HIGH POTENCY APIS phase revealed minimal differences between C18 and C8 stationary phases, while demonstrating a pronounced effect of the organic modifier on retention behaviour and solubility. Acetonitrile-based systems provided acceptable selectivity but introduced solubility constraints, whereas methanol-rich systems enabled higher crude solubility and loading capacity.
These are critical parameters for industrial productivity. Solubility assessment therefore represents a central element of our workflow. From an industrial standpoint, a method that cannot support practical loading levels is inherently limited, regardless of analytical resolution. Early qualitative solubility screening allows process constraints to be identified and addressed before significant development resources are committed.
Once a suitable chromatographic system has been selected, focused application of Design of Experiments( DoE) becomes the core optimisation tool. For gradient elution processes, the initial organic phase percentage and gradient slope are typically the most influential variables, affecting not only retention time but also impurity separation and process productivity.
By applying structured DoE designs at analytical scale, these parameters can be explored efficiently with a limited number of experiments. Multiple responses, including product retention window, separation from critical impurities and peak behaviour, are evaluated simultaneously and modelled using response surface techniques.
100 %
DoE Optimization of Purification
Optir,al-point
From response surfaces to robust operating windows
Response Surface Methodology shifts the development focus from identifying a single optimal condition to defining robust operating regions. This distinction is particularly important for ADC payload-linkers, where narrow optima are unlikely to translate reliably across scales.
In the case examined, initial modelling showed that part of the investigated space resulted in premature elution outside the target window. Rather than incremental trial adjustments, the design space was shifted and a second DoE iteration completed within a single day. This iterative capability is a key advantage of a structured statistical framework.
The refined response surfaces ultimately identified a broad region delivering consistent separation from critical impurities, while also highlighting conditions providing enhanced resolution. These insights offered additional flexibility for subsequent productivity optimisation.
With a statistically defined gradient structure in place, transfer to semipreparative scale enables evaluation of loading capacity and productivity with reduced development risk. In this programme, crude loadings exceeding 6 g / L were achieved, increasing product purity from approximately 75 % to > 98 %, with recoveries above 90 %.
The wide separation from lateeluting impurities allowed fraction
collection to be streamlined, reducing cycle times and improving overall throughput. These results illustrate a fundamental industrial principle: productivity gains are most sustainably realised through robust method design rather than aggressive fine-tuning.
Implications for ADC development programmes
For R & D and CMC decision-makers, the value of a statistically driven purification strategy extends beyond chromatographic performance. Predictable timelines, reduced iteration and clear scale-up pathways directly support accelerated progression toward clinical manufacture and reduce overall programme risk. Olon applies this approach systematically across multiple purification steps and diverse payload – linker chemistries, enabling the accumulation of organisational knowledge.
The effectiveness of this methodology is maximised when embedded within an integrated ADC development and manufacturing environment. Close interaction between synthetic development, analytical science and scale‐up capabilities ensures that chromatographic decisions are made with full visibility of downstream implications.
By combining structured statistical process development with an integrated ADC platform that spans payload-linker synthesis, bioconjugation and GMP manufacturing, complex purification challenges can be addressed proactively and predictably. For organisations facing similar payloadlinker purification challenges, a structured, statistical approach can make a decisive difference. ●
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Dr Mattia Stucchi
R & D CDMO MANAGER
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Figure 1 – DoE optimisation of purification |
Lab-scale confirmation < 2 weeks |
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COMPANY NAME mstucchi @ olonspa. it www. olonspa. com |
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