Techniques for Characterization of mAbs

Monoclonal antibodies (mAbs) are among the fastest growing drug classes with over 100 approved by at least one regulatory agency worldwide.¹ MAbs are successful because they accurately target specific proteins, making them highly effective with mild side effects.

Because mAbs are manufactured in living cells, quality control is more complex and tedious than with traditional small molecule drugs.^2,3 Understanding their physical, chemical, and other critical quality attributes is essential to ensuring the final product is safe and high quality. Impurities, for example, can cause patients to have a harmful immune response.^4,5

Critical quality attributes (CQAs) are defined during product development and monitored during manufacturing to ensure they remain within set limits.^3,5 Defining CQAs often requires state-of-the-art equipment and techniques for analyzing the structure, function, and purity of the mAb.³

Understanding the structure of mAbs

During early drug development, companies want a cell line that consistently produces the right mAb.⁶ To help, they often turn to comprehensive analytical tools, explains Steve Martin, Ph.D., VP of Research Portfolio Delivery at Waters. “They’re typically using high-end mass spectrometry, which has the ability, in high-level detail, to analyze the monoclonal antibody down to single amino acids.”

Mass spectrometry is often used alone, or in combination with reverse-phase high-performance liquid chromatography (LC-MS)⁷ to measure the molecular weight of the intact antibody—to ensure the cells have expressed the right protein. “The first step most people do in antibody characterization would be LC-MS [to see if] their gene sequence aligns with the right protein sequence,” Martin says.

Companies also use LC-MS for peptide mapping.³ By breaking down the antibody into constituent peptides, and comparing against a reference material, companies can tell if the antibody has the expected structure. They can also identify lot-to-lot variations in the mAb’s genetic stability, perhaps due to mistranslations or point mutations in a single amino acid.⁸

Other techniques, such as capillary electrophoresis (CE) are also suitable for mAb structural analysis. But, according to Martin Vollmer, Ph.D., Biopharma Program Manager at Agilent Technologies, although CE is “well known for its superior resolution, it has the [disadvantage] of being less robust and less frequently used.”

Analyzing higher-dimensional structures

An antibody’s DNA and amino acid sequence heavily determine how it folds into a 3D shape. However, how the protein works can be dramatically affected by post-translational enzymatic or chemical changes, or the mis-formation of disulfide bonds.⁷

Glycation, for example, is a nonenzymatic reaction, which typically occurs during manufacturing when antibodies are exposed to sugars—perhaps in cell culture. Glycation can potentially affect product safety and needs to be analyzed during early drug discovery but can be hard to detect as glycans have complex structures.⁹ As Dr. Martin explains, “the challenge with carbohydrates is that [two] can have the same molecular weight, but the linkages are different, and this impacts activity and changes the 3D structure.”

To characterize glycation and some other post-translational modifications, scientists often use high-sensitivity charge-based separation techniques, such as ion mobility spectrometry (IMS). IMS involves suspending molecules in a neutral carrier gas and applying a weak electric field, which causes the ions to move. As two antibodies with different glycation will drift differently within the gas, scientists can detect subtle differences in antibody structure.¹⁰

To analyze the disulfide bonds that provide structural stability to the protein, techniques such as collision-induced dissociation (CID) are commonly used.¹¹ Other aspects of protein structure, such as the random coil shapes that can form when proteins are denatured,¹² as well as β-sheet ribbons and α-helical fibers, are often studied with circular dichroism (CD). Proteins absorb circularly polarized light to a varying extent, depending on their symmetry,¹³ and this can be used to detect the spatial arrangement of amino acids along the protein backbone.

Moving to manufacturing

After the mAb is moved into manufacturing, companies typically turn to simpler characterization techniques for quality control (QC). “When you look at downstream QC, detailed characterization doesn’t need to be done,” says Dr. Martin. Identifying the best cell line, for example, is done during early drug development, and QC is mostly about ensuring no changes occur between product batches.

For these applications, Waters offers a more simplified LC-MS system. According to Dr. Vollmer, this is because economic factors come into play. “The level of pre-existing knowledge, training requirements and service support have a significant impact on timelines and costs,” he says.

Other techniques, such as size-exclusion chromatography, are used for QC to, for example, identify protein aggregation during downstream processing. “Aggregation is not good from a product standpoint as it can induce immune responses,” says Dr. Martin. Size-exclusion chromatography separates molecules based on their physical size as they pass through pores in a chromatography column.^14,2

Functional characterization of mAbs

Measuring the biological activity of a mAb, such as its binding strength to a target molecule, helps companies understand how the drug may work in a patient. Several techniques are commonly used, including fluorescence-based ELISA.

ELISA is convenient and cheap, and variants are commonly used. Cell-based assays help monitor how the mAb may affect cell activity, such as cytokine production, after binding occurs. Other assays typically involve coating a 96-well plate with the target molecule. Candidate mAbs either bind to the target molecule unhindered, or compete with a standard antibody. The competitive approach is typically used in late-stage drug development because it allows for a quantitative estimate of binding strength.⁷

Although ELISA is cheap, it can be cumbersome and time-consuming, and this is where label-free techniques for analyzing the biological activity of mAbs may help. According to Helge Schnerr, Ph.D., Global Campaign Manager at Sartorius, they offer a bio-layer interferometry system that provides real-time automated analysis of mAb behavior and binding activity. He believes this can replace ELISA and high-performance liquid chromatography, but companies need to make an initial capital investment and change their processes to realize its time and cost-savings.

References

1. Lyu, X. et al. (2022) The global landscape of approved antibody therapies Antibody Therapies, Vol. 5, Issue 4. pp.233 – 257. 

2. Ando, E., Fujimura, D., Matsumoto, K. Method Optimisation for the Analysis of Monoclonal Antibodies by Size-Exclusion Chromatography. Shimadzu Technical Report 

3. Characterisation of Therapeutic Monoclonal Antibodies 

4. Bio-Rad. Principles of ion exchange chromatography

5. Hamilton, What are quality control attributes 

6. De Palma, A. (2015) Insights on Streamlining Clone Selection. Lab Manager 

7. Wang, X. et al (2018) Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein & Cell 9 (1) pp 74-85

8. Allen, D. et al (1996) Validation of Peptide Mapping for Protein Identity and Genetic Stability. Biologicals, 24, 255-275

9. Mo, J, et al (2017) Quantitative analysis of glycation and its impact on antigen binding mAbs, vol. 10, issue 3, pp. 406-415.

10. Hofmann, J. and Page, K. (2017) Glycan Analysis by Ion Mobility–Mass Spectrometry. Angewandte Chemie International Edition. Vol. 56, Issue 29, pp. 8342-9349

11. Levels of Protein Structure 

12. Fitzkee, N.C. and Rose, G.D. (2004) Reassessing random-coil statistics in unfolded proteins. Biophysics and computational biology. PNAS, vol.101 (34), pp. 1249—12502

13. Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006; 1(6): 2876–2890.

14. Chakrabarti, A. Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography.