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Grafted MCM-41 materials are ordered mesoporous adsorbents suitable for reversed-phase liquid chromatography applications (RP-HPLC): they possess high surface area, which is a great advantage to enhance the thermodynamic behavior of the classical stationary phase by increasing solute retention. Hence, MCM-41s allow the separation of poorly retained solutes which are hard to separate on conventional silica gels. Furthermore, the ordered porosity in MCM-41 enhances the kinetic properties of the classical stationary phase. MCM-41s afford higher and more homogeneous molecular diffusivity, which increases the column efficiency for high flow rates as compared to that of classical silica-based columns. Besides, RP-HPLC provides a meaningful description of the particle size distribution at a macroscopic scale and of the pore ordering by probing the diffusivity of solutes. MCM-41s obtained by pseudomorphic synthesis of silica gel have successfully passed all the chromatographic tests and the results show that this new synthesis pathway really allows independent control of the pore size and the particle size of MCM-41. Spherical particles of 5 µm of MCM-41 without aggregation have been synthesized by this route and are revealed as very good candidates for stationary phases in RP-HPLC.

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There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quality control laboratories. Native MS offers structural insights into multiple ADC critical quality attributes, thanks to accurate mass measurement. However, both techniques can lead to misinterpretations or incomplete characterization when used as standalone methods. Online coupling of both techniques can thus potentially be of great interest, but the presence of large amounts of nonvolatile salts in HIC mobile phases makes it not easily directly compatible with native MS. Here, we present an innovative multidimensional analytical approach combining comprehensive online two-dimensional (2D)-chromatography that consists of HIC and size-exclusion chromatography (SEC), to ion mobility and mass spectrometry (IM-MS) for performing analytical characterization of ADCs under nondenaturing conditions. This setup enabled comprehensive and streamlined characterization of both native and forced degraded ADC samples. The proposed 4D methodology might be more generally adapted for online all-in-one HIC×SEC-IM×MS analysis of single proteins or analysis of protein complexes in nondenaturing conditions.

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Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.