Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products

Fekete, Szabolcs; Ritchie, Harald; Lawhorn, Jason; Veuthey, Jean‐Luc; Guillarme, Davy

doi:10.1016/j.chroma.2020.460901

Cited by 15 publications

(9 citation statements)

References 22 publications

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“…Resolution generally increases with column length (∼√L). However, with large molecules, solute molecules are completely bound (“on” mode) at the head of the column and therefore peaks are focused at the column inlet. , Then, when increasing the mobile phase strength, the solutes rapidly leave the column without further interactions.…”

Section: Resultsmentioning

confidence: 99%

“…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. Therefore, one can have the impression that infinitely short column and infinitely steep gradient are optimal for large solute separations from the band broadening point of view.…”

Section: Resultsmentioning

confidence: 99%

“…Finally, there are also a number of additional advantages when using ultrashort columns that need to be considered: shorter equilibration time, less protein hydrolysis thanks to lower residence times, reduced sample consumption, and more expedient turnaround of multidimensional chromatography. In addition, such short-column formats could be also used for column coupling to perform very fast on-column fractioning …”

Section: Discussionmentioning

confidence: 99%

“…In addition, such short-column formats could be also used for column coupling to perform very fast on-column fractioning. 28 ■ ASSOCIATED CONTENT * sı Supporting Information…”

Section: ■ Conclusionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…In addition, such short-column formats could be also used for column coupling to perform very fast on-column fractioning. 28 ■ ASSOCIATED CONTENT * sı Supporting Information…”

Section: ■ Conclusionmentioning

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.

Self Cite

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“…Such high S LSS values are one of the reasons why gradient elution is indispensable in RPLC of proteins. Selectivity could be improved by serially coupling columns with increasing retentive capacity and applying multistep gradients [49,50].…”

Section: Methodsmentioning

confidence: 99%

Recent applications of retention modelling in liquid chromatography

Uijl

Schoenmakers

Pirok

et al. 2020

J of Separation Science

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Recent applications of retention modelling in liquid chromatography (2015–2020) are comprehensively reviewed. The fundamentals of the field, which date back much longer, are summarized. Retention modeling is used in retention‐mechanism studies, for determining physical parameters, such as lipophilicity, and for various more‐practical purposes, including method development and optimization, method transfer, and stationary‐phase characterization and comparison. The review focusses on the effects of mobile‐phase composition on retention, but other variables and novel models to describe their effects are also considered. The five most‐common models are addressed in detail, i.e. the log‐linear (linear‐solvent‐strength) model, the quadratic model, the log–log (adsorption) model, the mixed‐mode model, and the Neue–Kuss model. Isocratic and gradient‐elution methods are considered for determining model parameters and the evaluation and validation of fitted models is discussed. Strategies in which retention models are applied for developing and optimizing one‐ and two‐dimensional liquid chromatographic separations are discussed. The review culminates in some overall conclusions and several concrete recommendations.

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A simple mathematical treatment for predicting linear solvent strength behavior in gradient elution: Application to biomolecules

Guillarme

Bouvarel

Rouvière

et al. 2022

J of Separation Science

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This paper describes an approach to rapidly and easily calculate the linear solvent strength parameters, namely log k 0 and S, under reversed-phase liquid chromatography conditions. This approach, which requires two preliminary gradient experiments to determine the retention parameters, was applied to various representative compounds including small molecules, peptides, and proteins. The retention time prediction errors were compared to the ones obtained with a commercial HPLC modeling software, and a good correlation was found between the values. However, two important constraints have to be accounted for to maintain good predictions with this new approach: i) the retention factor at the initial composition of the preliminary gradient series have to be large enough (i.e., log k i above 2.1) and ii) the retention models have to be sufficiently linear. While these two conditions are not always met with small molecules or even peptides, the situation is different with large biomolecules. This is why our simple calculation method should be preferentially applied to calculate the linear solvent strength parameters of protein samples.

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Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products

Cited by 15 publications

References 22 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

Recent applications of retention modelling in liquid chromatography

A simple mathematical treatment for predicting linear solvent strength behavior in gradient elution: Application to biomolecules

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