Glycosylation is one of the most consequential quality attributes in biopharmaceutical development. Regulatory bodies scrutinize it closely because it can directly influence a drug's safety, stability, and efficacy, and even minor variations in a glycan profile can have significant implications for how a therapeutic performs in the body.
In this conversation, Revvity scientist James White discusses the LabChip™ GXII Touch™ Glycan Assays, its practical implementation, and why capturing the full glycan picture matters at every stage of biotherapeutic development.
Beyond full and empty capsid characterization
Q: Can you explain why glycosylation is considered a critical quality attribute (CQA), and what can go wrong if it isn't properly monitored?
Glycosylation is classified as a CQA because the specific sugar structures attached to a therapeutic protein directly influence how the drug behaves in the body. These structures can affect the efficacy, safety, and overall performance of monoclonal antibodies and other glycoprotein therapeutics.
Variations in glycan profiles can arise from upstream process parameters, such as cell culture media and cell feeding, or through downstream purification steps. Even subtle changes in glycosylation can significantly impact target binding, pharmacokinetics, and how the drug interacts with the immune system.
If glycosylation is not properly monitored, these changes may go undetected, potentially reducing therapeutic efficacy, altering pharmacokinetics, or increasing the risk of an unwanted immune response. Consistent monitoring and control of glycosylation is therefore essential to help ensure product quality, safety, and performance.
Can you give us a concrete example of how a glycan profile could directly affect a drug's efficacy or a subject’s safety?
A good example is high mannose glycans, such as mannose-5 (Man5). Elevated levels of high mannose glycans have been associated with an increase in the rate at which a therapeutic is cleared from the body and a greater potential for immunogenicity. For these reasons, high mannose is ideally present at low levels. Man5 is one of the glycans we routinely assess with the Glycan Assay.
Sialylated glycans are another important example. Changes in sialylation patterns can also affect the pharmacokinetics and immune response, which in turn may affect how effective each dose is. This is why having the right tools to detect and monitor sialylated glycans is a key consideration when assessing the full glycan profile.
Q: Traditional HPLC and mass spectrometry methods remain widely used for glycan analysis but can take a day or more per analysis. What challenges does that create for bioprocessing teams that need timely glycosylation data during development and manufacturing?
Lengthy analysis time can make it more difficult to process data in real-time or conduct high-throughput experiments in a single day. Standardized sample preparation combined with rapid microfluidic separation means more upstream samples can be analyzed per day. An HPLC experiment may take 10-40 minutes to run, whereas the microfluidic separation occurs in under a minute. This enables design of experiment (DOE) studies for protein expression parameters, purification conditions, and stability assessments to be completed more efficiently.
Q: The Glycan Extended Range Assay introduces a novel lower marker that creates a broader detection window than conventional approaches. Can you walk us through what that means in practice, and why capturing the full glycan picture in a single run is significant for biotherapeutic development?
In the Revvity Glycan Assay, which measures N-linked neutral glycans, a sugar-based lower marker is used to define the detection window. However, mono and di-sialylated glycans often co-elute with this marker, making them difficult to distinguish, and, therefore, undetectable in the analysis. This is a significant limitation because sialylated glycans can play an important role in the stability, activity, and overall performance of therapeutic proteins. Without visibility of these glycans, critical information about a product’s glycosylation profile may be missed.
The Glycan Extended Range Assay addresses this challenge by replacing the traditional sugar-based lower marker with a small molecule marker. This extends the detection window, allowing monosialylated glycans (e.g., A1, A1f) and di-sialylated glycans (e.g., A2, A2f) to be detected within a single run.
Q: The assay has demonstrated detection of low-abundant neutral and sialylated glycans. Why is detecting low-abundance glycan species so important, and what are the consequences of missing them in a quality control setting?
Low-abundance glycan species may be present at only trace levels, but overlooking them can have real consequences in a quality control setting, from undetected shifts in product quality to batch failures and regulatory challenges. Even subtle changes in bioprocessing parameters can alter the glycan profile, making continuous monitoring essential from early development through commercial release.
The LabChip system is capable of detecting glycan species down to 2.5% of total glycan area, while maintaining coefficients of variation (CVs) below 10% at 2.5 ng/µL. This level of sensitivity can help development teams to characterize glycan profiles more comprehensively, identify meaningful shifts between batches, and generate robust data to support to support informed decision-making throughout development.
Q: Less than 1 minute per sample and 96 samples in ~90 minutes is an impressive throughput. How does the assay maintain analytical quality like resolution and sensitivity at that speed?
A common misconception about microfluidics is that it is simply about miniaturizing a conventional analytical workflow, but that is not the full story. When you are operating at nanoliter volumes, miniaturization changes the underlying physics of how these systems operate. Molecules diffuse over shorter distances, reducing transport times and diffusion-related band broadening, heat is dissipated more efficiently, and electric fields can be controlled with high precision. These characteristics enable rapid separations while maintaining high resolution and reproducibility. This means the technology can overcome many limitations associated with conventional-scale systems.
When engineered with high-quality glass substrates and purpose-built gel formulations, speed and analytical performance can be achieved simultaneously.
Q: In practice, biotherapeutic samples rarely have glycans at equal concentrations. How did you design the study to reflect that real-world complexity, and what did the results tell you about the assay's robustness?
To reflect real-world glycan heterogeneity in biotherapeutic samples, the study intentionally incorporated mixed glycan panels with unequal concentrations rather than uniform standards. Specifically, sample plate maps were designed to include different “All Mix” glycan mixture samples where glycans were present atdifferent concentration levels (e.g., 10 ng/µL adjacent to 1 ng/µL) to mimic the wide dynamic range and relative abundance differences typically observed in biologics.
These mixtures were constructed so that low-abundance glycans appeared alongside high-abundance species, often creating challenging conditions such as partially overlapping and shoulder peaks, which are representative of real sample complexity.
Q: Where in the biotherapeutic development journey, from cell line development and process development through to QC release, do you see this assay delivering the most value, and why?
Microfluidic glycan analysis delivers value for clone selection, process development, and comparability studies. This is where sample volume is often scarce and throughput pressure is high, so having an assay that delivers reliable glycan data from minimal sample volumes is particularly valuable. In process development, even subtle shifts in glycosylation can have real downstream consequences for product quality, while in comparability studies, regulators expect robust analytical evidence that the glycan profile remains consistent.
Q: For a lab considering adopting this assay, what does the practical implementation look like? Is significant method development required, or is it largely preconfigured?
The short answer is that, yes, it is straightforward to implement. Quick Guide and Full User Guide protocols are provided for glycan release and labeling, and the separation method is standardized, which means operators can perform the experiments and data analysis without significant method development. Once trained on LabChip operation and familiarized with the procedure, the workflow becomes user ready. The software also adds to its ease of use, with an intuitive data analysis platform available to quantitatively process results and easily display overlays of sample electropherograms.
Q: Where do you see microfluidic capillary electrophoresis-based glycan analysis heading over the next few years, and are there any glycan species or biotherapeutic modalities beyond monoclonal antibodies where you would expect this approach to make an impact?
There is a lot of room for the technology to grow. Beyond monoclonal antibodies, microfluidic capillary electrophoresis has the potential to make a significant impact on the characterization of glycans on more complex therapeutics, including bispecific antibodies, fusion proteins, antibody-drug conjugates, and gene therapy vectors, all of which present their own analytical challenges.
O-linked glycans are another area worth watching. They are also relevant to drug safety and efficacy, yet they have been more difficult to characterize with existing methods. As next-generation chip designs continue to evolve and push the boundaries of resolution, the most compelling capabilities of microfluidic capillary electrophoresis in this space may still be ahead of us.
For research use only. Not for use in diagnostic procedures.
