The choice between targeted and untargeted assays really depends on the analytical objective, and in practice, they are often complementary rather than mutually exclusive.

For global HCP profiling and process understanding, we generally recommend an untargeted approach, as it allows identification and quantification of a wide range of HCPs without prior knowledge. Within this, DIA is typically preferred over DDA, as it provides more comprehensive and reproducible coverage, particularly for low‑abundance HCPs in complex samples.

For GMP or routine monitoring, however, a targeted approach (e.g. MRM on triple quadrupole) is often used, especially when focusing on known or high‑risk HCPs. This is because targeted assays are easier to validate and provide high precision for predefined analytes. [Gene Thera...e brochure | PDF]

In terms of supporting data, studies aligned with USP <1132.1> demonstrate that:

• Untargeted LC‑MS (e.g. DIA) provides broad HCP coverage and is well suited for characterization and risk assessment
• Targeted LC‑MS (MRM) provides highly reproducible and robust quantification for specific HCPs and is more straightforward to implement in GMP settings

Thomas Kofoed, PhD, CEO of Alphalyse in Denmark, discussed key factors in implementing GMP-compatible LC-MS methods for release assays and highlighted successful cases where this approach has replaced traditional ELISA methods. Don’t miss his presentation and expert insights at the panel discussion.