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Antibody Drug Conjugates in Flow

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Figure 1: Left, General schematic of antibody-linker-payload. Middle, graphic of antibody structure and function. Right, benefits of ADC’s vs “standard” chemotherapy.

The Problem

The APC team was set the challenge to design a scalable, flexible, flow chemistry system capable of providing fast mixing and thermal control whilst being able to operate safely during the conjugation of highly toxic payload with monoclonal antibody (mAb). A combined modelling and “wet lab” experimental approach were used to demonstrate that the designed flow platform provided the required mixing for this reaction while not damaging the antibody due to the shear experienced in the flow system.


In-silico modelling:

Scientists in APC have undertaken numerous forms of in silico modeling studies to inform and predict various outputs of the flow chemistry system used for the antibody-payload conjugation. Using this experience, a kinetic model was developed for the reaction of payload and antibody. The model could accurately link pH and antibody concentration to the performance of the reaction. The mixing in Plug Flow Reactors (PFR’s), is dependent on a number of factors such as flowrate and tubing size and the degree of mixing can often be a limitation of the technology. The APC team utilised DynoChem modeling to determine the necessary parameters for an ideal mixing environment in this case. It was found that static mixing elements (Figure 2) of a specific length were required at the interface of the reactants input streams to ensure an ideally mixed system.

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Figure 2: An example of a static mixer used to create an ideal mixing environment.

In undertaking the modelling, we were mindful of the fact that antibodies are sensitive to sheer. Mechanical sheer forces can lead to the breakup (resulting in an access of undesired monomer state antibody) and agglomeration of the antibody (resulting in an excess of the undesired high molecule form of the antibody) resulting in reactions which are inconsistent and don’t reach the required QC standards. Thus, the team at APC modelled the sheer of the proposed flow rate within the flow chemistry set-up which informed the design and functionality of the flow chemistry system. This model was then validated by “wet-lab" experimental results.

“Wet lab” Process Optimisation:

Scientists at APC undertook flow conjugation experiments which gave very promising results in term of drug-antibody ratio (DAR) which were in agreement with experiments previously conducted in batch. An experimental plan was designed and executed to study the effect of reaction temperature, residence time, antibody concentration and number of payload equivalents. This enabled the identification of new optimal operating conditions. This led to the use of higher temperatures and shorter residence times compared to those in batch mode - which provided enhanced reaction rates, thus improving productivity whilst not damaging the antibodies.

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Figure 3: Representation of the reaction between cytotoxic payload and antibodies.


Upon finding the optimum experimental conditions, a flow chemistry rig fit for manufacturing was designed (Figure 4). Using an engineering-driven approach, a fundamental understanding of the PFR was developed utilising a residence time distribution (Figure 4) and disturbance study employing both modelling and experimental approaches including the degree of back mixing and disturbance. Secondly, the robustness of the flow chemistry rig and operating conditions (hydrodynamic and reaction), for enabling steady-state continuous manufacturing of the antibody-linker and payload conjugation reaction, was demonstrated over an extended operating time, to verify stability - meaning our client had confidence that their process was robust at large scale. Finally, a thorough Clean in Place (CIP) strategy was developed to demonstrate the removal of bioburden and endotoxins ensuring each flow chemistry run and its effluent were within manufacturing specifications.

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Figure 4:Left,  P&ID demonstrating an ADC flow chemistry system. Right, P&ID showing a proposed setup of a residence time distribution study.