The Heightened Role of HILIC in Biopharmaceutical Analysis

Jul 17, 2017
By LCGC Editors
Valentina D’Atri from the University of Geneva spoke to The Column about the evolving role of HILIC in biopharmaceutical analysis.

Q. Your group has been focusing on using hydrophilic interaction chromatography (HILIC) for biopharmaceutical analysis. What does HILIC offer that the more historically established techniques do not?
A: Size-exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) are non-denaturing and well-established techniques that allow the characterization of biopharmaceutical size-, charge-, and hydrophobic-related variants. However, these techniques are incompatible with mass spectrometry (MS), unless applied in exotic two-dimensional liquid chromatography (2D-LC) configurations, and they cannot offer any information about the glycan’s heterogeneity that characterize most biopharmaceuticals. In this context, hydrophilic interaction chromatography (HILIC) allows a comprehensive profiling of the glycosylation pattern to be obtained with the possibility of hyphenating with MS. This latter option also allows the possibility of interrogating domain specific post-translational modifications (PTMs) and streamlining the analytical characterization so that information from such micro-heterogeneity can be effectively assessed in a unique HILIC–MS analysis.  
Q. Do you think there are any misconceptions surrounding the use of HILIC generally?
A: HILIC robustness is often unfairly questioned. It is true that HILIC needs more care in conditioning and equilibration, but it should also be taken into account that HILIC has a multimodal interaction mechanism involving hydrophilic partitioning, ion-exchange, and adsorption, even though it is often considered as a mere opposite of reversed-phase LC. This could lead to inappropriate care of the column and consequent failure in an experiment’s reproducibility. It is known that HILIC columns are not so easy to equilibrate, and often equilibration times of > 20 column volumes are suggested. However, equilibration time really depends on the mobile phase composition and the type of analytes. Preliminary tests should always be performed before the analysis to assess the effective lasting of the equilibration step.
Finally, the incompatibility with aqueous samples that could lead to peak distortion. Indeed, an initial steep gradient can significantly decrease this injection effect and allow the proper focusing of the aqueous sample onto the column.
Q. Results from a paper you published in 2016 (1) using HILIC to analyze insulins, interferon -2b, and trastuzumab were resoundingly positive, particularly for the separation of glycoforms. What was novel about your approach?
A: That was the first contribution from our group in the field of biopharmaceutical analysis performed by HILIC. Thanks to purposely developed column technology (stationary phase based on wide-pore hybrid silica bonded with amide ligands), HILIC allowed the resolution of protein glycoforms at unprecedented levels of analysis; namely, at intact and subunits level. Then, the impact of the injected volume, the temperature of the mobile phases, and the initial gradient conditions on trastuzumab subunits separation were also evaluated to obtain the best method optimization.  
Q. Were there any other benefits to this approach you did not anticipate? Similarly, were there any downsides?
A: As benefits, I would mention a more rapid analysis allowing the assessment of the glycosylation profile, without releasing and labelling the glycans. As a downside, the column lifetime, which is roughly 300 injections. However, there are no other downsides of HILIC to be addressed here, at least in the context of mAbs analysis. Some possible issues deriving from the injection mechanisms or the needle wash solvents composition are already well known and they have been discussed several times. 
Q. A follow-up publication applied HILIC to mAb analysis and the impending market of “biosimilars” that are expected to emerge from the expiration of patents from several mAbs (2). What are the needs of this newly emerging market and what benefits does HILIC offer to the future of biosimilar analysis?
A: Comprehensive analytical characterization and comparability studies between originator and biosimilar candidates represent the main needs of this new emerging market. In this context, HILIC is already widely used for glycan profiling of released glycans, but it could be used at the protein level for streamlining the analysis. A routine use of HILIC could then be envisaged for such applications.  
Q. Glycans are an important target for analysis. Why is this?
A: Glycans can directly affect several mAb functions such as stability, efficacy, and safety. For this reason, the glycosylation pattern is considered as a critical quality attribute (CQA) and is carefully monitored during manufacturing and product development. For this application, HILIC really represents the gold standard.  
Q. A recently published article provides generic guidance to develop a HILIC method for mAbs analysis (3). Are there any special considerations required to use HILIC for the analysis of mAbs? And in what situations would HILIC be ideal for mAb analysis?
A: Column conditioning is extremely important for protein separation and mAb analysis by HILIC. In addition, the mAb recovery could be dependent on temperature and so it should be checked to maximize the performance of the separation. Regarding the second question, HILIC would be ideal for assessing the mAb glycosylation profile, eventually performed at several levels of the analysis, such as released glycans, glycopeptides, or protein subunits. It could also be ideal for batch-to-batch or originator–biosimilar candidate comparison, or for monitoring the tuning of the glycosylation profile. 
Q. Are there other areas of separation science where you think HILIC could be more commonly applied?
A: HILIC could be useful in peptide mapping analysis because it has highly orthogonal selectivity for peptides compared to reversed-phase LC and it is able to retain small polar peptides. Other applications could include bioanalysis (sample extracts from biological fluids and tissues) or dopants–drugs screening, because of the complementarity of HILIC to reversed-phase LC and the increasing sensitivity when coupled to MS.
Q. What are you currently working on?
A: We are currently investigating the possibilities offered by HILIC for the analytical characterization of more complex biopharmaceutical proteins, namely antibody–drug conjugates (ADC) and fusion proteins. 
1. D. Guillarme et al., Journal of Chromatography A 1448, 81–92 (2016).
2. D. Guillarme et al., Analytical Chemistry 89, 2086–2092, (2017).
3. S. Fekete et al., Journal of Pharmaceutical and Biomedical Analysis 145, 24–32 (2017).
Valentina D’Atri obtained her Ph.D. in industrial and molecular biotechnologies in 2013 at the University of Naples “Federico II”, Italy. She was then a postdoc researcher in the structural characterization of nucleic acids by performing ion mobility mass spectrometry and molecular modelling studies at the University of Bordeaux in France. In 2016, she joined the group of Jean-Luc Veuthey and Davy Guillarme (Analytical Sciences Department, University of Geneva, Switzerland). Her current research activity is in the field of biopharmaceutical proteins; in particular, her interest focuses on the analytical characterization of mAbs, ADCs, and fusion proteins by coupling HPLC, especially reversed-phase LC and HILIC, to high resolution mass spectrometry (HRMS).
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