阿柏西普类似药电荷异构体的药代动力学研究
|
摘要
阿柏西普类似药是一种全人源化的重组Fc融合蛋白,经过protein A柱纯化后分离制备得到3个等电点(pI)逐渐升高的电荷异构体组分,分别为Fr 1、Fr 2和Fr 3;采用成像毛细管等电聚焦电泳、分子排阻色谱、肽图分析、寡糖分析和唾液酸分析进行表征.从Fr 1到Fr 3,pI值升高可能是C端赖氨酸含量逐渐升高和唾液酸Neu5Ac含量依次降低的结果.单次静脉给药SD雄性大鼠进行药代动力学(PK)研究,通过与阿柏西普进行对照,观察不同电荷异构体的PK特性,并初步揭示了机理.结果表明:从Fr 1到Fr 3,组分的AUC_(0-t)和t_(1/2)逐渐降低,清除率增加,推测可能是由于唾液酸Neu5Ac的含量降低造成的,并建议阿柏西普类似药的研制过程中要控制唾液酸Neu5Ac的含量.
Aflibercept biosimilar is a fully humanized recombinant Fc fusion protein. After being purified by protein A column,the protein is isolated into three charge variant fractions with increasing isoelectric point( p I). The three fractions are named as Fr 1,Fr 2 and Fr 3,respectively,and characterized by imaged capillary isoelectric focusing,size exclusion chromatography,peptide mapping analysis,oligosaccharide analysis and sialic acid analysis.From Fr 1 to Fr 3,the increase of pI value may be resulted from a gradual increased content of C-terminal lysine and a decreased content of sialic acid Neu5 Ac. Then,a single intravenous administration to Sprague-Dawley( SD)male rats is performed for the pharmacokinetic( PK) study,in addition,the PK properties of various charge variants are observed and the mechanism is revealed initially by conducting a comparative study with Aflibercept. The results show that,the AUC_(0-t) and t_(1/2) of the fractions gradually decrease,the clearance rate increases from Fr 1 to Fr 3,presumably due to a decreased content of sialic acid Neu5 Ac,thus it is suggested that the content of sialic acid Neu5Ac should be controlled in the development of Aflibercept biosimilar.
Aflibercept biosimilar is a fully humanized recombinant Fc fusion protein. After being purified by protein A column,the protein is isolated into three charge variant fractions with increasing isoelectric point( p I). The three fractions are named as Fr 1,Fr 2 and Fr 3,respectively,and characterized by imaged capillary isoelectric focusing,size exclusion chromatography,peptide mapping analysis,oligosaccharide analysis and sialic acid analysis.From Fr 1 to Fr 3,the increase of pI value may be resulted from a gradual increased content of C-terminal lysine and a decreased content of sialic acid Neu5 Ac. Then,a single intravenous administration to Sprague-Dawley( SD)male rats is performed for the pharmacokinetic( PK) study,in addition,the PK properties of various charge variants are observed and the mechanism is revealed initially by conducting a comparative study with Aflibercept. The results show that,the AUC_(0-t) and t_(1/2) of the fractions gradually decrease,the clearance rate increases from Fr 1 to Fr 3,presumably due to a decreased content of sialic acid Neu5 Ac,thus it is suggested that the content of sialic acid Neu5Ac should be controlled in the development of Aflibercept biosimilar.
引文
[1] ZHU L,GUO Q,GUO H Z,et al. Versatile characterization of glycosylation modification in CTLA-Ig fusion proteins by liquid chromatography-mass spectrometry[J]. MAbs,2014,6(6):1474-1485.
[2] FOSS S,GREWS A,SAND K M,et al. Enhanced FcRn-dependent transepithelial delivery of Ig G by Fe-engineering and polymerization[J]. Contml Release,2016,10(223):42-52.
[3] HOLASH J,DAVIS S,PAPADOPOULOS N,et al.VEGF-Trap:a VEGF blocker with potent anti-tumor effects[J]. Proc Natl Acad Sci US,2002,99(17):11393-11398.
[4] MORE A S,TOPRANI V M,OKBAZGHI S Z,et al.Correlating the impact of well-defined oligosaccharide structures on physical stability profiles of Ig G1-Fc glycoforms[J]. Journal of Pharmaceutical Sciences,2016,105(2):588-601.
[5] PERKINS S L,COLE S W. Ziv-aflibercept(Zaltrap)for the treatment of metastatic colorectal cancer[J]. Ann Pharmacother,2014,48(1):93-98.
[6] HONG G Y,BAZIN-REDUREAU M I,SCHERRMANN J M. Pharmacokinetics and organ distribution of cationized colchicine-specific Ig G and Fab fragments in rat[J]. Pharm Sci,1999,88(1):147-153.
[7] RODWELL J D,ALVAREZ V L,LEE C,et al. Sitespecific covalent modification of monoclonal antibodies:in vitro and in vivo evaluations[J]. Proc Natl Acad Sci USA,1986,83(8):2632-2636.
[8] GANGOPADHYAY A,PETRICK A T,THOMAS P.Modification of antibody isoelectric point affects biodistribution of111-indium-labeledantibody[J]. Nucl Med Biol,1996,23(3):257-261.
[9] GOETZE A M,LIU Y D,ZHANG Z,et al. High-mannose glycans on the Fc region of therapeutic Ig G antibodies increase serum clearance in humans[J]. Glycobiology,2011,21(7):949-959.
[10] LIU L M,GOMATHINAYAGAM S,HAMURO L,et al. The impact of glycosylation on the pharmacokinetics of a TNFR2:Fc fusion protein expressed in glycoengineered pichia pastoris[J]. Pharm Res,2013,30(3):803-812.
[11] 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.
[12] KHAWLI L A,GOSWAMI S,HUTCHINSON R,et al.Charge variants in Ig G1:isolation,characterization,in vitro binding properties and pharmacokinetics in rats[J]. MAbs,2010,2(6):613-624.
[13] LIU H C,BULSECO G G,SUN J. Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody[J]. Immunol Lett,2006,106(2):144-53.
[14] SUEN K F,TURNER M S,GAO F,et al. Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure[J].Protein Expr Purif,2010,71(7):96-102.
[15] BUMBACA D,BOSWELL C A,FIELDER P J,et al.Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics[J]. AAPS J,2012,14(3):554-558.
[2] FOSS S,GREWS A,SAND K M,et al. Enhanced FcRn-dependent transepithelial delivery of Ig G by Fe-engineering and polymerization[J]. Contml Release,2016,10(223):42-52.
[3] HOLASH J,DAVIS S,PAPADOPOULOS N,et al.VEGF-Trap:a VEGF blocker with potent anti-tumor effects[J]. Proc Natl Acad Sci US,2002,99(17):11393-11398.
[4] MORE A S,TOPRANI V M,OKBAZGHI S Z,et al.Correlating the impact of well-defined oligosaccharide structures on physical stability profiles of Ig G1-Fc glycoforms[J]. Journal of Pharmaceutical Sciences,2016,105(2):588-601.
[5] PERKINS S L,COLE S W. Ziv-aflibercept(Zaltrap)for the treatment of metastatic colorectal cancer[J]. Ann Pharmacother,2014,48(1):93-98.
[6] HONG G Y,BAZIN-REDUREAU M I,SCHERRMANN J M. Pharmacokinetics and organ distribution of cationized colchicine-specific Ig G and Fab fragments in rat[J]. Pharm Sci,1999,88(1):147-153.
[7] RODWELL J D,ALVAREZ V L,LEE C,et al. Sitespecific covalent modification of monoclonal antibodies:in vitro and in vivo evaluations[J]. Proc Natl Acad Sci USA,1986,83(8):2632-2636.
[8] GANGOPADHYAY A,PETRICK A T,THOMAS P.Modification of antibody isoelectric point affects biodistribution of111-indium-labeledantibody[J]. Nucl Med Biol,1996,23(3):257-261.
[9] GOETZE A M,LIU Y D,ZHANG Z,et al. High-mannose glycans on the Fc region of therapeutic Ig G antibodies increase serum clearance in humans[J]. Glycobiology,2011,21(7):949-959.
[10] LIU L M,GOMATHINAYAGAM S,HAMURO L,et al. The impact of glycosylation on the pharmacokinetics of a TNFR2:Fc fusion protein expressed in glycoengineered pichia pastoris[J]. Pharm Res,2013,30(3):803-812.
[11] 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.
[12] KHAWLI L A,GOSWAMI S,HUTCHINSON R,et al.Charge variants in Ig G1:isolation,characterization,in vitro binding properties and pharmacokinetics in rats[J]. MAbs,2010,2(6):613-624.
[13] LIU H C,BULSECO G G,SUN J. Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody[J]. Immunol Lett,2006,106(2):144-53.
[14] SUEN K F,TURNER M S,GAO F,et al. Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure[J].Protein Expr Purif,2010,71(7):96-102.
[15] BUMBACA D,BOSWELL C A,FIELDER P J,et al.Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics[J]. AAPS J,2012,14(3):554-558.