title: Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein.
abstract: Fc fusion proteins are a new emerging class of molecules for immune-targeted delivery of therapeutic proteins. Biophysical and bioanalytical characterization is critical for clinical development and delivery of therapeutic proteins. Here we report molecular and functional characterization of a recombinant human fusion protein Mutant IL-15/Fc. MutIL-15/Fc has a molecular weight of ∼95 kDa as determined by multiangle laser light scattering with online size exclusion chromatography and migrated at a faster rate (lower retention time) in gel filtration column. The kinetics of binding of MutIL-15/Fc to Fcγ receptor is best fitted in a bivalent modal with K(D1) 5 μM and K(D2) 9 μM determined by surface plasmon resonance (BIAcore). N-Glycoprofiling analysis revealed extensive glycosylation of MutIL-15/Fc. The Fc and IL-15 components in the MutIL-15/Fc are detected using the dual mode ELISA. The HT-2 cell proliferation inhibition assay is qualified as a quantitative in vitro marker functional assay. Molecular state changes associated with forced stress analyzed by SEC-MALS resulted in changes in bioactivity and Fc:Fcγ receptor interaction affinity. These data provide a systematic approach to molecular and functional characterization of the MutIL-15/Fc to establish product consistency and stability monitoring during storage and under drug delivery conditions.
authors: Biopharmaceutical Development Program, SAIC-Frederick, Inc. , Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.
authors: Xiaoyi
authors: X
authors: Yang
authors: Abraham
authors: A
authors: Kallarakal
authors: Nirmala
authors: N
authors: Saptharishi
authors: Hengguang
authors: H
authors: Jiang
authors: Zhiwen
authors: Z
authors: Yang
authors: Yueqing
authors: Y
authors: Xie
authors: George
authors: G
authors: Mitra
authors: Xin Xiao
authors: XX
authors: Zheng
authors: Terry B
authors: TB
authors: Strom
authors: Gopalan
authors: G
authors: Soman
mesh_terms: D049109:Cell Proliferation; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D019409:Interleukin-15 / Q000037:antagonists & inhibitors*; D007700:Kinetics; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007141:Immunoglobulin Fc Fragments; D019409:Interleukin-15; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins