In this recombinant process using supposedly identical insect cell lines, analysis revealed subtle differences in N-glycosylation profiles suspected to be at the origin of variations in later protein-protein interactions.

An infection with a synthetic virus approach was applied to SF9 insect cells, from initially identical sources, one clone being recently ordered and banked from a commercial source while the second clone had been banked, maintained and used in-house for over 10 years.

After identical expression and purification processes where applied, both proteins were compared by SDS PAGE analysis and protein specific ELISA. While expression yields, purity, migration profiles were identical, differences were observed in the ELISA results : the protein expressed from the newly banked cell line was ineffective for antibody interaction.

Even if the general glycosylation profiles are typical for glycoproteins produced in SF9 insect cells, differences are observed

6 different N-glycan types are present on In-house SF9 produced protein and the ELISA tests are positive : Protein-protein interactions occur

3 additional N-glycan types are present in commercial SF9 produced protein and the ELISA tests are negative : Protein-protein interactions are quenched

Fig: MALDI TOF MS Analysis: Comparison of the relative intensities of purified permethylated N-glycans from glycoproteins produced in SF9 cells from two different origins

This result leads to 2 learning for recombinant projects in therapeutic as well as non therapeutic applications

– Commercial insect cell lines are heterologous. With banking and time, the dominant cell type may differ from one clone to the other. There is therefore an interest in testing several insect cell clones in parallel.
– Changes in Glycosylation profiles may impact on protein recognition by antibodies or other protein-protein interactions, or even on protein functionality or stability. Characterizing in detail the glycosylation profile of a candidate of interest increases robustness and result consistency

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