Structure Identification And Biological Potency Analysis Of PEGylation Of Recombinat Insulin Conjugates

Insulin, the only hormone in human body, is used to lower blood glucose. It isimportant for the treatment of diabetes, especially inhibiting complications. There areseveral problems accompanied with insulin when used as a clinical treatment for diabetes,such as frequent injections, the risk of hypoglycemia, weight gain and so on. Therefore, itis significant to develop a new kind of insulin analog with longer duration of action andflat or near-flat “peakless” time-action profile in order to reduce diabetics incurredinconvenience and suffered from injecting insulin frequently, and hypoglycemic events,especially nocturnal hypoglycemia.The insulin Conjugates (Code: PEG-DesB³⁰-5kDa, PEG-DesB³⁰-10kDa,PEG-DesB³⁰-20kDa, PEG-DesB³⁰-40kDa) retained the amino acid sequence of humaninsulin except for the deletion of ThrB³⁰(Code: DesB³⁰) and were attached differentmolecular weight （5kDa,10kDa,20kDa,40kDa） of polyethylene glycol to LysB29.Comparative analysis of process, pharmacodynamics, pharmacokinetics, security was used.The structure of PEG-DesB³⁰-20kDa was determined for preclinical studies.Objectives:to provide the basis for the design of new long-acting insulin analog and developinsulin analogs with longer duration of action and flat or near-flat “peakless” time-actionprofile, we modify recombinant human insulin by polyethylene glycol. Basic on thepreliminary work, we have carried out the following research: focus on the structure andmodification site of PEG-DesB³⁰-20kDa confirmed; Calibration of protein contein;Evaluation of in vitro and vivo biological potency. To provide the basis of preclinical andclinical studies,we need to get uniform and stable protein.Methods:①Primary and secondary structure of PEG-DesB³⁰-20kDa were studied by aminoacid composition analysis、N-terminal amino acid analysis、mass spectrometric molecularweight determination、Circular dichroism spectroscopy、disulfide bond analysis and massspectrometric peptide mapping analysis. The modification site of PEG-DesB³⁰-20kDa wasconfirmed by mass spectrometric peptide mapping analysis and modified peptide aminoacid analysis.②Calibration of protein concentration by Lowry method、UV method、Kjeldahlnitrogen method, comparative analysis the result. Evaluation of Lowry method and UVmethod as standard of the result of Kjeldahl nitrogen method,and then determine an accurate and simple method.③Determination of the biological potency by mouse method and insulin receptorbinding method and basic on the structure identification and protein contein calibration.Result:①Amino acid composition、 N-terminal amino acid sequence、 mass spectrometricmolecular weight and circular dichroism spectroscopy were consistent with the theoreticalvalue;Correct disulfide pairing（CysA6-CysA11、CysA7-CysB7、 CysA20-CysB19）;Thepolyethylene glycol attached to LysB29was confirmed by mass spectrometric peptidemapping analysis and modified peptide amino acid analysis.②Determination of protein concentration by Kjeldahl nitrogen method、UV method、Lowry method, the result is1.02mg/ml、1.03mg/ml、1.14mg/ml. Comparative to Kjeldahlnitrogen method, the deviation of Lowry method﹥10%, and UV method﹤5%. Socalibration of protein contein by UV method、Kjeldahl nitrogen method. The averageprotein content of PEG-DesB³⁰-20kDa was1.025mg/ml.③Determination of the biological potency by mouse method and insulin receptorbinding method, the result was8.39U/mg、8.04U/mg.Clusions:The primary and secondary structure of PEG-DesB³⁰-20kDa were consistent with thetheoretical value. The polyethylene glycol attached to LysB29. PEG-DesB³⁰-20kDa haslower biological potency, approximately8.39U/mg. Protein sample is uniform and stable,it has longer duration of48hours and flat or near-flat “peakless” time-action profile,PEG-DesB³⁰-20kDa could be a potential long-acting insulin analog for the treatment ofdiabetes.