N-glycosylation is a post-translational modification (PTM) that plays a crucial role in monoclonal antibody (mAb) effector functions and is, therefore, classified as a critical quality attribute (CQA) that requires comprehensive, precise and accurate characterization and monitoring.
N-glycosylation of mAbs (and other therapeutic proteins) has the potential to influence the pharmacodynamics (PD) and pharmacokinetic (PK) behavior, overall therapeutic efficacy, and safety of the product because certain N-glycan species can cause adverse immune reactions. Some glycoforms are capable of inducing such effects even at very low abundances, meaning a highly sensitive assay that can identify and quantify glycan species below 0.1% relative abundance is essential.
Typically, glycosylation in mAbs is isolated to a single site in the Fc-region, however, related products such as fusion proteins often exhibit more elaborate glycan profiles that extend beyond the Fc-region and across multiple N-glycosylation sites. Generally, the species observed at these sites are larger, with increased variation and complexity.
In this webinar, we will discuss our LC-MS approach to N-glycan characterization for a variety of biopharmaceutical products as well as best practices for overcoming some of the most common product-related challenges.
Specifically, the webinar will cover:
- Introduction to glycosylation, typically observed N-glycan species and their importance as CQAs
- Overview of platform LC-MS workflows for ‘first-look’ N-glycan profiling including intact mass, middle-up and peptide mapping analyses
- In-depth review of our current released N-glycan method using RapiFluor-MSTM labelling, with comparison to the traditional 2-AB technique, including:
- An example mAb glycosylation data from biosimilar studies using Covance Biosimilar Analytical Master Files (AMF) and the option of bespoke chromatography for the separation of specific glycan species
- An overview of the supplementary exoglycosidase linkage analysis used to provide additional confidence in N-glycan assignments and differentiate between isobaric species
- Focus on fusion proteins and products with additional N-glycan sites outside of the Fc-region, with:
- Example data from our Aflibercept AMF, highlighting the challenges associated with analyzing more complex N-glycan profiles
- Overview of a specific digestion and separation strategy for assigning N-glycan profiles to discrete regions of the protein
- Further use of peptide map LC-MS analysis to identify site-specific glycan occupancy
James Duffy, Assay Development Scientist, BioPharm CMC, Covance Inc.
James Duffy has worked for Covance for 11 years, with his time split over periods in small molecule LC-MS bioanalysis and large molecule biopharmaceutical CMC solutions. In his current role, as a manager of a team of assay development scientists within the mass spectrometry development team, Duffy specializes in LC-MS glycan analysis. This includes developing workflows for the characterization of N-glycan profiles for a range of mAbs and fusion proteins, as well characterizing O-glycan via middle-up LC-MS analysis. Duffy has also gained further experience in other biopharmaceutical characterization methods such as intact mass, middle-up and peptide mapping analysis during his time at Covance.Message Presenter
Who Should Attend?
Head, Director, CxO, Managing Director, Senior Director, VP, Consultant, Lead Scientist, Staff Scientist of:
- Quality Analytics/ Quality Control
- CMC Analytical
- Manufacturing Control
- Manufacturing Quality
- CMC Program Management
- CMC Leader
- CMC Regulatory Affairs
- CMC Consultant
- CMC Pharmaceutical Analysis
Covance is a business segment of LabCorp, a leading global life sciences company, which provides contract research services to the drug, medical device and diagnostics, crop protection and chemical industries. Employing over 21,000 people worldwide, we are the world’s most comprehensive CRO, dedicated to improving health and improving lives.
Covance is a global leader in nonclinical safety assessment, clinical trial testing and clinical trial management services. Our unique perspectives are based on decades of scientific, medical, and regulatory expertise. We generate more safety and efficacy data to support drug approvals than any other company, supporting our clients’ complete lifecycle management.
Together with our clients, Covance transforms today’s healthcare challenges into tomorrow’s solutions. Visit us at www.covance.com.