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Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.

ABSTRACT: Online comprehensive two-dimensional liquid chromatography (LC × LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC × LC comes at the price of higher dilutions (thus, reduced sensitivity) and, often, long analysis times (>100 min). The need to preserve the separation attained in the first dimension ((1)D) causes greater dilution for LC × LC, in comparison with 1D-LC, and long analysis times to sample the (1)D with an adequate number of second dimension separations. A way to significantly reduce these downsides is to introduce a concentration step between the two chromatographic dimensions. In this work we present a possible active-modulation approach to concentrate the fractions of (1)D effluent. A typical LC × LC system is used with the addition of a dilution flow to decrease the strength of the (1)D effluent and a modulation unit that uses trap columns. The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate phosphate surfactants, using a combination of hydrophilic interaction and reversed-phase liquid chromatography. The modified LC × LC system enabled us to halve the analysis time necessary to obtain a similar degree of separation efficiency with respect to UHPLC based LC × LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200 min, respectively), while at the same time reducing dilution (DF of 142, 299, and 1529, respectively) and solvent consumption per analysis (78, 120, and 800 mL, respectively).

SUBMITTER: Gargano AF

PROVIDER: S-EPMC5373567 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.

Gargano Andrea F G AF Duffin Mike M Navarro Pablo P Schoenmakers Peter J PJ

Analytical chemistry 20160108 3

Online comprehensive two-dimensional liquid chromatography (LC × LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC × LC comes at the price of higher dilutions (thus, reduced sensitivity) and, often, long analysis times (>100 min). The need to preserve the separation attained in the firs ...[more]

PMID: 26709410

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Project description:Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Dataset Information

Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.

Publications

Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.

Similar Datasets

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