Development Of Quality Control For PEGylated Urate Oxidase

Polyethylene glycol(PEG)is commonly used as modifier for protein drugs.Protein drugs after PEGylation can significantly improve the shortcomings such as short half-life and poor water solubility.There is no foreign quality standards for reference,domestic enterprises and the related quality control technology needs to be further improved.In this thesis,several key quality control projects was studied about PEGylated urate,including the determination of content?the measurement of modified rate and the determination of free PEG.Provide some technical support for the improvement of the quality standards of these drugs.Established the HPLC method to determine the content of PEG-UOX dope.HPLC method was performed on TSKG5000PWXL gel column(300 mm × 7.8 mm)with a temperature of 25℃;0.05 mol/L Tris-Hcl(pH 9.0)was used for mobile phase at a flow rate of 0.5mL/min under the wavelength of 280nm.Result:Both the two factories’UOX show a good linear in the range of 200μg/mL-1000μg/mL(R2=1 and R2=0.9999),the average recovery of factory M and factory J were geater than 98%at low,medium,high three concentration respectively.Result showed that this method have better specificity,accurate and repeatable.The second part,we studied the detemination of the extent of modification about PEG-UOX.Established the method to determine the content of PEG in the PEG-UOX though SEC-HPLC with RID in series,then we can know the average extent of modification in PEG-UOX accutately.HPLC method was the same as above.Result:In factory M,the concentration of the PEG portion of the PEG-UOX was 2.054mg/mL.The average recovery of factory M and factory J were geater than 98%at low,medium,high three concentration respectively.The number of modification in PEG-UOX was 7.2 and 7.3.The extent of modification was 29.2%and 28.8%respectively.This method can determine the average rate of modification of PEG-UOX accurately.Compare and establish a method for the detemination of polyethylene glycol(PEG)in PEGylated uricase.Using the methods high-performance liquid chromatographic with evaporative light scattering detector in series and SDS-PAGE stained with iodine reagents.The C4 with a column temperature of 30℃ was adopted;the mobile phase A was 0.1%trifluoroacetic acid-water(v/v)and mobile phase B was 0.1%trifluoroacetic acid-acetonitrile(v/v);gradiently with a flow rate of 1.0 mL· min-1.Iodine staining of SDS-PAGE using barium chloride fixed and iodine-potassium iodide staining.The developed high-performance liquid chromatographic method had a good binomial regression relationship in the ranges of 1～6 μg(R2=0.9993)and the sensitivity of PEG detected was 0.2 μg.The average recoveries at low,medium,high three concentration was 96.21%、97.38%、96.85%。The iodine staining sensitivity of SDS-PAGE was 0.1 μg.The former is accurate,reproducible,and the latter is simple,requiring no special equipment，but only can be used to limit test.