Excipient-Free Spray Drying of Bioactive Recombinant Proteins Produced in Plants

Abstract

Spray drying is an economical drying method for converting aqueous solutions into stable dry powders. This one-step continuous process generates a sustainable solution for long-term storage of various protein formulations. This study focuses on recombinant growth factors produced in a barley seed host. The retained bioactivity of the growth factor in the final solid form suggests that co-purified host components may have preserving effects throughout the optimized spray drying process. To identify the critical spray drying parameters, a customized response surface design of experiment was applied. The defined input spray drying parameters: feed flow rate, spray gas flow rate, and outlet temperature, as well as their interactions, were discovered to be the most critical in terms of product quality and yield. The best operating parameters were chosen after considering potential reduction in energy consumption of the process. Cell proliferation assay results, which show the bioactivity of the growth factors, reveal that the native host components seem to act as proper stabilizing agents that protect the fragile growth factors against various stresses during the drying procedure. This unique matrix composition therefore surpasses the time-consuming process optimization with excipients, allowing for a fully continuous process from purification to the final formulated powder.

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 cost-effective, 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.

Figure 1.

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

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 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 spray-dried 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.

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 (hEGF) was selected since it is the most studied growth factor internally at ORF Genetics. Although hEGF 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 hEGF, 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.

2.2.1 Design of Experiment (DoE)

DoE is a systematic approach to simultaneously evaluate the effects and interaction of multiple factors that influence the responses of a process. DoE is a component of Quality by Design (QbD), which is a recommended statistical practice in the formulation of drug products by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human use (ICH) [17, 36].

Quality characteristics for each product must be pre-determined and must be able to be accurately measured. The measured quality characteristic is called a response. The quality of spray-dried products is influenced by several factors. Arthur et al. [37] used DoE to optimize the spray drying process of beer powder and found that moisture content, water activity, solubility, pH, and product yield were the most influential factors. Ziaee et al. [38] successfully spray-dried solid dispersions of ibuprofen and used DoE to identify that residual moisture content and particle size were the critical factors for the final yield, but the API/excipient ratio was critical to formulating samples. For our case study, we chose the following factors to investigate and optimize: Feed flow rate (FFR), spray gas, and outlet temperature. Preliminary operation tests assisted in designing the study. The following factors were kept constant between trials: Starting feed solution was identical (constant temperature and product dry weight % w/v), and the drying gas flow rate (aspiration) was kept at maximum capacity.

JMP Pro Version 17.0 (SAS Institute Inc., Cary, NC) was used for statistical analysis and model building. We used Response Surface Methodology (RSM) with an I-Optimal, custom, DoE design, consisting of 16 runs. RSM is an effective tool to optimize process parameters quickly and efficiently. It can explore the effects of multiple factors simultaneously, investigate factor interactions, and predict the resulting response.

2.2.1.1 Factors and responses

The FFR, spray gas flow rate, and outlet temperature were defined, in this study, as the potentially critical process parameters, also known as factors in this study. The factors and investigated level ranges are listed in Table 1. The levels selected represent the experimental space of the DoE. The selection was based on previous in-house trials and technical restrictions.

Factor	Level
FFR (mL/min)	4–7
Spray gas (L/h)	600–1800
Outlet temperature (°C)	60–100

Table 1.

Input factors selected in the DoE custom design used for this case study.

The influence of the factors and their interactions resulted in varying powder qualities and characteristics. These effects were measured via the selected output responses: powder output, reconstitution performance, hEGF/powder, and total protein/powder to evaluate in this study; see Table 2.

Response	Unit of measurement
Powder output	% w/v
Reconstitution score	Scored from 1-4
hEGF/powder	μg/mg
Total protein/powder	μg/mg

Table 2.

Output responses selected in the DoE custom design used for this case study.

2.2.1.2 Powder output

The powder output (% w/v) was defined based on the spray-dried powder (g) collected per input liquid solution (mL). The amount of input liquid solution was 50 mL for each run; the exact volume was recorded and considered for the powder output calculations. The vessel collecting the dry powder from each run was weighted before and after the run, to determine the amount of collected powder from the run. For example, if 4.9 g of powder was generated from 50 mL of input liquid, the powder output was determined as 9.7%.

2.2.1.3 Reconstitution of powder

For the reconstitution score assessment, the solid powder sample was dissolved in the solvent (Milli-Q® water) to a dry weight percentage of 8% w/v. This value resembled the approximate dry weight (% w/v) of the input liquid, when comparing the product output across the samples. This was kept consistent between sample runs by dissolving 200 mg powder in 2.5 mL. Upon reconstitution, the solution was left undisturbed for 15 minutes and then vortexed briefly to obtain a homogenous state. The solution was then visually inspected and ranked based on the solubility performance, the intensity of cloudiness, and color formation. The solubility capability of the dissolved powder was ranked from 1 to 4, from bad (1) to good (4) solubility. Low scores (1) were given to cloudy solutions with visible particles, and high scores (4) were given to solutions that were clear and fully homogenous with no visible particles. Solutions from the reconstitution assessment were used further for the quantifications of the growth factor (see Section 2.2.1.4) and the total protein (see Section 2.2.1.5).

2.2.1.4 Growth factor quantification

For hEGF/powder (μg/mg) assessment, the amount of hEGF target protein was quantified by capillary-based nano immunoassay, JESS Simple Western™ (ProteinSimple®, Bio-Techne, Minneapolis, MN, USA). The hEGF amount was quantified within each reconstituted sample based on a generated standard curve of hEGF with a known hEGF concentration and the concentration of hEGF within the powder, which could then be calculated compared to the standard. The hEGF/powder (μg/mg) value indicates the product yield and stability of the growth factor after the spray drying step. Receiving a low concentration can, for example, indicate aggregation or irreversible protein denaturation as an indirect estimate, such as the hEGF not being able to dissolve after spray drying. Refer to Appendix A for a more detailed method description for the growth factor quantification.

2.2.1.5 Total protein quantification

Total protein/powder (μg/mg) was measured with Bradford assay. Since the growth factor is semi-purified, other barley proteins are also present in the final product, and these need to be considered. Therefore, native barley proteins were quantified, along with the recombinant growth factor. Reduction of the total protein within the powder can, for example, indicate that the protein is forming an insoluble material due to the spray drying process. Refer to Appendix A for a more detailed method description for the total protein quantification.

2.2.2 Optimization: Analysis of DoE data

The data were fit with an RSM model with linear regression (Eq. (1)), and model reduction was performed by enforcing a 95% confidence interval, including only factors and interactions with a p-value <0.05 and factors containing statistically significant effects; refer to Table 3. As with the factors, a 95% confidence level was chosen for the responses in the model. The responses in the RSM model that had p-value <0.05 were considered statistically significant; others were disregarded. This RSM model can only predict outcomes for the responses “powder output” and “reconstitution score.”

Factors and interactions	P value	Responses	P value
Outlet temperature (60, 100)	0.00057	Powder output	0.0010
FFR * Outlet temperature	0.00169	Reconstitution score	0.0029
FFR * Spray gas	0.00556	hEGF/powder	0.7066
Spray gas * Spray gas	0.00654	Total protein/powder	0.4137
Spray gas (600, 1800)	0.00730 ˆ	—	—
Outlet temperature * Outlet temperature	0.01846	—	—
FFR (4, 7)	0.05531 ˆ	—	—

Table 3.

Effect summary. Factors and interactions that influence the responses in the RSM model and their corresponding P values.

Outlet temperature (°C), Spray gas (L/h), and FFR (mL/min). ˆ denotes factors with containing effects above them.

$$Y=a+\sum _{i=1}^3{b}_i{X}_i+\sum _{i=1}^3{c}_i{X}_i^2+\sum _{i=1}^2\sum _{j=i+1}^3{d}_{\mathit{ij}}{X}_i{X}_j$$E1

Y is the yield; a is the intercept; b_i, c_i, and d_ij are model coefficients; and X_i and X_j represent the model regressors.

A single sample, sample #3, was eliminated from the DoE analysis due to a loss of powder to the cyclone, for technical reasons. However, since this error did not influence the bioactivity of the sample, sample #3 was included in the following bioactivity measurements and the SEM analysis. All other samples and data were included, and no outliers were detected. Factors and response values are summarized below in Table 4.

Factors				Responses
Sample run	FFR (mL/min)	Spray gas (L/h)	Outlet temperature	EGF/powder (μg/mg)	Total Protein/powder (μg/mg)	Powder output (% w/v)	Reconstitution (score)
1	4	1800	60	8.31	408.1	9.80	4
2	7	1800	100	7.99	411.8	9.68	2
3	5	1200	80	8.41	386.5	7.34	2
4	7	600	60	7.57	377.6	8.29	2
5	4	600	100	8.15	347.6	8.41	1
6	4	600	60	6.33	379.9	8.73	3
7	5	1200	60	6.46	390.6	9.88	3
8	5	1800	80	6.93	370.6	9.96	3
9	4	1800	100	7.58	370.5	9.86	2
10	7	1200	80	7.59	410.2	9.77	3
11	5	600	80	7.19	326.4	9.19	3
12	4	1236	80	6.66	355.4	9.92	3
13	7	600	100	7.39	331.4	9.47	2
14	7	1800	60	6.70	363.6	7.9	3
15	5	1200	100	7.06	361.0	9.79	2
16	5	1200	80	6.74	375.9	9.8	3

Table 4.

Experimental design matrix, containing input factors and output responses from all sample runs.

The variation in the amount of hEGF and total protein per powder, respectively, could not be explained by the factors included in the model. This confirms that the spray dryer settings, even at extremities, do not affect either the growth factor quantity or the overall total protein quantity. This data shows the robustness of the spray drying process within the tested ranges.

Powder output and reconstitution score were found to be statistically significant, and the model can therefore explain the variation in their responses. The outlet temperature affected reconstitution the most, followed by the interactions of FFR x outlet temperature and FFR x spray gas flow rate. For the powder output, the interactions of FFR x outlet temperature and FFR x spray gas flow rate were the most impactful. Figure 2a illustrates factor interactions.

Figure 2.

Effects of the factors on the responses. (a) Interaction profilers for powder and reconstitution show how the different factors interact to affect the responses. (b) Prediction profiler for maximized responses and optimal setting.

A quadratic effect was observed in the model for both the spray gas flow rate and the outlet temperature. A quadratic effect in a statistical model means that an optimum has been observed in the defined experimental space. This can be visualized by a curvature in their responses, as illustrated in Figure 2b. Optimum settings, within the tested range, were found for outlet temperature and spray gas flow rate, as curvature was observed in their responses. The quadratic effect for outlet temperature was only significant for powder output, and the quadratic effect for spray gas was only significant for reconstitution.

The optimized settings of the input factors to maximize the responses in this case study were found to be the following:

$$\begin{array}{c}\mathbf{\text{Feed Flow Rate (FFR)}}:4\mathrm{\text{mL/min}}\\ \mathbf{\text{Spray Gas Flow Rate}}:1400\mathrm{\text{L/h}}\\ \mathbf{\text{Outlet Temperature}}:60°\mathrm{\text{C}}\end{array}$$

The software calculated the maximized spray gas flow rate settings at 1800 L/h, but we took economic considerations into account when selecting the optimized settings and lowered the selected spray gas flow rate settings to 1400 L/h.

The RSM approach of the DoE in this case study was successful and provided optimum parameter settings for spray drying growth factors in a stabilizing barley matrix with high quality powder and had no impact on the quantity of the target protein or the barley proteins.

2.2.3 Particle morphology with scanning electron microscope (SEM)

Particle morphology analysis was carried out to investigate whether there was a correlation between microscopical particle shapes and the applied spray drying conditions. The surface morphology of the spray-dried particles was examined using a field emission scanning electron microscope (FE-SEM), Supra 25 by Zeiss (Oberkochen, Germany). Powder samples were mounted to a sample stub and gold coated. Samples were scanned at a voltage of 3.0 kV, and their images were captured at two magnification levels, 1000× and 5000×.

Selection criteria for the sample runs ultimately taken for SEM characterization were based on analyzing the morphology of the samples expected to have experienced the upper and lower limits for the outlet temperature, reconstitution rating, and particle size.

The analysis of the SEM images served as a qualitative assessment for the given range of parameters in the study. Morphology classification for spray-dried particles as suggested by Prinn et al. [39] can be divided into four categories: (I) smooth spheres, (II) collapsed or dimpled particles, (III) wrinkled or raisin-like particles, and (IV) highly crumpled or folded structures.

The study design range demonstrated a distribution of most of the above-mentioned different shapes but mostly showed smooth spheres and raisin-like structures. Shown in Figure 3 are the samples expected to exhibit the smallest particles (sample #9; 4 ml/min, 100°C and 1800 l/h) and the largest particles (sample #4; 7 ml/min, 60°C and 600 l/h). By comparing these two different particle formations, these reveal only raisin-like particles for the smallest ones (Figure 3a), whereas the largest ones show a combination of the two morphologies, smooth spheres and raisin-like particles (Figure 3b). The smaller particles seem to tend to clump together. The other tested formulations showed varying amounts of large smooth spheres with smaller particles always forming raisin-like structures with some indications that the higher outlet temperature results in more crumbled structures compared with lower outlet temperatures where smooth surface is more prevalent (data not shown). In other ways, the SEM analysis reveals that particle morphology is not susceptible to changes within the selected range of process parameters.

Figure 3.

SEM images of the spray-dried particles at magnification 1000×. Shown are captures from the following samples: (a) Sample #9, expected to have the smallest particles, here shown as several raisin-like particles crumpled together. (b) Sample #4, expected to have the largest particles, here shown as a mixture of larger, smooth spheres; collapsed particles; and wrinkled particles.

2.2.4 Bioactivity of growth factor

A cell proliferation assay for hEGF using 3 T3 fibroblast cells was performed by SBH sciences (Natick, MA) to measure the biological activity of the growth factor for selected samples. The cells were seeded on multi-well plates and incubated with a dilution series of a commercial growth factor standard. After a pre-defined incubation period, cell proliferation or cell death had been measured using a colorimetric assay. The biological activity of hEGF was expressed as ED50 (effective dose), which is the concentration of the growth factor that induces 50% of the maximum assay response. Thus, the lower the ED50 value, the higher the activity.

The sample selection for the bioactivity analysis is summarized in Table 5. This assay was performed to determine whether there was a correlation between biological activity and the other output responses investigated in the case study.

Sample run	Details	FFR (mL/min)	Spray gas (L/h)	Output temperature (°C)	ED50 value (ng/mL)
1	Reconstitution score significantly better than the others	4	1800	60	0.05–0.08
2	Upper extreme conditions for all factors	7	1800	100	0.08–0.11
3	Mid-point of factor levels	5	1200	80	0.06–0.09
5	Reconstitution score significantly worse than the others	4	600	100	0.05–0.07

Table 5.

Sample runs selected for the bioactivity measurement and the resulting ED50 values.

The bioactivity curves of all samples were tested for parallelism to determine whether the samples were statistically different from each other. Parallelism was determined by an F-test using JMP Version 17.0 (SAS Institute Inc., Cary, NC) software; see Appendix B.

All samples were found to retain their bioactivity after spray drying. Sample #2 was found to have the highest ED50 value, and it was found to be different from the other samples, except for sample #3. Sample #2 had the most extreme settings for all the factors. This indicates that the combination of the extreme process parameters might decrease the bioactivity of spray-dried growth factors. This, however, needs further investigation.

In summary, the biological activity of the growth factor was not disrupted for any of the applied process parameters. Spray drying is a robust process to dry recombinant growth factor solutions while preserving biological activity.

3. Continuous, sustainable, and cost-effective process

Spray drying presents clear advantages over freeze drying, especially regarding great scalability capabilities and automation options. It has the potential of making the preservation process more economical.

3.1 Continuous system

A continuous process system with a sample bag connection design at a laboratory scale that was set up in-house is shown below in Figure 4. The protein liquid in a sterile bag, retrieved straight from the final downstream process filtration, can be fed directly into the spray dryer, surpassing the need for further formulation with the addition of excipients. After the process, the output powder assembles into the collection vessel, which can then be aliquoted into smaller dosages of powder samples for storage.

Figure 4.

Continuous spray drying system with sample bag connection design.

3.2 Economical drying

Reducing the carbon footprint is unquestionably a big industrial focus. Making the shift from freeze drying to spray drying may, in fact, represent a significant step toward reduced energy consumption, thereby lowering the overall climate footprint. Baeghbali et al. [40] compared the energy consumption of spray drying and freeze drying, showing that the spray dryer required less than 10% of the energy consumed in the freeze drying process, even though noting that the spray drying process was suboptimal and improvements could be made [40].

The DoE approach, in this study, allowed evaluation of the process based on the estimated energy expenditure. Since drying air flow was kept constant throughout the experiments, the aim would be to minimize the inlet air temperature (energy usage per unit time) while maximizing the feed flow rate (reduced processing time) without affecting the quality attributes negatively. Other researchers have shown a strong correlation between the inlet temperature and the outlet temperature [39, 41, 42], whereas they show lesser [39] or even non-significant influence [42] of the feed flow rate on the temperature relationship despite testing much wider feed flow rate ranges than in the current study (3–20 ml/min and 7.3–17.5 ml/min, respectively). This indicates that to reduce the energy consumption in the process, a design space should be created with low outlet temperature and high feed flow rate. In the current study, the best results were achieved at low outlet temperature, which supports reduced energy consumption, whereas the optimized low feed flow rate increases the energy expenditure. The strongest drive in the model to keep feed flow rate at low levels comes, however, from the sharp drop in powder output at higher feed flow rates, meaning a loss in yield at higher feed flow rates. Therefore, considering that the spray drying operation is the last step in the manufacturing process, a drop in yield results in wasted energy and resources in all manufacturing steps upstream from the spray dryer, hence justifying the use of lower feed flow rate for overall reduction in energy consumption.

To evaluate the energy usage of the process, a compact energy meter, Energy-230 Micro LCD (Vemer, Italy), was connected to the spray dryer and the dehumidifier. This equipment is designed to display the consumption of active energy in a single-phase system. The electricity usage was roughly 10 kWh when drying one liter of hEGF solution. This is consistent with the results of Baeghbali et al. [40], showing that a lab-scale spray dryer coupled with a dehumidifier can manufacture high-quality, dry hEGF powder with only a fraction of the energy required for freeze drying [40].

3.3 Future considerations

Moisture content in spray-dried powder can influence the overall product stability. Higher feed flow rates potentially result in undesirably high water content. Furthermore, the glass transition temperature has been linked with feed flow rate in similar manners as the residual moisture acts as a plasticizer, increasing molecular mobility at lower storage temperatures [43]. Therefore, lower moisture content in dry powders exhibits better long-term stability for protein [44]. Further investigation is needed toward evaluating the powder quality, including comparison of the moisture content, which shall be considered as an added critical output response in future studies. Also, in later strategies and assessments, a long-term stability study of the storage capabilities of the powder should be investigated.

Spray drying is ideal for food-grade material production, like animal-derived recombinant growth factors that are a crucial component of the serum-free media for CCM production. Since cost reduction of growth factors is important for the ultimate success of cell-cultured meat [45], the production should be tailored to this given industry. Food-grade media with lowered associated costs that could still maintain cell proliferation and differentiation at a larger scale would be considered as a success. Later, translation of this application to pilot scale and then eventually to industrial scale will be needed to ensure feasibility to hand the process over to a larger spray drying production.

4. Conclusions

• The novelty of this study was to illustrate that the stabilizing effects, generally obtained from excipients, are suggested to be already present from the native barley background matrix, which is a part of the final product.

• The growth factor tolerates high outlet temperatures while retaining its stability in the powder and bioactivity and showing limited effects on the morphology scale.

• The amount of powder collected from the process, along with the ease of powder reconstitution after processing, had the largest effects on the resulting optimization model.

• The spray drying process in this study appears robust enough to surpass the need to add external excipients to the semi-purified growth factor formulation.

• This study demonstrates the feasibility of spray drying bioactive recombinant growth factors embedded in native barley matrix at a laboratory scale.

Acknowledgments

Thanks go Dr. Raphael Nir (SBH Sciences, Natick, MA) for biological activity measurements in cell proliferation assay.

Thanks go to Tæknisetrið, Reykjavik, Iceland for operational assistance and access to their field emission scanning electron microscope (FE-SEM) for the particle surface morphology characterization.

Conflict of interest

The authors declare the following interests: affiliation with ORF Genetics, either through employment¹ or paid consultancy services².

Notes

ORF Genetics owns a patent on the barley expression system, ORFEUS™. For this reason, confidential information cannot be disclosed relating to the exact purification procedures and full LC/MS data of the commercial products under the portfolio MESOkine.

Appendix A – Materials and Methods in Case Study

Jess Simple Western

JESS Simple Western™ instrument (ProteinSimple®, Bio-Techne, Minneapolis, MN, USA) is a fully automated and capillary-based system that performs all downstream steps of sample preparation [46]. For preparation of the separation module a 2–40 kDa separation module from ProteinSimple was used which includes a capillary cartridge, pre-filled microplates, wash buffer, 10× sample buffer, lyophilized fluorescent 5x master mix, lyophilized DTT and a lyophilized biotinylated ladder. Standard pack reagents were prepared per manufacturer recommendations, for the 400 mM DTT solution preparation, the fluorescent 5X master mix and the ladder. The samples and hEGF standard were prepared. The samples and the standard hEGF, with a known concentration, were serially diluted down to 3–104 ng/mL using 0.1× sample buffer. Then, fluorescent 5X master mix was added to all measured samples, so the final mixture is 1 part master mix and 4 parts sample. The samples and standards were heated at 95°C for 5 minutes. The samples were then mixed again by vortex and then spun down and stored on ice. For detection, an anti-rabbit detection module from ProteinSimple was used which includes anti-rabbit secondary antibody, Luminol S, Peroxide, Streptavidin-HRP and antibody diluent. For preparation of the primary antibody, polyclonal rabbit anti-hEGF antibody (ab9695, Abcam) was diluted in 1:50 ratio with antibody diluent. For the preparation of the chemiluminescence substrate, 200 μL of luminol-S and 200 μL of peroxide was mixed in a microcentrifuge tube. Then the prepared samples, the biotinylated ladder, antibody diluent for washing and blocking, the diluted primary antibody, the secondary antibody, Streptavidin-HRP for biotinylated ladder capillary only, and luminol-peroxide mix were pipetted into the microplate. The plate was spun down at 1000 ×g for 5 minutes followed by addition of wash buffer and all air bubbles removed with ethanol vapor and the plate placed into Jess and the right program selected in Compass Simple Western ™ software.

Bradford assay

A Bradford assay was performed to determine total protein concentrations. For each sample, standard or blank (Milli-Q water), 10 μL was pipetted into a microplate well in triplicate. Into each microplate well, 300 μL of Bradford reagent from Thermo Fisher was added and mixed for 30 seconds on a shaker and then incubated for 10 minutes at room temperature. Absorption was measured at 595 nm with a microplate reader (Thermo Fisher Multi-scan FC). The average from the triplicate Blank was then subtracted from all other measurements and a 4PL standard curve generated from a pre-diluted Pierce™ Bovine Gamma Globulin (BGG) from Thermo Fisher with a concentration range of 125 μg/mL – 2000 μg/mL. The standard curve is then used to determine the total protein concentration of each unknown sample.

Appendix B – Design of Experiment (DoE) Results

Parallelism test in bioassay curves

To test for Parallelism in the Bioassay results, curves were plotted from the Net OD at 490 nm against the log of the concentration (ng/mL). The curves were fitted with a Logistic 4PL fit and a Parallelism test was run between all the possible combinations of curves. A Parallelism F-test where the Prob > F was ≤0.05 means there is no parallelism and therefore the samples are statistically different from each other.

Bioactivity

See below analysis for bioactivity curves of all samples (Figure B.1), raw data of the bioactivity results (Table B.1) and results of statistical analysis on bioactivity curves (Table B.2).

Figure B.1.

Bioactivity curves of all samples.

Net O.D. (ng/ml)
Sample	1	2	3	4	5
	1.000	0.618	0.614	0.587	0.568
	0.333	0.611	0.526	0.542	0.568
	0.111	0.419	0.329	0.376	0.385
	0.037	0.204	0.150	0.181	0.202
	0.012	0.120	0.062	0.106	0.109
	0.004	0.038	0.011	0.033	0.069
	0.001	0.014	−0.005	−0.003	0.024
	0.000	0.008	−0.017	−0.004	0.024
	0.000	0.003	−0.009	−0.005	0.029
	0.000	0.000	0.020	0.012	0.000

Table B.1.

Raw data of the bioactivity results.

Sample	Prob > F	Difference
3 + 5	0.9917	No
3 + 1	0.0571	No
3 + 2	0.1564	No
5 + 1	0.0664	No
5 + 2	0.0205	Yes
1 + 2	0.0017	Yes
3 + 5 + 1	0.1	No
3 + 5 + 2	0.1953	No
3 + 1 + 2	0.0062	Yes
1 + 5 + 2	0.0042	Yes
3 + 5 + 1 + 2	0.0103	Yes

Table B.2.

Results of statistical analysis on bioactivity curves.

Abbreviations

CCM	Cell-cultured meat
cGMP	Current good manufacturing practice
DoE	Design of Experiments
EGF	Epidermal growth factor
FFR	Feed flow rate
FGF-b	basic Fibroblast growth factor basic
GRAS	Generally regarded as safe
GFs	Growth factor
ICH	International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human use
IGF-1	Insulin-like growth factor 1
QbD	Quality by Design
RSM	Response Surface Methodology
SD	Spray drying
w/v	Weight per volume