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specifications of most products on the market are limited to typically 200 bar to avoid particle deformation and the resulting loss of performance. Therefore, lower flow rates have to be used, which result in longer retention times which is counterproductive to higher throughput.
In order to overcome this drawback, a new stationary phase, (BioPro HIC HT, YMC) was developed which is based on a non-porous polymethacrylate particle. The butyl bonded particle is available with 2.3 µm particles and is designed for the analyses of biomolecules, especially ADCs. This very rigid phase is different to other phases as it allows much higher flow rates and therefore shorter run times thanks to its pressure stability of up to 400 bar (twice that of other commercially available phases).
Figure 4: Separation of Bevacizumab (Avastin® (bottom). Eluent: A) 20 mM NaH2PO4
-Na2 HPO4
) using non-porous BioPro IEX SF with 3 µm (top) and 5 µm (pH 6.8), B) 20 mM NaH2
PO4 -Na2 HPO4
0.2 M NaCl; gradient: 0-50 %B (0-30 min) 0 %B (30-45 min); flow rate: 0.5 mL/min; temperature: 25 °C; detection: UV at 215 nm; injection: 10 µL (0.25 mL/min).
SEC particle sizes of 2 µm or smaller for UHPLC are very challenging to produce. Similar to RP, typical approaches to provide higher pressure resistance are either to reduce the porosity or to use different base materials such as hybrid materials as described above for RP. In addition to the hurdles described previously, the second option has the disadvantage of reduced inertness, as the organic components of the base particles can provide undesirable secondary interactions [4]. An additional aspect of using small particle sizes of sub-2 µm also must be considered since elevated pressures can lead to an increased formation of aggregates during the chromatographic process, which alters the actual rate of aggregation in the sample [5].
Silica-based SEC phases typically with diol modification (e.g. YMC-Pack Diol or YMC-SEC MAB) provide the highest inertness. Different pore size and particle size combinations cover different analyte types from small peptides to antibodies and their aggregates. Particles are available in 3 and 5 µm for HPLC separations. The two most common pore size particles are 200 and 300 Å, Diol-200 and Diol-300, which are used e.g. MAbs, are also produced as 2 µm particles for UHPLC purposes. These 2 µm particles provide the same porosities and properties as their larger counterparts (Figure 2). Therefore, they not only allow for the development of new and improved
methods, but also a linear down scaling of existing HPLC methods to UHPLC due to the same particle properties being used over different particle sizes.
In order to further improve the column inertness, it is ideal to suppress any option of secondary interaction. Even if the stationary phase itself is inert, the hardware used might also have an influence. Therefore, using bioinert hardware such as PEEK-lined stainless steel column bodies can be a possible approach and is currently under evaluation.
HIC columns designed for high throughput
Hydrophobic interaction chromatography (HIC) is used as a standard technique to determine the drug-to-antibody ratio (DAR) of antibody-drug-conjugates (ADCs) [6]. Therefore, HIC methods are routinely used for quality control purposes of ADCs. Because quality control departments demand a high throughput, and shorter runtimes which can be achieved by the use of smaller particle sizes are required.
In HIC, polymer-based stationary phases separate the substances according to hydrophobicity, typically using a reverse salt gradient to re-hydrate proteins salted out by a high concentration of chaotropic salt. Even though more stable non- porous particles can be used, pressure
(pH 6.8) containing
Using BioPro HIC HT, analysis times for DAR determinations of ADCs such as Brentuximab vedotin, which are already quite fast, can be further reduced to 6 min by applying a 2.4 times higher flow rate, while maintaining the high resolution (Figure 3). This allows a 2-3 times higher sample throughput, which would not be possible with the competitor column due to the much lower pressure limit of only 200 bar.
Are smaller particles in IEX an ideal solution?
In general, polymer-based particles are also used in IEX, especially for charge variant analyses of MAbs. The same particle technology is used in this case as for the HIC columns, though optimised for IEX chromatography. The strong exchangers with either quaternary ammonium or sulfonic acid groups are available as porous (BioPro IEX QA/SP, YMC) or non-porous (BioPro IEX QF/SF, YMC) types. The porous type (5 µm) allows high sample loadings and high resolutions, while the non-porous phases provide outstanding efficiencies at low sample loads typically used in QC separations. The non-porous exchangers are available in 5 or 3 µm particles.
Although less pronounced, compared to the separation modes described above, a smaller particle size still improves resolution in IEX. On the other hand, the backpressure is much higher (Figure 4). Consequently, not all applications benefit from the use of smaller particles. Depending on the mobile phase, the backpressure can be close to the 3 µm column’s pressure limit and the separation cannot be improved much further [7].
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