**2. Case study: spray drying optimization**

The following case study investigated whether an economical spray drying procedure could be developed with a wide range of operating conditions, where the bioactivity of the growth factor would be preserved when the target protein is embedded in native barley background and the powder quality could be optimized.

### **2.1 Recombinant growth factors at ORF Genetics**

Barley plant (*Hordeum vulgare*) is used at ORF Genetics for the production of recombinant growth factors. The barley grain has a natural inert storage environment to preserve proteins and nutrients for the growing embryo [5]. At large, the nutritional profile of barley consists of starch (65%–68%), total protein (10%–17%), free lipids (2%–3%), β-glucans (4%–9%), and minerals (1.5%–2.5%) [35].

The recombinant growth factor is expressed in the endosperm tissue of the barley seed. The production expression system has been further developed and optimized at ORF Genetics, Iceland. After harvesting, dehulling, and milling of the seeds, the target protein is extracted along with the native barley proteins in an aqueous buffer solution. The suspension is then centrifuged, further purified, and concentrated. The semi-purified growth factor solution then undergoes buffer exchange in preparation for the final formulation.

The continuously expanding MESOkine® portfolio at ORF Genetics represents high-quality, plant-made, endotoxin-free animal recombinant growth factors available for the cell-cultured meat (CCM) industry (https://www.orfgenetics.com/). However, for this case study, human epidermal growth factor (*h*EGF) was selected since it is the most studied growth factor internally at ORF Genetics. Although *h*EGF is not a part of the MESOkine® portfolio, since it is from the human species, the growth factor displays a good model growth factor representative for the purpose of this study. Barley expresses EGF in high yield, and the protein remains stable after processing. The input liquid feed solution containing the extracted *h*EGF, with a native barley matrix that still holds some remaining barley proteins and polysaccharides, was spray dried into a powder form as described below.

### **2.2 Spray drying process**

Spray drying trials were executed using next-generation, laboratory-scale Büchi S-300 Advanced Pro Mini Spray Dryer (*Büchi Labortechnik AG, Switzerland).* The spray dryer was coupled with a Büchi S-396 dehumidifier to ensure consistent humidity of the drying air and equipped with a high-performance cyclone to improve collection of the smallest particles*.* A two-fluid nozzle was used in the trials using air as the drying medium. The spray drying operation was first tested with some feasibility pilot runs.
