重组人血管内皮生长因子抑制剂中异天门冬氨酸含量检测

目的: 建立对重组人血管内皮生长因子抑制剂进行异天门冬氨酸(Isoasp)含量检测的方法。方法: 利用ISOQUANT异天门冬氨酸检测试剂盒并结合HPLC的方法检测重组人血管内皮生长因子抑制剂原液和成品中的Isoasp含量,并对该方法进行加标回收率试验、精密度和准确性的验证。结果: 40、25、6.25 pmol · L^-1的3种浓度对照品的加标回收率均在80%~120%的范围内。对1批原液和1批成品的各3个样品分别进行测定,原液和成品中每摩尔蛋白的Isoasp含量平均值分别为(0.066±0.010)、(0.093±0.008)mol,RSD分别为15.2%和8.6%,原液和成品中各1个样品的25 pmol · L^-1对照品的加标回收率分别为90.2%和108.1%。结论: 经方法学验证,本文所建立的方法可作为该产品的Isoasp含量的常规检测方法,也为融合蛋白及单抗类制品的Isoasp含量检测提供借鉴。

Objective: To develop a method to determine the content of isoaspartic acid(Isoasp)in the recombinant human vascular endothelial growth factor inhibitor(VEGF Trap). Methods: The Isoasp content in the stock solution and the product of VEGF Trap was determined by ISOQUANT kit and HPLC method, and the test was verified by standard recovery rate test, precision test and accuracy test. Results: The recovery rates for the reference substances at three concentrations(40, 25 and 6.25 pmol · L^-1)were all in the range of 80%-120%.Two groups of samples taken respectively from a batch of stock solution and a batch of product were separately determined and the average Isoasp contents in per mol VEGF Trap of the stock solution and the product were(0.066±0.010)mol and(0.093± 0.008)mol, with RSDs of 15.2% and 8.6% respectively.The recovery rates for 25 pmol · L^-1 standard substances of a sample respectively from the bulk and the product were 90.2% and 108.1%. Conclusion: The method for determination of Isoasp content in VEGF Trap was successfully developed, which can be used as a routine detection method and provide a reference for detection of Isoasp content in fusion protein and monoclonal antibody products.

[1] TAKATA T, OXFORD JT, BRANDON TR, et al.Deamidation alters the structure and decreases the stability of human lens betaA 3-crystallin[J].Biochemistry, 2007, 46(30):8861
[2] HUANG L, LU J, Wroblewski VJ, et al. In vivo deamidation characterization of monoclonal antibody by LC/MS/MS[J].Anal Chem, 2005, 77(5):1432
[3] TAKATA T, WOODBURY LG, LAMPI KJ.Deamidation alters interactions of beta-crystallins in hetero-oligomers[J].Mol Vis, 2009, 15:241
[4] LIU YD, VAN Enk JZ, FLYNN GC.Human antibody Fc deamidation in vivo[J].Biologicals, 2009, 37(5):313
[5] WRIGHT HT.Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins[J].Crit Rev Biochem Mol Biol, 1991, 26(1):1
[6] CLARKE S.Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair[J].Ageing Res Rev, 2003, 2(3):263
[7] ROBINSON NE, ROBINSON AB.Amide molecular clocks in drosophila proteins:potential regulators of aging and other processes[J].Mech Ageing Dev, 2004, 125(4):259
[8] REISSNER KJ, ASWAD DW.Deamidation and isoaspartate formation in proteins:unwanted alterations or surreptitious signals[J].Cell Mol Life Sci, 2003, 60(7):1281
[9] SHIMIZU T, MATSUOKA Y, SHIRASAWA T.Biological significance of isoaspartate and its repair system[J].Biol Pharm Bull, 2005, 28(9):1590
[10] TREMOLADA G, LATTANZIO R, MAZZOLARi G, et al.The therapeutic potential of VEGF inhibition in diabetic microvascular complications[J].Am J Cardiovasc Drugs, 2007, 7(6):393
[11] STEWART MW, ROSENFELD PJ.Predicted biological activity of intravit real VEGF Trap[J].Br J Ophthalmol, 2008, 92(5):667
[12] ROBINSON NE.Protein deamidation[J].Proc Natl Acad Sci USA, 2002, 99(8):5283
[13] PALEARI R, PAGLIETTI E, MOSCA A, et al.Posttranslational deamidation of proteins:the case of hemoglobin J Sardegna [alpha 50(CD8)His→ Asn→Asp-BULSECO G, FALDU D, et al.Heterogeneity of monoclonal antibodies[J].J Pharm Sci 2008, 97(7):2426
[17] SCHIRCH V, DELLE Fratte S, DI SALVO M.Detection of isoaspartate residues as a posttranslational modification of proteins and peptides[J].Methods Mol Biol, 2002, 194:269
[18] WAKANKAR AA, BORCHARDT RT.Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization[J].J Pharm Sci, 2006, 95(11):2321