Proof of Concept To Achieve Infinite Selectivity for the Chromatographic Separation of Therapeutic Proteins

Abstract

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients.

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester, capillary‐channeled polymer fiber phase

“…After flushing the column with Buffer C (10 mM potassium phosphate buffer; pH = 7.4), it was equilibrated with Buffer A (1.8 M (NH 4 ) 2 SO 4 solution dissolved in PBS; pH = 7.4). As previously described in research regarding antibody purification using HIC, appropriate amounts of organic additives (such as methanol and acetonitrile) in the elution buffer demonstrate improved protein recovery, while maintaining their respective bioactivities . As suggested previously in this report regarding the case in a potential RP exosome separation, use of high organic solvent content may have delitereous effects on the integrity of the exosomes.…”

Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept

“…Based on scouting linear gradients ( t G = 10, 20, and 30 min), LSS model parameters were determined for intact ipilimumab (log k 0 = 42.2, S = 116.7), an HC fragment (log k 0 = 10.4, S 27.6), an LC fragment (log k 0 = 6.5, S = 18.8), and a small molecule (aspirin, 0.2 kDa). Data for aspirin was taken from a previous study …”

“…The band compression is stronger for large solutes (see eqs –, since G becomes smaller with a high b and thus peak width becomes smaller); however, their intrinsic band broadeningdue to their low diffusivity and thus high mass transfer resistanceis inherently high . Therefore, the common observation is that large molecules elute in broad peaks with flat gradients but can be eluted in very sharp peaks with steep gradients. ,, However, once the protein molecules desorbed and just travel with the mobile phase velocity on the remaining column segment, their peak shape begins to broaden due to dispersion (e.g., eddy dispersion) processes (longitudinal diffusion is negligible for large solutes) and due to the lack of further band compression. Therefore, one can have the impression that infinitely short column and infinitely steep gradient are optimal for large solute separations from the band broadening point of view.…”

Recent applications of retention modelling in liquid chromatography

“…Fekete et al. reported very large S LSS values for proteins (calculated from two scanning gradients) and a tenfold decrease in retention when the organic‐modifier concentration was increased by 0.8% [48]. Such high S LSS values are one of the reasons why gradient elution is indispensable in RPLC of proteins.…”