Method of synthesising adcs using affinity resins
a technology of affinity resins and adcs, which is applied in the direction of dipeptide ingredients, instruments, pharmaceutical non-active ingredients, etc., can solve the problems of large waste generated by solution phase methods, inability to synthesise adcs, so as to reduce the variation in the point at which the drug component is attached to the immobilised biomolecule, improve the regio-specificity, and reduce the variation in the resulting biomolecul
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example 1
se Antibody Drug Conjugate Screening
[0266]This example demonstrates that immobilized antibodies can be conjugated to a defined drug loading with a generic process that negates the need for process development. This approach is suitable for adapting to 96 well plate high throughput screening.
[0267]Herceptin (0.5 of 1 mg / ml in PBS, pH 7.4) was bound to 100l (settled resin volume) of Fabsorbant™ F1P HF resin equilibrated in PBS by mixing the resin slurry and antibody solution gently for 60 minutes. Unbound Herceptin was removed by washing the resin with PBS, 2 mM EDTA and the resin finally re-suspended in 0.5 ml PBS / EDTA.
[0268]The bound Herceptin (Her) was reduced by adding tris-(2-carboxyethyl)phosphine hydrochloride to a final concentration of 2 mM and then incubating the suspension at ambient temperature for 18 hours. The resin was washed with PBS / EDTA to remove unreacted TCEP and then re-suspended in 4751l PBS / EDTA.
[0269]vcMMAE (vcE), N-ethyl maleimide (NEM) and dimethylacetamide (...
example 2
se Partial TCEP Reduction in Batch Mode
[0274]This example shows that immobilized antibodies can be conjugated to a defined drug loading by partial reduction of the interchain disulphide bonds followed by conjugation with vcMMAE and that product quality is enhanced relative to the same conjugates made in solution.
[0275]Herceptin (0.5 ml of 2 mg / ml PBS, pH 7.4) was bound to 100l (settled resin volume) of Fabsorbant™ F1P HF resin equilibrated in PBS by mixing the resin slurry and antibody solution gently for 30 minutes. Unbound Herceptin was removed by washing the resin with PBS, 2 mM EDTA and the resin finally re-suspended in 0.5 ml PBS / EDTA.
[0276]The bound Herceptin was reduced by adding tris-(2-carboxyethyl)phosphine hydrochloride to a ratio of 1 to 4 moles of TCEP per mole of Herceptin and then incubating the suspension at ambient temperature for 2 hours.
[0277]vcMMAE and Dimethylacetamide (DMA) were added to achieve 2.5 to 10 moles of vcMMAE per mole of Herceptin and 5% v / v DMA and...
example 3
se Partial TCEP Reduction on Column
[0283]This example shows that immobilized antibody conjugation can be adapted to a chromatographic flow process with excellent reproducibility.
[0284]Herceptin (5 ml of 2 mg / ml PBS, pH 7.4) was bound to a 1 ml column of Fabsorbant™ F1P HF resin (previously equilibrated in PBS) by loading at 120 cm / hr. The bound Herceptin was prepared for reduction by equilibrating the resin with PBS, 2 mM EDTA.
[0285]A micro peristaltic pump was used to create a small volume PBS / EDTA recirculation loop through the column (approximately 200 μL external to the column) to which TCEP was added to give a molar ratio of 2 TCEP per mole of Herceptin. This was allowed to recirculate for 120 minutes at ambient to reduce the Herceptin.
[0286]The contents of the reservoir and column were flushed to waste and replaced with PBS / EDTA / 5% v / v DMA to which vcMMAE was added to give a molar ratio of 5 vcMMAE per mole of reduced Herceptin. This was allowed to recirculate for 60 minutes a...
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