Therapeutic Antibody Sialic Acid Analysis Services

Glycosylated proteins, including erythropoietin (EPO), monoclonal antibodies (MAb) and various hormones, constitute a large part of approved therapeutic biological drugs. Sialic acid is a negatively charged monosaccharide and exists as a terminal epitope on many glycans. The two most common sialic acids in biopharmaceuticals are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is found in both human and non-human cells. Neu5Gc is synthesized by all mammalian cells except human cells. Neu5Gc and its homologue Neu5Ac have only one oxygen atom difference, which is enough to make Neu5Gc immunogenic in humans (Figure 1). A common modification of sialic acid is acetylation. O-acetylation can reduce the kinetics of enzyme-catalyzed glycan desialylation, which may affect clinical efficacy. Since the amount and type of sialylation affect the clinical manifestations of therapeutic glycoproteins (serum half-life, immunogenicity, activity, etc.), it is considered a critical quality attribute (CQA). Therefore, characterizing sialylation throughout the product life cycle is a regulatory requirement (ICHQ6B) to monitor the following metrics:

1. Total sialic acid-quantification of Neu5Ac and Neu5Gc (nanmol/mg protein)
2. The amount of Neu5Gc
3. The presence of O-acetylated sialic acid

Fig.1 Structure of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).^{1, 2}

Key Benefits of Our Sialic Acid Analysis Services

The benefits of using Creative Biolabs' glycan profiling analysis scheme and related products for sialic acid analysis are:

• Sialic acid is released from glycoproteins through mild acid hydrolysis and labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) (CreTag DMB kit). The DMB-labeled sialic acid is then separated, and the amount of each sialic acid in the sample can be measured by comparing the response in the sample with the standard curve of each sialic acid.
• Combining several standards for analysis provides a reliable quantitative method. The standards used are:
  1. Qualitative Sialic Acid Reference Plate (SRP), which contains a mixture of sialic acids found in humans and animals. Used as a system suitability standard.
  2. Quantitative Neu5Ac and Neu5Gc standard (traceable to NIST). Use serial dilutions to make a calibration curve to calculate the absolute amount of Neu5Ac and Neu5Gc.
  3. Neu5,9Ac2 standard for relative quantification.
  4. qNMR quantitative sialylated glycopeptide (GPEP) process standard to evaluate the efficiency of release and labeling procedures.
  5. Fetuin glycoprotein standard positive control, which contains protein, Neu5Ac and Neu5Gc.
• Use in-house kits and methods for contract analysis – Creative Biolabs has continuous practical experience in this method and is used with many glycoproteins (EPO, MAb, etc.).

Workflow

Under the condition of maintaining N-acetyl, N-glycolyl and O-acetylation, sialic acid is released from glycoprotein by mild acid hydrolysis. For highly sialylated samples, the typical starting amount is 50 µg glycoprotein, and for low-sialylated glycoproteins, the typical starting amount is 100-500 µg. The DMB-labeled sialic acid was then analyzed and DMB-labeled standard solution for quantification.

With extensive experience in glycan characterization, our team can work on custom solutions to meet the unique requirements of each product. With flexible global capabilities, you can provide services where you need it most. Contact our expert scientists today to discuss how our range of glycan analytical services can improve your unique process.

References

1. Spruit, Cindy M., et al. "N-glycolylneuraminic acid in animal models for human influenza A virus." Viruses 13.5 (2021): 815.
2. under Open Access License CC BY 4.0, without modification.