In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution.
Keith J ArlottaAditya V GandhiHsiao-Nung ChenChristine S NervigJohn F CarpenterShawn C OwenPublished in: Antibodies (Basel, Switzerland) (2018)
Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.
Keyphrases
- raman spectroscopy
- mass spectrometry
- liquid chromatography
- cancer therapy
- capillary electrophoresis
- high throughput
- amino acid
- binding protein
- high resolution
- protein protein
- dna binding
- high resolution mass spectrometry
- diffusion weighted
- magnetic resonance
- high glucose
- adipose tissue
- solid state
- skeletal muscle
- drug induced
- magnetic resonance imaging
- insulin resistance
- epidermal growth factor receptor
- high performance liquid chromatography
- glycemic control
- optical coherence tomography
- molecularly imprinted
- room temperature
- simultaneous determination
- endothelial cells
- stress induced
- weight loss
- contrast enhanced