1.Protective effects of puerarin on radiation injury of experimental rats.
Lehong JIN ; Chuanfei LIU ; Yu ZENG
Journal of Integrative Medicine 2005;3(1):43-5
To observe the protective effects of puerarin on radiation injury of experimental rats and to discuss the possible mechanism of its radiation protection.
2.Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody.
Chunyu LIU ; Lan WANG ; Wei GUO ; Chuanfei YU ; Feng ZHANG ; Wenbo WANG ; Meng LI ; Kai GAO
Acta Pharmaceutica Sinica 2015;50(1):94-8
The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.
3.Comparative study of size and charge heterogeneities of anti-TNF-αantibodies by high performance liquid chromatography
Wei GUO ; Wenbo WANG ; Chuanfei YU ; Feng ZHANG ; Lan WANG ; Chunyu LIU ; Meng LI ; Kai GAO
Chinese Journal of Microbiology and Immunology 2014;(9):723-726
Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products ( SBP ) .Methods The size exclusion chromatography ( SEC-HPLC ) and weak cation exchange chromatography ( WCX-HPLC ) were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B (CpB) treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .
4.Size heterogeneity analysis of monoclonal antibody products
Chuanfei YU ; Wenbo WANG ; Meng LI ; Lan WANG ; Feng ZHANG ; Chunyu LIU ; Kai GAO
Chinese Journal of Microbiology and Immunology 2014;(9):718-722
Objective To compare the capability of capillary electrophoresis-sodium dodecyl sul-fate ( CE-SDS) and size exclusion-high performance liquid chromatography ( SE-HPLC) for analysis of size heterogeneity of monoclonal antibody products .Methods The size heterogeneity of one humanized anti-VEGF monoclonal antibody was analyzed by using non-reduced and reduced CE-SDS, and conventional , de-natured and denatured reduced SE-HPLC.Results The percentage of aggregates detected by non-reduced CE-SDS (0.82%±0.01%) was equal to that by using denatured SE-HPLC (1.05%±0.02%), but it was significantly lower than that by using conventional SE-HPLC analysis (5.08%±0.10%).With regard to fragments analyzed with non-reduced antibodies, its percentage was (7.12±0.04)% measured by non-re-duced CE-SDS analysis that was significantly higher than that by conventional SE -HPLC analysis (0.02%± 0.01%) and denatured SE-HPLC analysis (0.62%±0.01%).Using reduced antibodies , the percentage of fragments was (3.19±0.50)%tested by reduced CE-SDS analysis that was significantly higher than that by using denatured reduced SE-HPLC analysis (0.07%±0.01%).Conclusion Conventional SE-HPLC was more objective than CE-SDS for content analysis of aggregates , as both the covalent and non-covalent forms of aggregates could be detected .Non-reduced CE-SDS could demonstrate the content of clips , while reduced CE-SDS showed the degraded fragments .Therefore, CE-SDS had an advantage over conventional SE-HPLC for content analysis of fragments .The use of the two analytical methods in combination provided solid techni-cal supports for the quality control of size heterogeneity of monoclonal antibodies .
5.N terminal sequencing for practical detection of monoclonal antibody.
Wei GUO ; Chuanfei YU ; Meng LI ; Lan WANG ; Feng ZHANG ; Chunyu LIU ; Wenbo WANG ; Kai GAO
Chinese Journal of Biotechnology 2014;30(9):1473-1480
Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.
Amino Acid Sequence
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Antibodies, Monoclonal
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isolation & purification
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Chromatography, Ion Exchange
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Humans
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Isoelectric Focusing
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Mass Spectrometry
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Peptide Mapping
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Peptides
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Sequence Analysis, Protein
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methods
6.Determination of drug antibody ratio in an antibody-drug conjugate.
Chuanfei YU ; Meng LI ; Wei GUO ; Lan WANG ; Feng ZHANG ; Chunyu LIU ; Wenbo WANG ; Junzhi WANG ; Kai GAO
Acta Pharmaceutica Sinica 2014;49(3):363-7
This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.
7.Thromboembolism Risk and Treatment of Therapeutic Agents for Inflammatory Bowel Disease
Xinglian YU ; Chuanfei LI ; Lin LÜ
Chinese Journal of Gastroenterology 2023;28(7):441-448
Patients with inflammatory bowel disease(IBD)have an increased risk of thromboembolism.Recent reports on Janus kinases inhibitors and thromboembolic adverse events have revealed that IBD therapeutic drug play an essential role in modifying this risk in a pro or antithrombotic manner,in addition to the increased risk of thrombosis of IBD itself.In this review,we provide an overview of the current understanding on thrombosis risk,mechanism and anticoagulant therapy of IBD drugs.While controlling the activity of the disease with appropriate therapy,thromboembolism prophylaxis and personalized treatment o should be emphasized.
8.A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT.
Zhihao FU ; Hongchuan LIU ; Lan WANG ; Chuanfei YU ; Yalan YANG ; Meiqing FENG ; Junzhi WANG
Acta Pharmaceutica Sinica B 2021;11(12):3925-3934
T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.