Analysis of the glycosylation heterogeneity of recombinant human pro-urokinase using UPLC-MS / 药学学报

The glycosylation heterogeneity of recombinant human pro-urokinase (pro-UK) was assessed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Firstly, the source of heterogeneity was determined by measuring the Mr of intact protein before and after N-deglycosylation. Glycosylation sites and the proportion of O-glycopeptides then were determined at the peptide level. Finally, the N-glycans were confirmed and quantified using the N-glycan profile. Results show that the structural heterogeneity of pro-UK is mainly caused by glycosylation. All T18 were fucosylated, and 6.4% of S138/139 was O-glycosylated with two kinds of oligosaccharides with a ratio of 6.0% and 0.4% respectively. All N302 positions were N-glycosylated by more than ten types of glycans, among which A2F and A3F accounted for 80% of the total. The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.

Establishment and application of the quality control technology system for recombinant drugs in China: A review / 中国药学杂志

In order to ensure the safety and effectiveness of biotech drugs, the CFDA has released a series of regulations and guidelines, and the NIFDC established an effective quality control technology system for recombinant drugs. This paper briefly reviews the important development stage of pharmaceutical biotechnology and the research and development situation of recombinant drugs in China, introduces in detail the establishment and application situation of the quality control technology system for recombinant drugs in the last 30 years in China, including the research basis and regulatory requirements for the quality standard, the quality standards of recombinant drugs in Chinese Pharmacopoeia Part III and the quality control requirements for recombinant drugs in the current Pharmacopoeia, the support of national science and technology projects for establishing quality control system of biotechnology drugs by NIFDC and the establishment and application of the recombinant drug quality standards and all kinds of test methods such as the determination of biological activity and protein content, physical and chemical analysis and protein structural identification, determination of purity and impurity since 1986; development of national standards for biological activity and content determination, establishment and validation of the quality standards of the physical and chemical reference substances used for peptide mapping analysis and isoelectric point determination of recombinant drugs; analysis of inspection reports of a total of 5 920 batches of biotech drugs for all kinds of tests including registration inspection, import inspection, sample inspection, commission, and contracts tests completed by Division of Recombinant Biological Products, IBPC, and NIFDC since 2001.

Characterization and comparison of interferon reference standards using UPLC-MS / 药学学报

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.

Introduction of the related content of biotech drugs quality control in the Chinese Pharmacopoeia 2015 / 中国药学杂志

The Chinese pharmacopoeia 2015 has been issued. To better understand and implement the new pharmacopoeia, this article first briefly reviews the situation of biotech drugs quality control after the execution of the 2010 edition pharmacopoeia, lists related issues, and illustrates the necessity to study and understand the new pharmacopoeia and related documents in time. Then according to the relevant regulations and the Chinese pharmacopoeia 2015 Volume III, the contents of the draft of biotech drugs quality control are introduced, including research basis and regulations of quality standards, the general notice and related provisions of standard materials such as standard, reference, reference substance in the article 26 in the notice, general requirements and six requirements related to the production and quality control of biotech drugs, general monograph of human recombinant DNA technology products and general monograph of human recombinant monoclonal antibody products, the monograph and the China's 2013 annual valuation to sample vial recombinant human interferon alpha 2a as an example analysis for verification regulation related content, and appendices and the second method (reporter gene method) for determination of interferon biological activity in the new appendix. The advantages and disadvantages of the requirements on products contained in this edition of pharmacopoeia were discussed, as well as the regulations and technical background when the quality standards were formulated. Focus was put on the importance of peptide mapping analysis in the structure analysis of recombinant protein product and the stability evaluation of production process, key technical requirements in new general monograph of human recombinant DNA technology products, and the application of new technologies, such as LC-MS in structure analysis of biotech drugs protein and identification of reference substance, etc.

Characterization of the primary structure of TNK-tissue plasminogen activator using LC-MS / 药学学报

The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.

Two-dimensional ultrasound imaging of atrial appendages in normal fetuses / 中华超声影像学杂志

Objective To understand demonstration methods and ultrasonographic features of left and right atrial appendages in normal fetuses.Methods 200 consecutive normal fetuses during gestation age 19-28 weeks from November 2011 through April 2012 entered the study.Ultrasonographic features and the demonstrated rates of atrial appendages at different incidence angle of ultrasonography and different gestational weeks were recorded and calculated,respectively.Results Atrial appendages of normal fetuses can be demonstrated on atrial appendages plane,which was little lower the view of parasternal great artery short axis.The display rate of left,right and left-right atrial appendages was 92％,68％,65.5％,respectively.When ultrasound beam entranced into chest from the right side,the display rate of left-right atrial appendages was highest(94.3％).The best time to observe atrial appendages was 22 to 24 week of pregnancy.The majority of left atrial appendages were fingerlike hook shapes,with narrower bases and longer bodys,and the endocardial surfaces were relatively smooth.Sometimes with incisures on external edges.Whereas the majority of right atrial appendages were obtuse-angled triangle shapes,with broad bases and shallower bodys.On endocardial surface the pectinate muscles were often seen,which looked like serrated echoes,sometimes prominent taenia sagittalis were noted in right atrial appendages.Conclusions Atrial appendages of normal fetuses can be demonstrated using two-dimensional ultrasonography on atrial appendages plane.The demonstrated rates of atrial appendages were different according to different incidence angle of ultrasonography and gestational weeks.There were some differences in ultrasonographic features between left and right atrial appendages,which is very helpful in determinating atrial situs.

Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS / 药学学报

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

Quality control of recombinant oncolytic adenovirus/p53 / 药学学报

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody / 药学学报

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.

Factors influencing the diagnosis and treatment of chronic prostatitis among urologists in China / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To identify the factors influencing diagnosis and treatment of chronic prostatitis (CP) among Chinese urologists.</p><p><b>METHODS</b>A sample of 656 urologists from 29 provinces of China were asked to complete a questionnaire that explored attitudes towards CP as well as diagnosis and treatment patterns in the management of CP. Both univariate and multivariate logistic regression analysis schemes were used to determine the factors that influence the diagnosis and treatment of CP.</p><p><b>RESULTS</b>A total of 656 questionnaires were given out. All were returned and 410 of those were included in the final univariate and multivariate analysis. Multivariate logistic regression analysis indicated that belief of bacterial infection in the etiology of CP (odds ratio [OR], 2.544; 95% confidence interval [CI], 1.650-3.923; P < 0.001) was the most significant factor influencing the routine performance of bacterial culture test. Using the same model, the type of hospital (OR, 2.799; 95% CI, 1.719-4.559; P < 0.001) and the routine use of the 4- or the 2-glass test (OR, 3.194; 95% CI, 2.069-4.931; P < 0.001) were determined to be significant factors influencing the use of the National Institutes of Health (NIH) new classification system. According to the same model, belief of bacterial infection in the etiology of CP (OR, 3.415; 95% CI, 2.024-5.762; P < 0.001) and the routine use of bacterial culture test (OR, 2.261; 95% CI, 1.364-3.749; P < 0.01) were important factors influencing the routine prescription of antibiotics.</p><p><b>CONCLUSION</b>Our findings suggest that attitudes towards CP, and the characteristics of individual urologists' practices may influence the diagnosis and treatment of CP among Chinese urologists.</p>

Expression of Ki-67, Bcl-2,Bax and caspase-3 in benign prostatic hyperplasia combined with prostatitis and their significances / 中南大学学报(医学版)

OBJECTIVE@#To detect the expression of Ki-67, Bcl-2, Bax and caspase-3 in simple benign prostatic hyperplasia (BPH) and BPH combined with prostatitis,and to evaluate the effect of inflammation on the development and progression of BPH.@*METHODS@#All specimens were obtained from patients undergoing surgical resection of the prostate. The paraffin section of the specimens was stained with hemotoxyline and eosin, and observed under light microscope to examine the inflammation hispathological changes. Sixteen patients with simple BPH (Group A) and 42 patients with BPH combined with prostatitis (Group B) were included. Immunohistochemical analysis and Western blot were used to examine the expression of Ki-67, Bcl-2, Bax and caspase-3.@*RESULTS@#The expression of Ki-67 and Bcl-2 was significantly higher in Group B than that in Group A (P0.05).@*CONCLUSION@#Prostatitis can up-regulate Ki-67, Bcl-2 expression, and down-regulate the expression of caspase-3 in BPH. Prostatitis appeared to play an important role in the development of BPH by affecting the proliferation and apoptosis of the prostatic cells.

Preliminary Study of Bio-transformation of Phytosterol by HPLC-MS / 微生物学通报

The method for analysis and determination the cleavage of soybean sterol, in which the soybean sterol was degraded and the products androst-1,4-diene-,17-dione (ADD) and androst-4-ene-3,17-dion (AD) were developed by Liquid Chromatography-mass Spectrometry. The HPLC conditions adopted were: a All- tima ODS-2 column (250 mm?4.6 mm, 5 ?m), a mobile phase consisted of menthanol-water (70:30), a flow rate of 1.0 mL/min, a room column temperature. and the detective wavelength was 244 nm.The ZMD Micromass electrospray ionization (ESI)-mass spectrometer was employed. In such conditions the corre- sponding HPLC chromatogram and MS spectrum were obtained. The method has a linear ranger of 0.01 mg/mL ~ 0.09 mg/mL, R2 =0.9999, the recoveries of ADD and AD were 102.6% and 105.90%, the RSD of ADD and AD were 3.02%, 3.5% and 3.08%, 3.24%. This method showed high sensitivity, accuracyand easy to perform. It is suitable to analysis the process cleavage of soybean sterol as well as quality control of product.

Quality control methods and requirements for recombinant human lymphocyte function associated antigen 3 IgG1 fusion protein (rhLFA3-IgG1) / 药学学报

To establish methods and requirements for quality control of rhLFA3-IgG1, biological potency of rhLFA3-IgG1 was determined by CD2 molecule competitive binding assay on Jurkat cell surface. Purity of rhLFA3-IgG1 was analyzed by SEC-HPLC and IEC-HPLC. Peptide mapping was preformed by tryptic digestion and RP-HPLC after sample reduced and carboxymethylation by DTT and indoacetic acid, respectively. CHO host cell protein and Protein A residual were detected by ELISA separately. The quality control methods and requirements, such as biological potency, the physical-chemical characteristic of rhLFA3-IgG1 had been established. The methods and requirements for quality control of rhLFA3-IgG1 showed advantages of assuring the products safety and efficacy, which can be used for routine quality control of rhLFA3-IgG1.

Peptide mapping analysis of recombinant human interleukin-11 with HPLC-ESI-Q-TOF/MS spectrometry / 药学学报

<p><b>AIM</b>To analyze the peptide mapping of recombinant human interleukin-11 (rhIL-11) by HPLC-ESI-Q-TOF/MS spectrometry.</p><p><b>METHODS</b>The trypsin digested rhIL-11 at 37 degrees C over night, and the peptide mapping was performed by HPLC. The relative molecular weight of the peptides fragments was measured by ESI-Q-TOF/MS, and amino acid sequence was analyzed by MS/MS.</p><p><b>RESULTS</b>The peptide fragments of rhIL-11 in the peptide mapping were assigned by analyzing the retain time, relative molecular weight and amino acid sequence. And 97% of the expected peptides were detected in this way.</p><p><b>CONCLUSION</b>The study proves that HPLC-ESI-Q-TOF/MS is a good method to analyze peptide mapping of protein with the advantage of sensitivity, high speed and accuracy.</p>

Expression,Purification and Enzymatic Characterization of Klebsiella sp.Glycerol Dehydrogenase in E.coli / 中国生物工程杂志

The dhaD gene encoding glycerol dehydrogenase(GDH) from Klebsiella sp.was amplified,and was inserted into expression vector pET-28a(+),the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21(DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21.Then GDH was purified by Ni-NTA affinity chromatography,the results showed a single band about 39kDa on SDS-PAGE gel,and the specified activity was about 156U/mg.The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%.The optimum reaction pH was 11.0,and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0.The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃～45℃.The Km value was 0.54mmol/L and Vmax was 0.49 ?mol/ml?min in the glycerol.

Research in Extraction of GPDH with Ultrasonic from Tobacco Leaf / 中国生物工程杂志

Nicotiana tabacum is an important and classical model plant which can respond to the change of environmental conditions by accumulating osmoprotectants, such as glycerol and proline which contribute to the re-establishment of homeostasis when exposed to various adverse environmental stresses, such as drought, salinity, high and low temperatures. The optimization of ultrasonic extraction (UE) conditions of glycerol-3-phosphate dehydrogenase (GPDH) of tobacco leaf have been built by orthogonal test. It showed that optimum of the powers, treatment times, slot times and leaf-to-solvent ratios of UE was 75w, 2h, 2s, and 1[DK]∶12 g/ml, respectively. Under these conditions, the activity of GPDH has been tested as 0.3937U/mg protein, which was higher than other extraction methods such as liquid nitrogen and grinding on ice bath. According to investigation, it is the first description of determination of content of GPDH with ultrasonic in tobacco. It could provide basis for the further research in the relation of content of glycerol and osmotic pressure in tobacco.

Determination of Dihydroxyacetone in Fermentation Broth by HPLC / 中国生物工程杂志

A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on a Alltima C18(5?m,250?4.6mm). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 ml/min and the detective wavelength was 200 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC, which was in agreement with the result by spectrophotometric method.The method was applicable for DHA determination in the fermentation process.

Effect on the Synthesis of 1,3 propanediol of Organic acids in the Two-stage Fermentation / 中国生物工程杂志

Effect of organic acids on the synthesis of 1,3 propanediol was studied.The adsorption of organic acids from glycerol fermentation liquor by ion-exchange resins was investigated.The results showed that organic acid and 1,3 propanediol production was in negative relationship.The static adsorption showed that ion-exchange resin 005 had the best adsorption abilities of the organic acids in the glycerol fermentation liquor.It was showed that the yield of 1,3propanediol increased by 166% after the extraction of organic acids from glycerol fermentation liquor and the convertion rate increased by 34%.

Secretory Expression of the Fusion Protein PTH-HSA in Pichia pastoris / 中国生物工程杂志

The fused gene (PTH-HSA) of parathyroid hormone (PTH) gene and Human Serum Albumin(HSA) gene was amplified without linker by Overlapping PCR technology. The spliced gene was clone into Pichia pastoris secretory vector pPIC9K. With the help of promoter AOX1 and mat ? signal peptide, the PTH-HSA gene was designed to secretory expression.Linearized by restriction enzyme SalI, The recombinant plasmid pPIC9K/PTH-HSA was transformed into Pichia pastoris KM71 by electroporation. The recombinant strains which were identified by G418 and PCR analysis were induced by methanol to express protein PTH-HSA. The target protein was expressed in fermentation supernatant. Western blot analysis of the fusion protein showed that the expressed fusion protein PTH-HSA had the antigenicity of HSA.adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis The specific activity of broth was about 318IU/ml.

Activity Study of Ciliary Neurotrophic Factor(CNTF)Mutant / 中国生物工程杂志

To study still further the activity of CNTF mutant designed by computer molecular modling,the methods of dissociated cultures of chick dorsal root ganglion、TF-1 prolification and the normal mice'weight loss tests weve used.The results indicated that the mutant protein promoted the survival of dorsal root ganglion、induced TF-1 prolification and made the normal mice lose weight,decrease appetite and reduce fat index.The weight loss effect was dependant with its administration dosage,ED50 was 150.986?g/kg/d.To TF-1,the specific activity reached 2.0?106U/mg against international reference reagent.In a word,CNTF mutant had excel bioactivity.So it provided clues for its development and application.