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.
Amino Acid Sequence ; Chromatography, High Pressure Liquid ; methods ; Interferons ; standards ; Mass Spectrometry ; methods ; Molecular Weight ; Oxidation-Reduction ; Peptide Mapping ; Protein Processing, Post-Translational ; Reference Standards

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 M_r 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 S_138/139 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.

AIMTo establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).

METHODSIdentification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT.

RESULTSIdentity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements.

CONCLUSIONThe methods and requirements had been established for quality control of rAAV-2/hFIX.

Animals ; Dependovirus ; genetics ; Factor IX ; biosynthesis ; genetics ; Female ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Genome, Viral ; Humans ; Male ; Mice ; Mice, Knockout ; Quality Control ; Random Allocation ; Recombinant Proteins ; biosynthesis ; genetics

AIMTo establish methods and requirements for the quality control of recombinant human neu epitope peptide 12.

METHODSBiological activity of recombinant human neu epitope peptide 12 was evaluated in FVB/N transgenic mice (TgN MMTV neu 202 Mul, Jackson Lab., USA) administered with samples. The percentage of antibody-positive mice detected by ELISA was used in the biological activity evaluation. The peptide map was performed by peptic digestion. The antigen content was determined by SEC-HPLC.

RESULTS AND CONCLUSIONThe quality control methods, such as biological activity, peptide map, antigen content, and the requirements for the quality control of recombinant human neu epitope peptide 12 were established. The established methods and requirements were already used for the quality control of recombinant human neu epitope peptide 12 products.

Animals ; Antibodies, Monoclonal ; analysis ; Biotechnology ; methods ; Epitopes ; analysis ; chemistry ; immunology ; Female ; Genes, erbB-2 ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Peptide Fragments ; chemistry ; immunology ; Peptide Mapping ; Quality Control ; Receptor, ErbB-2 ; analysis ; chemistry ; immunology ; Recombinant Fusion Proteins ; analysis ; chemistry ; immunology ; Technology, Pharmaceutical ; standards

AIMTo investigate the quality and optimized test methods and establish the quality specification of recombinant adenovirus-IL2.

METHODSThe titer of Adv-IL2 was measured by cytopathic effect (CPE). Hela cells were infected with Adv-IL2 in vitro, the expressed IL-2 and its bioactivity in Hela cell were determined by enzyme-linked immunosorbent assay (ELISA) and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) respectively. The purity of Adv-IL2 was analysed by UV and IE-HPLC method. The molecular weight and enzyme digestive map of Adv-IL2 genome were analysed by electrophoresis. The characteristic gene E2B, IL-2 expression casseter and foreign factors (RCV, HIV, HBV, HCV) were detected with polymerase chain reaction (PCR). Other tests were carried out according to the Chinese Requirements for Biological Products.

RESULTSAdv-IL2 was generated efficiently with a titer of 3 x 10(10) pfu.mL-1. The expressed IL-2 and its bioactivity were 25 ng.mL-1 and 700 u.mL-1 respectively. A260 nm/A280 nm was 1.23. The purity determined by IE-HPLC was higher than 98%. The molecular weight, enzyme digestive map of Adv-IL2 genome, the characteristic gene E2B and IL-2 expression casseter conformed to expected values.

CONCLUSIONThe specification for Adv-IL2 is established and can be used for the quality control of the product.

Adenoviridae ; genetics ; Cell Line ; Embryo, Mammalian ; Genetic Vectors ; genetics ; HeLa Cells ; virology ; Humans ; Interleukin-2 ; biosynthesis ; genetics ; Quality Control ; Recombinant Proteins ; biosynthesis ; Transfection

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.

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.

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.

Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-? and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-? expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 ?mol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-?,and its fermentation parameters are confirmed.

AIMTo establish methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein (rhTNFR-Fc).

METHODSBiological potency of rhTNFR-Fc was determined by neutralizing the bioactivity of TNF-alpha. rhTNFR-Fc samples were reduced by beta-mercaptoethanol and the peptide map was performed by tryptic digestion. Residual protein A and the host cell protein content were detected by ELISA. Anti-TNFR and anti-Fc antibodies were used in ELISA for detection of the rhTNFR-Fc content.

RESULTSThe quality control methods, such as bioassay, peptide map, residual protein A detection, were established and used for quality control of rhTNFR-Fc. The unit of rhTNFR-Fc (AU) was defined according to the international unit of TNF-alpha. The specific activity was up to 8 x 10(4) AU.mg-1. The requirements for quality control of rhTNFR-Fc were established.

CONCLUSIONThe methods and requirement were used for quality control of rhTNFR-Fc products.

Animals ; Biotechnology ; methods ; Cell Division ; drug effects ; Etanercept ; Immunoglobulin G ; biosynthesis ; chemistry ; pharmacology ; Mice ; Peptide Mapping ; Quality Control ; Receptors, Tumor Necrosis Factor ; biosynthesis ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; pharmacology ; Technology, Pharmaceutical ; standards ; Tumor Cells, Cultured