Rising Stars of Separation Science: Sara Carillo

ColumnJuly 2022
Volume 18
Issue 07
Pages: 24–27

This month we interview Sara Carillo, Bioanalytical Research Lead at NIBRT, about her work developing new analytical approaches in biopharma, what attracted her to this research, and the benefits of a multi-attribute method (MAM) workflow in her work.

Q. When did you first encounter chromatography and what attracted you to the subject?

A: Chromatography has been a fundamental part of my research since my undergrad research project, when I worked in the Structure and Synthesis of Carbohydrates Naples (SSCN) group at the University “Federico II” in Naples, Italy, under the supervision of Professor Antonio Molinaro. The isolation of bioactive macromolecules of bacterial origin, such as lipopolysaccharides, biofilms, and polysaccharides, requires extensive purification from very complex matrices, such as cell lysates or bacteria spent culture media. In this intricate mix of small and big molecules with different features, preparative liquid chromatography (LC) is the best weapon researchers have to purify active compounds and then proceed to their biological and structural characterization.

Q. Can you tell us more about your Ph.D. thesis?

A: For my Ph.D thesis I continued my work in the SSCN group under the mentorship of Professor Maria Michela Corsaro. I worked on the structural characterization of bioactive glyco-conjugates of bacterial origin, mainly derived from extremophiles bacteria. The knowledge of the adaptive mechanisms of these bacteria is yet to be uncovered and they constitute an endless reservoir of bioactive compounds (1). From the bacteria cell culture there is extensive work that is needed to purify these compounds from their matrices, and the approach needed varies a lot depending on the nature of the carbohydrates or lipids constituting the macromolecules. Indeed, the length of lipids, or the presence of deoxysugars and lately the size of the molecule, may influence your purification strategy. In general, the very first purification steps based on liquid–liquid extractions (LLE) are followed by a series of preparative size-exclusion chromatography (SEC) runs. The purified compound can be intrinsically heterogeneous, often requiring further analytical-scale purification. One very interesting technique I used during my Ph.D. was high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC–PAD), which allowed the separation of very close and isobaric oligosaccharides. Every step of the characterization of these complex macromolecules was supported by derivatized monosaccharides or lipids analysis by gas chromatography–mass spectrometry (GC–MS) and, more recently, structural characterization by nuclear magnetic resonance (NMR).

Q. What chromatographic techniques have you worked with?

A: During my research journey, I have worked with large preparative-scale purification based on SEC or ion-exchange chromatography (IEC), with columns taller than me, and columns thicker than my wrist! GC has also been key in my research for many years.

In the last seven years, I have moved to biopharmaceutical analysis. In this exponentially expanding field, the chromatographic techniques required are multiple and are mainly based on LC. A plethora of stationary phases are involved—often used to obtain orthogonal analysis, such as SEC or IEC again, but also hydrophobic interaction chromatography (HIC), reversed phase, hydrophilic interaction liquid chromatography (HILIC), affinity chromatography—and they can vary from preparative- to nanoscale.

Q. Your primary research focus is the development of new analytical approaches in biopharma—what specifically attracted you to this area
of research?

A: The development of biopharmaceuticals has not stopped for a single day in the last decade and is actually accelerating. It started with recombinant proteins such as insulin and then monoclonal antibodies, bispecifics, antibody-drug conjugates, new modalities such as gene therapy, and now the explosion of mRNA-based vaccines after the pandemic, which has paved the way for this powerful modality for many diseases. As a consequence, there is a huge push coming from industry to develop new analytical tools, as well as from vendors, regulatory bodies, and academic research groups like the Characterization and Comparability Laboratory in NIBRT (Dublin, Ireland) where I work under the supervision of Professor Jonathan Bones. I cannot say what attracted me initially, but I can say what drives me every day in my job; there is an amazing connection between all the entities above, trying to solve real problems that people in the biopharma industry face in their work. Every day I witness the practical application of our research for the improvement of the production process and quality control of these complex drugs, and this is exciting. We know our work can provide a concrete difference in making drugs safer and more efficient through the deeper knowledge we gain from analytical tools such as liquid chromatography and mass spectrometry (MS).

Q. You have recently published a paper on a multi-attribute method (MAM) workflow for the analysis of biopharmaceuticals (2). Can you detail exactly what a MAM workflow is? Why is this approach important and what advantages does it offer over existing techniques?

A: A MAM workflow is an analytical approach initially developed for quality control (QC) laboratories to strictly monitor a large number of biopharmaceutical features using a single analysis. The workflow is based on peptide mapping analysis performed through reversed-phase chromatography and high-resolution mass spectrometry. A MAM workflow can be divided into two phases. The first phase is a discovery phase that is based on LC–MS/MS analysis of a reference standard to build a library of components that need to be monitored to ensure product quality (these features are known as critical quality attributes or CQAs); the library is based on accurate mass and chromatographic retention time of each component. When the library is ready and implemented in an automated processing method, a QC laboratory can monitor all following samples (batch release, stress studies) on the basis of this processing method using LC–MS analysis with full MS only acquisition, and ensure CQAs are within the established and accepted ranges. This second phase is called the monitoring phase and is usually easier to implement in a regulated environment compared with more complex MS/MS techniques; it can provide a totally compliant data acquisition and processing. A third phase (called new peak detection) based on a nontargeted search of the new data compared with the reference sample is then performed to highlight any new or missing component that may indicate a potentially critical deviation of the manufacturing process. The approach promises to replace a large number of assays while also providing more detailed information compared with methods based on UV or fluorescence detection. Nevertheless, MAM can be used for different scopes, such as monitoring biopharmaceutical features during upstream processing. In our recent paper (2), we demonstrated the suitability of a MAM approach for upstream monitoring, host-cell protein analysis, and biosimilar comparisons.

Q. Are there any challenges involved with using this approach?

A: The implementation of this workflow has several challenges, mainly related to the transferability of the workflow across different instruments or different laboratories, as well as challenges deriving from different aspects of the software, such as data integrity, compliance of the whole workflow, and strategies to obtain useful and relevant data from the new peak detection phase. Sample preparation reproducibility can also be challenging, and for this reason automation is often part of the workflow. Challenges of a different nature are present when MAM is implemented in different steps of the bioproduction, such as during upstream or product development, where the availability of sample may be lower and the dynamic range of the CQAs to be monitored higher.

Q. What advice would you offer to anyone thinking of using a MAM workflow for the first time?

A: MAM is a complex workflow, but it has the power to be based on a simple approach, such as peptide mapping. Robustness of the sample preparation and LC separation play the most critical part, together with a deep knowledge of the product. Those aspects are surely the first that need to be addressed by anyone approaching a MAM workflow. Beyond that, the development of user-friendly mass spectrometers and software solutions is a key challenge for vendors and big steps have been made recently to support the implementation of MAM in every QC environment.

Q. Another of your recent papers discusses the use of microchip-based capillary electrophoresis for native mass spectrometry (MS) analysis of biopharmaceuticals (3). What is novel about this research and what advantages does it offer the analyst?

A: Native MS of intact proteins plays a critical role in the characterization of biopharmaceuticals, as it has the potential to uncover and monitor some attributes only visible at the intact level. A good example of this is the presence of protein fragments derived from degradation of monoclonal antibodies (mAbs). In addition, some of the newer modalities based on mAb scaffolds require the preservation of the intact structure of the protein in order to obtain critical information, such as drug‑antibody ratio in the case of cysteine‑linked antibody-drug conjugates, or to monitor the right assembly in the production and development of bispecific antibodies. All of these examples explain the increased use of native MS analysis that needs to be aided as much as possible by proteoform separation before MS detection to simplify the characterization. Although LC-based methods are reliable and offer an increasing number of solutions, we found microchip‑based capillary electrophoresis (CE) to be almost unique in the depth of information returned—plus the dynamic range of the analysis is incredible. The separation obtained using CE is based on charge variants separation, which, combined with the nanoflow used to introduce the sample in the mass spectrometer, increases the amount of proteoforms that can be detected (3). We observed this difference on several molecules; in our lab any new molecule is first analyzed with this technique to get an overall deeper knowledge of its features before proceeding with further analysis.

Q. What projects are you currently working on?

A: Both MAM and native MS are in continuous development, so we keep working on these two areas almost every day to optimize sample preparation, separation, and analysis. For example, there is huge interest in bringing analytical tools closer to the production pipeline for the development of online tools or process analytical technology (PAT).

We have recently started working on oligonucleotides. The approval of mRNA vaccines for the prevention of Sars-Cov2 infection has paved the way for this class of biotherapeutics, and it is interesting to see how the whole scientific community is coming together to develop analytical techniques for the analysis of mRNA or the lipid nanoparticle delivery system. In the future there will be an exponential growth in the analytical tools available for this class, and this will surely bring a deeper knowledge of this new technology and its production process.


  1. S. Carillo, A. Casillo, G. Pieretti, E. Parrilli, F. Sannino, M. Bayer-Giraldi, S. Cosconati, E. Novellino, M. Ewert, J.W. Deming, R. Lanzetta, G. Marino, M. Parrilli, A. Randazzo, M.L. Tutino, and M.M. Corsaro, J. Am. Chem. Soc.137(1), 179–89 (2015).
  2. C. Jakes, S. Millán-Martín, S. Carillo, K. Scheffler, I. Zaborowska, and J. Bones, J. Am. Soc. Mass Spectrom.32(8), 1998–2012 (2021).
  3. S. Carillo, C. Jakes, and J. Bones, J. Pharm. Biomed. Anal.185, 113218 (2020).

Sara Carillo completed her Ph.D. in chemical sciences in 2013 at the University of Naples “Federico II” in Italy. Under the guidance of Professor Corsaro, she focused on the structural characterization of polysaccharides and glyco-conjugates from Gram-negative bacteria via NMR and mass spectrometry techniques, focusing on the immunological properties and potential of extremophiles endotoxins. After a period at the University College of Dublin, in 2015 she joined Jonathan Bones’s research group at NIBRT, working on the understanding of the effects of extractables and leachables from single-use bioreactors on CHO cells N-glycome and produced monoclonal antibodies. She is now working at NIBRT in collaboration with Thermo Fisher Scientific as Bioanalytical Research Lead for the development of new analytical approaches in biopharma.

Related Videos
Robert Kennedy
John McLean | Image Credit: © Aaron Acevedo
Related Content