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.

Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

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.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

Objective To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Ma-din-Darby canine kidney ( MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination ( HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99. 62％ and 98. 1％, and the HA antigen recovery rate was 66. 96％. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

Objective@#To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus.@*Methods@#Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison.@*Results@#Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%.@*Conclusions@#This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.

Comparison of three types of residual Chinese hamster ovary cell protein detection kits / 中国药学杂志

OBJECTIVE: To compare the detection ability of three types of residual Chinese hamster ovary(CHO) host cell protein (HCP) ELISA kits (Cat No. A, B, C) from one manufacturer. METHODS: Residual CHO-HCP in the bulks of therapeutic monoclonal antibody (mcAb) produced with different strains of CHO cells, extracted proteins from CHO-K1 cultural supernatant and standards from different kits were determined with three types of kits to evaluate the CHO-HCP detection ability of different kits. Furthermore, the capture antibody coated microplates and enzyme conjugated detection antibody of three kits were re-assembled to detect standards and extracted supernatant proteins to analyze the possible reasons of differentiated detection ability. RESULTS: For the detection of mcAb samples, the relative recoveries of kit A and B to kit C were 1.89% and 15.34%, respectively; for the detection of extracted proteins from CHO-K1 cultural supernatant, the recoveries of kit A, B, and C were 4.05%, 21.52%, and 35.98%, respectively; for the detection of self-standards, the recoveries of three kits were all within the range of (100±15)%. For the detection of standards from other kits, there was a great discrepancy among the three kits. For kit A detection, the recoveries of standard B and C were 31.28% and 7.99%; for kit B detection, the recoveries of standard A and C were 55.76% and 25.08%; for kit C detection, the recoveries of standard A and B were 266.65% and 73.31%, respectively. By analyzing the results from reassembled capture and secondary antibodies, it was inferred that the detection ability of the capture antibodies of the three kits was sequenced as A>C>B, while the detection ability of enzyme conjugated secondary antibodies was sequenced as B>C>A. CONCLUSION: The detection abilities of three CHO-HCP ELISA kits from the investigated manufacturer were determined as C>B>A.

Improved protein-A chromatography for monoclonal antibody purification / 生物工程学报

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.

Screen and Identification of The Protein-protein Interactors in The Host Cell With The SARS Coronavirus Nucleocapsid Protein / 生物化学与生物物理进展

15 SARS-CoV N Protein Interacting Protein (NPIP) were selected from host cells using Yeast Two-hybrid system (Y2H). These are Angiogenin, acyglycerol kinase, cytochrome oxydase subunit I, CXC chemokine ligand 16, epidermal growth factor receptor pathway substrate 15, glutathione S-transferase kappa 1,integrin beta 1, jun oncogene, NIMA (never in mitosis gene a)-related kinase 10, protein tyrosine kinase 2 beta,homo sapiens SH3KBP1 binding protein 1 and ubiquitin specific peptidase 53. With the aid of immunological co-precipitation (CO-IP), it was confirmed that chemokine CXCL16 was the interactor with SARS-CoV N protein in host cells.