Some promising reversed-phase widepore stationary phases (organic monolithic, fully porous and core-shell type) were recently
introduced for the fast and high resolution separations of large biomolecules. It was demonstrated that kinetic performance,
retention capability, selectivity for closely related proteins and loading capacity, as well as recoveries, were satisfactory
with recent core-shell and sub-2 μm fully porous materials. This article focuses on the achievable throughput and resolution
for the characterization of biomolecules. As shown, fast separation of a therapeutic protein (~19 kDa) and some of its degradants
(related proteins) was achieved in less than 1.5 min with a 50-mm column length while a peak capacity of more than 150 was
obtained in about 15 min using two columns of 150 mm coupled in series. Similar results were also attained with intact therapeutic
monoclonal antibodies (mAbs) of ~150 kDa and their fragments by varying the column length and gradient time. However, the
recovery of intact mAbs was found to be unacceptable in reversed-phase liquid chromatography (RPLC) except when using elevated
mobile phase temperature (80–90 °C).
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The importance of therapeutic proteins and monoclonal antibodies is growing in the pharmaceutical field and expectations are
high for this new class of compounds (1,2). Indeed, it has been established that such large biomolecules could present some
significant benefits for the patient such as high efficacy, specificity, wide therapeutic range and limited side effects.
All biopharmaceuticals are inherently variable as they are produced from living organisms. This variability exists within
batches, from batch to batch, and when production processes are improved or changed between manufacturers. The variability
of biopharmaceuticals is greater than that typically observed for conventional pharmaceuticals and applies to originator reference
products as well as biosimilars. There is consequently a need to develop analytical tools that are able to characterize such
complex molecules (3,4). The analytical step is quite difficult and is based on the combination of various analytical strategies
to determine identity, impurity content, heterogeneity and activity before release. Even if capillary electrophoresis and
biophysical techniques (for example, circular dichroism, light scattering and fluorescence spectrophotometry) are considered
as reference methods, liquid chromatography (LC) is also widely applied in analytical laboratories (5,6). Today, the analyst
has the choice between (i) historical techniques, such as size-exclusion chromatography (SEC) and ion-exchange chromatography
(IEC) that provide suitable selectivity for charge variants and aggregates, respectively, and (ii) reversed-phase liquid chromatography
(RPLC) that produces lower selectivity between closely related proteins but compensated by the sharpness of the peak because
of the fast kinetic interactions in RPLC. In addition, RPLC is more directly compatible with electrospray ionization mass
spectrometry (ESI–MS) detection than SEC or IEC (7).
Recently, some noteworthy improvements were brought to conventional RPLC columns to improve the performance achieved with
large proteins. This includes the commercialization of various types of widepore phases such as organic monoliths with macropore
sizes of 1–5 μm, core-shell particles of 2.7–5 μm and fully porous sub-2 μm UHPLC columns, which have been described in the
literature. (8). In this article, two recent technologies of widepore RPLC columns were evaluated and compared for the fast
and high resolution analysis of therapeutic proteins and mAbs (9,10). 
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