Scaling hempseed protein isolation from laboratory to pilot scale: Reproducibility, downstream processing challenges, and scale-dependent functionality shifts

The reliable scale-up of plant protein isolation from laboratory to industrially relevant processing conditions remains a major challenge, particularly due to differences in separation efficiency, residence times, and process robustness. Hempseed ( Cannabis sativa L.) press cake is a promising underutilized raw material for sustainable protein ingredient production, yet systematic studies on the reproducibility and engineering aspects of scale-up, including material distribution and downstream separation, are lacking. In this study, hempseed protein isolation by alkaline extraction and isoelectric precipitation was transferred from laboratory scale (6–16 L) to pilot scale (1,000–1,500 L) and evaluated using multiple independently conducted pilot-scale trials, enabling a reproducibility-aware assessment of technical feasibility, process variability, and scale-dependent product functionality. Pilot-scale trials yielded lower, more variable protein recovery (53.8–69.0% dw) than laboratory-scale processing (76.1–77.3% dw), indicating losses largely occurred during downstream processing. Reduced separation efficiency led to lipid carry-over, lower crude protein contents (82.8–84.4% dw), and increased residual fat contents (14.4–17.0% dw) compared with laboratory-scale ingredients. The apparent structural consequence of scale-up was increased protein denaturation associated with prolonged alkaline residence times during continuous pilot-scale separation. This scale-dependent shift reduced acidic protein re-solubility, while simultaneously preserving least gelation concentration and enhancing water holding capacity. Differences associated with the applied downstream powdering were observed in particle size distribution and color, but did not primarily determine functional performance. This study demonstrates a reproducibility-aware engineering framework for hempseed protein isolation scale-up and provides broader guidance for the valorization of oilseed press cakes into functional protein ingredients.