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;.

주 : 적절한 포유 동물 발현 벡터는이 프로토콜을 사용해야합니다. 여기서, 두 발현 카세트를 포함하는 하나의 구조가 사용되는 (즉, 중쇄 및 경쇄 발현 별도 프로모터에 의해 구동된다). 트라 스투 주맵 중쇄 및 경쇄 이전 벡터에 클로닝 하였다. 이 벡터는 비영리 플라스미드 저장소 (13)를 통해 얻은 앤드류 Beavil의 선물이었다. 1. 복구 및 벡터 DNA의 스케일 업 참?...

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이 프로토콜은 HEK-293 세포에 형질 감염 안정 발현 및 치료 용 항체의 정제 사항. 항체 유전자의 안정한 발현은 치료 용 항체의 개발 및 제조에 대한 항체 생산 세포주를 생성하는 첫 번째 단계이다. 중국어 햄스터 난소 (CHO) 세포 치료 용 단백질에 대한 선택의 발현 플랫폼 남아 있지만, HEK-293 세포주 게시물의 측면에서 이들 세포에서 생성 된 단백질이 자연적으로 인간 단백질을 발생하는 가까운 ?...

치료 용 항체의 성공은 차세대 치료제의 물결이 시작으로 항체 개발에 상당한 투자를 구동하기 위해 계속된다. 항체 시장은 유리한 속성 4 항체 단편 1 항체 - 약물 접합체 (2) 특이 적 항체 (3)과 조작 항체로 재 성형 될 것으로 예상된다. 제약 관심을 얻고 또 다른 클래스는 바이오시 밀러입니다. 바이오시 밀러 항체는 이미 규제 당국의 승인을받은 치료 항체의 제품을 복제 &#39;매우 유사한&#39;이다. 제안 된 바이오시 밀러는 구조, 기능, 동물 독성, 임상 적 안전성과 유효성, 인간의 약동학 (PK), 약력학 (PD) 및 면역 원성 5, 6에 대한 송신자 항체와 비교해야합니다. 승인 연구바이오시 밀러 항체의 ATES 인해 제품의 최종 품질에 엄격한 제약으로 느린왔다. 이러한 최종 처리 단계에 이르기 세포주 및 배양 조건과 정확히 특정 제조 공정 독자적인 남아있을 수있다. 무엇보다, 항체의 제조는 본질적으로 매우 유사한 제품을 생산하는 도전에 추가 할 수있는 변화의 정도를 포함한다. 포괄적 물리 및 생물 리 학적 특성화 및 비교는 매우 곤란하고, 또 밀러 항체의 특성을 입증하는 연구들이 문헌 7, 8, 9에 나타나고있다. 치료 항체를 생성하는 것은 각각의 항체 유전자를 운반하는 벡터를 포유 동물 숙주 세포의 형질 감염과 함께 시작된다. 벡터 디자인, 세포주 및 배양 조건은 예를 설정하기위한 주요 고려 사항은Pression의 시스템입니다. 항체의 DNA 서열은 약 은행 (www.drugbank.ca), IMGT (www.igmt.org) 또는 특허 공보를 포함한 연구에서 공급 될 수있다. 예를 들어, 트라 스투 주맙의 순서는 약물 은행 (: DB00072 DB의 ID)를 통해 사용할 수 있습니다. 가변 영역의 아미노산 서열은 바람직한 숙주 종에서 합성 유전자의 디자인 및 최적화를 겪을 수있다. 그것은 더 변형되는 아미노산 서열로 이루어지지 않은 밀러 항체 중요하다. 합성 후, 항체 유전자는 선택의 적절한 벡터 내로 클로닝 할 수있다. 인간 IgG 항체는 두 개의 동일한 중쇄 2 개의 동일한 경쇄로 구성되어 있습니다. 두 체인의 단단히 규제 발현은 포유류 세포 (10)의 이종의 IgG 단백질의 최적 생산을 위해 필수적이다. 사슬 간 이황화 결합을 형성 할 수 있고 번역 후 변형의 개수에 있어야 할뿐만 아니라 인트라단백질 생합성 동안 scaling과 shearing. 벡터의 수는 항체 유전자 (재료의 표 참조) 표현하기 위해 특별히 설계되었습니다이 가능합니다. 이 항체 고유 벡터는 일반적으로 각 체인 그렇게 만 가변 영역 복제를 필요로 무거운 가벼운 모두 체인의 일정 영역을 표현한다. 두 개의 독립적 인 구조 (공동 트랜)와 세포의 형질 감염은 중쇄 및 경쇄를 코딩하는 유전자를 전달하기위한 가장 일반적인 방법이다. 즉, 각각의 유전자가 소포체에 조립되기 전에 자신의 프로모터에 의해 구동되는 분리 된 항체 사슬로 전사된다. 한편, 멀티 cistronic 벡터는 mRNA를 (11)의 내부 영역에서 허용 번역 된 단일의 mRNA 전 사체 여러 유전자의 발현을 허용 혼입 내부 리보솜 진입 사이트 (IRES) 요소를 가지고있다. 이 경우, 중쇄 및 경쇄를 코딩하는 유전자는 ARRANG로 연결된다두 항체 쇄 (10) (12)의 공동 발현을 달성 장담. 일시적으로 형질 감염된 세포는 실험 제한을 수행하기에 충분한 단백질을 수득하지만, 게놈 통합을위한 선택을받은 안정적으로 형질 감염된 세포주는 높은 수율을 제공 할 수있다. 높은 단백질 량은 시험 관내 특성화 관련 분석법 개발을 허용하며 클론 세포주 리드 후보 선택으로 하류 애플리케이션을 고려하여 항체의 품질의 표시를 제공 할 수있다. 이 문서의 목적은 포유 동물 발현 시스템에서 생산 된 치료 용 항체의 안정적인 발현과 정제를 설명한다. 실제로,이 방법은 밀러 항체의 발현에 적용될 수있다. 이 방법은 시간이 많이 소요 인트이라도 임계로 진행하기 전에 항체의 초기 특성에 사용될 수있다대규모 생산을위한 바람직한 클론을 식별 PS. 또한,이 방법은 다른 단백질뿐 아니라 항체를 발현하는데 사용될 수있다. 상세한 프로토콜은 치료 용 항체 트라 스투 주맵의 발현을 설명한다. 이 자동화 된 크로마토 그래피 방법에 의해 HEK-293 세포주 및 항체 단백질의 정제 안정한 형질 뒤에 벡터 DNA의 준비로 구성된다.

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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.

도 1에 제시된 바와 같이 형질 감염된 HEK-293 세포에 의해 트라 스투 주맙의 안정적인 생산이 바이오 - 층 간섭 (BLI)을 이용하여 확인 하였다. IgG의 표준 곡선은 IgG 항체 표준 단백질 바이오 센서 (도 1a) 사이의 결합 비율을 측정하여 생성 하였다. 조 상등액 시료를 유사하게 측정 한 후, 그 농도는 표준 곡선 (도 1b)에서 보간. 상등액의 농...

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

이 프로토콜은 포유 동물 발현 시스템에서의 치료 용 항체의 생산을 설명한다. 설명하는 방법은 친 화성 크로마토 그래피를 사용하여 대규모 문화와 정화의 설정 벡터 DNA, 안정적인 형질 전환 및 인간 배아 신장 293 세포주의 무 혈청 적응의 준비를 포함한다.

실험실 규모의 생산과 치료 용 항체의 정제

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

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

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Expression, Purification, and Antimicrobial Activity of S100A12

S100A12의 발현, 정제 및 항균 활성

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;

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생산, 결정 화, 및 인간 IKK1/α의 구조 결정에 대 한 안내

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

효율적인 Refolding 및 Chemoreceptor Ligand 바인딩 도메인의 정화 방법

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

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Nicotiana Benthamiana 에 과도 식 보관 생산 대리점 규모에 대 한 나뭇잎

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

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뉴클레아제 없는 산소 제거제 프로토카테의 발현 및 정제 3,4-디옥시게나아제

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