.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293 Expression Medium</td>
			<td>12338-018</td>
			<td>Life Technologies</td>
			<td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Kolliphor P188</td>
			<td>K4894</td>
			<td>Sigma-Aldrich</td>
			<td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose&#160;</td>
			<td>11995-065</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat-Inactivated Foetal Bovine Serum</td>
			<td>10082-147</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Polyethylenimine, Linear, MW 25,000&#160;</td>
			<td>23966</td>
			<td>Polysciences, Inc.</td>
			<td>Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 &#956;m filter. Store at -80oC until use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiPro SFM</td>
			<td>12309-050</td>
			<td>Life Technologies</td>
			<td>Transfection formulated serum-free media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B Solution</td>
			<td>ant-hg-1</td>
			<td>Jomar Life Research</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethylsulphoxide (DMSO)</td>
			<td>AJA2225</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tryptone (casein peptone)</td>
			<td>LP0042B</td>
			<td>Thermo Fisher Scientific</td>
			<td>Prepare to 20% in PBS and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td>
			<td>09-2051-100</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiTrap Protein A High Performance, 1 x 5 ml column</td>
			<td>GE17-0403-01</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AKTApurifier 100</td>
			<td>28406266</td>
			<td>GE Healthcare</td>
			<td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine-HCl</td>
			<td>G2879</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric Acid, monohydrate</td>
			<td>BIOC2123</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Citrate, trisodium salt dihydrate&#160;</td>
			<td>BIOCB0035</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M Tris-HCl solution pH 9.0</td>
			<td>BIOSD8146</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal Filters (30 MWCO)</td>
			<td>UFC803008/UFC903008</td>
			<td>Merck Millipore</td>
			<td>Used to buffer exchange and concentrate purified protein.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Bicinchoninic Acid (BCA) Assay Kit</td>
			<td>23227</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BLItz System</td>
			<td>45-5000</td>
			<td>fort&#233;BIO</td>
			<td>Instrument used for bio-layer interferometry (BLI) measurements.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein A biosensors</td>
			<td>18-5010</td>
			<td>fort&#233;BIO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide/Bisacrylamide (37.5:1), 40% solution</td>
			<td>786-502</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Persulfate (APS)</td>
			<td>AM0486</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>AM0761</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Brilliant Blue R-250</td>
			<td>786-498</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Precision Plus Dual-Color Protein Standard</td>
			<td>1610374</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory scale production and purification of a therapeutic antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

図1に示すようにトランスフェクトされたHEK-293細胞によるトラスツズマブの安定生産は、バイオレイヤー干渉法（BLI）を用いて確認しました。 IgGの標準曲線は、IgG抗体標準及びタンパク質バイオセンサー（ 図1A）との間の結合速度を測定することによって作製しました。粗上清のサンプルを同様に測定し、その濃度を、標準曲線（ 図1B...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

このプロトコルは、哺乳動物発現系における治療抗体の産生を記載しています。記載されている方法は、ベクターDNAの調製、安定なトランスフェクションおよびアフィニティークロマトグラフィーを用いた大規模な文化や精製の設定ヒト胚性腎臓293細胞株の無血清適応を含みます。

治療用抗体の実験室規模の産生および精製

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

からの外膜小胞内の指向性タンパク質包装<em&gt;エシェリヒア・コリ</em&gt;：デザイン、生産および精製

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

の生産、結晶化と構造決意<em&gt; C。ディフィシル</em&gt; PPEP-1 Microseeding及び亜鉛SADを介しました

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Expression, Purification, and Antimicrobial Activity of S100A12

S100A12の発現、精製および抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

生産、結晶化、および人間 IKK1/α の構造決定へのガイド

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

精製と TRPV1 の分光学的解析のための再構成

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

一過性発現タバコにおける葉トリテルペン用分取スケールで

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

エシェリヒア属大腸菌のウイロイド派生システムを使用して円形の足場に組換え Rna の大量生産

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

ヌクレアーゼフリー酸素スカベンジャープロトカテク酸プロトカテクエート3,4-ジオキシゲナーゼの発現と精製

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

ジテルペノイド天然物の酵素生産と精製のためのカスタマイズ可能なアプローチ

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...