Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

Sandra, Koen; Vanhoenacker, Gerd; Vandenheede, Isabel; Steenbeke, Mieke; Joseph, Maureen; Sandra, Pat

doi:10.1016/j.jchromb.2016.04.040

Cited by 50 publications

(23 citation statements)

References 38 publications

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“…Selectivity may also be modified by changing the pH or adding an ion-pairing agent in one of the two dimensions. An example of the first is the separation of the antibodydrug conjugate ado-trastuzumab emtansine by LC × LC-MS [88]. The method developed by Sandra et al combined two RPLC separations, one at high pH and the other at low pH, with shifting gradient assemblies to obtain good separation.…”

Section: Rplcmentioning

confidence: 99%

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Pirok

Gargano

Schoenmakers

2017

J of Separation Science

198

139

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Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.

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Section: Rplcmentioning

confidence: 99%

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Pirok

Gargano

Schoenmakers

2017

J of Separation Science

198

139

View full text Add to dashboard Cite

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“…Those samples clearly necessitate 2D separations and MS detection. Therefore, a combination of multiple heart-cutting IEX-RPLC performed at the intact protein and sub-unit levels, and RP x RP performed at the peptide level (using high and low pH gradients) was used since it provides valuable information on the drug load and its distribution on the protein backbone via lysine linkages (ado-trastuzumab emtansine) [74]. This approach also allowed the assessment of drug conjugation sites.…”

Section: Multidimensional Separationmentioning

confidence: 99%

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Bobály

Fleury-Souverain²,

Beck

et al. 2018

Journal of Pharmaceutical and Biomedical Analysis

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Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DAR), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

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“…ADC analysis and characterization can be performed at different levels such as topdown, middle-down and bottom-up levels. Coupling of different techniques such as LC-MS or multidimensional LC separations may offer additional benefits over one-dimensional methods [14,15].…”

Section: Introductionmentioning

confidence: 99%

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

Sarrut

Corgier

Fekete

et al. 2016

Journal of Chromatography B

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Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.

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Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

Cited by 50 publications

References 38 publications

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

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