Ultrafiltration behavior of monoclonal antibodies and Fc-fusion proteins: Effects of physical properties

2017 ◽  
Vol 114 (9) ◽  
pp. 2057-2065 ◽  
Author(s):  
Youngbin Baek ◽  
Nripen Singh ◽  
Abhiram Arunkumar ◽  
Michael Borys ◽  
Zheng J. Li ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Physical Properties ◽  
Fusion Proteins

Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies

1992 ◽  
Vol 147 (1) ◽  
pp. 1-11 ◽  
Author(s):  
D. Wunderlich ◽  
A. Lee ◽  
R.P. Fracasso ◽  
D.V. Mierz ◽  
R.M. Bayney ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Recombinant Fusion Proteins ◽  
Rapid Characterization

scholarly journals The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Pia Gattinger ◽  
Shiva Izadi ◽  
Clemens Grünwald-Gruber ◽  
Somanath Kallolimath ◽  
Alexandra Castilho
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Degradation Products ◽  
Therapeutic Proteins ◽  
Cost Effective ◽  
Full Length ◽  
Peptide Sequence ◽  
Reporter Protein ◽  
The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

Monoclonal antibodies and fusion proteins

2008 ◽  
pp. 1405-1416
Author(s):  
Susan J. Lee ◽  
Arthur F. Kavanaugh
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins

scholarly journals Monoclonal Antibodies to Escherichia coli-Expressed P46 and P65 Membranous Proteins for Specific Immunodetection of Mycoplasma hyopneumoniae in Lungs of Infected Pigs

2003 ◽  
Vol 10 (3) ◽  
pp. 459-468 ◽  
Author(s):  
K. Cheikh Saad Bouh ◽  
F. Shareck ◽  
S. Dea
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Genomic Sequence ◽  
Clinical Signs ◽  
Mycoplasma Hyopneumoniae ◽  
Mycoplasma Species ◽  
Antigenic Determinants ◽  
Recombinant Fusion Proteins ◽  
Species Specific

ABSTRACT The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65c (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-β-d-thiogalactopyranoside), both GST-P46 and GST-P65c recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65c moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65c monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65c MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65c MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.

scholarly journals Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells

2020 ◽  
Vol 26 (4) ◽  
pp. 393-398
Author(s):  
Ehsan Naghneh ◽  
Es'hagh Pourmaleki ◽  
Azam Rahimpour
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Chinese Hamster ◽  
Vascular Endothelial ◽  
Cell Clones ◽  
Fc Fusion Protein ◽  
Endothelial Growth Factor

Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

Sign in / Sign up

Export Citation Format

Share Document