Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein

Xiaoyi Yang; Abraham Kallarakal; Nirmala Saptharishi; Hengguang Jiang; Zhiwen Yang; Yueqing Xie; George Mitra; Xin Xiao Zheng; Terry B. Strom; Gopalan Soman

doi:10.1021/mp300513j

Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein

Xiaoyi Yang, Abraham Kallarakal, Nirmala Saptharishi, Hengguang Jiang, Zhiwen Yang, Yueqing Xie, George Mitra, Xin Xiao Zheng, Terry B. Strom, Gopalan Soman

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

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.

Original language	English
Pages (from-to)	717-727
Number of pages	11
Journal	Molecular Pharmaceutics
Volume	10
Issue number	2
DOIs	https://doi.org/10.1021/mp300513j
State	Published - 4 Feb 2013
Externally published	Yes

Keywords

Fc receptor binding
N-glycoprofiling
SEC-MALS
bioactivity
fusion protein
surface plasmon resonance

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10.1021/mp300513j

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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 KD1 5 μM and KD2 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.",

keywords = "Fc receptor binding, N-glycoprofiling, SEC-MALS, bioactivity, fusion protein, surface plasmon resonance",

author = "Xiaoyi Yang and Abraham Kallarakal and Nirmala Saptharishi and Hengguang Jiang and Zhiwen Yang and Yueqing Xie and George Mitra and Zheng, {Xin Xiao} and Strom, {Terry B.} and Gopalan Soman",

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doi = "10.1021/mp300513j",

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T1 - Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein

AU - Yang, Xiaoyi

AU - Kallarakal, Abraham

AU - Saptharishi, Nirmala

AU - Jiang, Hengguang

AU - Yang, Zhiwen

AU - Xie, Yueqing

AU - Mitra, George

AU - Zheng, Xin Xiao

AU - Strom, Terry B.

AU - Soman, Gopalan

PY - 2013/2/4

Y1 - 2013/2/4

N2 - 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 KD1 5 μM and KD2 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.

AB - 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 KD1 5 μM and KD2 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.

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KW - bioactivity

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KW - surface plasmon resonance

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JF - Molecular Pharmaceutics

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ER -

Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein

Abstract

Keywords

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