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Avoiding Deadly Aggregates in Bio-therapies: Part 1 of a Monoclonal Antibody Characterization Series

Team TFS
Team TFS
shutterstock_93256651I’ve got a personal interest in Alzheimer’s research because I have family members with the disease, so I’m always looking for breakthroughs and new studies.   I recently read about a Trinity College Dublin study that may indicate the mere presence of protein aggregates may be as important as their form and identity in inducing cell death in brain tissue when it comes to neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The article got me thinking about how complex large bio-molecules are and how important it is for scientists to be able to characterize them.

Because these diseases can affect any one of us, it is amazing to watch the growth in the development of Monoclonal antibody therapeutics. I thought I’d share a series of articles focused on the different characterization challenge that are currently  helping scientist move their discovery and development of these beneficial drugs quickly through the process, and  provide information about the most current HPLC techniques and tools available.  Today’s topic is aggregates, as it is the most common characterization assay. And  over the next weeks I’ll follow with other techniques related to charged variants, oxidation variants, and accurate mass analysis.

What are mAbs?

The body’s first line of defense against infectious diseases are antibodies. Monoclonal antibodies (mAbs) are being investigated (and used) as therapies for many health issues and diseases today. They are used as a form of immunotherapy where specific mAbs are designed and used to bind to target cells or proteins to stimulate the patient’s immune system to attack disease cells or modulate signaling pathways. Scientists are working hard to create mAbs specific to cell targets for various serious diseases such as Hodgkin disease, prostate cancer, asthma, flu, HIV and Ebola to name a few. Clearly, the potential seen for monoclonal antibodies is significant.

What are aggregates and why characterize them?

Monoclonal antibodies have very complex structures. The most common physical degradation pathway is aggregation. Aggregates are clusters of mAb units that form due to two or more mAbs attaching to each other. Studies show that these aggregates present in drug products may result in enhanced desired response, but may also cause severe immunogenic and anaphylactic reactions (it can kill the patient). This is why biopharmaceutical manufacturers are required to develop analytical methods to monitor size heterogeneity and control levels of dimer (2 mAbs stuck together) and higher-order aggregates.

What are the latest techniques and tools to characterize aggregation?

High pressure liquid chromatography (HPLC) is established as an essential tool in the characterization of mAb and other protein bio-therapeutics. Size-exclusion chromatography (SEC) is an ideal choice for characterization of mAb aggregates. SEC separation is based solely on the size of the molecule which does not interact with the stationary phase (HPLC column packing). Molecules of different sizes penetrate the pores of the stationary phase to varying extents. The mAbs and aggregates travel through the media at different speeds depending on their size. Larger molecules do not diffuse deep into the pores and elute first, while smaller entities diffuse deeper in the pores and elute later.

Hydrophobic interaction chromatography (HIC) is a complimentary (orthogonal) method which can be used as a confirmatory tool to SEC for aggregation analysis. The HIC method separates analytes based on hydrophobicity but can often detect changes in biomolecule structure and aggregates.

What is available for aggregation analysis?

The most common technique is still SEC. Aggregate quantitation by SEC can be improved by changes to the mobile phase composition and detection methodology, and there is no doubt that SEC exhibits better precision than most analytical methods. UV absorbance detection is the most commonly used. The use of a diode array detector or comparison or two wavelengths, such as 214nm and 280nm, is a good strategy to improve sensitivity and quantitation of aggregation. mAb aggregates and their fragments can also be determined by coupling SEC and mass spectrometry.


Column Technology for Monoclonal Antibody Analysis

Analysis of Monoclonal Antibodies, Aggregates, and Their Fragments by Size Exclusion Chromatography ...

Classification and Characterization of Therapeutic Antibody Aggregates The Journal of Biological Chemistry, Marisa K Joubert, Q. Luo, Y. Nashed-Samuel, J. Wyoych and L. Narho

Monoclonal antibodies to treat cancer

Biophysical Approaches to the Detection and Characterization of Particles in an Antibody Solution

Orbitrap in Biopharma