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

Antibody–drug conjugates (ADCs) are heterogeneous biotherapeutics and differ vastly in their physicochemical properties depending on their design. The number of small drug molecules covalently attached to each antibody molecule is commonly referred to as the drug-to-antibody ratio (DAR). Established analytical protocols for mass spectrometry (MS)-investigation of antibodies and ADCs often require sample treatment such as desalting or interchain disulfide bond reduction prior to analysis. Herein, the impact of the desalting and reduction steps—as well as the sample concentration and elapsed time between synthesis and analysis of DAR-values (as acquired by reversed phase liquid chromatography MS (RPLC–MS))—was investigated. It was found that the apparent DAR-values could fluctuate by up to 0.6 DAR units due to changes in the sample preparation workflow. For methods involving disulfide reduction by means of dithiothreitol (DTT), an acidic quench is recommended in order to increase DAR reliability. Furthermore, the addition of a desalting step was shown to benefit the ionization efficiencies in RPLC–MS. Finally, in the case of delayed analyses, samples can be stored at four degrees Celsius for up to one week but are better stored at −20 °C for longer periods of time. In conclusion, the results demonstrate that commonly used sample preparation procedures and storage conditions themselves may impact MS-derived DAR-values, which should be taken into account when evaluating analytical procedures.

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Separation of Liquiritin by Two-Dimensional Liquid Chromatography

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.455-456.1232 ◽

2012 ◽

Vol 455-456 ◽

pp. 1232-1238 ◽

Cited By ~ 1

Author(s):

Jing Xiang Cong ◽

Shao Yan Wang ◽

Hong Gao

Keyword(s):

Liquid Chromatography ◽

Reversed Phase ◽

Normal Phase ◽

Size Exclusion ◽

Two Dimensional ◽

Target Compound ◽

Reversed Phase Chromatography ◽

Complex Samples ◽

Exclusion Chromatography ◽

Licorice Extract

Two-dimensional liquid chromatography (2DLC) is an important technology for the separation and analysis of complex samples. Liquiritin, an important active component in licorice, was chosen as the target compound and it was separated by three kinds of off-line 2DLC, i.e. size exclusion chromatography × reversed phase chromatography, normal phase × reversed phase chromatography and reversed phase chromatography × reversed phase chromatography (SEC×RP, NP×RP and RP×RP). The chromatographic conditions were selected and the 2D systems were combined. The results show that it is feasible to separate Liquiritin from licorice extract using 2DLC. Among the 2D modes mentioned above, the highest purity of Liquiritin was obtained in the RP×RP mode, and the concentration of Liquiritin was increased most significantly in the NP×RP mode.

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Antibody–Drug Conjugates and Small Molecule–Drug Conjugates: Opportunities and Challenges for the Development of Selective Anticancer Cytotoxic Agents

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.5b00457 ◽

2015 ◽

Vol 58 (22) ◽

pp. 8751-8761 ◽

Cited By ~ 85

Author(s):

Giulio Casi ◽

Dario Neri

Keyword(s):

Small Molecule ◽

Cytotoxic Agents ◽

Antibody Drug Conjugates ◽

Small Molecule Drug ◽

Drug Conjugates ◽

Antibody Drug

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An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates

mAbs ◽

10.1080/19420862.2016.1151590 ◽

2016 ◽

Vol 8 (4) ◽

pp. 698-705 ◽

Cited By ~ 23

Author(s):

Yi Li ◽

Christine Gu ◽

Jason Gruenhagen ◽

Peter Yehl ◽

Nik P. Chetwyn ◽

...

Keyword(s):

Small Molecule ◽

Antibody Drug Conjugates ◽

Drug Conjugates ◽

Antibody Drug

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What Can We Learn about Antibody-Drug Conjugates from the T-DM1 Experience?

American Society of Clinical Oncology Educational Book ◽

10.14694/edbook_am.2015.35.e117 ◽

2015 ◽

pp. e117-e125 ◽

Cited By ~ 8

Author(s):

Francisco J. Esteva ◽

Kathy D. Miller ◽

Beverly A. Teicher

Keyword(s):

Small Molecule ◽

Antibody Fragment ◽

Cytotoxic Agents ◽

Specific Cell ◽

Malignant Cells ◽

Antibody Drug Conjugates ◽

Drug Conjugates ◽

Protein Toxin ◽

Antibody Conjugates ◽

Antibody Drug

Antibody conjugates are a diverse class of therapeutics that consist of a cytotoxic agent linked covalently to an antibody or antibody fragment directed toward a specific cell surface target expressed by tumor cells. The notion that antibodies directed toward targets on the surface of malignant cells could be used for drug delivery is not new. The history of antibody conjugates has been marked by hurdles identified and overcome. Early conjugates used mouse antibodies, drugs that either were not sufficiently potent, were immunogenic (proteins), or were too toxic, and linkers that were not sufficiently stable in circulation. Four main avenues have been explored using antibodies to target cytotoxic agents to malignant cells: antibody-protein toxin (or antibody fragment–protein toxin fusion) conjugates, antibody-chelated radionuclide conjugates, antibody-small molecule conjugates, and antibody-enzyme conjugates administered along with small molecule prodrugs that require metabolism by the conjugated enzyme to release the activated species. Technology is continuing to evolve regarding the protein and small molecule components, and it is likely that single chemical entities soon will be the norm for antibody-drug conjugates. Only antibody-radionuclide conjugates and antibody-drug conjugates have reached the regulatory approval stage, and there are more than 40 antibody conjugates in clinical trials. The time may have come for this technology to become a major contributor to improving treatment for patients with cancer.

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