Analysis of factors affecting stability of high concentration VHH-Fc fusion protein / 中国生物制品学杂志

@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.

The serum levels of FCGBP, SAA1, CXCL10 in children with mycoplasma pneumoniae pneumonia and their relationship with prognosis / 中国医师杂志

Objective:To investigate the serum levels and clinical significance of Fc fragment of the IgG-binding protein (FCGBP), serum amyloid protein A1 (SAA1), and CXC chemokine ligand 10 (CXCL10) in children with mycoplasma pneumoniae pneumonia (MPP) and their relationship with prognosis.Methods:A prospective study was conducted on 122 children with MPP admitted to the department of pediatrics of the 970th Hospital of the Joint Logistics Support Force of the Chinese People′s Liberation Army from January 2019 to December 2021. According to the severity and prognosis of MPP, they were divided into mild and severe groups, good prognosis group, and poor prognosis group. Forty healthy children who underwent physical examination during the same period were set as the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of FCGBP, SAA1, and CXCL10 in each subject, and to compare the differences in serum levels of FCGBP, SAA1, and CXCL10 among different groups. Multivariate logistic regression analysis was used to investigate the influencing factors of poor prognosis in MPP patients. The diagnostic value of individual and combined detection of serum procalcitonin (PCT), FCGBP, SAA1, and CXCL10 for poor prognosis in MPP children by analyzing the receiver operating characteristic (ROC) curve.Results:The levels of serum FCGBP [(115.68±10.57)ng/ml, (78.41±6.73)ng/ml, (12.55±3.25)ng/ml], SAA1 [(34.18±3.72)mg/L, (25.54±2.63)mg/L, (6.74±0.82)mg/L], and CXCL10 [(714.26±55.64)ng/L, (353.74±42.67)ng/L, (106.25±12.92)ng/L] in the severe MPP group were significant higher than those in the mild MPP group and the control group, with statistical significance (all P<0.05). The white blood cell (WBC), neutrophil percentage, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), PCT, lactate dehydrogenase (LDH), D-dimer (D-D), FCGBP, SAA1, CXCL10 of the children in the poor prognosis group were significantly higher than those in the good prognosis group, and the differences were statistically significant (all P<0.05). Multivariate logistic regression analysis showed that increased PCT ( OR=1.603, 95% CI: 1.190-2.160), FCGBP ( OR=1.757, 95% CI: 1.115-2.770), SAA1 ( OR=1.900, 95% CI: 1.327-2.720) and CXCL10 ( OR=1.704, 95% CI: 1.212-2.397) were independent risk factors for poor prognosis of MPP children (all P<0.05). The combined detection of serum PCT, FCGBP, SAA1, and CXCL10 had a significantly higher diagnostic value for the risk of poor prognosis in children with MPP than a single indicator. Conclusions:The elevated levels of serum FCGBP, SAA1, and CXCL10 in children with MPP are associated with the severity of MPP and are independent risk factors for poor prognosis in MPP patients.

Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Development of the method for determinating ADCC biological activity of IVIG / 中国输血杂志

【Objective】 To establish a method for determinating the antigen-dependent cell-mediated cytotoxicity (ADCC) of human immunoglobulin (pH4)for intravenous injection (IVIG) on luciferase reporter gene-modified cell assay. 【Methods】 As effector cells, Jurkat-NFAT-Luc-CD16 cells were used in the assay, and PLC/PRF/5 cells were used as target cells. After incubation of effector cells and target cells with IVIG, the method for determinating ADCC biological activity of IVIG was established by detecting luciferase released by activated T nuclear factor after binding of IVIG Fc fragment to effector cells. Meanwhile, the experimental assay conditions were optimized, and the methodology was verified subsequently. 【Results】 IVIG had a dose-response relationship in this method, which was consistent with four parameter logistic model. And the PLC/PRF/5 cells were finally determined as the target cells. The initial dilution concentration of antibody was 20 mg/mL, and the ratio dilution was 1∶2, and the effector to target ratio was 1∶3, and co-incubation time of two cells and IVIG was 24 hours. Within-run and between-run analysis including three independent tests, initial working concentration relative light unit (RLU) and the relative standard deviation (RSD) of the concentration for 50% of maximal effect(EC50) were less than 11%. The relative titers of the recovery samples of the two different dilution groups were (23.50±1.69)% and (49.30±2.97)%, respectively, and the corresponding recovery rates were (93.50±6.30)% and (96.24±5.43)%, respectively, with RSD less than 11%. 【Conclusion】 The method for determinating ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established. It could be applied in determinating the ADCC biological activity of IVIG, and has the advantages of satisfactory linearity, accuracy, precision and specificity.

Proteomics study of Mycoplasma pneumoniae pneumonia reveals the Fc fragment of the IgG-binding protein as a serum biomarker and implicates potential therapeutic targets / 医学前沿

Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.

Effect of IgG fragments in IVIG(pH4)on phagocytosis of sensitized erythrocytes by macrophage / 中国输血杂志

【Objective】 To research the effect of the Fc, Fab and F(ab′)2 fragments of immunoglobulin G, the main components of Human Immunoglobulin(pH4) for Intravenous Injection(IVIG), on the phagocytic function of macrophages derived from THP-1 cells. 【Methods】 First of all, IVIG was digested with papain and pepsin to obtain Fc, Fab and F(ab′)2, and these components were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, propylene glycol monomethyl ether acetate (PMA) was used to induce THP-1 cells to differentiate into M0 macrophages. Finally, the sensitized erythrocytes were labeled with carboxy fluorescein succinimidyl ester (CFSE), and the effect of the above components on the phagocytic ability of M0 macrophages to engulf sensitized erythrocytes was detected by flow cytometry. 【Results】 The identification results of SDS-PAGE showed that the prepared IgG fragments met the requirements of subsequent experiments. Flow cytometry performs showed that the phagocytosis model of M0 macrophages had been successfully established. When the concentration of Fc increased from 0.1μg/ mL to 10μg/ mL, the phagocytosis rate of erythrocytes sensitized by M0 macrophages decreased from (24.21±0.58) % to (12.27±0.19) %. When the concentration of IVIG protein increased from 0.1 μg/ml to 10 μg/ml, the phagocytosis rate decreased from (20.57±0.39) % to (0.20±0.03) %. Meanwhile, at the same protein concentration (10 μg/ml), the inhibitory effect of Fc on phagocytosis was only half that of IVIG. In addition, Fab, F(ab′)2, and human serum albumin could not inhibit phagocytosis of M0 macrophages. 【Conclusion】 IVIG can effectively inhibit the phagocytosis of THP-1 derived M0 macrophages, which is mainly dependent on the Fc, but not related to the Fab of IgG and F (ab′)2.

Micro-determination on activating complement in detecting biological properties of Fc fragment of IVIG / 中国输血杂志

【Objective】 To improve method for determinating of biological potency of IgG Fc fragment in Chinese Pharmacopoeia and establish a method which is suitable for enzyme-labeled instrument. 【Methods】 The biological properties of FC fragment of IVIG in Chinese Pharmacopiea(General 3514) was adjusted, including sensitization process, sample treatment and dynamic curve parameters. Meanwhile, the kinetic curve of hemolysis was fitted with Origin, which made the method more suitable for enzyme plate. The biological properties of Fc fragment of IVIG was calculated by kinetics curve reaction of reference and sample. 【Results】 The method are stable, the RSD of repeatability and intermediate is 10%, also with significant improvement in efficiency. 【Conclusion】 The detection of properties of Fc fragment of IVIG is stable and efficient, it can be used for properties of Fc fragment of IVIG.

Immune complex inhibits toll-like receptor 9-activated JNK and p38 pathways in B lymphocytes / 第二军医大学学报

Objective To explore the inhibitory effect of immune complex (IC) on the signal pathways of high-expressed CD40 and CD80 induced by Toll-like receptor (TLR9) agonist CpG oligodeoxynucleotide (ODN) in B lymphocytes. Methods The mice were intraperitoneally injected with CpG ODN or IC plus CpG ODN, and the spleen CD19+ B lymphocytes were sorted by magnetic-activated cell sorting (MACS). The expressions of CD40 and CD80 on the B lymphocytes were detected by flow cytometry. The spleen B lymphocytes were isolated from wild type and immunoglobulin G Fcγ receptor Ⅱb (FcγRⅡb) knockout mice by MACS. After the isolated cells were stimulated with CpG ODN or IC plus CpG ODN in vitro, the phosphorylation levels of related protein kinases were detected in the B lymphocytes by Western blotting. Following CpG ODN stimulation, the B lymphocytes were treated with JNK p38 inhibitor SP600125 (50 μmol/L) or p38 inhibitor SB203580 (20 mg/L), and then the CD40 and CD80 expression levels on the CpG ODN-activated B lymphocytes were detected by flow cytometry. Results IC inhibited CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes in vivo (both P0.05). IC inhibited the phosphorylation levels of JNK and p38 induced by CpG ODN in B lymphocytes, but did not inhibit them in the B lymphocytes from FcγRⅡb－/－ mice. The CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes were significantly decreased after treated with SP600125 and SB203580 (both P0.01). Conclusion IC can inhibit the CD40 and CD80 expressions induced by TLR9 agonist CpG ODN through inhibiting the JNK and p38 pathways in B lymphocytes.