Development of a process to manufacture PEGylated orally bioavailable insulin.
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.
Hazra
Partha
P
Research and Development, Biocon Limited, Electronic City, Bangalore 560100, India. partha.hazra@biocon.com
Adhikary
Laxmi
L
Dave
Nitesh
N
Khedkar
Anand
A
Manjunath
H S
HS
Anantharaman
Ramya
R
Iyer
Harish
H
D002851:Chromatography, High Pressure Liquid; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007328:Insulin / Q000008:administration & dosage / Q000302:isolation & purification* / Q000627:therapeutic use; D011092:Polyethylene Glycols / Q000737:chemistry*; D013607:Tablets
D007328:Insulin; D013607:Tablets; D011092:Polyethylene Glycols