کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7616937 | 1494053 | 2015 | 28 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach
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کلمات کلیدی
AExTICmAbESIMSNSRMIgG2N-ethyl maleimideUPLCCEXTFAACNTris (2-Carboxyethyl) phosphinePQALC–MS - LC-MSMonoclonal antibody - آنتی بادی مونوکلونالAcetonitrile - استونیتریلTrifluoroacetic acid - اسید TrifluoroaceticCho - برایChinese Hamster Ovary - تخمدان هامستر چینیtotal ion current - جریان یونی کلDisulfide - دی سولفیدlight chain - زنجیره سبکheavy chain - زنجیره سنگینTCEP - ساکتsulfhydryl - سولفیدریلMulti-stage mass spectrometry - طیف سنجی جرم چند مرحله ایMass spectrometry - طیف سنجی جرمیselected reaction monitoring - نظارت بر واکنش انتخاب شدهNEM - نهLiquid chromatography/mass spectrometry - کروماتوگرافی مایع / طیف سنج جرمیUltra-performance liquid chromatography - کروماتوگرافی مایع فوق العاده عملکردیhigh-performance liquid chromatography - کروماتوگرافی مایعی کاراHPLC - کروماتوگرافی مایعی کاراAnion-exchange chromatography - کروماتوگرافی مبادله آنیونcation-exchange chromatography - کروماتوگرافی کاتیون مبادلهelectrospray ionization - یونیزاسیون الکترو اسپری
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 997, 1 August 2015, Pages 30-37
Journal: Journal of Chromatography B - Volume 997, 1 August 2015, Pages 30-37
نویسندگان
Yuling Zhang, Robert Bailey, Nancy Nightlinger, Alison Gillespie, Alain Balland, Richard Rogers,