生物制药的现状和未来(一):历史与现实市场

以基因工程产品、抗体工程产品和细胞工程产品为主要代表的生物制药产业是发展最快的高技术产业之一 ,最近 6年全球年销售额连续每年以 1 5 %～ 33%的速度增长 ,并在 2 0 0 4年突破 40 0亿美元。生物技术药物在治疗肾性贫血、白细胞减少、癌症、器官移植排斥、类风湿关节炎、糖尿病、矮小症、心肌梗死、乙肝、丙肝、多发性硬皮病、不孕症、粘多糖病、戈谢病、法布莱氏病、囊性纤维化、血友病、银屑病和脓毒症等发挥了重要乃至关键作用。综述生物制药的历史、现状和市场。     

浅谈我国生物制药产业的现状和发展趋势

本文讲述了我国生物制药产业的目前形势,阐明了我国生物制药产业存在的问题和国外企业的差距逐渐拉大,指出了今后我国生物技术制药的发展方向,并且规划了我国生物制药产业的行业前景。     

哺乳动物细胞中重组蛋白瞬时表达技术（TGE）的研究进展

近年来越来越多的重组蛋白,尤其是单克隆抗体,作为生物药应用于医疗。临床及实验室研究中,经常要求在短时间内生产一定量的候选蛋白供应研究需求。经典的建立稳定细胞系生产重组蛋白过程复杂冗长,而作为替代方法,瞬时基因表达技术在数周内即可生产数十至数百毫克重组蛋白,得到广泛应用。本文将总结近年来工业及学术上,在哺乳动物细胞尤其是人胚胎肾细胞（HEK293）TL中国仓鼠卵巢细胞（CHO）中瞬时表达重组蛋白的一系列研究,概述瞬时表达技术在宿主细胞改造、表达载体最优化设计、瞬时转染条件等方面的研究进展,并展望其未来发展方向。     

Hedgehog（Hh）信号：未来治疗骨质疏松的新靶点

成年哺乳动物通过精细调控骨形成和骨重吸收之间的平衡进行骨组织更新和重塑,一旦这种平衡被打破,骨的重吸收超过骨形成,则有可能导致骨质疏松。成骨细胞主要负责骨的形成,而破骨细胞主要负责骨的重吸收,     

治疗性单克隆抗体药物产品与技术发展现状

抗体治疗已有很长的历史,可以追溯到几千年以前.早在公元前200年,中国就出现了以疫苗接种形式对抗感染性疾病的方法.19 世纪后期,含有抗体的人源或动物血清被广泛用于病毒或细菌性疾病的预防和治疗.随着分子生物学的出现,用哺乳动物细胞制备重组抗体已成为可能.     

马槟榔甜蛋白基因（MBL11）的剪切重组和结构分析

马槟榔甜蛋白（mabinlin II）是我国所特有且唯一的植物甜蛋白,在体外至今没有得到具有甜味的基因表达产物。本文采用基因工程手段对基因进行剪切重组,将重组基因构建成植物表达载体转入拟南芥中,通过RT-．PCR检测导入基因的表达,同时采用生物信息学方法对MBL II基因及其重组基因进行分析和甜味检测显示,转基因拟南芥不具有明显的甜味,但RT-PCR的结果显示,MBL II基因及其重组基因可在转基因的拟南芥中表达。根据生物信息学方法分析结果推测,导入拟南芥中的重组马槟榔甜蛋白可能是具有甜味的蛋白。     

治疗性抗体的市场考量和靶点分析

<正>近年来,单抗药物因其独特的分子靶向性和良好的临床疗效成为全球研发的热点,治疗性抗体成为产业界及学术界合作研发的令人振奋的领域。目前,在美国和欧盟有约33种市场在售和约30种处于临床Ⅲ期的治疗性抗体药物,主要用于肿瘤、炎症和自身免疫病及其他一些疾病的治疗。主要对近几年单抗药物的市场状况及治疗靶点进行了概述。     

Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures

“Molecular farming” in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative “factory”. However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.     

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

From our recent publications, it was found that the deimmunization method (Dharshanan et al. (2012) Sci Res Essays 7:2288–2299) should be applied for the development of humanized anti-C2 monoclonal antibody (H1C2 mAb). However, the overlapping-PCR mutagenesis procedure used to insert the variable regions into cloning vectors was laborious and time-consuming. Additionally, the expression of H1C2 mAb in NS0 cells was low in static culture vessels. Therefore H1C2 mAb was redeveloped by deimmunization method with the following modifications in order to optimize the production of H1C2 mAb. First, instead of the overlapping-PCR mutagenesis procedure, synthetic DNA coding the variable regions were used to express the mAb. Second, two expression vectors, pFUSE and UCOE, were used to express H1C2 mAb in NS0 cells and CHO cells in order to investigate the combination that gave the highest number of high producing stable clones. This will provide the highest chance of finding clones with the requisite high productivity and stability required for manufacturing. We found that transfection of UCOE in CHO cells generated the highest number of high producing stable clones. To our knowledge, this is the first time that H1C2 mAb has been expressed in CHO cells.     

Prospects and limitations of the rational engineering of fibrillar collagens

Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large-scale production of nonmodified recombinant collagens; however, engineering of novel collagen-like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen-like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi-D4 collagens defined as collagen-like proteins, consisting primarily of a tandem of the collagen II D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase 1 cleavage site present in the D4 period. We demonstrated that the recombinant collagen alpha-chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N-proteinase and procollagen C-proteinase. The nonmutated multi-D4 collagen had a normal melting point of 41 degrees C and a similar carbohydrate content as that of control. In contrast, the mutant multi-D4 collagen had a markedly lower thermostability of 36 degrees C and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi-D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences.     

主要发达国家生物技术关键领域SCI计量对比研究

生物技术是我国战略性高新技术发展领域,也是全球各国在生物经济时代能否获胜的关键技术。本文通过科学引文索引数据库（SCI）中论文收录文献的文献计量学研究,对单克隆抗体、疫苗、重组蛋白、基因治疗、干细胞研究、组织工程、基因组学、蛋白质组学、芯片技术九类关键技术领域的全球研究分布格局进行了探讨,并结合生物医药产业链进行定标比超研究,以期发现我国在全球生物技术领域所处的位置和产业差距。研究发现美国在以上关键技术领域均处于全球绝对领先地位,而我国在某些技术领域如芯片、组织工程、组学等领域已具有一定比较优势和国际竞争能力,但在较长时期内研发和产品仍将处于跟随地位。     

Use of Hydrocyclones for Mammalian Cell Retention: Separation Efficiency and Cell Viability (Part 1)

A hydrocyclone with a volume of 2.56 cm³ was studied as a potential cell retention device for mammalian cell cultures (6 L volume). For the feasible operation range (0.9 to 1.6 L/min flow corresponding to pressure drops of 0.4 to 1.3 bar) the hydrocyclone was characterized with regard to flow split (underflow‐to‐overflow ratio) and flow ratio (underflow to supply). Cultures of BHK and HeLa cells (with low cell concentrations) were applied to measure separation efficiency and cell viability for a hydrocyclone operation period of 3 min corresponding to a cell suspension throughput of 2.7 to 4.8 L. Cell separation efficiencies ranged from 0.77 to 0.97 and cell viability was not affected except for BHK cells in the overflow at the highest pressure drop (1.3 bar). As the overflow is commonly used for product harvest and cells are discarded, the application of the hydrocyclone has no detrimental effect on the reactor perfusion system. The results indicate that only cells passing from the primary vortex downwards into the inner secondary vortex and from there upwards could be damaged. Evidence for this hypothesis is obtained from operating the hydrocyclone with closed overflow (only centrifugal forces acting) for a period of 3 h. In these studies no significant effect on cell viability could be detected for HeLa and CHO cells. Hence, the results indicate that the hydrocyclone can be appropriately used for cell retention and separation in perfusion cultures. Application at higher pressures is recommended whereby separation efficiencies of 0.97 without any loss in viability can be achieved.     

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

抗人P185~(erbB2)嵌合抗体在CHO细胞中的高效表达及其活性分析

为降低人抗鼠抗体 (HAMA)反应并在CHO细胞中高效表达抗人P185 erbB2 人 /鼠嵌合抗体 ,将抗人P185 erbB2 单抗C2 5的轻、重链可变区基因分别克隆入具有人抗体恒定区基因组序列和弱化启动子驱动的选择标志基因的真核表达载体中 ,共转染CHO dhfr-细胞 ,经G418及氨甲喋呤 (MTX)梯度加压筛选进行了嵌合抗体的高效表达。采用RT PCR、ELISA、细胞ELISA、免疫荧光细胞化学等实验证实了所表达的抗人P185 erbB2 嵌合抗体的人源性及抗原特异性。培养上清中的抗体产量可达 10 0mg/L ,所表达的嵌合抗体具有抑制P185 erbB2 高表达肿瘤细胞增殖的作用     

The structure of an antitumor C(H)2-domain-deleted humanized antibody

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.