Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.     

广东优势群落甜槠林多样性研究

采用每木调查法,对广东粤北石门台和粤中象头山地区甜槠( Castanopsis eyrie (Champ.) Tutch.)林样方内胸径≥ 1 cm的乔、灌木进行测量,记录其种名、胸径、高度和在样方中的坐标位置等指标,对群落的植物组成、区系成分、优势种及物种多样性进行分析。结果显示:石门台样地有维管植物112种,隶属36科54属;象头山样地有维管植物109种,隶属36科59属。两个群落均具有明显的热带向亚热带过渡的性质。其中,石门台样地与华东、华中地区联系紧密;象头山样地则与中国台湾、日本的岛屿联系更多。比较两个地区的群落特征发现,他们的相似性较高,物种多样性指数接近,纬度并不是决定群落植物多样性高低的因素,小环境、群落演替进程及人为因素对群落植物多样性的影响更大。     

Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents

During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.     

广东优势群落甜槠林多样性研究

采用每木调查法,对广东粤北石门台和粤中象头山地区甜槠（Castanopsis eyrie（Champ.） Tutch.）林样方内胸径≥ 1 cm的乔、灌木进行测量,记录其种名、胸径、高度和在样方中的坐标位置等指标,对群落的植物组成、区系成分、优势种及物种多样性进行分析。结果显示：石门台样地有维管植物112种,隶属36科54属;象头山样地有维管植物109种,隶属36科59属。两个群落均具有明显的热带向亚热带过渡的性质。其中,石门台样地与华东、华中地区联系紧密;象头山样地则与中国台湾、日本的岛屿联系更多。比较两个地区的群落特征发现,他们的相似性较高,物种多样性指数接近,纬度并不是决定群落植物多样性高低的因素,小环境、群落演替进程及人为因素对群落植物多样性的影响更大。     

Ion chromatographic quantification of cyanate in urea solutions: estimation of the efficiency of cyanate scavengers for use in recombinant protein manufacturing

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 microM, with LOQ/LOD values of 6 and 2 microM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5-10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.     

High prevalence of irritable bowel syndrome with poor sleep quality in children and adolescents in Shanghai

Sleep and Biological Rhythms - This study explored the prevalence of irritable bowel syndrome (IBS) and investigated the relation-ship between IBS and disturbed sleep in school-age children in...