Szabolcs Fekete

Comparative study of new shell-type, sub-2 μm fully porous and monolith stationary phases, focusing on mass-transfer resistance

Today sub-2 microm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 microm shell particles (1.9 microm nonporous core surrounded by a 0.35 microm porous shell, Kinetex, Core-Shell), packed with other shell-type particles (Ascentis Express, Fused-Core), totally porous sub-2 microm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW=270-430) and a high molecular weight one (MW approximately 900) was conducted. This study proves that the Kinetex column packed with 2.6 microm shell particles is worthy of rivaling to sub-2 microm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.

Theory and practice of size exclusion chromatography for the analysis of protein aggregates

Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates. The main advantage of this approach is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure and local environment. Despite the fact that the chromatographic behavior and peak shape are hardly predictable in SEC, some generic rules can be applied for SEC method development, which are described in this review. During recent years, some improvements were introduced to conventional SEC that will also be discussed. Of these new SEC characteristics, we discuss (i) the commercialization of shorter and narrower columns packed with reduced particle sizes allowing an improvement in the resolution and throughput; (ii) the possibility of combining SEC with various detectors, including refractive index (RI), ultraviolet (UV), multi-angle laser light scattering (MALLS) and viscometer (IV), for extensive characterization of protein samples and (iii) the possibility of hyphenating SEC with mass spectrometry (MS) detectors using an adapted mobile phase containing a small proportion of organic modifiers and ion-pairing reagents.

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: theory and applications.

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis

In the last decade, an important technical evolution has occurred in pharmaceutical analysis with numerous innovative supports and advanced instruments that have been proposed to achieve fast or ultra-fast separations in LC with an excellent sensitivity and ease of operation. Among the proposed strategies to increase the throughput, the use of short narrow-bore columns packed with sub-3 μm core-shell and porous sub-2 μm particles have emerged as the gold standards. Nevertheless, to take the full benefits of these modern supports, a suitable chromatographic system has to be employed. This review summarizes the instrumental needs and challenges in terms of extra-column variance, dwell volume, maximum system pressure, detector data acquisition rate, and injection cycle time. In addition, because of their reasonable pressure drop, the use of columns packed with sub-3 μm core-shell particles on a conventional LC instrument is discussed in detail. A methodology is proposed to check the compatibility between stationary phase and instrument, and some solutions are proposed to improve the performance of standard instruments. Finally, because the column technology is evolving faster than instrumentation, it is nowadays possible to purchase short, narrow-bore columns packed with 1.3 μm core-shell particles. Micro columns (1 mm I.D.) packed with 1.7-1.9 μm porous particles are also available from several providers, which limit frictional heating effects and reduce solvent and sample consumption. However, it remains difficult to find instruments compatible with such column geometries and a severe loss of performance may be observed due to the system itself.

Efficiency of the new sub-2 μm core-shell (Kinetex™) column in practice, applied for small and large molecule separation.

At present sub-2 μm packed columns are very popular to accomplish rapid and efficient separations. Applying particles with shortened diffusion path to improve the efficiency of separation performs higher efficiency than it is possible with the totally porous particles having the same size. The advantages of sub-2 μm particles and shell particles are combined in the new Kinetex 1.7 μm particles. In this study a systematical evaluation of the efficiency and achievable analysis time obtained with 5 cm long narrow bore column packed with sub-2 μm core-shell particles (1.25 μm core diameter and 0.23 μm porous silica layer), and other type very efficient columns is presented. The efficiency of separation was investigated also for small pharmaceutical and large molecules (proteins). Van Deemter, Knox and kinetic plots are calculated. The results obtained with low molecular weight polar neutral analytes (272 g/mol, 875 g/mol), with a polypeptide (4.1 kDa) and with different sized proteins (18.8 kDa, 38.9 kDa and 66.3 kDa) are presented in this study. Moreover, particle size distribution, and average pore size (low-temperature nitrogen adsorption, LTNA) of the new very fine core-shell particles were investigated. According to this study, increased flow rates can be applied on sub-2 μm core-shell columns to accomplish very fast separations without significant loss in efficiency. The new sub-2 μm shell particles offer very high efficiency both for small and large molecule separation.

Maximizing kinetic performance in supercritical fluid chromatography using state-of-the-art instruments

Recently, there has been a renewed interest in supercritical fluid chromatography (SFC), due to the introduction of state-of-the-art instruments and dedicated columns packed with small particles. However, the achievable kinetic performance and practical possibilities of such modern SFC instruments and columns has not been described in details until now. The goal of the present contribution was to provide some information about the optimal column dimensions (i.e. length, diameter and particle size) suitable for such state-of the-art systems, with respect to extra-column band broadening and system upper pressure limit. In addition, the reliability of the kinetic plot methodology, successfully applied in RPLC, was also evaluated under SFC conditions. Taking into account the system variance, measured at ∼85μL(2), on modern SFC instruments, a column of 3mm I.D. was ideally suited for the current technology, as the loss in efficiency remained reasonable (i.e. less than 10% decrease for k>6). Conversely, these systems struggle with 2.1mm I.D. columns (55% loss in N for k=5). Regarding particle size, columns packed with 5μm particles provided unexpectedly high minimum reduced plate height values (hmin=3.0-3.4), while the 3.5 and 1.7μm packing provided lower reduced plate heights hmin=2.2-2.4 and hmin=2.7-3.2, respectively. Considering the system upper pressure limit, it appears that columns packed with 1.7μm particles give the lowest analysis time for efficiencies up to 40,000-60,000 plates, if the mobile phase composition is in the range of 2-19% MeOH. The 3.5μm particles were attractive for higher efficiencies, particularly when the modifier percentage was above 20%, while 5μm was never kinetically relevant with modern SFC instruments, due to an obvious limitation in terms of upper flow rate value. The present work also confirms that the kinetic plot methodology could be successfully applied to SFC, without the need for isopycnic measurements, as the difference in plate count between predicted and experimental values obtained by coupling several columns in series (up to 400mm) was on average equal to 3-6% and with a maximum of 13%.

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach

Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.

Kinetic evaluation of new generation of column packed with 1.3 μm core–shell particles

The goal of this study was to critically evaluate a new generation of columns packed with 1.3 μm core-shell particles. The practical possibilities and limitations of this column technology were assessed and performance was compared with other reference columns packed with 1.7, 2.6 and 5 μm core-shell particles. The column efficiency achieved with 1.3 μm core-shell particles was indeed impressive, Hmin value of only 1.95 μm was achieved, this would correspond to an efficiency of more than 500,000 plates/m. The separation impedance of this column was particularly low, Emin=2000, mostly due to a reduced plate height, h of 1.50. Comparing the kinetic performance of 1.3 μm core-shell particles to that of other particle dimensions tested in this study revealed that the 1.3 μm material could provide systematically the shortest analysis time in a range of below 30,000 theoretical plates (N<30,000).Despite its excellent chromatographic performance, it was evident that this column suffers from the limitations of current instrumentation in terms of upper pressure limit and extra-column band broadening: (1) even at 1,200 bar, it was not possible to reach an optimal linear velocity showing minimal plate height value, due to the low permeability of this column (Kv=1.7×10(-11)cm(2)), and (2) for these short narrow bore columns packed with 1.3 μm core shell particles, which is mandatory for performing fast-analysis and preventing the influence of frictional heat on column performance in UHPLC, it was observed that the extra-column band broadening could have a major impact on the apparent kinetic performance. In the present work, significant plate count loss was noticed for retention factors of less than 5, even with the best system on the market (σ(2)ec=2 μL(2)).

Evaluation of a new wide pore core–shell material (Aeris™ WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins

The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2 μm particles or shell particles. In this context, a new wide-pore core-shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2 μm solid inner core surrounded by a 0.2 μm porous layer (total particle size of 3.6 μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h(min) values of the Aeris WIDEPORE column were determined for small model compounds. The h(min) values were equal to 1.7-1.8 and 1.4 for the 2.1 and 4.6 mm I.D. columns, respectively, which are in agreement with the values reported for other core-shell materials. In the case of a small protein Insulin (5.7 kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7-66.8 kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8 kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t(grad)<6 min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.

Shell and small particles; Evaluation of new column technology

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 microm particles in gradient and isocratic elution separations of hormones (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone). Peak capacities around 140-150 could be achieved in 25 min with the 5 cm long columns. The Ascentis Express column (packed with 2.7 microm shell particles) showed similar efficiency to sub-2 microm particles under gradient conditions. Applying isocratic separation, the column of 2.7 microm shell particles had a reduced plate height minimum of approximately h=1.6. It was much smaller than obtained with totally porous particles (h approximately = 2.8). The impedance time also proved more favorable with 2.7 microm shell particles than with totally porous particles. The influence of extra-column volume on column efficiency was investigated. The extra-column dispersion of the chromatographic system may cause a shift of the HETP curves.