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Development of a process to manufacture PEGylated orally bioavailable insulin
Author(s) -
Hazra Partha,
Adhikary Laxmi,
Dave Nitesh,
Khedkar Anand,
Manjunath H. S.,
Anantharaman Ramya,
Iyer Harish
Publication year - 2010
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.487
Subject(s) - conjugated system , chemistry , bioavailability , insulin , covalent bond , peg ratio , polyethylene glycol , pegylation , amino acid , peptide , molecule , residue (chemistry) , chemical structure , chromatography , combinatorial chemistry , organic chemistry , biochemistry , polymer , medicine , bioinformatics , finance , economics , biology , endocrinology
To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short‐chain methoxy polyethylene glycol (mPEG) derivative to the ε‐amino group of a specific amino acid residue (LysB 29 ). During the conjugation process, activated PEG can react with any of the free amino groups, the N‐terminal of the B chain (PheB 1 ) , the N‐terminal of the A chain (GlyA 1 ), and the ε‐amino group of amino acid (LysB 29 ), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy‐PEG 3 ‐propionyl—insulin at LysB 29 :IN‐105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN‐105) was purified to homogeneity using RP‐HPLC. The purified product, IN‐105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI‐TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose‐dependent glucose lowering effect in Type 2 diabetes patients. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010