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MOLNÁR-INSTITUTE DryLab® applied to determine efficacy and safety of ADC oncology products
Berlin: – Molnár-Institute for Applied Chromatography’s DryLab® software platform, used in conjunction with Waters Corporation’s Empower 3 Chromatography Data System, has been used to develop a faster method of calculating the average drug to antibody ratio (DAR) of any antibody drug conjugate (ADC) product.
This innovation is significant for oncology in enabling superior quantification of the critical quality attributes (CQAs) of an ADC, which are largely determined by average DAR as the critical measure of active “payload” that can be delivered to a tumor cell, thus affecting both efficacy and safety.
Using RPLC to separate ADC species
The study ‘Separation of antibody drug conjugate species by RPLC: A generic method development approach’ was led by Professor Szabolcs Fekete, assisted by Davy Guillarme, from the School of Pharmaceutical Sciences, Universities of Geneva/Lausanne, with participation by Dr. Imre Molnár, founder and President of the Berlin-based Molnár-Institute.
The team set out to harness the power of advanced liquid chromatography (LC) modelling software for the method development of IgG1 cysteine conjugated (ADC) in reverse phase LC, using a generic method development strategy that included optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. Their ultimate goal was to be able to calculate the average drug to antibody ratio (DAR) of any ADC product.
Pioneering application of 3D Modelling
This was the first time a 3D retention modelling approach has been applied to a large therapeutic protein.
Antibody drug conjugates (ADCs) are chemotherapeutics constituted of a cytotoxic chemical drug linked covalently – via peptide linker – to a monoclonal antibody (mAb). They are used for the treatment of cancer by combining the proven antigen-specific selectivity and activity of mAbs with the potency of highly cytotoxic small molecules. Drug conjugation can be achieved via reactions at different amino acid residues such as at lysine side chains amines (lysine conjugation), at cysteine thiol groups after reduction of the interchain disulfide bonds (cysteine conjugation), or at engineered cysteine residues at specific sites on a mAb. The addition of the drugs results in a heterogeneous population of ADCs that differ in the number of drugs per antibody, and this mixture can be described by an average drug to antibody ratio (DAR).
In the past, a variety of analytical methods have been used to measure the average DAR, including UV–vis spectroscopic, liquid chromatographic, and native mass spectrometric methods. [Among liquid chromatographic methods, hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPLC) are routinely used. The former approach separates the intact DAR species under non-denaturating conditions, while RPLC is mostly used to separate the DAR species of reduced ADC sample related to the H or L chains as L0, L1 and H0, H1, H2 and H3 species.
The team succeeded in achieving fast and efficient separation of the DAR species of a commercial ADC sample, brentuximab vedotin, based on a limited number of preliminary experiments. The predictions offered by the modelling software were found to be highly reliable in both 2D or 3D retention models, with an average error of retention time prediction always lower than 0.5%. Just four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model.
“RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample,” the authors concluded.
“The developed RPLC method can be considered for determining the average DAR of an ADC, based on the observed peak area ratios of the reduced ADC sample. The native ADC separation can also be useful for multidimensional separations in the second dimension to separate the positional isomers and different species and enabling the MS identification,” the study notes.
“It also worth mentioning that – by understanding the retention behavior of ADC peaks – analysis time could be shortened into the 20–25 min range while 60–70 min long separations were reported previously,” the team adds.
- Fekete, S., Molnár, I., Guillarme, D., Separation of antibody drug conjugate species by RPLC: A generic method development approach, Journal of Pharmaceutical and Biomedical Analysis 137 (2017) pp. 60–69. DOI: http://dx.doi.org/10.1016/j.jpba.2017.01.013
Founded in 1981, Molnár-Institute develops DryLab®4, a software for UHPLC modeling for a world-wide market. Its powerful modules allow for the most sophisticated method development as required across pharma industries. Analytical scientists use DryLab® to understand chromatographic interactions, reduce runtimes, increase robustness, and conform to Analytical Quality by Design (AQbD) standards.
The Molnár-Institute is a registered vendor to the US FDA, CDC and many other regulatory bodies. DryLab® has pioneered AQbD long before regulatory agencies across the world encouraged such submissions. Widely implemented by thought leaders, the software contributes substantially to the paradigm shift towards a science and risk driven perspective on HPLC Quality Control and Assurance.
Further information at: http://www.molnár-institute.com/
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