单抗药物A003的影响因素试验研究
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摘要
本研究对单抗药物A003进行了影响因素试验研究,包括光照、高温、剧烈振荡、低温循环、冻融循环、酸碱破坏、氧化破坏等试验研究,考察外观、蛋白质含量、pH、渗透压摩尔浓度、电荷异质性、纯度、生物活性等质量属性的变化。结果表明:(1)单抗药物A003对光照、高温、氧化破坏等比较敏感,电荷异质性结果显示主峰比例明显降低、酸性峰比例明显升高,纯度明显降低,聚体比例明显升高,其中氧化破坏导致抗原结合活性、细胞水平活性结果由107%、110%分别降低至43%、42%;(2)剧烈振荡、碱破坏(pH9.5)对A003有轻微影响,CEX、cIEF检测主峰比例均有下降趋势,酸性峰比例均有升高趋势,纯度有轻微降低;(3)A003在低温循环、冻融循环、酸破坏(pH3.0)等试验条件下,比较稳定,无明显变化。提示该产品在生产、储存及运输中,应避免接触空气,高温、光照及剧烈振荡。
This study is designed to evaluate the effect of stress conditions on therapeutic recombinant monoclonal antibody A003. Stress conditions including photostability, high temperature, shaking, low temperature cycle, freezethaw cycle, pH stressing and oxidation were tested. Changes of appearance, concentration, pH, osmotic pressure,charge heterogeneity, purity, bioactivity were evaluated for the assessment of drug stability. The results indicate A0003 is sensitive to light, high temperature andoxidation. CEX and cIEF results showed significant decrease of Main peak percentage and increase of acid peak percentage. Reduced purity and increased proportion of aggregation were also observed. Affinity between antibody and antigen from ELISA and cell-based assaydropped from 107% and 110% to43% and 42% after oxidative treatment, respectively. Shaking and pH stress(pH 9.5) showed slight damage to A003.Almost no significant change was observed in Low temperature test, Freeze/Thaw test and pH stress(pH 3.0) test. In conclusion, A003 should avoid light, high temperature, shaking and exposure to oxidative environment, in the process of production, storage and transportation.
This study is designed to evaluate the effect of stress conditions on therapeutic recombinant monoclonal antibody A003. Stress conditions including photostability, high temperature, shaking, low temperature cycle, freezethaw cycle, pH stressing and oxidation were tested. Changes of appearance, concentration, pH, osmotic pressure,charge heterogeneity, purity, bioactivity were evaluated for the assessment of drug stability. The results indicate A0003 is sensitive to light, high temperature andoxidation. CEX and cIEF results showed significant decrease of Main peak percentage and increase of acid peak percentage. Reduced purity and increased proportion of aggregation were also observed. Affinity between antibody and antigen from ELISA and cell-based assaydropped from 107% and 110% to43% and 42% after oxidative treatment, respectively. Shaking and pH stress(pH 9.5) showed slight damage to A003.Almost no significant change was observed in Low temperature test, Freeze/Thaw test and pH stress(pH 3.0) test. In conclusion, A003 should avoid light, high temperature, shaking and exposure to oxidative environment, in the process of production, storage and transportation.
引文
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[17]Joubert M K,Hokom M,Eakin C,et al.Highly Aggregated Antibody Therapeutics Can Enhance the in Vitro,Innate and Late-stage T-cell Immune Responses[J].Journal of Biological Chemistry,2012,287(30):25266-25279.
[2]国家药典委员会.9001原料药物与制剂稳定性试验指导原则-中国药典四部[M].北京:中国医药科技出版社,2015:354-356.
[3]食品药品监管总局.生物制品稳定性研究技术指导原则(试行)[Z].2015.
[4]ICH.Q1A(R2).Stability Testing of New Drug Substance and Produces[S].2003.
[5]ICH.Q5C.Quality of Biotechnological Produces:Stability Testing of Biotechnological/Biological Produces[S].1995.
[6]陈海峰.药物使用期间稳定性试验的技术要求探讨[J].中国新药杂志,2010,19(3):184-189.
[7]EMEA.Note for Guidance on In-use Stability Testing of Human Medical Products[S].2001.
[8]ICH.Q1B.tability Testing:Photostability Stability Testing of New Drug Substance and Produces[S].1996.
[9]Yi Du,Alison Walsh,Robin Ehrick,et al.Chromatographic Analysis of the Acidic and Basic Species of Recombinant Monoclonal Antibodies[J].mAbs 2012,4(5):578-585.
[10]SCALLON B J,TAM S H,MCCARTHY S G,et al.Higher Levels of Sialylated Fc glycans in Immunoglobulin G Molecules can Adversely Impact Functionality[J].MolImmunol,2007,44(7):1524-1534.
[11]HUANG L,LU J,WROBLEWSKI V J,et al.In Vivo Deamidation Characterization of Monoclonal Antibody by LC/MS/MS[J].Anal Chem,2005,77(5):1432-1439.
[12]张坤明,潘勇兵,张爱华,等,人用重组单克隆抗体电荷异质性的研究进展[J].中国生物制品学杂志,2016,29(2):206-212.
[13]BOSWELL C A,TESAR D B,MUKHYALA K,et al.Effects of Charge on Antibody Tissue Distribution and Pharmacokinetics[J].Bioconjug Chem,2010,21(12):2153-2163.
[14]Wang W.Protein Aggregation and Its Inhibition in Biopharmaceutics[J].Int J Pharm,2005,289:1-30.
[15]Christopher J.Roberts Therapeutic Protein Aggregation:Mechanisms,Design,and Control[J].Trends Biotechnol,2014,32(7):372-380.
[16]Rosenberg AS.Effects of protein aggregates:an immunologic perspective[J].AAPS J,2006,8:E501-507.
[17]Joubert M K,Hokom M,Eakin C,et al.Highly Aggregated Antibody Therapeutics Can Enhance the in Vitro,Innate and Late-stage T-cell Immune Responses[J].Journal of Biological Chemistry,2012,287(30):25266-25279.