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  1. Regulatory Science in Action

A novel method for rapid glycan profiling of therapeutic monoclonal antibodies

CDER researchers have made use of lectins as probes to devise a method for analyzing a critical quality attribute of antibody-based therapeutics.

Background and regulatory challenge

Monoclonal antibodies (mAbs) and antibody-based therapeutics have become integral to modern medicine, and account for over 160 approved new molecular entities since the first approval of muromonab-CD3 (trade name Orthoclone OKT3) in 1986. These include monoclonal antibodies, antibody-drug conjugates, and other bioengineered antibody forms. Although about half of these products are used to treat cancer, they also play crucial roles in treating other conditions, including immune-mediated disorders and viral infections such as Ebola and HIV. The majority of therapeutic mAbs belong to the IgG class, as shown in Figure 1. They consist of four protein subunits, forming a “Y” shaped molecule with pairs of identical heavy and light chains. The specificity of antibody binding to a target molecule, or antigen1, originates from the distinct combination of amino acids found in the variable regions of the heavy and light chains at the ends of the Y arms. These amino acids create a binding site that attaches to a specific structural domain, known as an epitope, on an antigen. 

 

Figure 1: The basic structure of an antibody is shown, with two identical heavy chains (blue) and two identical light chains (brown) forming a Y-shaped structure.

Figure 1. The basic structure of an antibody is shown, with two identical heavy chains (blue) and two identical light chains (brown) forming a Y-shaped structure. The variable domains of the Fab region are responsible for the antibody’s highly specific affinity for an epitope on an antigen’s surface. The Fc region of the antibody interacts with various kinds of immune cells to mediate immune responses. Glycans formed by the addition of diverse carbohydrate molecules (colored geometric shapes) on each heavy chain influence Fc-mediated processes like antibody-dependent cell-mediated cytotoxicity and other immune effector mechanisms, thus serving as critical quality attributes in evaluating antibody-based therapeutics.

Antibodies undergo extensive modifications in a cellular process called glycosylation. This process initiates at a specific amino acid on the Fc portion of each heavy chain, where multiple carbohydrate molecules are sequentially added to form branched structures known as glycans. Glycans are critical quality attributes of antibody therapeutics because they can affect the complex interactions between antibodies and receptors on various immune cells in the body, ultimately influencing functions such as antibody-dependent cell killing and the patient’s immune responses. Moreover, novel glycans that may arise in bioengineered antibody therapeutics have the potential to trigger adverse immune reactions in patients.

The scientific challenge

The glycosylation profile of a particular antibody-based therapeutic may vary due to several factors. These include the genetic sequences used to express the protein in cells, the conditions present during large-scale cell culture (such as nutrient levels, pH, oxygen availability, and cell density), and the purification methods employed. Therefore, it is crucial to continually monitor and analyze   glycosylation patterns across different batches of mAbs to ensure consistent product quality and optimize therapeutic effectiveness. One challenge in this ongoing monitoring process has been the complexity of current analytic methods used to characterize the glycan constituents. Many of these methods involve removing the glycan from the protein backbone and then utilizing a combination of various separation techniques and mass spectrometry to identify the carbohydrate molecules based on their precise molecular masses. 

A new way to assess the glycosylation of antibody therapeutics

To address the need for a simple, rapid, and high-throughput method for analyzing glycosylation patterns in antibody-based therapeutics, CDER researchers have recently explored the use of lectins to detect glycan epitopes in therapeutic antibodies. Lectins are a diverse class of naturally occurring or bioengineered proteins that share a key property with antibodies, i.e., an affinity for specific structures. In the case of lectins, these structures are typically found on the surface or terminus of glycans, which, borrowing from the term used in antibody-antigen interactions, are also referred to as epitopes. 

The investigators first analyzed the data in applications submitted to the FDA’s Electronic Common Technical Document (eCTD) system for more than 150 FDA-approved therapeutic antibody products from December 1994 to May 2023. These antibodies were produced through diverse mammalian cell expression systems. They identified nine universal glycan epitopes present across all therapeutic mAbs. Following this, they immobilized 74 different lectins - each exhibiting distinct binding affinities to various glycan epitopes - on glass chips. These lectins were then incubated with fluorescently labeled mAbs, for which there was prior knowledge about their glycan structures.  By measuring the binding of various fluorescently labeled glycoproteins, the investigators identified nine different lectins, each selectively binding to one of the nine common epitopes. They constructed a microarray of these nine lectins (see Figure 2) and validated the selectivity of each one for a particular glycan by conducting binding experiments with commercial antibodies possessing well-documented glycosylation profiles, as well as two non-glycosylated therapeutic proteins. Furthermore, the specific binding of each of the nine lectins was confirmed by using enzymes (glycosidases) to remove particular carbohydrate groups from the glycans and assessing whether changes in binding were consistent with the removed epitopes. Additional experiments with an innovator antibody and its three biosimilars confirmed the ability of the array to detect different glycosylation patterns that were consistent with proprietary data about these products. 

Figure 2

Figure 2. The method developed by CDER researchers to rapidly characterize glycosylation patterns during the production of mAbs relies on lectins, a class of proteins that bind to various glycan structures within mAbs. Through CDER’s research, nine lectins were identified, each specifically binding to one of nine common epitopes found on mAb therapeutics. These lectins were then immobilized on a glass chip. Glycosylated antibodies sampled are labelled with a fluorescent molecule, Cy3. Incubating them on the nine-lectin chip and measuring fluorescent intensity of the well containing each lectin with a fluorometer provides quantitative information about the specific glycan epitopes present on the antibody.

The glycan epitopes that the CDER-developed tool can analyze were found in all FDA-approved mAb products. Hence, the CDER method for assessing glycosylation of antibody-based therapeutics may have broad applicability to new therapeutic products. A significant advantage of the lectin microarray over traditional methods is its capacity to analyze intact glycoprotein samples, eliminating the imprecision linked to conventional methods that require glycans to be released from the protein structure. Overall, the procedure is straightforward, offers high throughput capacity, and allows for comparative testing of numerous samples within a short timeframe.

How can this work advance drug development and evaluation?

Glycosylation serves as a critical quality attribute of therapeutic antibodies, demanding thorough analysis and precise control at every stage of development and throughout the product’s lifecycle. The lectin microarray for glycan profiling, developed by CDER researchers, presents a solution characterized by simplicity, speed, and high-throughput capacity. As a result, it holds the potential to enhance the development and manufacturing processes of these increasingly indispensable therapeutics.

[1] The National Cancer Institute defines an antigen substance that causes the body to make an immune response against that substance. Antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body. Body tissues and cells, including cancer cells, also have antigens on them that can cause an immune response.

References

  1. Shen Luo & Baolin Zhang (2024) Benchmark Glycan Profile of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems. Pharmaceutical Research 41, 29–37.
  2. Shen Luo & Baolin Zhang (2024) A tailored lectin microarray for rapid glycan profiling of therapeutic monoclonal antibodies. MABS 16 (1), 2304268. 
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