Gradient reversed-phase liquid chromatography of proteins on very short columns.  Influence of particle size on peak dispersion

Verzele, M.; Yang, Yang Bo.; Dewaele, C.; Berry, V.

doi:10.1021/ac00164a017

Cited by 14 publications

(7 citation statements)

References 13 publications

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“…The rest of the column will broaden the peak due to dispersive and diffusive processes. 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.…”

Section: Resultsmentioning

confidence: 99%

“…For large solutes, the length of the column has only a minor or negligible impact on the separation. − Experimental results suggested that longer columns mostly increase loading capacity, but not necessarily resolution; therefore, very short columns can be applied for protein separations. − Another important finding was that the recovery of proteins improved by the use of short columns. Microporous adsorptive membranes and short monolithic beds have been also applied for protein separations. − All of the above-mentioned studies concluded that short columns could potentially be beneficial for protein separations since operating pressure can be significantly decreased, while resolution remains pretty much the same.…”

Section: Introductionmentioning

confidence: 99%

“…Most notably, older light-chain (LC) systems exhibited comparatively large extra-column band dispersion and gradient delay volumes, which might have significantly impacted retention and resolution data. In addition, most of the above-mentioned experiments were performed at a temperature ranging from ambient to 35 °C, ,, which is not ideal for peak shape, band broadening, and protein recovery because of strong secondary (mixed mode) interactions. Today, we have learned that a high temperature (70–90 °C) is generally required to limit unwanted band broadening and also to improve the mass transfer kinetics of slowly diffusing large solutes, especially when performing a denaturing separation of a protein.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Fekete

Bobály²,

Nguyen

et al. 2020

Anal. Chem.

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Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columnsin theoryjust broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25–150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 μm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody–drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Fekete

Bobály²,

Nguyen

et al. 2020

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…Such behavior is often referred to as “on/off” or “bind and elute” mechanism. Those solutes show an important change in their retention with only a minor change of mobile phase composition. − At a given composition, the retention factor is so high that the molecule does not move anymore in the chromatographic column. With a small increase in the eluent strength, the retention factor quickly drops to practically zero and the molecules move and apparently travel through the column without further interaction.…”

mentioning

confidence: 99%

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

et al. 2019

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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.

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“…Early work suggested that column length has a minor or negligible impact on the separation of large molecules in gradient liquid chromatography. − This can be explained by the phenomenon known today as an “on–off” elution mechanism. This mechanism involves only a small number of adsorption–desorption steps instead of a multiple-step partitioning process, which is usually the case for small molecules .…”

Section: Introductionmentioning

confidence: 99%

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography

et al. 2020

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In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 μL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns’ extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (e.g., high-throughput screening) of proteins.

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Gradient reversed-phase liquid chromatography of proteins on very short columns. Influence of particle size on peak dispersion

Cited by 14 publications

References 13 publications

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

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

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

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography

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