Adsorption and recovery issues of recombinant monoclonal antibodies in reversed-phase liquid chromatography†

Abstract

The poor recovery of large biomolecules is a well-known issue in reversed-phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state-of-the-art reversed-phase wide-pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150,000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene-bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42,000 and 443,000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.

Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals

“…With RPLC ( Figure 4A), a peak area reduction of 15% was noticed in decreasing the mobile phase temperature from 80 to 60 °C, and at 40 ºC, 2% or less of the trastuzumab was recovered. This trend is in good agreement with previous findings [25,26]. In contrast, this recovery issue was much less pronounced in HILIC.…”

“…Indeed, at 40 and 60 ºC, around 80% of the original peak area (observed at 80 ºC) was recovered. Even if recovery from stationary phases is mAb dependent [25], this first example on trastuzumab illustrates what may be one of the most important benefits of HILIC, namely the possibility to work at reasonable mobile phase temperatures, without appreciable sample loss. This characteristic is appealing since the use of high column temperatures (i.e.…”

“…An important issue related to RPLC of mAbs is that the adsorption process becomes especially problematic at mobile phase temperatures lower than 60 ºC, to a point that very poor recoveries are observed at such temperatures [25,26]. Since its interaction mechanism is entirely different, it was of interest to study whether HILIC presented a similar phenomenon.…”

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease

“…The ratio of hydrophobic amino acid residues to protein/peptide length may significantly affect the adsorption predisposition as illustrated the conclusions of their paper [38]. We set up a microscale RP-LC experiment using the core-shell C18 solid phase for an indirect testing of the hydrophobic character of the adhesive fragment [39]. We repeatedly confirmed its hydrophobic behavior by exhibiting the greatest affinity to the hydrophobic C18 solid phase.…”

Ion-exchange chromatography for the characterization of biopharmaceuticals

“…Besides the separation of mAbs' charge variants, pH-gradient based IEX chromatography can also be applied to evaluate the pI of intact mAbs [74].…”