From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium Pseudoalteromonas haloplanktis TAC125

Marine bacteria are increasingly explored as alternative microbial platforms for the production of high-value biopharmaceuticals. In this study, we investigate the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), an unconventional host capable of yielding soluble and biologically active human cyclin-dependent kinase-like 5 (hCDKL5). This serine/threonine kinase plays a crucial role in neuronal development, and its deficiency causes CDKL5 Deficiency Disorder, a severe and currently untreatable neurodevelopmental disease. Recombinant production of hCDKL5 is a prerequisite for the development of enzyme replacement therapy; however, current manufacturing processes remain insufficient for industrial translation, particularly in terms of product quality and functional consistency. To address these limitations, we developed dedicated analytical strategies: protein accumulation was quantified using a customised sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to selectively detect full-length hCDKL5, while protein functionality was assessed by mass spectrometry-based quantification of autophosphorylation, a critical determinant of kinase activation. These complementary tools were applied to characterise hCDKL5 production under different growth conditions. Overall, this work establishes an integrated analytical framework aligned with a Quality by Design approach, enabling the simultaneous assessment of yield, structural integrity, and functional activation, and providing a robust basis for rational process optimisation towards scalable hCDKL5 manufacturing.