Identifying NGNA Sialylation in Antibodies with NGS

August 5, 2026
Craig Blyth

When developing therapeutic antibodies, even subtle changes in cell line behavior can alter product characteristics. One such change is the unexpected presence of N‑glycolylneuraminic acid (NGNA) instead of the human-compatible N‑acetylneuraminic acid (NANA) in antibody glycosylation. Since NGNA is not naturally produced in humans, its presence raises concerns about product quality and suitability for therapeutic use.

Understanding the cause of these glycosylation changes requires more than detecting that a change has occurred. Traditional analytical methods can reveal what has changed, but not why. Next-generation sequencing (NGS), combined with genomic and transcriptomic analysis, enables deeper investigation into the molecular mechanisms underlying such deviations.

In this article, we show how researchers at Boehringer Ingelheim used NGS, supported by Genedata Selector^®, to identify the molecular cause of abnormal NGNA sialylation in Chinese hamster ovary (CHO) cell–derived antibodies.¹

What is NANA?

NANA is the most common form of sialic acid found in humans.² It typically appears at the terminal positions of glycan structures on glycoproteins, including therapeutic antibodies.

These terminal residues influence key aspects of antibody structure and function. In biopharma development, NANA is considered the human-compatible form of sialic acid because it reflects typical human glycosylation patterns.

Maintaining NANA-based glycosylation affects therapeutic properties such as circulation half-life and receptor interactions. For these reasons, NANA glycosylation is considered a critical quality attribute in biologics development and manufacturing. ³

Unexpected NGNA Sialylation in CHO Cell-Derived Antibodies

Successful cell line development requires routine screening to ensure consistent production of the desired therapeutic product. Because these biological molecules are intended for clinical use, it is essential to detect any changes that could affect their function, quality, or consistency.

During routine screening at Boehringer Ingelheim, Fischer et al. identified an unusual characteristic in monoclonal antibodies produced by one of their proprietary CHO cell lines. ¹ Antibodies from this clone exhibited elevated NGNA sialylation instead of the expected NANA pattern.

Because NGNA is not naturally produced in humans, its presence prompted further investigation into the underlying cause. Using NGS-based analyses, the team identified a novel microRNA (miR-111) and a promoter point mutation in the SDK1 (sidekick cell adhesion molecule 1) gene, hosting the miR-111. This created a binding site for the repressor HINF-P (histone H4 transcription factor), silencing both SDK1 and miR-111.

The loss of miR-111 led to increased expression of cytidine monophospho‑N‑acetylneuraminic acid hydroxylase (CMAH), an enzyme that converts NANA into NGNA, resulting in elevated NGNA incorporation into the antibody product.

Investigating the Molecular Cause of NGNA Sialylation

To understand why certain CHO‑derived antibodies exhibited NGNA instead of the expected NANA pattern, the researchers conducted a systematic investigation combining genomic and transcriptomic analysis. The aim was to identify the molecular differences underlying the observed glycosylation shift.

Using NGS-based analyses, the team generated mutation profiles and gene expression data for the affected and reference cell lines. This enabled the identification of sequence variation and transcriptional changes associated with the abnormal glycosylation pattern.

Experimental Validation Using CRISPR Gene Editing

To confirm the role of CMAH in driving NGNA sialylation, the researchers applied targeted clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing. Knocking out the CMAH gene in the affected CHO cell line eliminated NGNA incorporation, confirming its role in the altered sialylation pattern.

In parallel, reintroducing miR-111 into high-NGNA cell line suppressed CMAH expression and reduced NGNA levels. The results demonstrated that the loss of miR-111 was a key upstream driver of CMAH activation and the resulting glycosylation shift.

Together, these experiments provided functional validation of the sequencing-derived findings, linking the identified mutation to the observed phenotype.

A cascade of events leading to an unusual protein product characteristic triggered by a single-point mutation

The Role of NGS in Biologic Troubleshooting

In cases like this, where subtle regulatory changes drive significant shifts in product characteristics, identifying the root cause requires more than conventional analytical methods. NGS and genomic analysis provide the resolution needed to uncover the underlying genetic and regulatory mechanisms.

By integrating genomic, transcriptomic, and regulatory data, NGS enables a comprehensive understanding of production cell lines. This allows researchers to identify mutations, regulatory changes, and gene expression differences linked to observed phenotypes, supporting a clearer connection between genetic variation and product characteristics.

In Fischer et all's study, this approach enabled the identification of a mutation-driven regulatory mechanism linking miR-111 downregulation to CMAH expression and abnormal NGNA sialylation.

How Genedata Selector Supports NGS Analysis of CHO Cell Lines

In the study, identifying the root cause of the NGNA sialylation shift required integrating sequencing data across multiple layers of analysis. Genedata Selector supported this process by enabling the systematic analysis and comparison of genomic and transcriptomic datasets derived from the production cell lines.

The solution was used to generate mutation profiles and analyse gene expression patterns, helping researchers identify regulatory changes associated with the observed phenotype. The analysis revealed a single-point mutation affecting a regulatory binding site, which contributed to the downregulation of miR-111 and subsequent upregulation of CMAH expression. These regulatory changes provided a mechanistic explanation for the increased NGNA sialylation observed in the affected clone.

By integrating these data within a unified analytical environment, Genedata Selector helped researchers interpret complex datasets and connect genetic variation to changes in product characteristics.

Conclusion

Controlling glycosylation patterns is critical for ensuring the safety, efficacy, and consistency of therapeutic antibodies. As this example shows, even subtle genetic changes can lead to shifts in product characteristics, such as the unexpected presence of NGNA instead of the human‑compatible NANA.

By combining NGS with genomic analysis, researchers can identify the underlying molecular causes of these deviations. In this case, this approach enabled the identification of a mutation-driven regulatory mechanism linking miRNA-111 downregulation to CMAH expression and abnormal NGNA sialylation. Such insights support a more detailed understanding of production cell lines and help explain unexpected changes in product characteristics.

As biologics pipelines continue to increase in complexity, approaches that support efficient interpretation of genomic and regulatory data will play an increasingly important role in maintaining product quality and supporting informed decision‑making across development and manufacturing.

To find out more, read the publication,¹watch the talk by author Simon Fischer (PhD), or read our case study.

FAQs

What is NGNA and why is it a concern in therapeutic antibodies?
How does NGNA differ from NANA?
Why is it difficult to Identify the root cause of abnormal glycosylation?
How does NGS help in biologics troubleshooting?
Why is controlling glycosylation important in biologics development?
What is developability?
Why is developability important for therapeutic antibodies?

N-glycolylneuraminic acid (NGNA) is a form of sialic acid that is not naturally produced in humans. Its presence on therapeutic antibodies may raise concerns regarding immunogenicity and product consistency. As a result, NGNA is considered an undesirable modification in biologics development.

N-acetylneuraminic acid (NANA) is the predominant form of sialic acid in humans and is typically found on glycoproteins, including therapeutic antibodies. Unlike NGNA, NANA is considered human-compatible and supports normal biological function. The substitution of NANA with NGNA represents a deviation from expected sialylation patterns and may affect product quality.

Traditional analytical methods can detect changes in glycosylation profiles but often do not provide insight into the underlying molecular mechanisms. Root cause identification requires understanding genetic, regulatory, and expression-level changes within the production cell line, which are not accessible through standard analytical approaches alone.

Next-generation sequencing (NGS) enables comprehensive analysis of genetic and regulatory elements within production cell lines. By integrating genomic, transcriptomic, and regulatory data, it helps identify mutations, gene expression changes, and regulatory disruptions associated with unexpected product characteristics.

Glycosylation affects key properties of therapeutic antibodies, including stability, efficacy, pharmacokinetics, and immunogenicity. Maintaining consistent and human-compatible glycosylation patterns is essential for ensuring product safety, quality, and regulatory compliance.

Developability assesses whether a biologic candidate can be reliably manufactured with consistent quality, focusing on attributes such as stability, glycosylation, and expression.

It helps identify risks early—such as abnormal glycosylation or instability—reducing late-stage failures and ensuring product quality and consistency.

References

Fischer, S., Mathias, S., Stadermann, A., et al. (2022). Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N‑glycolylneuraminic acid sialylation on monoclonal antibodies (Biotechnology and Bioengineering).https://pubmed.ncbi.nlm.nih.gov/34935124/ [ncbi.nlm.nih.gov]
Elsevier. (n.d.). N‑acetylneuraminic acid (topic overview). https://www.sciencedirect.com/topics/medicine-and-dentistry/n-acetylneuraminic-acid
American Pharmaceutical Review. Glycosylation: A Critical Quality Attribute for Biopharmaceutical Products. December 1, 2016. https://www.americanpharmaceuticalreview.com/Featured-Articles/331626-Glycosylation-A-Critical-Quality-Attribute-for-Biopharmaceutical-Products/