(T1430-06-32) High Throughput N-glycan Analysis of Heavily Glycosylated Protein Therapeutics to Support Real-Time %sialylation Monitoring for Upstream Bioreactors

Purpose: Protein based therapeutics are rapidly growing, offering a promising approach to fight against cancer, autoimmune diseases, and other conditions. The N-glycosylation, especially mannosylation and sialylation of proteins and fusion proteins, play crucial roles in maintaining important therapeutic attributes such as thermal stability, pharmacokinetics (PK), and antibody effector functions. However, the bioreactor environment is variable, and the sialylation period changes throughout the upstream process. Therefore, a high throughput method is necessary to maintain consistent and optimal sialylation levels while monitoring glycoprotein production.

Methods: In this work, we utilized the Agilent instant-PC-N-glycan kit ® to carry out the N-glycan analysis of heavily glycosylated proteins. The fluorescence (FLR) detection-based iPC-HILIC method was successfully employed to analyze cell culture fluid (CCF) samples without interference from the process-related impurities.

Results: The results obtained from the CCF samples helped guide immediate decisions to harvest and forward the process during the upstream bioengineering. Interestingly, the %sialylation of both CCF and pro-A purified samples provided a similar trend, indicating no impact on the method performance. Later, a RAMAN model was developed by inputting %sialylation of small-scale reactors as a calibration standard. The partial least squared (PLS) regression model was generated using the calibration data, whereas SIMCA (soft indicated modeling by class analogy) was used for method development and implementation.

Conclusion: This model aims to obtain real-time %sialylation of upstream reactors through an inline RAMAN probe. Furthermore, the iPC-N-glycan study was performed on the desialylated mAbs to understand the efficacy of the Glyko sialidase enzyme in removing the sialylated glycans. Finally, it is worth mentioning that the day-to-day, instrument-to-instrument, and operator-to-operator variability was successfully evaluated for %sialylation of heavily glycosylated proteins by the iPC-N-glycan method.