Forced Degradation Study of Monoclonal Antibody Using Two- Dimensional Liquid Chromatography

A simple, fast, precise and accurate ultra high performance liquid chromatography method was developed for degradation study of eletriptan hydrobromide (EH) under exaggerated conditions. An Inertsil ODS C18 (250 x 4.6 mm, 5µm) column in isocratic mode was used with mobile phase comprising of water, methanol and trifluoroacetic acid mixed in the ratio 55:45:0.1 % V/V/V, maintained at pH 3.5. The flow rate was set at 0.4 mL per minute with UV detection at 225 nm. The retention time of EH was found to be 3.7 minutes. Linearity for EH was found in the range of 3.5- 200 µg per mL and percentage recoveries were obtained in the range of 100.2 % to 100.6 %. The method was capable of resolving all degradants and principle component in sample. The proposed method is accurate, precise, selective, reproducible, and rapid for detection of degradation of eletriptan hydrobromide.

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Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-014-8299-1 ◽

2014 ◽

Vol 407 (1) ◽

pp. 355-366 ◽

Cited By ~ 47

Author(s):

Gerd Vanhoenacker ◽

Isabel Vandenheede ◽

Frank David ◽

Pat Sandra ◽

Koen Sandra

Keyword(s):

Monoclonal Antibody ◽

Liquid Chromatography ◽

Two Dimensional ◽

Therapeutic Monoclonal Antibody

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Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

10.1101/2020.03.23.003608 ◽

2020 ◽

Cited By ~ 1

Author(s):

MK Knight ◽

RE Woolley ◽

A Kwok ◽

S Parsons ◽

HBL Jones ◽

...

Keyword(s):

Monoclonal Antibody ◽

Structural Heterogeneity ◽

Forced Degradation ◽

Degradation Study ◽

Highly Active ◽

Extreme Sensitivity ◽

The Stability ◽

Energy Dependent ◽

Stable Molecule

AbstractAmong the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a ‘fingerprint’ of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (μg–mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

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The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection

Journal of Chromatography A ◽

10.1016/j.chroma.2017.06.052 ◽

2017 ◽

Vol 1523 ◽

pp. 283-292 ◽

Cited By ~ 32

Author(s):

Koen Sandra ◽

Mieke Steenbeke ◽

Isabel Vandenheede ◽

Gerd Vanhoenacker ◽

Pat Sandra

Keyword(s):

Monoclonal Antibody ◽

Liquid Chromatography ◽

Two Dimensional ◽

Clone Selection

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Bridging size and charge variants of a therapeutic monoclonal antibody by two-dimensional liquid chromatography

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2020.113178 ◽

2020 ◽

Vol 183 ◽

pp. 113178

Author(s):

Zhi Chen ◽

Ming Zeng ◽

Sung-Joo Park ◽

Gurusamy Balakrishnan ◽

Kaimeng Zhou ◽

...

Keyword(s):

Monoclonal Antibody ◽

Liquid Chromatography ◽

Two Dimensional ◽

Therapeutic Monoclonal Antibody ◽

Charge Variants

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STABLE LIQUID CHROMATOGRAPHY METHOD FOR THE RESOLUTION AND QUANTIFICATION OF RELATED IMPURITIES OF FOSAMPRENAVIR IN BULK AND PHARMACEUTICAL FORMULATIONS

INDIAN DRUGS ◽

10.53879/id.56.09.11896 ◽

2019 ◽

Vol 56 (09) ◽

pp. 37-42

Author(s):

G. V. S. Singh ◽

D. R. S Reddy ◽

T. E Divakar ◽

Keyword(s):

Liquid Chromatography ◽

Mobile Phase ◽

Pharmaceutical Formulations ◽

Forced Degradation ◽

Mobile Phase Composition ◽

Chromatography Method ◽

Degradation Study ◽

Liquid Chromatography Method ◽

Bulk Drug ◽

Isocratic Condition

Highly resolved and validated Liquid Chromatography method was developed for the separation and quantification of fosamprenavir and its related impurity 2 and 5 in bulk and pharmaceutical formulations. Separation of fosamprenavir and its impurities was achieved on prontosil ODS C18 column using mobile phase composition of methanol and 0.1 m sodium acetate in the ratio of 40:60 (v/v) at pH 5.9 as mobile phase at a flow rate of 0.9 mL/min in isocratic condition. Uv detection of the eluents was monitored at a wavelength of 246nm. In these conditions, well resolved peaks were observed at a retention time of 8.67, 5.73 and 4.00 min for fosamprenavir, Impurity 2 and 5, respectively. Calibration curve was plotted in the concentration range of 75-450 µg/mL for fosamprenavir and 1-6 µg/mL for impurity 2 and 5. Forced degradation study confirms that the method can separate the known and unknown impurities of fosamprenavir and the % degradation was found to be very less in all the stress conditions. Hence the method is suitable for the identification and quantification of impurities 2 and 5 along with fosamprenavir in bulk drug and formulations.

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Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Biochemical Journal ◽

10.1042/bcj20200580 ◽

2020 ◽

Vol 477 (18) ◽

pp. 3599-3612

Author(s):

Michael J. Knight ◽

Rachel E. Woolley ◽

Anthony Kwok ◽

Stuart Parsons ◽

Hannah B. L. Jones ◽

...

Keyword(s):

Monoclonal Antibody ◽

Structural Heterogeneity ◽

Forced Degradation ◽

Degradation Study ◽

Highly Active ◽

Extreme Sensitivity ◽

The Stability ◽

Energy Dependent ◽

Stable Molecule

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a ‘fingerprint’ of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg–mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

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