Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies.

Authors:
Samuel D Stimple
Samuel D Stimple
The Ohio State University
Columbus | United States
Sibel Kalyoncu
Sibel Kalyoncu
School of Chemistry and Biochemistry
Columbus | United States
Jesper E Mogensen
Jesper E Mogensen
Aalborg University
Denmark
Arne Staby
Arne Staby
Rensselaer Polytechnic Institute
United States
Peter M Tessier
Peter M Tessier
Rensselaer Polytechnic Institute
United States

Biotechnol Bioeng 2019 Apr 14. Epub 2019 Apr 14.

Department of Pharmaceutical Sciences, Biointerfaces Institute, University of Michigan, Ann Arbor, MI.

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay-which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates-can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5-1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.

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https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.26994
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http://dx.doi.org/10.1002/bit.26994DOI Listing
April 2019
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References

(Supplied by CrossRef)
Trastuzumab, a humanized anti‐HER2 monoclonal antibody, for the treatment of breast cancer
Albanell J. et al.
Drugs Today 1999
A novel, stable, aqueous glucagon formulation using ferulic acid as an excipient
Bakhtiani P. A. et al.
Journal of Diabetes Science and Technology 2015
Optimization of the native glucagon sequence for medicinal purposes
Chabenne J. R. et al.
Journal of Diabetes Science and Technology 2010
Protein misfolding, evolution and disease
Dobson C. M. et al.
Trends in Biochemical Sciences 1999
A bihormonal closed‐loop artificial pancreas for type 1 diabetes
El‐Khatib F. H. et al.
Science Translational Medicine 2010
Conformational states of glucagon
Gratzer W. B. et al.
Biochemical and Biophysical Research Communications 1967

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