Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production

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 Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) 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 Galbeta1-4 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 Galbeta1-4 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.

Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell‐free system

“…Although liquid chromatography-mass spectrometry methods are available, we used a lectin-based assay system of the type used for doping analysis where lectins bind specifically to the carbohydrate structures present on EPO (Nagano et al, 2005;Storring et al, 1996Storring et al, , 1998Xu, Chen, Yamasaki, Murphy, & Mei, 2010). Although liquid chromatography-mass spectrometry methods are available, we used a lectin-based assay system of the type used for doping analysis where lectins bind specifically to the carbohydrate structures present on EPO (Nagano et al, 2005;Storring et al, 1996Storring et al, , 1998Xu, Chen, Yamasaki, Murphy, & Mei, 2010).…”

“…In recent years, several methods have been developed to identify the presence of glycan moieties attached to the glycoprotein. Although liquid chromatography-mass spectrometry methods are available, we used a lectin-based assay system of the type used for doping analysis where lectins bind specifically to the carbohydrate structures present on EPO (Nagano et al, 2005;Storring et al, 1996Storring et al, , 1998Xu, Chen, Yamasaki, Murphy, & Mei, 2010). The absence and presence of glycan structures are determined by comparing the signal obtained with unglycosylated and glycosylated EPO (Table 3, and Supplementary Figure S3).…”