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Purification of TNFR-Fc produced in recombinant CHO cells: Characterization of product-related impurities

Paper ID Volume ID Publish Year Pages File Format Full-Text
34321 45017 2015 5 PDF Available
Title
Purification of TNFR-Fc produced in recombinant CHO cells: Characterization of product-related impurities
Abstract

•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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Keywords
CHO cells; TNFR-Fc; Product-related impurity; Disulfide bond
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Purification of TNFR-Fc produced in recombinant CHO cells: Characterization of product-related impurities
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 8, August 2015, Pages 1313–1317
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us