Reaching the “Peak” of Recovery: Improving Antibody Separations with the Inclusion of Organic Alcohols

By: Hillel Brandes, Supervisor, Principal R&D Chemist and Cory E. Muraco, Senior R&D Scientist, Reporter US Volume 34.4

Monoclonal antibodies (mAbs) are a promising class of biologics for the treatment of several autoimmune diseases and cancers. An additional application of mAbs, however, is when a cytotoxic payload (i.e., drug) is attached to the mAb, allowing the mAb to target a certain cell type or tissue and deliver the payload to the specific target. This combination of mAb plus cytotoxic drug, connected through an organic linker, is known as an antibody-drug conjugate (ADC). As of July 2016, there are approximately 60 ADCs in the pharmaceutical pipeline; of those, 25% are in late Phase II or Phase III clinical trials.10

One downside of mAb-based drugs, however, is that, due to their structural complexity, there is significant heterogeneity. This heterogeneity can arise due to the presence of charge variants, glycosylation variants, phosphorylation variants, and/or payload variants, that arise by nature of the biological and chemical production processes.

Several different chromatographic strategies are applied to investigate and resolve the different structural and chemical variants of mAbs. Size exclusion chromatography is used as one method to assess the aggregation of a mAb sample. Ion-exchange chromatography is a method to conduct charge variant analysis. Both of these chromatographic modes can present issues of compatibility with electrospray ionization mass spectrometry (ESI-MS), which is routinely used for protein characterization. Hydrophilic interaction chromatography is routinely employed for analysis and characterization of protein glycans. Reversed-phase chromatography (RPC) has long been a method of choice for analyzing proteins due to its high resolution and compatibility with MS. RPC of proteins, however, has its own issues. Of primary significance is that protein structures can be flexible in comparison to structures of small organic molecules. This fact may present a chromatographic challenge as various structural conformations may differentially interact with the stationary phase. With proteins, peak shape in RPC is generally enhanced by parameters that stabilize a single denatured state.1-3 Temperature is one parameter that can dramatically affect the tertiary and quaternary structure of proteins and thus a “denatured state”.

Another aspect of protein and peptide reversed-phase chromatography is that, for most applications, elution must be by utilization of a solvent strength gradient. This requirement is due to at least two reasons: 1) polypeptides are generally polyionic, and, therefore, can present problems of secondary interactions with the silica surface, potentially causing issues of peak tailing and 2) partitioning of polypeptide analytes between the mobile and stationary phase occurs over a narrow window of solvent strengths (as compared to most small molecules), therefore exhibiting much more of an on-off adsorption phenomenon. With the requirement for gradient elution comes the requirement for column reequilibration prior to injection of a sample. Column re-equilibration can be shorten by reducing changes to the solvation state of the silica surface as has been shown by inclusion of low levels of small, primary alcohols in the mobile phase.4,5 How this mechanistically takes place has not been defined, but computer modelling of short, primary alcohols in binary mobile phase systems is consistent with intercalation of the alcohol into the stationary phase, with the hydroxyl hydrogen-bonding to the surface silanols or an adsorbed water layer.6 This phenomenon may have additional benefits in masking silanols, therefore improving peak shape. Scott & Simpson reported that 1-butanol can form a simple monolayer on a C18-bonded silica surface;7 this too fits with a model of a small, primary alcohol hydrogen-bonding to the surface silanols.

High temperature has been shown to be necessary in achieving optimal recovery and peak shape in RPC of mAbs.8 This fact has been confirmed to be the case, irrespective of the column used or the specific mAb sample.9 Additionally, Fekete et al. have shown that inclusion of low levels of 1-butanol reduced the temperature optimum for the RPC of the mAb.8 Thus, the mechanism of any conferred benefits of inclusion of low levels of primary alcohols in the mobile phase is not entirely clear. These previously published data suggest a primary mechanism of masking of the silica surface; another possibility might be imagined to explain the effects on antibody chromatography in which the intercalated 1-butanol is oriented with the hydroxyl facing the bulk mobile phase, thus lending some polarity to the environment at the antibody-stationary phase interface. Such models might be elucidated by inspecting results with analogs of 1-butanol.

This hypothesis was investigated further using a mAb standard, SiLu™ Lite SigmaMAb, catalog number MSQC4. Initially, the goal was to at least confirm previous reports on the chromatographic effects of 1-butanol on the RP chromatography of mAbs. Two primary alcohols, 1-propanol and 1-butanol, were investigated. Figure 1 shows chromatographic results of the recovery of SigmaMAb at varying percentages of 1-butanol at 55 °C.

As noted in Figure 1, the peak area and height of the analyte increased as the concentration of 1-butanol increased. In addition, as the concentration of 1-butanol increased, one can begin to resolve impurities from the main analyte peak. This phenomenon was further investigated by looking at how temperature played a role in the recovery of SigmaMAb. The results of this analysis are displayed in Figure 2.

As can be seen in Figure 2, it should become obvious that one of the main advantages of including 1-butanol in the mobile phase is the much lower temperature required to achieve maximum recovery of the analyte. The data, however, cannot differentiate effects due to possible mitigation against thermal degradation or impacts on the actual chromatography of the mAb. The experiment was repeated, this time with 1-propanol as the mobile phase modifier. Figure 3 shows the results of this analysis.

Analysis of SigmaMAb by RPC with Varying Amounts of 1-Butanol

Recovery, Measured by Peak Area, as a Function of Temperature and 1-Butanol Concentration

Recovery, Measured by Peak Area, as a Function of Temperature and 1-Propanol Concentration

Recovery, Measured by Peak Area, as a Function of Temperature and 2-Butanol Concentration

Recovery, Measured by Peak Area, as a Function of Temperature and 1,4-Butanediol Concentration


As shown in Figure 3, the temperature required to achieve the maximum recovery of the analyte was much higher (around 80 °C) in comparison to 1-butanol. While 1-propanol has been shown to have similar benefits as 1-butanol for keeping the silica surface solvated,4 and shielding surface silanols,5 it clearly did not provide the same chromatographic benefits in this case, as compared to 1-butanol.

Continuing the investigation, the effect of type of alcohol (primary, secondary, etc.) on analyte recovery was examined. As noted in Figure 4, the secondary alcohol elicits good recovery of the antibody albeit not as good as 1-butanol. It could be inferred that, due to steric effects, 2-butanol should be not as effective as primary alcohols in hydrogen-bonding with surface silanols or an adsorbed water layer. However, 2-butanol achieved higher recovery of the analyte at low to moderate temperatures than with 1-propanol, suggesting that the mechanistic explanation is not as simple as hydrogen-bonding to the silica surface.

A final test was to try 1,4-butanediol. The idea is that this alcohol could possibly play a dual role of hydrogen-bonding to the silica surface as well contributing additional polarity to the interface where protein adsorption takes place on the stationary phase. The results are shown in Figure 5.

As can be deduced from Figure 5, there appears to be no advantage in adding 1,4-butanediol to the mobile phase. Perhaps the two terminal hydroxyls render it sufficiently polar such that it no longer readily distributes within the stationary phase to provide any performance benefit to the reversed-phase chromatography.

Despite these seemingly, conflicting results, what emerges is a picture in which the role of the alcohol in conferring a chromatographic benefit to the RPC of mAbs (or maybe most any other IgG molecule) is not as simple as has been suggested from other chemical and computer modeling studies – that something other than masking the silica surface is at play here. Perhaps there is a separate effect on the thermal stability of the mAb in this common mobile phase. Nevertheless, as shown in Figures 1 and 2, the addition of 1-butanol to the mobile phase can elicit excellent recovery of an antibody standard at far lower temperatures than in its absence and can serve as a general method for RPC of antibodies, or perhaps any other proteins that exhibit poor peak shape at moderate temperatures.  




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