Structure-Function Engineering of Interferon--1b for Improving Stability, Solubility, Potency, Immunogenicity, and Ph
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- 作者：Amartya Basu ; Karen Yang ; Maoliang Wang ; Sam Liu ; Ramesh Chintala ; Thomas Palm ; Hong Zhao ; Ping Peng ; Dechun Wu ; Zhenfan Zhang ; Jack Hua ; Ming-Ching Hsieh ; John Zhou ; Gerald Petti ; Xiguang Li ; Ahsen J
- 刊名：Bioconjugate Chemistry
- 出版年：2006
- 出版时间：May 2006
- 年：2006
- 卷：17
- 期：3
- 页码：618 - 630
- 全文大小：315K
- 年卷期：v.17,no.3(May 2006)
- ISSN：1520-4812
文摘
Recombinant interferon-beta-1b (IFN-
-1b) is used clinically in the treatment of multiple sclerosis. In commonwith many biological ligands, IFN--1b exhibits a relatively short serum half-life, and bioavailability may befurther diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increasethe blood residency time of protein therapeutics, there is a further requisite for molecular engineering approachesto also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins.We investigated these five parameters of recombinant human IFN--1b in over 20 site-selective mono-PEGylatedor multi-PEGylated IFN--1b bioconjugates. Primary amines were modified by single or multiple attachments ofpoly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternateapproaches, site-directed mutagenesis was independently employed in the construction of designed IFN--1bvariants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugatepreparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and acomparison of the structural and functional properties of the IFN--1b proteins and their PEGylated counterpartswas conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sitesof the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferationbioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusionHPLC, and immunoassays demonstrated that the functional activities of the designed IFN--1b conjugates weremaintained, while the formation of soluble or insoluble aggregates of IFN--1b was ameliorated. Immunogenicityand pharmacokinetic studies of selected PEGylated IFN--1b compounds in mice and rats demonstrated bothdiminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. Theresults demonstrate the capacity of this macromolecular engineering strategy to address both pharmacologicaland formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN--1b, were further investigated in formulation optimization and biologicalstudies.
-1b) is used clinically in the treatment of multiple sclerosis. In commonwith many biological ligands, IFN--1b exhibits a relatively short serum half-life, and bioavailability may befurther diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increasethe blood residency time of protein therapeutics, there is a further requisite for molecular engineering approachesto also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins.We investigated these five parameters of recombinant human IFN--1b in over 20 site-selective mono-PEGylatedor multi-PEGylated IFN--1b bioconjugates. Primary amines were modified by single or multiple attachments ofpoly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternateapproaches, site-directed mutagenesis was independently employed in the construction of designed IFN--1bvariants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugatepreparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and acomparison of the structural and functional properties of the IFN--1b proteins and their PEGylated counterpartswas conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sitesof the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferationbioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusionHPLC, and immunoassays demonstrated that the functional activities of the designed IFN--1b conjugates weremaintained, while the formation of soluble or insoluble aggregates of IFN--1b was ameliorated. Immunogenicityand pharmacokinetic studies of selected PEGylated IFN--1b compounds in mice and rats demonstrated bothdiminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. Theresults demonstrate the capacity of this macromolecular engineering strategy to address both pharmacologicaland formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN--1b, were further investigated in formulation optimization and biologicalstudies.