Quite the character

As biosimilars and biobetters cut in on the traditional biotherapeutics model, one thing is very clear says Kim Colson – the ability to quickly and accurately characterise monoclonal antibodies will be vital, which is where NMR comes in...

It has taken around ten years for biotherapeutics, and monoclonal antibodies (mAbs) in particular to move from ‘early promise’ to become an established clinical option for many diseases. Back in 2006, just four biologic license applications were approved by the FDA; however, by 2015 this figure had grown fivefold with twenty FDA approvals granted to biological drugs.

Today, the market has moved on again, with much talk focused on biosimilars (the generic alternatives to the innovative early biotherapeutics) and biobetters. However, innovative products are still breaking new ground – with major players (including AbbVie and Roche) announcing that they will continue to innovate rather than supplement any loss of market share from biosimilars by making their own. mAb-based therapeutics is one area of biopharmaceutical research that is very popular. ‘First generation’ mAb-based therapeutics have already proven themselves as exceptionally valuable treatment options in the fields of gastroenterology, rheumatology, oncology, hematology and transplantation, and now there are newer mAb-based therapeutics currently in clinical trials.

Today, the market has moved on again, with much talk focused on biosimilars (the generic alternatives to the innovative early biotherapeutics) and biobetters

The continued interest in mAb-based therapeutics has mainly been driven by our improved understanding of diseases at a molecular level, and the advances in technology to generate the molecules for study. Additionally, as mAbs can be engineered to recognise almost any antigen they are highly specific. Furthermore, mAbs are generally well-tolerated, and the risk of unexpected safety issues in human clinical trials is considerably lower than with other pharmaceutical therapeutics. Despite their success, the development and manufacture of mAbs is a lengthy and complex process. Researchers are always searching for improvements in data content and quality as they move potential drugs through R&D and into trial and commercial production. After all, mAbs are complex biological macromolecules with multiple post-translational modifications, several potential function domains, and size and charge variants.

Accurate characterisation remains crucial, early in the development process and in production. Analysis can be a challenge however, because the higher order structure of a mAb is very closely related to its function. Correct folding is critical for drug efficacy, and incorrect folding can impact safety by causing unwanted immune or off-target responses. As a result, one of the critical steps involved in the analysis of a mAb is the successful characterisation of the 3D structure. As mAbs are heterogeneous, several analytical techniques are required for full characterisation, and the main challenge that arises is selecting an approach that will provide robust and reliable results.

To characterise mAbs, multiple analytical techniques are used, the most common being size exclusion chromatography, ion-exchange chromatography, and reversed-phased liquid chromatography. In comparison, methods such as nuclear magnetic resonance (NMR) spectroscopy were originally deemed largely unfeasible due to the size of protein molecules and the need for isotope labelling, despite the technique arguably being the most direct method of characterising the higher-order structure of proteins, whilst being non-invasive and non-destructive.

Correct folding is critical for drug efficacy, and incorrect folding can impact safety by causing unwanted immune or off-target responses.

In recent years, NMR has proven itself as a valuable tool in profiling and mapping complex diseases and this has, in turn, provided the driving force for a number of advances in NMR, which are now opening up new possibilities for the technique in mAb characterisation. These advances in 2D-NMR spectroscopy have included improvements in cryogenic probe technologies which allow the analytical technique to be completed at natural isotopic abundance – greatly enhancing the sensitivity of NMR. Also, a greater accessibility of high spectrometer field strengths along with pulse sequence development has enabled the technique to be more quantitative and faster.

[caption id="attachment_56837" align="alignnone" width="450"] NMR allows for detailed analysis of biotherapeutics.[/caption]

Following the advances within the field of NMR, a number of studies have been conducted, which have investigated 2D-NMR techniques for antibody characterisation. Recent research, conducted by Dr Luke Arbogast and colleagues at the University of Maryland’s Institute for Bioscience and Biotechnology Research, demonstrated the success of recent advances in 2D-NMR using methyl residues as a way to “fingerprint” the structure of a candidate mAb. By taking advantage of methyl resonances, the researchers were able to take advantage of the natural abundance of the 13C isotope, eliminating isotopic labelling. Using advanced spectrometers and cryogenic probe technologies allowed researchers to achieve resolution that was better than expected and analyse spectrum peaks. Furthermore, researchers were then able to reduce the experimental time down by more than half by using the mAb, enzymatically cleaved into its Fab and Fc domains and new sampling techniques.

With the global biopharmaceuticals market forecast to reach a total of $497.9 (USD) billion by 2020, and most large pharmaceutical companies now devoting around 40% or more of their R&D budget to biopharmaceuticals, it is clear that research in this field in not slowing down any time soon. So, it is an easy prediction to say that the importance of analysis will continue to grow. In addition to the developments in NMR, I imagine that researchers will start to look at alternative techniques that can be utilised, either alone or in combination, to further improve characterisation and streamline workflow and reduce time to market.

Author: Kimberly L. Colson PhD, is Business Development Manager for Bruker BioSpin