GlycoSenseTM based reaction monitoring kits are being developed to monitor glycoenzyme engineering reactions.
- GlycoSenseTM Sialidase Reaction Monitoring Kit
- GlycoSenseTM Galactosidase Reaction Monitoring Kit
- GlycoSenseTM Sialyltransferase Reaction Monitoring Kit
- GlycoSenseTM Galactosyltransferase Reaction Monitoring Kit
The use of glycan-processing enzymes to engineer glycoproteins with uniform glycosylation would clearly benefit from a convenient method for monitoring glycosylation changes during the reaction. One approach to producing biologics with more-uniform glycosylation is to subject the biologic to treatment with glycoenzymes post-production. In vitro glycoengineering proceeds in two stages. Firstly the glycans are trimmed down to a common core with glycosidases, and then elaborated through the action of glycosyl transferases to achieve a uniform level of complexity. This approach has the potential to overcome much of the variability that naturally arises in cell culture, and enables biopharmaceutical companies to treat glycosylation in a Quality by Design (QbD) strategy, consistent with the recommendations of the FDA.
In order to achieve uniform glycosylation, each enzyme reaction must go to completion, before the next reaction is initiated. However, each glycoenzyme typically exhibits unique kinetic properties (turnover rates, etc), and the same enzyme may display activity differences depending on the structure and accessibility of the glycan substrate. Thus, reaction times can vary from as brief as a few hours to several days. While approximate estimates of the reaction conditions may be formed on the basis of known enzyme properties, actual performance will vary for each glycoprotein and for each glycoform. To achieve consistency requires that the progress of the reactions be monitored at regular time points (Figure 1). Currently, a user follows the protocol recommended by the enzyme distributor, but is unable to routinely confirm that the reaction has gone to completion. Using GlycoSense TM reaction monitoring kits for specific enzymes, a user can take aliquots of the glycoenzyme reaction mixture at various time points and monitor reaction progress using a standard flow cytometer.
Figure 1. Hypothetical reaction time course for the treatment of a glycan with a sialidase, showing the loss of sialic acid (purple, substrate) and the exposure of underlying galactose residues (yellow, product). The graph assumes that the glycan is fully sialylated at time zero (no free terminal Gal). Lectenz® Bio is developing the GlycoSenseTM glycoenzyme reaction monitoring method to monitor this process using multiplexed glycan-specific reagents that simultaneously detect the loss of substrate and the formation of product.