INTRODUCTION

Analysis of the asparagine (Asn-) linked oligosaccharide (also known as the N-linked oligosaccharide or N-glycan) composition of selected recombinant therapeutic proteins has become necessary for product characterization or release specifications of these proteins. For information on the characterization and assessment of protein glycosylation, see Glycoprotein and Glycan Analysis—General Considerations 〈1084〉 . Chapter 〈1084〉 covers general analytical strategies and also highlights criteria for selecting appropriate methods for specific analytical challenges, thereby providing the context for this chapter. This chapter focuses on the analysis of N-linked oligosaccharides that are released from glycosylated recombinant therapeutic proteins. Therefore, the chapter provides 1) a generic procedure for enzymatic release of N-glycans using peptide N-glycosidase F (PNGase F), 2) two different approaches for fluorophore labeling of the released N-linked oligosaccharides, and 3) five analytical procedures based on chromatographic and electrophoretic separation, as well as performance criteria for the separation. Alternative analytical strategies are discussed in 〈1084〉 . Although the procedures in this chapter are validated, the procedures must be verified with the individual specific product (see Verification of Compendial Procedures 〈1226〉 ). Furthermore, validation is required when the procedure is optimized for a specific product [e.g., outside the allowed parameter limits defined in Chromatography 〈621〉, System Suitability (see Validation of Compendial Procedures 〈1225〉 )]. Data analysis, quantitation, and lot release specifications, which are expected to be product specific, will be found in individual product monographs; therefore, these aspects are not covered in this chapter.