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. 2020 Aug 28.
doi: 10.1002/prot.25995. Online ahead of print.

Modulation of granulocyte colony stimulating factor conformation and receptor binding by methionine oxidation

Sumit Kumar Singh  1 , Avinash Mishra  1 , Gaurav Goel  1 , Narendra Chirmule  2 , Anurag S Rathore  1
Affiliations

Modulation of granulocyte colony stimulating factor conformation and receptor binding by methionine oxidation

Sumit Kumar Singh et al. Proteins. .

Abstract

Biosimilars offer an avenue for potential cost savings and enhanced patient access to various emerging therapies in a budget neutral way. Biosimilars of the granulocyte colony stimulating factor (GCSF) are an excellent example in this regard with as many as 18 versions of the drug being currently approved across globe for treatment of neutropenia. Here, we identified oxidation of the various methionine residues in GCSF as a key heterogeneity that adversely impact its efficacy. In agreement with earlier studies, it was found that oxidation of Met 122 and Met 127 significantly contributes toward reduction of GCSF efficacy, measured using binding affinity to the GCSF receptor. The combination of molecular dynamics simulation along with structural characterization studies established that oxidation of Met 127 and Met 122 brings about a small local conformational change around the B-C loop in GCSF structure due to slight displacement of Asp113 and Thr117 residues. The simulation studies were validated using fluorescence quenching experiments using acrylamide as quencher and site-directed mutagenesis by replacing Met 122 and Met 127 residues with alanine. The results of this study lead to an enhanced mechanistic understanding of the oxidation in GCSF and should be useful in protein engineering efforts to design stable, safe, and efficacious GCSF product. In addition, the structure-function information can provide targets for protein engineering during early drug development and setting specifications of allowable limits of product variants in biosimilar products.

Keywords: MD simulations; biosimilars; early drug development; methionine oxidation; receptor binding; structure-function relationship.

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References

REFERENCES

    1. Eon-Duval A, Broly H, Gleixner R. Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach. Biotechnol Prog. 2012;28(3):608-622. https://doi.org/10.1002/btpr.1548.
    1. Shacter E. Quantification and significance of protein oxidation in biological samples*. Drug Metab Rev. 2000;32(3-4):307-326.
    1. Krishnamurthy R, Madurawe RD, Bush KD, Lumpkin JA. Conditions promoting metal-catalyzed oxidations during immobilized cu-iminodiacetic acid metal affinity chromatography. Biotechnol Prog. 1995;11(6):643-650.
    1. Kerwin BA. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J Pharm Sci. 2008;97(8):2924-2935.
    1. Torosantucci R, Schöneich C, Jiskoot W. Oxidation of therapeutic proteins and peptides: structural and biological consequences. Pharm Res. 2014;31(3):541-553.