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Glycoengineering is growing in importance as a technique to improve antibody therapeutic efficacy, safety, and product quality.
In glycosylation, carbohydrates (sugars) can naturally attach to an antibody, and the pattern of attachment can have a significant impact on the antibody’s therapeutic activity. Approximately 50% of human proteins are naturally glycosylated (1). Although in some cases glycosylation can be a challenge to biologic drug development, certain attachment patterns can have a beneficial effect, such as making the antibody more easily recognizable in vivo. This has led to the field of glycoengineering, where an antibody therapeutic is deliberately glycosylated with a preferred pattern.
Meanwhile, a growing market for glycoproteins makes glycosylation an increasingly important factor in drug development. Glycoform heterogeneity has so far demonstrated that different glycoforms have different biological activity, which means that control over the production process to produce specifically defined glycoforms is also becoming more important. Glycoengineering is motivated by an increased understanding of the functions of individual glycoforms in the pursuit of generating more enhanced, or “bio-better” therapeutics (1).
It is well established that glycosylation plays important roles in the biological functions and therapeutic efficacy of antibodies, Claus Kristensen, CEO of GlycoDisplay, confirms. “For example, immunoglobulin G (IgG), the most common isotype found in humans, carries single N-linked oligosaccharides (N-glycans) at its Fc domain, which helps to maintain the quaternary structure of Fc and the stability of the antibody,” Kristensen says.
Kristensen further explains that the fine structures of N-glycans, such as the status of core fucosylation or terminal sialylation, are critical in defining an antibody’s effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) and anti-inflammatory activities.
“Glycans may vary with different production conditions, so, during manufacturing, the glycan composition becomes an important quality parameter for antibody therapeutics,” Kristensen adds.
Detecting glycosylation patterns is also of crucial concern. The most common analytical methods for detection are based on mass spectrometry (MS). The traditional approach has been to glycoprofile released glycans, which involves a chemical release of the glycans and derivatization before MS analysis. Alternatively, MS analysis of glycans at specific sites in glycoproteins using glycopeptides—released by proteolytic digestion of the antibody—may be performed. “More recently, MS analysis of intact glycoproteins has become possible, and the obstacle of heterogeneity of glycans can be overcome using glycoengineering to make more homogeneous glycans and simplify the analysis,” Kristensen says.
As the biopharmaceutical industry strives to develop more effective biotherapeutics, using a glycoengineering approach may be a key to improving some therapeutics, such as monocloncal antibodies (mAbs), which have become the most common type of biotherapeutic in the market. The most common glycoengineering technique of antibodies involves the removal of what is known as the “core-fucose sugar group” from the antibody glycans, Kristensen highlights.
“Antibodies without the core fucose have 10–50-fold higher ADCC effect than antibodies with fucose, so antibodies without the fucose become more efficient in cancer treatment,” Kristensen points out as an example.
“Antibodies may also be glycoengineered to obtain more homogeneous glycans, which will simplify characterization and analysis and give higher product quality with respect to reduced batch-to-batch variation and more predictable bioassay results,” he adds. “This approach is still in early phases but will expectedly become the next level in antibody glycoengineering.”
Kristensen also explains that the full range of biological activities, which different glycan structures confer to antibodies, is not fully known, making it difficult to ascertain how much of a benefit or hindrance a naturally occurring glycosylated antibody may be in a therapeutic setting. “For example, the degree of sialic acid sugars on antibodies clearly have anti-inflammatory effects, but we do not understand the full range of effects in, for example, intravenous Ig treatment,” he says.
However, what has been shown is that removing fucose on antibodies improves ADCC and, therefore, provides the antibodies with higher potency against cancers. “Different technologies have been employed to reduce core fucose on therapeutic antibodies, these approaches differ in complexity and efficiency. If decided early in development, one may employ engineered cell lines during production cell-line development, and then development timelines are not delayed because of glycoengineering,” Kristensen states. In general, the afucosylated antibodies should be easier to characterize, requiring less effort from analytical and quality control operations, and possibly meaning less cost for the glycoengineered antibody, he adds.
Meanwhile, as the field of glycoengineering moves forward, many different strategies have been explored to modulate the glycosylation of antibodies, Kristensen notes. However, at this time, it seems that the most optimal approach is the stable engineering of glycosylation capacities of the host cells used to produce recombinant glycoprotein therapeutics. “This approach has been applied to produce optimized glycans on therapeutic antibodies, providing improved therapeutic efficacy. Especially with the emergence of the precise gene editing technologies, it is now possible to develop host cell lines with a high degree of stable, custom-designed glycosylation capacities,” Kristensen states. He notes that, in the future, such designed cell lines will provide further refined antibody glycodesigns. This will serve to improve efficacy, safety, and quality features that would have an overall benefit to patients.
1. Creative BioLabs, “Glycoengineering Service,” creative-biolabs.com, accessed June 5, 2020.