Lectin bio‐layer interferometry for assessing product quality of Fc‐ glycosylated immunoglobulin G

Abstract

Glycosylation, as the most prominent posttranslational modification, is recognized as an important quality attribute of monoclonal antibodies affected by various bioprocess parameters and cellular physiology. A method of lectin‐based bio‐layer interferometry (LBLI) to relatively rank galactosylation and fucosylation levels was developed. For this purpose, Fc‐glycosylated immunoglobulin G (IgG) was recombinantly produced with varying bioprocess conditions in 15 L bioreactor and accumulated IgG was harvested. The reliability, the robustness and the applicability of LBLI to different samples has been proven. Data obtained from LC–MS analysis served as reference and were compared to the LBLI results. The introduced method is based on non‐fluidic bio‐layer interferometry (BLI), which becomes recently a standard tool for determining biomolecular interactions in a label‐free, real‐time and high‐throughput manner. For the intended purpose, biotinylated lectins were immobilized on disposable optical fiber streptavidin (SA) biosensor tips. Aleuria aurantia lectin (AAL) was used to detect the core fucose and Ricinus communis agglutinin 120 (RCA120) to determine galactosylation levels. In our case study it could be shown that fucosylation was not affected by variations in glucose feed concentration and cultivation temperature. However, the galactosylation could be correlated with the ratio of mean specific productivity (qP) and ammonium (qNH4+) but was unrelated to the ratio of mean qP and the specific glucose consumption (qgluc). This presented method strengthens the applicability of the BLI platform, which already enables measurement of several product related characteristics, such as product quantity as well as kinetic rates (kd,kon) and affinity constants (kD) analysis.

Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production

“…Process parameters, such as temperature, pH, p O 2 , p CO 2 , osmolality, media, and feed composition can impact the glycan structure. [ 58–60 ] Our data suggest that bioprocess parameters might importantly contribute to the higher sensitivity of fucosylation to Fut8 expression in batch mode than in fed‐batch mode, although this requires further study.…”

Label-free methods for opticalin vitrocharacterization of protein–protein interactions

“… 53 A similar approach was used by Wallner et al to quantify glycosylation of Fc-glycosylated IgGs via immobilized lectins as measure for product quality. 54 Loomis and Steward-Jones et al used BLI to evaluate the antigenicity of their candidate vaccines in structure-based design of Nipha virus vaccines. 55 …”

“…53 A similar approach was used by Wallner et al to quantify glycosylation of Fc-glycosylated IgGs via immobilized lectins as measure for product quality. 54 Loomis and Steward-Jones et al used BLI to evaluate the antigenicity of their candidate vaccines in structure-based design of Nipha virus vaccines. 55 In summary, BLI is widely used for binding affinity and kinetics measurements of protein-protein interactions, and is increasingly applied as a complementary method to SPR.…”

Quantification of glycated IgG in CHO supernatants: A practical approach

“…Generation of the cell line was conducted by applying the Rosa26 bacterial artificial chromosome (BAC) expression strategy to a serum‐free adapted host cell line derived from CHO‐K1 (ATCC CCL‐61). 17 The procedure for cell‐line propagation and the parameters for the fed‐batch process in 15 L scale have previously been described by Wallner et al 18 The experimental setup, including temperature (37°C, switched to 34°C on Day 3) and feed strategy (constant), was kept identical for every three consecutive experiments shown in this article. As batch medium Dynamis AGT (Thermo Fisher) was used.…”