重组溶葡萄球菌酶成品蛋白含量测定方法的建立

Establishment of A Method for Determination of Protein Content in the Final Product Containing Recombinant Lysostaphin

重组溶葡萄球菌酶(recombinant lysostaphin,rLysn)对金黄色葡萄球菌具有良好的抗菌活性,是一种新型抗菌药物,含有该酶的药用制剂在临床上能有效控制金葡菌感染。为在rLysn成品中增加蛋白含量测定以控制制剂的质量,建立了成品中rLysn蛋白含量测定方法,并进行验证。采用体积排阻高效液相色谱法(size exclusion high performance liquid chromatograph,SEHPLC)测定rLysn理化对照品及成品蛋白质含量。色谱柱:TSK gel 2000SWXL(7.8 mm×30 cm,5μm);流动相:20 mmol/L磷酸缓冲液(pH7.0)+0.3 mol/L NaCl;流速:0.5 mL/min;柱温:25℃;检测波长:280 nm。结果显示,rLysn在2-32μg范围内与峰面积呈良好的线性关系(r2=1);低、中、高3种浓度的rLysn理化对照品空白加标平均回收率为102.9%,成品加标平均回收率为102.1%;3种不同上样量在不同时间内测定,蛋白含量RSD<1%;每隔1 h测定同一支供试品溶液,蛋白含量RSD为0.98%;同一批供试品溶液取6份进样,蛋白含量RSD为1.71%。实验结果表明,采用SE-HPLC方法简便,准确性、精密度、稳定性及重复性好,可用于rLysn成品蛋白质含量的测定。

Recombinant lysostaphin（rLysn）,duo to its solid antibacterial activity to Staphylococcal aureus,is novel antibacterialmedicine,and therapeutic agent containing this enzyme may efficiently control the infection of S.aureus.In order to control the quality ofpharmaceutical preparations with rLysn,a method of measuring the protein content in the final product with rLysn was established and verified.Size exclusion high performance liquid chromatograph（SE-HPLC）was employed to measure the protein content in both physico-chemicalcontrol and the final product with rLysn under the conditions as below ：a TSK gel 2000 SWXL column（7.8 mm×30 cm,5 μm）,the mobilephase composed of phosphate buffer（20 mmol/L,pH7.0）and Na Cl（0.3 mol/L）,the flow rate was 0.5 mL/min,the temperature was 25℃,and the detection wave length was 280 nm.As results,the rLysn（within 2-32 μg）was linearly correlated with the area of the elution peak（r^2 =1）.The weighted average recovery rate for physico-chemical controls in 3 rLysn concentrations of low,medium and high was 102.9%,whilethe weighted average recovery rate was 102.1%.Three supernatants were measured at different time,and RSD of protein content 1%;whilemeasurement was done for each supernatant in 1 h interval,RSD of the protein content was 0.98%;and 6 samples from one targeted solutionwere measured,RSD of the protein content was 1.71%.Above results reveal that SE-HPLC is simple and convenient,accurate,precise,stable and repeatable approach,thus may be used for the determination of protein content in the final product of rLysn.