A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates

Li, Yi; Gu, Christine; Gruenhagen, Jason; Zhang, Kelly; Yehl, Peter; Chetwyn, Nik P.; Medley, Colin D.

doi:10.1016/j.chroma.2015.03.027

Cited by 61 publications

(47 citation statements)

References 25 publications

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“…Therefore, SEC has been extensively employed for the analysis of antibody-drug conjugates and the assessment of drug purity. 18,19 However, SEC has conventionally been considered a low-resolution chromatographic method accompanied by sample dilution, 20 and therefore, has not been widely used for the fractionation of highly complex protein mixtures. Although a previous study coupling SEC with RPC for the analysis of cell lysate allowed for the identification of over 370 proteins with low MW (<40 kDa), 21 top-down analysis of high MW proteins (>60 kDa) remains challenging due to the low resolution and separation power of the conventional SEC methods.…”

Section: Introductionmentioning

confidence: 99%

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

et al. 2017

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Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules, but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10–223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that 2D sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to 1D RPC. Notably, effective sSEC-RPC separation of proteins significantly enhanced the detection of high MW proteins up to 223 kDa, and also revealed low abundance proteoforms that are post-translationally modified. This sSEC method is MS-friendly, robust and reproducible, and thus, can be applied to both high-efficiency protein purification and large-scale proteomics analysis of cell or tissue lysate for enhanced proteome coverage, particularly for low abundance and high MW proteoforms.

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Section: Introductionmentioning

confidence: 99%

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

et al. 2017

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“…By combining hydrophobic interaction chromatography (HIC) and RPLC, great structural detail was obtained in interchain cysteine conjugated ADCs [23]. Heart-cutting the small molecule peak observed in the size exclusion chromatography (SEC) analysis of a lysine conjugated ADC and subsequent analysis on RPLC provided insight in the quantity and identity of free drug and its impurities [33]. Similarly, the analysis of the sampled small molecule SEC peak on mixed-mode LC combined with evaporative light scattering detection (ELSD) allowed the identification and quantification of the excipients of mAbs and ADCs [34].…”

Section: Introductionmentioning

confidence: 99%

“…Compared to comprehensive LC × LC, first and second dimension run times are de-coupled meaning that there are no time constraints on the second dimension separation. Heart-cutting LC has been applied to the analysis of ADCs and mAbs [23,[33][34][35]. By combining hydrophobic interaction chromatography (HIC) and RPLC, great structural detail was obtained in interchain cysteine conjugated ADCs [23].…”

Section: Introductionmentioning

confidence: 99%

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

Vanhoenacker

Vandenheede

et al. 2016

Journal of Chromatography B

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“…[9][10][11] In this article we focus on two commonly utilized cosolvents and three small molecules (see Table 1) that are frequently present in the UF/DF unit operation. [9][10][11] In this article we focus on two commonly utilized cosolvents and three small molecules (see Table 1) that are frequently present in the UF/DF unit operation.…”

Section: Introductionmentioning

confidence: 99%

Clearance of solvents and small molecule impurities in antibody drug conjugates via ultrafiltration and diafiltration operation

Gates

Fang

Liao

2019

Biotechnology Progress

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Ultrafiltration and diafiltration (UF/DF) processes by tangential flow filtration (TFF) are frequently used for removal of solvents and small molecule impurities and for buffer exchange for biopharmaceutical products. Antibody-drug conjugates (ADCs) as an important class of biological therapeutics, carry unique solvents and small molecule impurities into the final UF/DF step as compared to standard antibody preparation. The production process of ADCs involves multiple chemical steps, for example, reduction and conjugation. The clearance of these solvents and small molecules by UF/DF, specifically the DF step, has been assessed and described herein. The rates of clearance for all the impurities in this study are close to the ideal clearance with no apparent interaction with either the protein or the TFF membrane and system. The effect of process variables during DF, such as pH, temperature, membrane loading, transmembrane pressure, and cross flow rate, has also been evaluated and found to have minimal impact on the clearance rate. These results demonstrate efficient clearance of solvents and small molecule impurities related to the ADC process by the DF process and provide a general data package to facilitate risk assessments based on the sieving factors and program specific needs.

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A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates

Cited by 61 publications

References 25 publications

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

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

Clearance of solvents and small molecule impurities in antibody drug conjugates via ultrafiltration and diafiltration operation

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