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.

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 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.

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.

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.

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.

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.

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>

Study on methods for quality control of recombinant human neu epitope peptide 12 / 药学学报

<p><b>AIM</b>To establish methods and requirements for the quality control of recombinant human neu epitope peptide 12.</p><p><b>METHODS</b>Biological 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.</p><p><b>RESULTS AND CONCLUSION</b>The 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.</p>

Expression of human nerve growth factor beta gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor beta (hNGFbeta) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.</p><p><b>METHODS</b>rAAV-2 containing hNGFbeta gene was constructed. The ability of hNGFbeta gene mediated by rAAV-2 vector (rAAV-2/hNGFbeta) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFbeta in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFbeta. rAAV-2/hNGFbeta and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFbeta concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.</p><p><b>RESULTS</b>After 48 hours, hNGFbeta content in supernatant was up to (188.0 +/- 28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFbeta at multiplicity of infection (MOI) 1.0 x 10(6) vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFbeta. Whole brain hNGFbeta content in rAAV-2/hNGFbeta transferred group was up to (636.2 +/- 140.6) pg/ml. hNGFbeta content of BBB disruption in rAAV-2/hNGFbeta infused group increased significantly compared to the control group (P <0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.</p><p><b>CONCLUSION</b>rAAV-2/hNGFbeta successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.</p>

Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX / 药学学报

<p><b>AIM</b>To establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).</p><p><b>METHODS</b>Identification 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.</p><p><b>RESULTS</b>Identity 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.</p><p><b>CONCLUSION</b>The methods and requirements had been established for quality control of rAAV-2/hFIX.</p>

Study on methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein / 药学学报

<p><b>AIM</b>To establish methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein (rhTNFR-Fc).</p><p><b>METHODS</b>Biological 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The methods and requirement were used for quality control of rhTNFR-Fc products.</p>

Quality control for recombinant adenovirus-IL2 / 药学学报

<p><b>AIM</b>To investigate the quality and optimized test methods and establish the quality specification of recombinant adenovirus-IL2.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Adv-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.</p><p><b>CONCLUSION</b>The specification for Adv-IL2 is established and can be used for the quality control of the product.</p>

Quality control methods for recombinant human endostatin / 药学学报

<p><b>AIM</b>To establish the quality control methods for recombinant human endostatin.</p><p><b>METHODS</b>Biological activity was determined by endothelial cell migration assays. Peptide mapping was tested by trypsin digestion and RP-HPLC. Purity was determined by non-reduced SDS-PAGE and RP-HPLC. Other tests including molecular weight, isoelectrical point, etc. were done according to the National Requirements for Biological Products (2000).</p><p><b>RESULTS</b>The method of bioassay was established and used for determining activity of endostatin. Specific activity of the three batchs of drug substance was 1.45 x 10(6), 1.57 x 10(6) and 2.73 x 10(6) u.mg-1 proteins. Peptide mappings of the three batches of drug substance were completely identified. Both purity results of the products tested by SDS-PAGE and RP-HPLC were more than 99%.</p><p><b>CONCLUSION</b>The established methods can effectively control the quality of recombinant human endostatin.</p>