CN102344926A - 真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 - Google Patents
真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 Download PDFInfo
- Publication number
- CN102344926A CN102344926A CN2011102744192A CN201110274419A CN102344926A CN 102344926 A CN102344926 A CN 102344926A CN 2011102744192 A CN2011102744192 A CN 2011102744192A CN 201110274419 A CN201110274419 A CN 201110274419A CN 102344926 A CN102344926 A CN 102344926A
- Authority
- CN
- China
- Prior art keywords
- vegf
- growth factor
- vascular endothelial
- endothelial growth
- mono
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010041308 Endothelial Growth Factors Proteins 0.000 title claims abstract description 16
- 230000002137 anti-vascular effect Effects 0.000 title claims abstract description 16
- 238000000746 purification Methods 0.000 title claims abstract description 7
- 238000005516 engineering process Methods 0.000 title abstract description 6
- 230000009465 prokaryotic expression Effects 0.000 title abstract 2
- 239000000126 substance Substances 0.000 title abstract 2
- 230000014509 gene expression Effects 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 3
- 238000003759 clinical diagnosis Methods 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 2
- 210000001236 prokaryotic cell Anatomy 0.000 abstract 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 11
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 9
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 238000001890 transfection Methods 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102000003666 Placenta Growth Factor Human genes 0.000 description 1
- 108010082093 Placenta Growth Factor Proteins 0.000 description 1
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000004862 vasculogenesis Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化方法。本发明的目的在于实现人源化抗血管内皮生长因子单克隆重组抗体在真核细胞中的大量表达，并通过简化的纯化步骤，获得纯度较高抗体。在工业生产中满足生产周期短，生产成本低，产量高的总体要求。
Description
技术领域
本发明涉及真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化方法。
背景技术
血管内皮生长因子(VEGF)是以二硫键连接的二聚体糖蛋白，分子量为35～40kDa。VEGF是一个细胞因子家族，包括VEGF-A、-B、-C、-D、-E和胎盘生长因子等多个成员。VEGF-A基因的选择性剪切可以产生至少5种蛋白形式，包括分泌型可溶性蛋白VEGF121、VEGF145、VEGF165。血管内皮生长因子通过特异性与3个血管内皮生长因子受体结合，启动下游细胞信号级联，刺激内皮细胞增殖，促进细胞迁移，并抑制细胞凋亡。血管内皮生长因子主要表达于血管内皮细胞、肿瘤细胞、某些炎症细胞和间质细胞。血管内皮生长因子的表达水平和肿瘤内的血管生成情况和肿瘤的转移风险。因此，抑制血管内皮生长因子信号转导可以抑制多种肿瘤的发生和发展。
如今，随着基因工程技术的发展，抗VEGF抗体已经可以由多种方式获得。目前市面上多为多克隆抗体或单克隆抗体，鲜见重组抗体。传统的抗VEGF抗体制剂生产的纯化步骤繁琐，纯度也较低，使得其经济意义大为降低。本发明通过基因工程技术，获得了人源化抗血管内皮生长因子单克隆重组抗体。
发明内容
本发明的目的在于实现人源化抗血管内皮生长因子单克隆重组抗体在真核细胞中的大量表达，并通过简化的纯化步骤，获得纯度较高抗体。在工业生产中满足生产周期短，生产成本低，产量高的总体要求。
本发明通过体外人工合成双链DNA分子的技术，分别合成人源化抗血管内皮生长因子单克隆抗体重链和轻链的编码基因，通过EcoRI和XhoI限制性酶切位点与表达载体pCDNA3.1连接。随后将质粒转染293T细胞系获得高效表达。
附图说明
图1是转染后人源化抗血管内皮生长因子单克隆抗体表达SDS-PAGE电泳分析结果图(M为marker；1.转染空载体的阴性对照；2.转染48小时后2ml培养基抗体表达情况(还原条件下)；3.转染96小时后2ml培养基抗体表达情况(还原条件下))。
图2是Anti-VEGF SDS-PAGE分析纯化纯度结果图。(A非还原条件，B还原条件)。
具体实施方式
1、人源化抗血管内皮生长因子单克隆重组抗体的真核表达
编码人源化抗血管内皮生长因子单克隆重组抗体重链和轻链的编码基因通过EcoRI和XhoI限制性酶切位点与表达载体pCDNA3.1连接。随后将质粒转染CHO-K1细胞系。
转染前48小时，将10ml活力大于95％、细胞密度为1.5*105/ml的CHO-K1细胞用含DMEM完全培养基(无抗，含10％胎牛血清)平铺于10cm细胞培养皿，在37℃，5％CO2培养箱中培养。当细胞汇合度达到70-80％时，开始转染。将含抗血管内皮生长因子单克隆抗体重链和轻链基因的表达载体各1μg，加入到200μl 37℃预热的PBS中，混匀后加入4μl1mg/ml PEI(聚乙烯亚胺)，立即震荡混匀，在室温下放置15分钟。将质粒-PEI混合液加入到细胞培养液中，轻轻混匀后在37℃，5％CO2培养箱中培养。6小时后，吸弃细胞培养液，并用37℃预热的PBS洗细胞3次去除残留的血清之后；加入10ml无血清培养基，同时加入终浓度分为2mM和1mM的谷氨酰胺(L-Glutamine)和丙酮酸钠继续培养。分别于48小时和96小时收集细胞上清。
人源化抗血管内皮生长因子单克隆抗体表达SDS-PAGE电泳分析结果见附图1。
2、Protein A亲和层析纯化抗体
将共计10个细胞培养皿收集所得上清200ml；10000rpm，4℃离心10min去除细胞等沉淀物，并用等体积PBS稀释1倍。将0.5ml ProteinA纯化树脂用5ml PBS平衡后，将细胞培养上清以流速0.5ml/min上柱。5ml PBS洗涤去除非特异结合蛋白后，用100mM pH3.0甘氨酸溶液洗脱蛋白。用1M Tris-HCl pH9.0将蛋白洗脱液pH调至中性，之后在PBS透析至少4小时。用PEG20000浓缩至浓度为1mg/ml，经液氮冷激后于-80℃冰箱保存。最终获得蛋白的量为0.8mg。
SDS-PAGE验证获得的是高纯度的蛋白，蛋白纯度＞95％见附图2。
Claims (4)
1.一种采用真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化方法，其特征在于该方法通过如下步骤用真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)：人源化抗血管内皮生长因子单克隆重组抗体的真核表达，Protein A亲和层析纯化抗体。
2.如权利要求1所述的采用真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)，特征在于所述的Anti-VEGF是通过真核表达并纯化的。
3.如权利要求1所述的采用真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)，特征在于所述的Anti-VEGF的表达为分泌性的可溶性蛋白。
4.如权利要求1所述的采用真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)，特征在于所述的Anti-VEGF作为生物制剂应用于研究和临床诊断。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102744192A CN102344926A (zh) | 2011-09-16 | 2011-09-16 | 真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102744192A CN102344926A (zh) | 2011-09-16 | 2011-09-16 | 真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102344926A true CN102344926A (zh) | 2012-02-08 |
Family
ID=45544000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102744192A Pending CN102344926A (zh) | 2011-09-16 | 2011-09-16 | 真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102344926A (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1445242A (zh) * | 2002-03-20 | 2003-10-01 | 上海中信国健药业有限公司 | 人源化抗血管内皮生长因子单克隆抗体及其制法和药物组合物 |
| CN102167740A (zh) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | 一种全人源抗vegf单克隆抗体、其制备方法及用途 |
-
2011
- 2011-09-16 CN CN2011102744192A patent/CN102344926A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1445242A (zh) * | 2002-03-20 | 2003-10-01 | 上海中信国健药业有限公司 | 人源化抗血管内皮生长因子单克隆抗体及其制法和药物组合物 |
| CN102167740A (zh) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | 一种全人源抗vegf单克隆抗体、其制备方法及用途 |
Non-Patent Citations (1)
| Title |
|---|
| LEONARD G ET AL: "Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal Antibody for the Therapy of Solid Tumors and Other Disorders", 《CANCER RESEARCH》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102607285B1 (ko) | 항체 중쇄불변부위 이종이중체 (heterodimeric Fc)에 융합된 사이토카인 (heterodimeric Fc-fused cytokine) 및 이를 포함하는 약제학적 조성물 | |
| WO2019154349A1 (zh) | 抗pd-1/抗vegf天然抗体结构样异源二聚体形式双特异抗体及其制备 | |
| US11591409B2 (en) | Anti-PD-L1/anti-PD-1 natural antibody structure-like heterodimeric bispecific antibody and preparation thereof | |
| CN102448987B (zh) | 一种抗vegf的单克隆抗体及含有该抗体的药物组合物 | |
| JP7262597B2 (ja) | 二重特異性抗体及びその作製方法と使用 | |
| CA3068841A1 (en) | Fusion molecules targeting immune regulatory cells and uses thereof | |
| TW202003569A (zh) | 多價抗體 | |
| CN111615521A (zh) | 抗pd-l1/抗cd47天然抗体结构样异源二聚体形式双特异抗体及其制备 | |
| CN114127111B (zh) | 与nkp30结合的抗体分子及其用途 | |
| KR20180012860A (ko) | 이종 이량체 다중 특이적 항체 포맷 | |
| CN109641949A (zh) | 促进选择性轻链配对的多特异性抗体 | |
| TW201934582A (zh) | 抗pd-1/抗her2天然抗體結構樣異源二聚體形式雙特異抗體及其製備 | |
| CN104829725A (zh) | 一种双特异性抗体cd133×cd3的构建及应用 | |
| KR20230160294A (ko) | Upar 항체 및 이를 갖는 융합 단백질 | |
| CN114929863B (zh) | 共表达趋化因子受体的免疫效应细胞 | |
| CN111094552A (zh) | 抗体制备新方法 | |
| WO2022206753A1 (zh) | GARP/TGFβ1抗体及其应用 | |
| CN105916883B (zh) | 双功能融合蛋白及其制备方法和用途 | |
| CN117222672A (zh) | 抗cldn4-抗cd137双特异性抗体 | |
| ES2960329T3 (es) | Proteínas de unión triespecíficas y/o trivalentes | |
| CN102344926A (zh) | 真核表达并纯化人源化抗血管内皮生长因子单克隆重组抗体(Anti-VEGF)的简便化工业技术 | |
| CN113667004A (zh) | 一种白介素2突变体 | |
| US20230093169A1 (en) | Combinations of antibody constructs and inhibitors of cytokine release syndrome and uses thereof | |
| CN1891716B (zh) | 一种无丝裂原活性抗cd3小分子抗体的设计方法 | |
| EP4436992A1 (en) | Binding domain molecules on cell surfaces |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120208 |