With the growing adoption of viral vectors in gene therapy and vaccine development, the focus on glycosylation as a critical quality attribute (CQA) is sharper than ever. As regulators tighten expectations around product consistency and safety, laboratories are under pressure to implement workflows that not only capture the complexity of N-glycan structures but do so reliably, quickly, and at scale.
In the Streamlining N-Glycan Analysis: A Complete Workflow from Agilent webinar, presented by Separation Science and Agilent, Product Manager Tom Rice and R&D Scientist Dr. Randall Robinson showcase an end-to-end workflow for N-glycan analysis, highlighting advances in sample prep, separation, and data interpretation that enable fast, reproducible characterization of viral vector glycosylation profiles.
The Importance of Glycosylation in Viral Vector Characterization
“Glycans affect many aspects of biotherapeutic drug performance, including pharmacokinetics, pharmacodynamics, and immunogenicity,” Rice explains. “Whether on AAV capsids or lentiviral envelope proteins, glycosylation influences how vectors behave in the body, from half-life to immune response.”
Terminal sialic acids, for example, play a role in anti-inflammatory response and drug half-life. Meanwhile, high-mannose structures can signal immunogenicity or suggest incomplete protein processing, and non-human glycan motifs such as Neu5Gc or α-Gal are routinely scrutinized for their potential to trigger immune reactions in patients.
Regulatory guidelines, including ICH Q6B, increasingly recognize glycosylation as a CQA, prompting more detailed analytical workflows to evaluate it at various stages of development and production.
Analytical Challenges in Glycan Profiling of Viral Vectors
Analyzing glycosylation in viral vectors presents a complex set of challenges that require precise and adaptable analytical strategies. Different expression systems introduce variability in glycan structures, including potentially immunogenic non-human epitopes. The need to differentiate closely eluting species, such as mono- versus di-sialylated forms, adds to the analytical complexity.
“Glycans such as Neu5Gc or α-Gal are not human-compatible, and their presence can lead to serious immunogenic responses,” notes Rice. “Regulators are particularly focused on structures like Neu5Gc or α-Gal because they’re not found in humans and can be immunogenic.”
These analytical hurdles are compounded by limitations in traditional workflows. “Those older workflows take a couple of days and don’t scale well in QC settings,” says Rice. “They weren’t built for speed or automation.” He explains that such delays can hinder the ability to correlate glycosylation patterns with potency. Moreover, these workflows are often disconnected from other critical assays, such as viral titer testing, used to evaluate consistency in viral vector preparations.
Viral Vector Characterization Workflow: From Glycoprotein to Data in Under an Hour
The experts present a workflow that significantly reduces the sample prep timeline while maintaining the integrity of glycan structures, an important factor for lab efficiency and reproducibility.
According to Rice and Robinson, enzymatic release of N-glycans is achieved in five minutes, and fluorescent labeling, using either InstantPC or traditional 2-AB dyes, can be completed in as little as one minute.
“From a purified glycoprotein to labeled glycans ready for liquid chromatography (LC), you’re looking at about an hour total,” says Rice.
Cleanup is equally efficient, employing vacuum-driven steps rather than ethanol precipitation, and the system accommodates both fluorescence and mass spectrometry detection workflows.
High-Resolution Separation with HILIC for Complex Glycans
Building on the foundation of streamlined sample prep, Robinson discusses Agilent’s AdvancedBio Amide hydrophilic interaction liquid chromatography (HILIC) column, highlighting its design for high-resolution separation of labeled N-glycans and its relevance to routine and advanced viral vector characterization workflows.
Robinson notes that HILIC remains the gold standard for released glycan separation. The column's amid-based stationary phase supports efficient resolution of neutral and charged glycans and performs reliably even on systems with standard 600-bar pressure limits.
“By adjusting the concentration of salt in the mobile phase, we’re able to modulate how well we separate sialylated structures, like S1 and S2 species,” says Robinson. “That kind of fine control really matters when you're working with charged glycans in complex samples.”
“We’ve run over 500 injections with this column and still see excellent retention time stability,” says Robinson. “That kind of reliability is exactly what QC labs need.”
Viral Vector Characterization Tools: Automated Peak Assignment and Expert Interpretation
After separation, the next analytical hurdle in viral vector characterization is accurately identifying glycan species, especially when mass spectrometry (MS) data is limited or unavailable.
To address this, the experts introduce the Glycan Peak Assignment Tool, which streamlines interpretation by using glucose unit (GU) normalization and a web-accessible glycan library.
“This is particularly useful for labs that need to operate efficiently without high-end instrumentation,” says Robinson. Robinson explains that the tool is especially useful in labs without MS, as it enables glycan assignment using established GU values and chromatographic behavior.
The tool also supports method refinement by allowing users to input known reference peaks to improve accuracy over time. Its label-specific compatibility, supporting both InstantPC and 2-AB dyes, adds flexibility for labs with varying protocols, making it a practical extension of the upstream workflow.
Degradation Study Highlights: the Value of Rapid Sample Preparation
To evaluate how sample prep methods affect glycan stability under stress, the Agilent team ran a thermal degradation study using their Gly-X InstantPC system. The goal was to mimic manufacturing and transport conditions where heat can impact glycan structure and reproducibility.
They compared traditional multi-day protocols to the InstantPC workflow, which completes prep in under an hour.
Using HILIC, they saw that traditional prep under thermal stress degraded glycan profiles, altering peak shape and reducing signal intensity. In contrast, InstantPC-labeled glycans retained consistent peaks. “Speed in sample prep isn’t just about throughput,” says Robinson. “It’s about preserving the native glycan profile before degradation or modification can occur.”
The study also assessed sialic acid profiling. Using DMB derivatization and RP-LC, researchers detected Neu5Gc in stressed samples, flagging immunogenicity risk. This reinforced the need to integrate sialic acid quantification in glycan workflows.
Key Findings:
- InstantPC labeling minimized degradation under thermal stress.
- HILIC chromatograms revealed distinct peak shifts.
- RP-LC detection of Neu5Gc flagged immunogenicity risks.
These results demonstrate that faster preparation not only saves time but also enhances analytical reliability. When combined with robust separation and detection, they ensure glycosylation profiles reflect product safety and stability, which are key to regulatory success. “The more accurately we reflect the true glycan state, the stronger our case for product consistency during regulatory review,” advises Rice.
“We really designed this to bridge discovery and GMP,” says Rice. “You can use the same tools for early research and rely on them for release testing.”
From Discovery to GMP: Viral Vector Characterization at Every Stage
As viral vector-based therapeutics move from experimental stages to widespread clinical use, robust analytical tools are essential to ensure their quality and safety. The webinar highlights how well-integrated glycan workflows can support the evolving analytical demands of viral vector development, from research to regulatory submission.
Meet the Experts
Tom Rice is the Bioconsumables Product Manager at Agilent Technologies, coming to Agilent through the acquisition of ProZyme in 2018. Prior to joining Agilent, he held various sales and marketing roles within ProZyme beginning in 2014. Much of Tom’s work, both at ProZyme and Agilent, has been focused on bringing innovative glycobiology solutions to biopharma customers. Tom is based in California and holds a B.S. from Sonoma State University.
Dr. Randall Robinson is a Research and Development Scientist in the Chemistries and Supplies Division at Agilent. Prior to joining Agilent in 2021, Randall completed his PhD in food chemistry with a focus on developing analytical techniques to characterize bioactive oligosaccharides in milk and dairy streams. Since joining Agilent, he has contributed to the design of numerous products utilized for N-glycan analysis, including the HILIC column described in this presentation.