کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
34321 | 45017 | 2015 | 5 صفحه PDF | دانلود رایگان |
• TNFR-Fc was produced in CHO cell cultures with undesired multiple impurities.
• The product-related impurities of TNFR-Fc were identified as N-terminally deleted fragments and scrambled disulfides.
• The product-related impurities showed greatly reduced TNF-α neutralizing activity.
Because of its large size and structural complexity, preparations of the cysteine-rich tumor necrosis factor receptor-Fc (TNFR-Fc) that are produced using recombinant CHO cells contain many undesirable impurities. In this study, to purify TNFR-Fc, cell culture supernatant was first clarified using a pre-filter and sterilization filter and then subjected to a series of purification steps consisting of protein A affinity column chromatography, anion exchange chromatography, and hydrophobic interaction chromatography (HIC). To characterize the presence of TNFR-Fc-related impurities after the HIC step, the HIC eluates were further fractionated using analytical HIC and then separated by size exclusion chromatography (SEC). Several product-related impurities were detected during SEC, including low molecular weight (LMW) species, high molecular weight aggregates, and species with a size equivalent to authentic TNFR-Fc. N-terminal sequence analysis of the LMW species indicated that N-terminal amino acids had been partially deleted from the protein sequence at amino acid positions 1–185 or 1–223. Peptide mapping analysis followed by quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and MS/MS indicated that the species that was equivalent in size to authentic TNFR-Fc contained scrambled disulfide bonds linked as Cys98-Cys115 or Cys104-Cys112. These product-related impurities, which led to a marked reduction in TNF-α neutralizing activity during cytotoxicity neutralization assays in L929 cells, should be removed from the final product during purification.
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Journal: Process Biochemistry - Volume 50, Issue 8, August 2015, Pages 1313–1317