Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.     

马铃薯杂种F1的SSR鉴定

为选育抗黑痣病、高产优质的马铃薯新品种,选用引进品种‘大西洋’分别与‘陇薯6号’、‘陇薯7号’杂交,获得了杂种F1代,利用SSR标记技术对‘大西洋’与‘陇薯6号’的42个杂种F1、‘大西洋’与‘陇薯7号’的9个杂种F1单株进行了鉴定。从59对SSR引物中筛选出2对在亲本间存在差异、扩增稳定、条带清晰的引物S184和STM1049,用于‘大西洋’ב陇薯6号’杂种F1、‘大西洋’ב陇薯7号’杂种F1及其亲本的基因组DNA扩增。SSR带型分析显示,杂种F1的SSR带型呈双亲互补型、缺失型、父本型和母本型4类,依据带型特征鉴定出供试的51个马铃薯杂种F1单株均为真杂种,表明SSR分子标记技术用于马铃薯杂种真实性鉴定是可行的。该研究可为进一步开展马铃薯杂交后代目标性状优异株系选育提供依据。     

Hash subgraph pairwise kernel for protein-protein interaction extraction

Extracting protein-protein interaction (PPI) from biomedical literature is an important task in biomedical text mining (BioTM). In this paper, we propose a hash subgraph pairwise (HSP) kernel-based approach for this task. The key to the novel kernel is to use the hierarchical hash labels to express the structural information of subgraphs in a linear time. We apply the graph kernel to compute dependency graphs representing the sentence structure for protein-protein interaction extraction task, which can efficiently make use of full graph structural information, and particularly capture the contiguous topological and label information ignored before. We evaluate the proposed approach on five publicly available PPI corpora. The experimental results show that our approach significantly outperforms all-path kernel approach on all five corpora and achieves state-of-the-art performance.     

玉米根系分布空间和空间密度分布的数学模型

应用重积分研究了土壤层中散根型和直根型玉米根系的分布空间,同时研究了玉米根系的空间密度分布．     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

食线虫真菌是植物寄生线虫的重要天敌,它们所产生的胞外水解酶(蛋白酶、几丁质酶和胶原蛋白酶等)能够降解线虫体壁和卵壳中的蛋白质及几丁质等结构成分并在侵染过程中发挥着重要的作用。本文中,我们发现刀孢轮枝菌Lecanicillium psalliotae对南方根结线虫Meloidogyne incognita卵具有较强的侵染能力。为了进一步研究刀孢轮枝菌胞外几丁质酶的性质,我们通过简并引物设计和DNA walking方法从刀孢轮枝菌的基因组中成功地克隆得到一个内切几丁质酶基因Lpchi1,该几丁质酶编码基因含有3个内含子,编码423个氨基酸。同源性和系统发育分析表明,不同生防真菌来源的几丁质酶具有较高的同源性并根据分子量的大小形成三个不同的进化分枝。     

药用旱生植物刺山柑的植物学特性研究进展

刺山柑是一种冬季落叶的、抗旱能力比较强的药用植物。虽然刺山柑的群落结果比较简单。但它是一种比较优良的防风固沙植物。国内外对刺山柑的种子萌发和组织培养等方面进行了大量研究,目前已经成功实现了刺山柑的快速繁殖。植物解剖学的结果表明:刺山柑根茎和叶片都表现出了旱生植物的典型特征。本文对刺山柑的生物学特性、种子萌发和组织培养以及适应干旱环境的机制进行了综述。     

唇受精卵的皮层反应及其引发机制

唇皮层小泡1-4层,在光镜和透射电镜下均具5种形态,由外及内,小泡内颗粒直径逐渐减少。在H．E染 色中,动物极低纬度区和精孔器附近的卵膜和质膜之间,具少量均匀的着色非常深的紫色斑点,与Ⅰ型皮层小泡内 容物形态结构相似,在透射电镜下,这些斑点和卵膜、质膜有明显的界限,其外没有包被膜相结构,我们称之皮层反 应引发斑,这是在鱼类受精卵中发现的一个新的结构。扫描电镜下,引发斑成絮状。引发斑对皮层反应的引发具 有重要作用。皮层反应可分为潜伏期、始发期、高潮期、衰退期四个时期,潜伏期没有皮层反应发生,始发期只是位 于受精卵外围的少量皮层小泡释放,高潮期为多个皮层小泡相互融合形成一个大的泡状体,泡状体再与质膜接触、 融合后,随后破裂,释放内容物,可分为两个阶段,衰退期释放Ⅴ型皮层小泡和其他残存的皮层小泡以及未完全降 解卵黄颗粒碎屑;皮层反应是由Ⅰ型皮层小泡和引发斑诱导的爆发性的链式反应,卵子外侧的皮层反应可以诱导 内侧皮层反应。皮层反应有两个起始区域,在受精后35s开始于动物极低纬度区,稍后出现在精孔器前庭附近,随 后在这两个始发区向四周扩散,并在前庭以外的区域愈合、打通。皮层小泡分批多次释放,质膜多次重组。精子入 卵位点附近没有皮层小泡,不发生皮层反应,这提示皮层反应对鱼类多精受精的抑制效应有限。     

中华须鳗嗅觉器官形态学观察

利用光学显微镜和扫描电镜观察了10尾不同体长中华须鳗嗅觉器官的结构.结果表明:中华须鳗嗅囊呈楔型;嗅囊膜和嗅囊腹面的透明膜共同围成嗅囊腔;嗅囊长径与眼径的平均比值为2.2倍;每侧嗅囊嗅板数变化范围在30～44之间;嗅板远轴端有一纤毛和嗅孔密集的舌状游离突;嗅板上皮纤毛密集,纤毛细胞表现为3种类型:纤毛感觉细胞、纤毛非感觉细胞和微绒毛感觉细胞;纤毛非感觉细胞和微绒毛细胞也出现在嗅囊壁.嗅板上大量的纤毛表明,中华须鳗嗅囊的水动力机制应属嗅板纤毛搅动型(isosmates).除观察到嗅囊壁表面有两种类型的微嵴外,还首次在嗅板上观察到一种呈荸荠状的杆状细胞.     

Structure of YciA from Haemophilus influenzae (HI0827), a hexameric broad specificity acyl-coenzyme A thioesterase

The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated "hypothetical protein" in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase "hot dog" fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.     

Crystal structure of Saccharomyces cerevisiae 3'-phosphoadenosine-5'-phosphosulfate reductase complexed with adenosine 3',5'-bisphosphate

Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.