Specialist Snapshot: Biopharmaceutical Analysis

The Experts

Anurag S Rathore

Anurag is familiar with both academic and industry perspectives in biopharma characterization. Today, he is Professor in the Department of Chemical Engineering at the Indian Institute of Technology in New Delhi, but he has previously held roles at Amgen and Pharmacia Corp. His main areas of interest include process development, scale-up, technology transfer, process validation, biosimilars, continuous processing, process analytical technology and quality by design.

Koen Sandra

Koen is currently the Scientific Director of the Research Institute for Chromatography (RIC). He is also the co-founder and co-owner of anaRIC  biologics, a company that offers a complete range of analytical solutions for characterization, quality control, release and stability testing of biological drugs As a non-academic scientist, he is the author of over 40 highly cited scientific papers and has presented his work at numerous conferences as an invited speaker.

Hermann Wätzig

Hermann has spent his career in academia and is today Professor at the Technische Universitat Braunschweig in Germany. Since 2001, he has been the chair of the pharmaceutical analysis/quality control division of the German Pharmaceutical Society. He is a scientific committee member of Germany’s Federal Institute for Drugs and Medical Devices (BfArM) and an expert of the European Pharmacopoeia.

Why is deep biopharma characterization so important for the discovery, development, and manufacture of new biologic drugs?

Anurag Rathore: The importance, as well as significance, of characterization for biopharma arises from the complexity of the product. Biotherapeutics are complex nano-machines, designed to work at a specific rate, for a specific function. This specificity can only be assured if all the parts of the nano-machines are intact and aligned accurately. For this, it is important to first understand how different stresses impact the assembly. Moreover, as it is a product used in bulk (millions of molecules per dose), the range of contaminants and their effect on product function will vary.

Characterization helps define all of the above features in minute detail – and this understanding can then be used in all aspects of development and manufacturing as a signature of the molecule’s behavior. In the drug discovery phase, anomalies identified during characterization of a biotherapeutic for a certain target might also help identify treatments for other disorders. Characterization, to some extent, also helps understand and manage the risk involved with manufacturing, and can help alleviate the cost attached to clinical trials. In my opinion, there are very few industries where quality of the product matters so much to the consumers. Ultimately, regulation of this quality comes down to efficient and accurate characterization.

Koen Sandra: Anurag summed that up very nicely. Biopharmaceutical products come with enormous structural complexity. The molecules are large (monoclonal antibodies have a molecular weight of 150,000 Da) and heterogeneous as a result of the biosynthetic processes, subsequent manufacturing steps and final storage. Despite the fact that typically only one product is cloned, the final drug substance or drug product is composed of a mixture of hundreds of variants that differ in post-translational modifications and higher order structure. These different variants can have an impact on function, stability, and efficacy, as well as safety. During development, these characteristics need to be determined in great detail using state-of-the-art methodologies and closely monitored prior to clinical or commercial release. For that, a wide range of analytical techniques and methodologies must be used.

What analytical advances have had the biggest impact in terms of developing biologics?

AR: The field of analytical characterization of biotherapeutics has definitely seen major developments in the last decade; there are two significant advances I would highlight. The first is mass spectrometry (MS). When hyphenated with separation tools such as electrophoresis and chromatography, MS has made it possible to probe the molecular structure of complex biomolecules in previously uncharted ways. Combinations such as LC-MS-MS (liquid chromatography-tandem mass spectrometry) allow us to accurately identify the mass of a molecule to the fifth decimal place and pin-point not only the type but also the exact location of a range of chemical and enzymatic modifications. Even modifications as complex as glycosylation are now being increasingly profiled using characterization tools. If there is a modification that can be separated via a specific mode of chromatography, it can be identified by MS.

The second set of tools that are becoming increasingly promising are surface plasmon resonance (SPR) and biolayer interferometry (BLI). These tools have made it easier to perform binding assays and have significantly boosted productivity. They are gradually becoming the industry gold standard for measuring drug specificity and kinetics. 

KS: The enormous advancements in MS and chromatography have had the biggest impact. New mass analysers have been introduced with improved robustness, sensitivity, resolution and mass accuracy, along with powerful software tools to mine all the data. Next to primary structural features such as amino acid sequence and post-translational modifications, we can even study higher order structures using MS (see tas.txp.to/0118/Landmark2). It is important to mention that, despite the many developments in software algorithms, data analysis still requires substantial manual intervention and there is a lack of trained people able to read the spectra.

In biopharmaceutical analysis, MS and chromatography go hand-in-hand. In parallel with MS, many advances have been noticed in chromatography, with the introduction of highly efficient columns with chemistries tailored towards the analysis of biopharmaceuticals and instrumentation capable of successfully operating these columns. Separations are nowadays even performed in multiple dimensions, i.e. two-dimensional liquid chromatography (2D-LC). It does not come as a surprise that instrument and column manufacturers as well as software and consumable providers are extensively focusing on biopharmaceutical analysis. The industry is booming. Looking back to the characterization of the first recombinant therapeutic protein (insulin) in the late 1970s/early 1980s, chromatography and MS were a far cry from the current state-of-the-art. Though fast atom bombardment was used to introduce insulin into low resolution mass spectrometers, today electrospray ionization has become the standard to introduce small peptides and large proteins into high resolution mass spectrometers equipped with a variety of fragmentation modes, providing sequence information and allowing modifications to be detected and localized at very low levels. While HPLC separations were performed on columns packed with 5-10 µm porous particles and pumps operated at 400 bar, one now witnesses the use of sub 2 µm porous and superficially porous particles and system pressures up to 1500 bar allowing rapid resolution of minor structural differences.

There was a time when scientists had to identify all peaks in a peptide map using Edman degradation – a very lengthy task – but now we can easily acquire and process 24 peptide maps a day thanks to the many developments in chromatography, MS and accompanying software tools.

Hermann Wätzig: We are constantly improving our understanding about the quality of the biologics being produced and how aspects such as charge variance and size variance play an important role. UHPLC and capillary electrophoresis continue to deliver better separations. MS, of course, is a much newer technology – and I must admit that the advances in this field continue to surprise me!

How does the characterization of biosimilars differ?

KS: Regulatory agencies evaluate biosimilars based on their level of similarity to the originator. In demonstrating similarity, an enormous weight is placed on analytics – and both the biosimilar and originator need to be characterized and compared in extensive detail. The analytical package for a biosimilar submission is considerably larger than that of an originator. The structural differences highlighted define the number of clinical studies required. When biosimilar developers re-characterize blockbuster products developed 20 years ago using the current state-of-the-art analytical tools, many more details are revealed that pose enormous challenges to position a product within the originator specifications.

What are the biggest discussion points in biopharma characterization? Where are there unmet needs?

AR: We have come a long way in understanding protein molecules as products – but this understanding has also led us to appreciate the limitations of our knowledge. In most cases, these gaps in our understanding are because of current technical limitations, which I am certain will be resolved in the near future. One example is aggregation; there are already established immunogenic effects of the presence of this class of contaminant, making it a Critical Quality Attribute (CQA), but we still need to understand – in greater detail – the specific effects of individual aggregate species on immune profiles. The mechanism of anti-drug antibody formation is poorly understood; whether the response pathway is generic to aggregates or species specific still needs to be resolved. Understanding this would greatly help in defining specific ranges for this class of contaminants. It would also help in predicting drug behavior more accurately during storage conditions and, ultimately, the quality of the product at the time of patient-administration.

A similar gap exists in our mapping of the glycan profile of complex biomolecules, such as monoclonal antibodies. Given the wide range of possible combinations of glycans that can attach to the antibody backbone, complete profiling of these variants becomes a technical challenge. Moreover, given the acute sensitivity of biotherapeutics to their environment, it becomes even harder to ascertain how true a given profile is and what changes have been introduced because of the analysis itself.

Should sensitivity always be a priority?

KS: Better sensitivity is not necessarily what biopharma companies want, but it is a consequence of recent advances in analytical tools. Today, it is remarkable that we can detect individual host cell proteins (HCPs) at 0.1 ppm levels and product variants at levels below 0.1 percent. In project meetings, we often hear the comment “we don’t want to know about all these low level variants!” or “we hope you have not found new liabilities.” As analytical scientists, we feel it is our duty to reveal all the details of the molecules we are studying. At the HPLC 2016 meeting in San Francisco, Reed Harris (Genentech) showed an interesting graph plotting the number of modifications revealed in a molecule versus popularity within the project team. When discovering the first set of modifications, the popularity within the team increases substantially. After having shared yet another set of modifications, popularity declines – and at a certain point you are “Doctor Doom” because of the consequences that your findings can have on the timeliness of a project! Sensitivity is important, but in the development of new techniques and technologies, I think our priority should lie in robustness.

HW: Being from academia, my opinion is that sensitivity is always beneficial! Sensitivity allows you to see and understand more – and I think scientists from commercial biopharma should share this view. Sensitivity, however, is not the only important feature of a system – separation efficiency and robustness, as Koen says, are equally important, depending on what you are trying to achieve. System reliability is also crucial. Interestingly, I think that standard analytical equipment can sometimes be more reliable than newer, sophisticated instruments. For example, I find that standard HPLC equipment can be slightly more reliable than highly sophisticated HPLC, electrophoresis or MS systems, though I expect to see this change as the technology becomes more established.

What advances do you see in the pipeline?

KS: Real-time monitoring of product attributes during manufacturing increasingly looks like the future, but the complexity of biopharmaceuticals makes it a challenge. Various groups within the biopharmaceutical industry have, nevertheless, made enormous progress in real-time monitoring of CQAs directly from the process. In vitro and in vivo CQA monitoring is also on the rise.

We furthermore expect MS, the workhorse in R&D, to find its way into routine environments as a release tool and we have high hopes for 2D-LC, where two different separation mechanisms are combined, with the aim of increasing overall resolution and thereby providing the next level of product detail.

AR: Numerous hybrid MS-based analytical techniques, including ion mobility-MS, capillary electrophoresis-MS, hydrogen-deuterium exchange-MS (HDX-MS), and size-exclusion chromatography coupled to native MS are yet to make their way into routine use. Also, real-time efficacy assessment platforms have been proposed (for example, CANScript technology), which I believe will greatly enhance effective biologic development.

HW: I expect considerable progress to come from automation, particularly sample preparation steps. Less error by dilution or extraction steps will certainly improve analytical precision. Miniaturization also has great potential to speed up analyses, and improve precision by multiple measurements and using the obtained average values as reportable results.