Development of methods and quality standard for quality control of recombinant human GLP-1 analogue fusion protein / 中国药学杂志

OBJECTIVE: To establish the quality control system for the recombinant human GLP-1 analogue fusion protein. METHODS: The potency of the fusion protein was determined by luciferase reporter gene assay. The purity was analyzed by non-reduced SDS-PAGE and SEC-HPLC respectively. RP-HPLC was used for the peptide mapping. ELISA was used to analyze the identification of the final products. The molecular mass and peptide mass spectra were analyzed by LC-ESI-MS technique. Other control tests were performed according to the requirements in the Chinese Pharmacopoeia (Volume III, 2010 edition). RESULTS: Control tests were performed on three different lots of bulks and final products of recombinant GLP-1 analog fusion protein by the developed methods. The results showed that all the indexes met the requirements in the Guideline for Quality Control of Recombinant DNA Products for Human Use and Chinese Pharmacopoeia (Volume III, 2010 edition). The molecular weight of the recombinant human GLP-1 analogue fusion protein was 71 361.0, which was in conformity with the theoretical value. CONCLUSION: The developed methods and standard may assure the safety, effectiveness, and controllability of the recombinant human GLP-1 analogue fusion protein, which might be used for the routine quality control of products of the same kind.

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.

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.

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.

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 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 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>