Objective To develop and verify an anion-exchange high-performance liquid chromatography(AEX-HPLC)method for the determination of empty capsid ratio of recombinant adeno-associated virus type 9(rAAV9).Methods AEXHPLC based on the differences in surface charge was used to establish a method for detecting the ratio of empty and full capsid rAAV9 by optimizing the elution gradient of mobile phase,pH,column temperature,flow rate,sample concentration,injection volume and detection wavelength of fluorescence detector. The specificity,linearity,limit of detection(LOD),limit of quantitation(LOQ),precision and accuracy of the method were verified to confirm the feasibility.Results Using a CIMac AAV full/empty-0. 1 mL column,20 mmol/L BIS-Tris propane(BTP)as mobile phase A and 20 mmol/L BTP+1 mol/L NaCl as mobile phase B,gradient elution was performed with pH of 9.0,column temperature of 20 ℃,flow rate of1 mL/min,sample concentration of 4×10~(12)vg/mL,injection volume of 10 μL,excitation wavelength of 280 nm and emission wavelength of 330 nm,which realised the baseline isolation and quantitative detection of empty and full capsid rAAV9. The verification results of the method showed that the preparation buffer had no interference with good specificity;rAAV9 showed a good linear relationship in the range of(1.6-8)×10~(12)vg/mL,r = 0. 993;the LOD was 5×10~(10)vg/mL,and the LOQ was 1×10~(11)vg/mL;the RSD of repeatability and intermediate precision were 2. 95% and 2. 10%,respectively;the accuracy rates were not less than 80%.Conclusion A highly sensitive and rapid AEX-HPLC method for determination of the ratio of empty capsid to full capsid rAAV9 was developed,which could be used for the analysis of empty capsid rate and quality control in gene therapy products.

Objective To validate a method,ion chromatography(IC)coupled with pulsed amperometric detector(PAD),for the analysis of recombinant human erythropoietin(rhEPO)N-glycan profile in several laboratories.Methods The buffer solution of rhEPO sample was replaced by ultrafiltration with 25 mmol/L phosphate buffer solution,and the concentration was adjusted to 2 mg/mL. After enzymatic digestion with glycosidase F,the supernatant was precipitated with ethanol,and freeze-dried to obtain free N-glycan of rhEPO. The chromatographic conditions were as follows:The analytical column was Dionex CarboPac PA200,and the protection column was Dionex CarboPac PA200. Mobile phase A was 50 mmol/L sodium hydroxide solution,mobile phase B was 200 mmol/L sodium hydroxide solution,and mobile phase C was 250 mmol/L sodium acetate solution. The injection volume was 25 μL with the flow rate of 0. 5 mL/min and the column temperature of 30 ℃,and gradient elution was performed. The retention time of the highest peak in the peak cluster and the percentage area of the peak cluster were obtained with the analysis software of the instrument. Three laboratories(L1-L3)were selected for joint validation of the method,including accuracy,precision,linearity,limit of detection(LOD)and limit of quantification(LOQ),as well as stability.Results The peaks of rhEPO N-glycan were clearly visible. The area percentage of each peak cluster of the samples with different concentrations in three laboratory ranged from 84% to 116%,and the accuracy of the method was good. The RSDs of the highest peak retention time in the peak clusters were all less than 5%,the RSDs of the percentage area were all less than 10%,and the reproducibility of the method was good. When the protein concentration was in the range of 1. 0-3. 0 mg/mL,the linearity was good,R2> 0. 98. The LOD and LOQ were approximately 0. 10 mg/mL and 0. 32 mg/mL respectively. The stability was good at 2-8 ℃ for 48 h.Conclusion The IC analysis methodology for rhEPO N-glycan profile was established,and the interlaboratory validation indicators were good,which will provide technical support for improving the quality standard of rhEPO.

Objective To investigate the effect of occupation of modified site of N-terminal site-specific mono-PEGylated recombinant human granulocyte colony-stimulating factor(rh G-CSF)(PEG-G for short) on N-terminal amino acid sequencing based on Edman degradation method.MethodsAccording to the standard sequencing procedure of the N-terminal amino acid sequencer, the N-terminal amino acid sequence of the reference substance for N-terminal site-specific mono-PEGylation of human granulocyte stimulating factor was detected for four cycles, with a sample loading amount of 500 fmol. The waste liquid and mobile phase from the N-terminal amino acid sequencer were collected and evaporated to dryness under low pressure. After dissolving in 2 m L of water, PEG was detected by reversed-phase high performance liquid chromatography(RPHPLC) combined with an evaporative light scattering detector(ELSD).[conditions: mobile phase A, 0. 1% trifluoroacetic acid(TFA)-aqueous solution; mobile phase B, 0. 1% TFA-acetonitrile solution; chromatographic column: C4 column; flow rate: 1. 0 m L/min; injection volume: 100 μL; column temperature: 25 ℃; elution gradient: 0 min→20 min with mobile phase B of 5%→95%, 20 min→30 min with mobile phase B of 95%, 30 min→40 min with mobile phase B of 5%. ELSD conditions: default parameters for carrier gas; temperature 90 ℃; gain 100]. After confirming the PEG retention time, the mobile phase was collected according to the retention time and mass spectrometry analysis was performed.(mass spectrometry collection mode: cation mode; m/z: 3 800-6 000; deconvolution mass tolerance: 20 ppm). N-terminal amino acid sequencing analysis was performed on PEG-G from other companies.ResultsIn the cleaning waste liquid of the N-terminal sequencer, an evaporative light detection chromatographic peak with a retention time close to HPLC was detected. After mass spectrometry analysis, the peak exhibited typical PEG mass spectrometry characteristics, proving that the N-terminal cleaning program(i.e. the first cycle) could cleave the first peptide bond at the N-terminus of some PEG-G, causing the modified PEG to detach. In the sequencing of similar products from other enterprises, it was found that the cracking efficiency was related to the sulfur(S) element near the modified site.ConclusionThe cleaning and sequencing program of N-terminal sequencer can break the N-terminal site-specific PEGylated molecules, which provides technical supports for the establishment of the detection method for N-terminal modified sites in N-terminal site-specific single-modified PEG-G.

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.

A 32-year old male who had liver cirrhosis accompanying with chronic renal insufficiency (uremia) was suffered from homochronous allograft transplantation of liver-kidney. The blood and other tissue typing were in concord between the donor and recipient. The liver transplantation took typical orthotopic transplantation technique while the kidney transplantation took general operation method to place the transplanted kidney at the recipient's right lilac fossa. Daclizumab was taken for the immunity induction treatment before transplantation while the trigeminy of tacrolimus+mycophemocate mofeil+methylprednisolone were taken as immunosuppressant after transplantation. The transplanted liver and kidney recovered well which could work at once without any serious complications after transplantation, suggesting that combined liver-kidney transplantation was an effective treatment method for homochronous function failure of liver and kidney characterizing by perfect tissue typing, excellent operation skill, reasonable usage of immunosuppresant being the key point of success for transplantation.

Objective To explore clinical feasibility of liver transplant from child of brain death to adult, to summarize the clinical experiences that a child of brain death transplants liver to an adult. Methods The recipient was a 39-year-old woman patient with primary hepatic carcinoma and posthepatitis cirrhosis (decompensation stage); while the donor was a 8-old-year child of brain death because of brain neoplasms. Donated liver was gained by the method of en bloc multivisceral procurement in a short time; the operative method was classic orthotopic liver transplantation. The postoperative managements included immunosuppression, prevention of infection, hepatic protection, and other relevant supports etc. Results The transplantation operative duration was 6 hours, after which not only did the recipient survive but also her body functioned well including the liver part, with no severe postoperative complications. Conclusions The technology of transplanting livers from children to adults is feasible. The key to ensure the success of transplant operation is systematic preoperative evaluation, excellent operative technique, and perfect postoperative treatment.

Objective To investigate the growth inhibition of human osteosareoma cell line(MG-63) intervened by Hydralazine and 5'-aza-2'-deoxycytidine (5-Aza-CdR),and the effect on the mRNA expression of gene WW domain-containing oxidoreductase (WWOX).Methods Certain volume of 5 × 104/ml of human osteosarcoma cell line MG-63 in logarithmic growth phase were added into 96-well plate.There were Hydralazine group (drug concentration,0.1,1.0,10 μmol/L),5-Aza-CdR group (drug concentration,5,10,20 μmol/L),Hydralazine combined with 5-Aza-CdR group (drug concentration,0.1 μmo/L + 5 μmol/L,1.0 μmol/L + 10 μmol/L,10 μmol/L + 20 μmol/L) and control group (culture medium).Methyl thiazol tetrazolium(MTT) colorimetric methods were used to test the growth inhibition of MG-63 cells intervened by different concentrations of Hydralazine and 5'-aza -2'-deoxycytidine (5-Aza-CdR).Flow cytometry AnnexinV-FITC/PI methods were used to assay the effects of Hydralazine and 5-Aza-CdR inducing apoptosis in osteosarcoma cells in vitro.Real-time polymerase chain reaction (Real-Time PCR)methods were used to detect amplification of WWOX mRNA induced by Hydralazine combined with 5-Aza-CdR or alone.Western-blotting methods were used to examine the expression of WWOX in MG-63 cells.Results Hydralazine and 5-Aza-CdR effectively inhibited the growth of MG-63 cells in a concentration and time-dependent manner.Combined effect was more obvious.Further more the expression levels of WWOX mRNA and protein were increased significantly in combined groups as compared with other groups.Conclusion Hydralazine and 5-Aza-CdR could effectively inhibit the proliferation of MG-63 cells and induce apoptosis which is concurrent with the promotion of the expression of WWOX.The mechanism may be that Hydralazine/5-Aza-CdR effectively cause the demethylated of WWOX gene CpG-rich promoter regions,leading to the high expression of WWOX and inhibit the growth of MG-63 cells.The use of hydralazine in the treatment of osteosarcoma is worthy of further investigation.

Objective To evaluate the quality status of recombinant human interferon α1b injection and find out some quality problems.Methods Totally 31 batches of recombinant human interferon α1b for injection and 11 batches of recombinant human interferon α1b injection from two enterprises were examined according to Chinese Pharmacopoeia Volume Ⅲ (2010),and the quality status of recombinant human interferon α1b injection was evaluated by statistical analysis of the results.Results All 42 batches of samples were qualified.The production process of each enterprise was steady.Conclusion At present the quality of recombinant human interferon αlb injection is generally good.The current standards are feasible,but the specified standard of osmolality needs to be improved.

Objective To explore the clinical efficacy and complications of the surgical treatment of sternal tumor resection and titanium mesh reconstruction.Methods From 2008 January to 2012 June,there were 8 cases of sternal tumor patients in our hospital,including 5 male and 3 female,with an average age of 50.4 (37-66) years old.The histological morphology included 2 cases of chondrosarcoma,1 case of osteosarcoma,2 cases of malignant fibrous histiocytoma,eosinophilic granuloma in 1 case,and 2 cases of sternal metastasis of breast cancer.Tumor invasion sites included the sternal manubrium in 3 cases,the body in 2 cases,and both in 3 cases.All patients had undergone preoperative puncture or incision biopsy.8 cases of sternal tumor patients were treated with sternal tumor resection and reconstruction of the thorax using titanium mesh.The clinical effect and complications were retrospectively analyzed.Results All patients were followed up for 9 months to 4 years.The operations went well in all cases,with no intraoperative crisis or operative death.Deep wound hematoma occurred in 1 patient 1 week postoperatively,who healed 2 weeks after drainage and debridement.There was no abnormal breathing,subcutaneous emphysema,pneumothorax,infection or other complications in other cases.1 case of malignant fibrous histiocytoma died of lung metastasis at 9 months follow-up,and 1 died of liver metastasis at 14 months,while other patients got no tumor recurrence,with good thoracic shape,free breathing,no titanium mesh loosening,dyspnea,chest tightness,pain,or abnormal respiratory discomfort during follow-up period.The chest radiograph showed no chest deformity,no loosening or fracture of the fixation device.Conclusion Sternal tumor resection and reconstruction with titanium mesh has the advantages of good shaping effect,fewer complications,and simple operation,showing that titanium mesh is an ideal material for the reconstruction of sternum.

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor?40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p＝0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.