Development and validation of online SPE purification coupled to HILIC-fluorescence-MS analysis for the characterization of N-glycans.

ABSTRACT

N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide (ProcA) labelling and online solid phase extraction (online SPE). N-glycans were enzymatically released, then labeled with ProcA and cleaned up via the online SPE using HILIC chemistry (online HILIC SPE). Two preparation protocols were optimized a short one (1 h 30) and a long one (18 h). Furthermore, the developed approach was compared to RapiFluor-MS (RFMS) kit (from Waters) and to InstantPC kit (from Agilent) which both include a classical HILIC µElution plate SPE purification. Samples were analyzed using HILIC separation coupled to fluorescence and MS detection (HILIC-FLD-MS) with or without the online HILIC SPE. During the validation, repeatability, intermediate precision, stability, response function and injection volume were tested. Human IgG mix (Multigam®) and NIST mAb standard were used as references as their glycoprofiles are well described. A comparison of three batches of a rituximab biosimilar (Truxima®) and one batch of its originator (MabThera®) was also performed. Online HILIC SPE sample cleanup shows a higher sensitivity and repeatability compared to the classical HILIC µElution SPE. Our online HILIC SPE approach also offers the highest MS signal compared to both commercial kits. However, InstantPC shows the highest FLD signal. The analyses of rituximab samples were in line with the literature showing the efficiency of the method for N-glycan monitoring of biotherapeutics. In conclusion, the results demonstrated the usefulness and ease of application of the developed protocol with the online HILIC SPE purification.