کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5136494 | 1494013 | 2017 | 29 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Identification and characterization of monoclonal antibody fragments cleaved at the complementarity determining region using orthogonal analytical methods
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کلمات کلیدی
TFASEC-HPLCEICsLMWDTTCQACDRmAbCIDm/z - m / zMonoclonal antibody - آنتی بادی مونوکلونالCollision-induced dissociation - اختلاف ناشی از برخوردTrifluoroacetic acid - اسید TrifluoroaceticFormic acid - اسید فرمیکCapillary electrophoresis - الکتروفورزمویرگیCho - برایChinese Hamster Ovary - تخمدان هامستر چینیFragmentation - تقسیم بندیTics - تیکسdithiothreitol - دیتیوتریتولlight chain - زنجیره سبکheavy chain - زنجیره سنگینLiquid chromatography mass spectrometry - طیف سنجی جرمی کروماتوگرافی مایعIAM - من هستمcomplementarity determining region - منطقه تعریف مکملmass to charge ratio - نسبت جرم به شارژlow molecular weight - وزن مولکولی کمCritical quality attribute - ویژگی کیفی بحرانیSize exclusion chromatography - کروماتوگرافی اندازهای طردیextracted ion chromatograms - کروماتوگرافی یونی استخراج شده استtotal ion chromatograms - کل کروماتوگرافی یون کلiodoacetamide - یووداکتامید
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40 °C for 28 days. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and trypsin digestion peptide mapping followed by LC-MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser-Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40 °C for 28 days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP-LC-MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP-LC-MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 1048, 24 March 2017, Pages 121-129
Journal: Journal of Chromatography B - Volume 1048, 24 March 2017, Pages 121-129
نویسندگان
Wenhua Li, Bin Yang, Dongmei Zhou, Jun Xu, Wenjia Li, Wen-Chen Suen,