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Fields & Applications Pharma & Biopharma, Mass Spectrometry

mAb Aggregation: Knowledge Is Power

Monoclonal antibodies (mAbs) are prone to protein aggregation during production, which can impact efficiency and lead to drug safety concerns for patients – high levels of aggregates, for example, can over-stimulate the patient’s immune system. To better understand of how protein aggregation occurs – which in turn could improve bioprocessing efficiency and safety – Brandon Ruotolo, Professor of Chemistry, and his colleagues from the University of Michigan, used ion mobility-mass spectrometry to capture the higher-order structure of mAbs at the millisecond timescale (1). 

To find out more about how the work could benefit biopharmaceutical manufacturing, we spoke with Ruotolo, who also discusses the role of analytical science in biopharma more broadly.

What is mAb aggregation and why is it important for biopharma? 
 

Antibody aggregation involves the self-association of individual mAbs to form larger oligomers. In some cases, these oligomers can be soluble but, in other cases, they can lead to the formation of larger insoluble aggregates. Antibody aggregation is most often produced when mAbs are misfolded or otherwise structurally compromised when exposed to stress (for example, heating), and thus the process is studied extensively in the context of development phase efforts for biotherapeutics and in formulation testing. In general, mAb aggregation is a complicated and poorly understood process. The complexity is derived from the fact that mAbs are large molecules (~150kDa) that can adopt an exceptionally wide array of structures, each of which is capable of producing different aggregate ensembles when studied under different conditions. 

An improved understanding of mAb aggregation would undoubtedly lead to biotherapeutics possessing improved qualities such as efficacy and safety, and could also ultimately lead to new biotherapeutic modalities.  

Why did you use ion mobility-mass spectrometry?
 

Ion mobility-mass spectrometry (IM-MS) is an exceptionally useful technology platform for studying mAbs. Our approach uses IM-MS in a native mode, whereby we attempt to capture native-like structural ensembles and non-covalent assemblies directly from sample solutions for measurements that can simultaneously assess the size, shape, stability, and molecular weight of mAbs on the millisecond timescale. Such higher-order structure (HOS) aspects of mAbs can be studied by other means, but they are typically quite slow and demand large amounts of purified sample. 

Native IM-MS techniques represent a rapid platform capable of producing rich datasets across thousands of unpurified, low-concentration samples in a short amount of time. Antibody aggregation, although starting from purified samples, creates a mixture of HOS states and oligomers present across a range of concentrations within a sample. Our native IM-MS methods characterized the HOS of mAb monomers, dimers, and other oligomers individually, as well as quantified the relative amounts of these different states in solution.  

What were your main findings? 
 

Our collaborative work with Abbvie (1) has revealed that native IM-MS can quantitatively track the effect of heat and pH stress on mAb HOS; different forms of stress lead to different mAb monomer and aggregate structures; mAb complementarity-determining regions (CDRs) significantly influence mAb aggregation; and heating produced highly-overlapped dimer structures, while pH stress produced more elongated (perhaps inverted and overlapped) dimer topologies for the mAb studied in our report.  

What impact could your findings have for the biopharma field? 
 

The native IM-MS methods we discuss could directly impact the way antibody stress studies are conducted when moved to an automated framework. Furthermore, the information we’ve gathered so far on mAb aggregation and oligomer structures will likely be useful in the continued development and discovery of next-generation therapeutics. 

Ultimately, the effectiveness of mAb therapeutics is built primarily upon their selectivity, which is linked to their HOS. Without deep knowledge of biotherapeutic HOS, mAb discovery, development, and manufacturing would be much more challenging than it already is.

What are the biggest discussion points and unmet needs in biopharma characterization today? 
 

There are vast unmet needs in biopharma that are regularly discussed in both peer-reviewed literature and at scientific meetings. These needs span from next-generation materials characterization to comprehensive in vivo analyses of protein function and high-throughput technologies capable of predicting clinical outcomes for an ever-growing list of complex therapeutic modalities. These needs are positioned throughout the pharma pipeline, and are best addressed by the growing list of industry/academic partnerships that bring together scientists throughout measurement science.

What is the single biggest challenge facing biopharmaceuticalanalysis?
 

As there are so many, it’s hard to select just one! I am personally concerned by the challenges surrounding the discovery and development of biotherapeutics (and biosimilars) that target proteins associated with protein misfolding diseases, including Alzheimer’s Disease. Currently, we lack the structurally sensitive screening technologies capable of assessing the polydisperse ensemble of structurally distinct proteoforms that must be prevented from forming cytotoxic oligomeric species through mAb binding. My group believes that native IM-MS can add significantly to this space and are working diligently on solutions.

Are there any cutting-edge advances that are poised to have a big impact over the next five years or so?
 

Beyond the development of high-throughput native IM-MS technologies aimed at solving problems in the pharmaceutical sciences, continuing advances in separation science, structural biology, sensor technologies, and machine learning/artificial intelligence are all poised to revolutionize the way that drugs are discovered. Of these, I am particularly excited by the continual advances in AI, which now offers many enabling tools to evaluate protein HOS, automate data analysis, and even manage entire discovery campaigns.

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  1. DD Vallejo et al., “Ion Mobility–Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates,” Anal Chem, 94, 18, 6745-6753 (2022). DOI: 10.1021/acs.analchem.2c00160.
About the Author
James Strachan

Over the course of my Biomedical Sciences degree it dawned on me that my goal of becoming a scientist didn’t quite mesh with my lack of affinity for lab work. Thinking on my decision to pursue biology rather than English at age 15 – despite an aptitude for the latter – I realized that science writing was a way to combine what I loved with what I was good at.

From there I set out to gather as much freelancing experience as I could, spending 2 years developing scientific content for International Innovation, before completing an MSc in Science Communication. After gaining invaluable experience in supporting the communications efforts of CERN and IN-PART, I joined Texere – where I am focused on producing consistently engaging, cutting-edge and innovative content for our specialist audiences around the world.

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