Discovery, characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells

MAbs
Chris SpahrStone D-H Shi

Abstract

Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequ...Continue Reading

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Methods Mentioned

BETA
glycosylation
size-exclusion chromatography

Software Mentioned

Xtract
Amgen
PEAKS
Thermo
Agilent MassHunter
SignalP
MassAnalyzer
Mascot
Xcalibur

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