Development and verification of an ELISA method for detection of process-specific residual host protein in biological preparation / 中国生物制品学杂志

@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.

Application of Design of Experiment in development of purification process of recombinant human growth hormone-Fc fusion protein / 中国生物制品学杂志

@#ObjectiveTo optimize the condition for anion exchange chromatography in purification process of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein by Design of Experiment(DOE)so as to decrease the content of host cell protein(HCP)in bulk of protein.MethodsA complete factorial experimental design with four factors at two levels was established by the DOE in Minitab 19 software.The condition(flow rate,sample load,pH value of buffer and salt concentration of eluent)for anion exchange chromatography in purification process of rhGH-Fc was optimized by DOE to find out the operating space.ResultsThe experimental results were predicted accurately by the established DOE model.The sample load,pH value of buffer,salt concentration of eluent as well as the interaction of pH value of buffer and salt concentration of eluent showed significant influence on the HCP content in the harvest.The optimal sample load,flow rate as well as pH value and salt concentration of eluent were 15 mg/mL,120 cm/h,7.0 ～ 8.0 and 0.1 mol/L respectively.The HCP contents in eluents under the optimal condition were less than 400 ng/mg,which met the requirements for verification within the range of results in determined operating space.ConclusionThe condition for removal of HCP by anion exchange chromatography was successfully optimized by DOE,which provided a reference for further application of DOE in the biopharmaceutical field.

Down-regulation of HCP5 inhibits cell proliferation, migration, and invasion through regulating EPHA7 by competitively binding miR-101 in osteosarcoma

Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.

Kangaroo mother care for low birth weight babies: supportive factors and barriers

Background: Kangaroo mother care (KMC) is skin-to-skin contact between mother and low birth weight (LBW) baby. It keeps the baby warm, increases accessibility to breast feeding, and protects the baby from infections. This study was done to identify supportive factors and barriers in practicing KMC as perceived by mothers of LBW babies and health care personnel (HCP).Methods: It was a questionnaire based descriptive study. Mothers of LBW babies and HCP were enrolled in study. Mothers and HCP were sensitized regarding KMC and after practicing KMC for 3 days, mothers were interviewed with the help of a predefined proforma. Feedback from the HCP was also taken. Data analysis was performed by using IBM SPSS ver. 20 software.Results: Most common factor in initiation and practice of KMC were, knowledge regarding KMC after training (100%), environmental factors (privacy and resources) (87.27%) and support from HCP (94.54%). Most common barriers perceived during performance of KMC were lack of knowledge about KMC during pregnancy (80%), pain due to LSCS/episiotomy (64.54%) and lack of support from family members (51.81%). Majority of the HCP strongly agree that parents must be encouraged to adopt KMC (82.92%), KMC is hampered due to presence of visitors in the ward (73.17%). KMC needs separate room (68.29%) and it is difficult due to LSCS (51.21%).Conclusions: To increase KMC practice, mother's knowledge about KMC can be improved by educating them in antenatal clinics and all HCP should receive training on KMC.