Application of DoE in development and robustness testing of a CE-SDS method
Capillary Electrophoresis SDS (CE-SDS) is a standard test for performing purity analysis and provides very useful size heterogeneity data when performing analysis of ADCs under reduced and non-reduced conditions. Platform methods are available for antibody testing and can be applied to ADCs but caution should be taken in the direct application of these conditions as individual antibodies / ADCs may be more prone to further reduction / fragmentation during sample preparation. In ADC testing, the fact that the antibody may have undergone manipulations such as partial reduction during processing may add additional complexity.
In Non-Reduced CE-SDS testing, the sample preparation involves the incubation of the ADC together with an alkylating agent and SDS buffer at high temperature for a period of a few minutes. The sample is then injected electrokinetically by application of a voltage for a period of several seconds. Separation then occurs by application of a set voltage over a period to ca 35min. As such there are a number of parameters that can affect the result, in particular the sample preparation. Use of Design of Experiments (DoE) is an ideal tool to help support the optimisation of sample handling conditions over One Factor At a Time (OFAT) development.
DoE was employed by Piramal staff to optimise the conditions for:
- [Alkylating agent]
- Incubation Temp
- Incubation Time
- Injection Time
- Injection Voltage
Optimal settings were achieved by performance of 3 experiments which started by looking at an experimental design that considered all parameters. After the first experiment, data was analysed and settings fixed for parameters that were not critical to: overall peak response, resolution, %Intact, HHL, HH, HL, H and L chains. A second and third experiment then narrowed down on the optimal settings. The 2nd and 3rd experiments were performed with less testing as the number of factors involved in the experiment was reduced each time.
After 3 experiments the optimal conditions were selected and a set of “Pre-Qualification” assays performed based on parameters required by ICH Q2 to show that the method was fit for purpose prior to formal GMP qualification. In addition a further intra-assay robustness experiment was performed by varying all critical factors around the selected centre points and this data was then used to identify if the method required tighter control around any of the critical factors.
The use of DoE as a tool to drive development lends itself very well to this technique and allowed a focussed approach to parameter selection and method robustness, allowing for accelerated development time.