Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production |
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Authors: | Weiduan Xu Jianmin Chen Glenn Yamasaki John E Murphy Baisong Mei |
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Institution: | (1) Expression Technologies, Global Biological Development, Bayer HealthCare, 800 Dwight Way, Berkeley, CA 94710, USA; |
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Abstract: | Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation
factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal
sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation
can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but
this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop
assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream
product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional
to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII
producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based
ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors
and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment.
To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing
bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation
between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be
rapidly performed, thereby making them effective for in-process monitoring of protein sialylation. |
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