CAJ | 학술논문

目的：研究陶瓷羟基磷灰石（ceramic hydroxyapatite，CHT） I型和II型对2种单克隆抗体（monoclonal Ab，mAb）1和2聚集体的去除工艺。方法层析仪为AKTA AVANT 150，层析柱为Tricon 10／50，先进行CHT I、CHT II 2种填料的动态载样量研究，然后选取合适的载量进行分离纯化研究。上样条件为5 mmol／L磷酸二氢钠（ NaH2 PO4），pH 6．5，上样，然后用10 mmol／L NaH2 PO4，pH 6．5和10 mmol／L NaH2 PO4，2 mol／L NaCl，pH 6．5梯度洗脱来分离单体和聚集体。用分子尺寸排阻高效液相色谱（ SEC－HPLC）测定上样蛋白溶液和洗脱峰单体及聚集体的含量。使用20 cm高的XK 16／40的层析柱进行工艺放大研究。结果 mAb 1在CHT I型的载量为40 mg／mL，去除后单体含量为98．6％，工艺收率为92．5％；mAb 2在CHT I型的载量为45 mg／mL，去除后单体含量为98．8％，工艺收率为91．5％；mAb 1在CHT II型载量为16 mg／mL，去除后单体含量为99．8％，工艺收率为91．8％；mAb 2在CHT II型载量为20mg／mL，去除后单体含量为99．9％，工艺回收率为92．2％。结论2种类型的陶瓷羟基磷灰石填料在聚集体含量高于10％的情况下，都有很好的去除能力，去除结果符合法规要求。该方法操作简单，能够很顺利的进行工艺放大，满足中试和生产需求。
목적：연구도자간기린회석（ceramic hydroxyapatite，CHT） I형화II형대2충단극륭항체（monoclonal Ab，mAb）1화2취집체적거제공예。방법층석의위AKTA AVANT 150，층석주위Tricon 10／50，선진행CHT I、CHT II 2충전료적동태재양량연구，연후선취합괄적재량진행분리순화연구。상양조건위5 mmol／L린산이경납（ NaH2 PO4），pH 6．5，상양，연후용10 mmol／L NaH2 PO4，pH 6．5화10 mmol／L NaH2 PO4，2 mol／L NaCl，pH 6．5제도세탈래분리단체화취집체。용분자척촌배조고효액상색보（ SEC－HPLC）측정상양단백용액화세탈봉단체급취집체적함량。사용20 cm고적XK 16／40적층석주진행공예방대연구。결과 mAb 1재CHT I형적재량위40 mg／mL，거제후단체함량위98．6％，공예수솔위92．5％；mAb 2재CHT I형적재량위45 mg／mL，거제후단체함량위98．8％，공예수솔위91．5％；mAb 1재CHT II형재량위16 mg／mL，거제후단체함량위99．8％，공예수솔위91．8％；mAb 2재CHT II형재량위20mg／mL，거제후단체함량위99．9％，공예회수솔위92．2％。결론2충류형적도자간기린회석전료재취집체함량고우10％적정황하，도유흔호적거제능력，거제결과부합법규요구。해방법조작간단，능구흔순리적진행공예방대，만족중시화생산수구。
Objective To compare the purification process of two types of ceramic hydroxyapatite(CHT I and CHT II)to remove the aggregates from two monoclonal antibodies(mAb 1 and mAb 2).Methods All the chromatography runs were performed on AKTA AVANT 150 with Tricon 10/50 column.The dynamic binding capacity( DBC) of two types of CHT was studied firstly, and then purification research was carried out selecting the suitable DBC.The column was equilibrated with 5 mmol/L sodium dihydrogen phosphate pH 6.5, and then was eluted with gradient buffers which were 10 mmol/L sodium dihydrogen phosphate pH 6.5 and 2 mol/L sodium chloride pH 6.5.Aggregate content in loading and elution pool was evaluated by size exclusion chromatography.Scale-up process was carried on 20 cm height chromatography column XK16/40.Results DBC of CHT I for mAb 1 was 40 mg/mL and mAb 2 was 45 mg/mL.After purity, monomer content of mAb 1 reached 98.6% and yield was 92.5% and monomer content of mAb 2 reached 98.8%and yield was 91.5%.DBC of CHT II for mAb1 was 16 mg/mL and mAb 2 was 20 mg/mL.After purity, monomer content of mAb 1 reached 99.8% and yield was 91.8% and monomer content of mAb 2 reached 99.9% and yield was 92.2%.Conclusion Two types of CHT both can remove aggregates effectively from monoclonal antibodies when aggregate content reaches more than 10%, and results conform to the regulations.CHT I has higher dynamic binding capacity than CHT II, and CHT II is superior to CHT I in removing aggregate efficiency.The purification process is simple and can be easily scaled up in pilot and manufacture.Therefore, it meets the requirement pilot and scale production.