**1. Introduction**

Plant molecular farming has grown and advanced immensely into a viable platform to produce commercial proteins [1]. Plant systems have many advantages over their counterparts, mostly in terms of ease in process scale-up, but they are also costeffective, versatile, and robust. After harvest, plant hosts like the barley seed can be stockpiled under ambient conditions for many years, which largely benefit protein production and enable separation of downstream from upstream processing. Crude

materials can therefore be collected and stored long term, and purification can occur at convenience, reinforcing the flexibility of the system [2].

Expression hosts for the production of recombinant proteins, like animal growth factors, mostly range from prokaryotic to eukaryotic systems, such as bacteria, yeast, insects, and mammalian cell cultures. The reason being, these already established expression host systems have been well-defined with current good manufacturing practice (cGMP) and generally offer high production capacity at low cost [3]. Transgenic plants, however, are more unconventional but have in the past two decades emerged in biopharma as an alternative production system [4]. One of those innovative plant systems is the barley plant, which displays a biologically contained expression host that is convenient in various ways. Barley has a generally regarded as safe (GRAS) status and constitutes an endotoxin-free host expression system, as it avoids bacterial host cell-derived endotoxins [5]. Also, barley does not contain secondary metabolites or mammalian-derived pathogens, such as virus infections [6].

The downstream processing involves extracting and purifying the recombinant growth factors from the barley seed, resulting in an aqueous protein solution. The recombinant growth factor is co-purified along with beneficial barley components thought to enhance the final product, as well as ease the purification procedure. The inherent instability of proteins in aqueous solutions is caused by the molecular mobility in solutions. This has been overcome by, for example, storing and transporting the proteins under frozen conditions [7], but dry powder formulations of protein simplify all handling, storage, and distribution, offering an intriguing alternative [7]. The final formulation of purified growth factors is therefore more conveniently given as dried powder with preserved bioactivity for effective storage, easier handling, and more economic shipping options. Dried formulations can circumvent the need for cold chain transport, thereby offering an economically feasible solution for storing and transporting more growth factor per weight at ambient conditions [8].

Spray drying transforms liquid feed into dry particles. However, unlike freeze drying, spray drying is a continuous process that dries the product material in a single manufacturing step. During the spray drying of proteins, the feed solution is atomized into heated air, and the solvent evaporates quickly, leaving behind dry particles that need to be separated from the airstream and collected [9]. This rapid solidification prevents the molecules from arranging into crystal lattices, allowing mainly just amorphous particles to form. Homogenous powders are produced by the spray drying process as a result [10]. This process is illustrated by Büchi (*Büchi Labortechnik AG, Switzerland)* in **Figure 1**.

Some of the process parameters that can be optimized in spray drying include the inlet/outlet temperature, spray gas flow rate (atomizing gas), drying gas flow rate (aspirator rate), liquid feed flow rate (FFR), and concentration and composition of the solution [12]. The outlet temperature is usually considered as a dependent variable, resulting from the combination of the other parameters, although the droplet/ particle temperature will never exceed the value of the outlet temperature [13], making it a critical parameter on its own for thermosensitive molecules, such as growth factors. The process is optimized by testing different parameter combinations. The resulting product quality is evaluated based on particle characteristics, including size, shape, flowability, density, and moisture content. All these output effects depend on the selected combination of the process parameters, the solvent, and whether any excipients have been added.

Although freeze drying has been recognized as the gold standard of drying methods [14], traditionally being considered the process of choice for improving the *Excipient-Free Spray Drying of Bioactive Recombinant Proteins Produced in Plants DOI: http://dx.doi.org/10.5772/intechopen.112944*

**Figure 1.** *Spray drying functional principle. With permission from Büchi Labortechnik AG, Switzerland [11].*

long-term storage of manufactured protein, there are some essential challenges to consider. Among others, those are the process scalability, batch-to-batch variance, occurrence of crystallization, and high associated costs [15]. Freeze drying is also restricted by an economic drawback that relates to long and energy-consuming processing time [16]. Consequently, spray drying has started gaining more prevalence as a reliable drying formulation method. That includes protein formulations in established and highly regulated industries such as biopharma [17]. Even though spray drying is considered to be a gentle drying process [17], it is intrinsically more aggressive than freeze drying, since the product is introduced to hot gas during evaporation of the droplets that are sprayed during the process. Therefore, the spray drying process still needs to be carefully examined, especially for the more sensitive proteins, like growth factors.

Drying and dehydration, in general, whether it is spray or freeze drying, puts additional stress on proteins. During drying, hydrogen bonds supplied by water are broken, which may cause conformational changes and thereby inactivity of the protein. The process can expose the product to various interface and shear stresses, which could result in reduced product stability during storage [8, 18, 19]. To counteract this, excipients may be introduced into the formulations. These excipients must replace the hydrogen bonds formerly supplied by water (water replacement theory) and form a viscous matrix around the protein molecules to hinder any molecular motion (vitrification theory), preferably with a high glass transition temperature to increase the storage stability [15, 17]. Traditionally, these excipients have been *i)* non-reducing sugars, such as trehalose and sucrose [15]; *ii)* sugar alcohols such as mannitol and sorbitol [15]; *iii)* oligo- and polysaccharides, such as dextrins and dextrans [15]; and *iv)* single amino acid, such as arginine, leucine, and glycine [15]. For example,

disaccharides are excipients thought to have a stabilizing effect on proteins by forming direct hydrogen bonding with them in solid state, thus stabilizing the structure, in addition to forming a highly viscous matrix around the proteins, slowing down molecular movements and thereby degradation [20, 21]. They assist in maintaining the homogeneous and native protein structure, resulting in a stable formulation [20, 22, 23]. Additionally, surfactants may be required to eliminate protein surface adsorption to the abundant air liquid interface created in the atomization step [15, 17].

*Pinto et al.* [15] recently published a comprehensive discussion on the latest development of modern-day trends and the contemporary progress in protein pharmaceutical formulation. Matrix forming excipients are commonly used for protecting protein molecules during the spray drying process and storage [15]. Knowledge about formulation and selection of excipients for freeze-drying proteins can often be applied for spray drying as well [24]. Although excipients are widely recognized for effectively stabilizing many protein solids for freeze-drying formulation [25], *Chen et al.* [26] recently pointed out that a systematic examination of excipient effects on protein stability in spray-dried solids, specifically, is still limited. Critical understanding of the respective interrelation of protein-matrix is still lacking, in addition to a deficient understanding of the storage stability of spray-dried material [26].

For spray drying of proteins, other proteins, such as various albumins, have been studied as excipients in spray-dried formulations [15]. They have even been shown to competitively occupy the particle surface, thereby protecting the protein of interest against surface accumulation and concomitant deactivation [27]. Barley proteins are interesting excipients for the food industry and are considered valued external excipients for the encapsulation of some bioactive ingredients both in spray drying [28] and in freeze drying [29]. It was shown by *Wang et al.* that encapsulation of fish oil with barley proteins had a protective effect against oxidation [28] and by *Meira et al.* that barley residue proteins from beer waste could be used as coating material in microencapsulation of β-carotene [29]. This makes the use of background barley proteins for growth factor dry powder stabilization interesting to investigate further.

Limited published work exists for the spray drying of growth factors explicitly. Growth factors are bioactive proteins that stimulate cell proliferation and differentiation. They have a collective function to expand, maintain, and differentiate cells. There is precedent for spray drying basic fibroblast growth factor (FGF-b) and insulin-like growth factor 1 (IGF-1). In these studies [30, 31], the growth factors are expressed in different host systems than barley. Industry-wide, FGF-b is notorious for being problematic to work with and is constrained by its lack of stability, especially in aqueous solutions. Due to its rapid degradation rate, formulating FGF-b into a reliable product has remained a great challenge [32]. *Ibrahim et al.* [30] developed a spraydried FGF-b using lactose and leucine as excipients, among others, lactose being a well-known matrix former [15] and leucine producing a hydrophobic surface due to its surfactant properties. IGF-1 promotes cell growth by resulting in a higher cell density and reducing cell death [33]. *Schultz et al.* [31] showed for IGF-1, encapsulated in trehalose, that the bioactivity remained unaffected after spray drying. In terms of recombinant proteins, a recent study by *Vilatte et al.* [34] illustrates spray drying as a viable preservation technology for recombinant proteins produced in microalgae.

Current study aims to investigate the suitability of spray drying recombinant growth factors generated in the barley seed host, co-purified with other host barley components. Generally, recombinant growth factors are fully purified to exclude other components derived from the expression system, but here, other barley

components are still present during the preparation of the final product. The goal of this research is to provide usable findings to other scientists working with plant expression systems to produce recombinant proteins. This is the only existing case study that covers the spray drying of recombinant protein expressed in barley. To the best of our knowledge, co-purifying barley has no precedence.
