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.

Single-domain antibody - advances in research and application / 生物工程学报

Conventional IgG is composed of heavy and light chains. The light chain has one variable region (VL) and one constant region (CL) domain, whereas the heavy chain has one variable region (VH) and three constant region domains (CH1, CH2 and CH3). Single domain antibody (sdAb) is a kind of antibody that is composed of a variable domain of heavy chain and devoid of the light chain completely. Due to its small size, it is also called as nanobody. Although the sdAb has a simple structure, it can exhibit a comparable even better antigen-binding affinity than conventional antibody. Compared with conventional antibody, the small size, high stability and simplicity in recombinant expression are representative advantages of sdAb. In recent years, scientists are becoming increasingly interested in the roles of sdAb in fundamental biomedical research and clinical application. In this review, we summarized the structural features, physicochemical properties, screening strategies and recent advances in application of sdAb.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1 / 国际生物医学工程杂志

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen,and to provide experimental basis for subsequent functional studies.Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times.The peripheral blood was collected and the lymphocytes were isolated.Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH),and to construct a phage display library.The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA).The PD-1 antigen,which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml),was coated in an ELISA plate,and the phage display library was subjected to 3 rounds of affinity selection.Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA.According to the results of DNA sequencing,three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector,and transformed into E.coli BL21 (DE3) competent cells,and then induced by isopropyl-β3-D-thiogalactoside.The recombinant VHH antibody protein was purified by nickel column affinity chromatography,and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA.Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc,high titer specific antibody was stimulated,and the immune serum titer reached 1∶32 000.A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes.After 3 rounds of affinity selection,46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA.Among them,three clones of VHH-B7,VHH-H5 and VHH-H12 had higher repeats,indicating that significant enrichment was obtained.The results of Western Blot and ELISA showed that the purified B7,H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity.Their affinity constants were 1.19×1011 and 1.63×1011,1.59×1011 L/mol,respectively.Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library,which can bind to the PD-1 antigen with high affinity.This study can provide an experimental basis for subsequent functional studies.

Single domain antibodies from camel:research advances / 国际药学研究杂志

Camelidae possess a special type of antibody,namely heavy-chain antibody. It consists of a homodimer comprising three domains:CH2,CH3 and one variable region of heavy chain(VH),but CH1 and light chain are absent. The VH of heavy-chain an-tibody is named nanobody. Compared to the traditional antibody,nanobody has many advantages. For example it has small molecular weight,better stability and can cross blood brain barrier(BBB). More and more application of nanobodies have been reported in scien-tific fields,such as biological detection,diagnosis,and pharmacy. In this paper we review structural characteristics and physicochemi-cal properties of nanobodies,and investigate their development in biomedicine researches as well.

Single domain antibodies from camel: Research advances / 国际药学研究杂志

Camelidae possess special type of antibody, namely heavy-chain antibody. It consists of a homodimer comprising three domains: CH2, CH3 and one variable region of heavy chain (VH), but CH1 and light chain are absent. The VH of heavy-chain antibody is named nanobody. Compared to the traditional antibody, nanobody has many advantages. For example it has small molecular weight, better stability and can cross blood brain barrier(BBB). More and more application of nanobodies have been reported in scientific fields, such as biological detection, diagnosis, and pharmacy. In this paper we review structural characteristics and physicochemical properties of nanobodies, and investigate their development in biomedicine researches as well.