Attaining an optimum formulation for a recombinant protein could be influenced by different factors [23, 24]. Since one of the biggest concerns of the biopharmaceutical manufacturing is the cost-effectiveness and repeatability of the recombinant protein production process, chemically defined-minimal media as excellent solutions can be used to achieve these two goals [25, 26]. Several studies have attempted to achieve maximal production under optimal conditions. Despite the constructive benefits of utilizing E. coli, high-level synthesis of soluble recombinant proteins can be challenging. Aggregated proteins, known as inclusion bodies, are generally misfolded and physiologically inactive [27]. In many cases, solubilization and refolding of insoluble proteins are time-consuming and labor-intensive procedures that add to the complexity of downstream processing [28, 29]. Several methods have been developed to address these challenges to increase the solubility of recombinant proteins. For instance, an efficient and practical method is to modify culture conditions [30]. According to the accumulated evidence to accelerate the expression of target genes and develop solubility in E. coli, remarkable methods have been developed. Four substantial factors highly affect the production of soluble protein: timing of induction, inducer concentration, temperature, and duration of induction. Nonetheless, efforts to identify optimal buffer conditions frequently rely on re-purification or functional assays, a time- and protein-intensive trial-and-error strategy. An optimal time window exists for the production of proteins. The correlation between the growth rate and the specific protein production rate for the induced batch and fed-batch cultures suggests that a sluggish growth rate under induced conditions produces little or no product. In addition, the duration of expression was limited in batch cultures. This provides information on protein aggregation in the cell and protein quality [31]. Induction is typically conducted during the early mid-log phase, although there are reports of induction occurring during the late-log phase or even during the stationary phase [32].

The recombinant nature of protein drugs allows intervening at upstream stages through protein engineering to produce analogue protein versions with higher stability and enhanced therapeutic values. This highlights the importance of protein engineering and structural analysis in improving the stability of recombinant proteins such as BlaR-CTD. In a study performed by Jianan et al., the stability of BlaR-CTD and its mutant proteins has been compared, revealing insights into their structural and functional properties. The wild-type BlaR-CTD was found to exhibit poor stability, while certain mutant proteins showed enhanced stability [7]. However, changing the recombinant protein structure using point mutations is not favorable in industry as this mutant/ modified protein is not anymore biosimilar therapeutic.

Another approach is to alter process conditions to maximize the wild-type construct stability based on a specific protein stability profile (PSP). A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production [33].

Protein-based therapies are complicated products with obstacles in obtaining stability from manufacturing to patient administration. To obtain stability of recombinant proteins, various strategies can be employed. Several solutions have been investigated, ranging from formulation modification to the invention of delivery systems, in an attempt to solve biopharmaceutical stability limits and safety issues as well as increase product quality and patient compliance.

Additionally, protein aggregation during purification can lead to remarkable solubility issues. Furthermore, proteins are susceptible to solution concentration, whereas recombinant protein aggregates can be solubilized during purification using a variety of buffer conditions [34]. Thus, an effective screening buffer should be considered at various concentrations and under optimal conditions to obtain specific results.

Excipients play a crucial role in stabilizing protein formulations and minimizing potential aggregation. They are additives that help stabilize the protein's structure and reduce unwanted aggregation cascade. Excipients can range from simple buffers to more complex components such as amino acids, sugars, surfactants, and antioxidants. Their mechanisms of action include strengthening protein-stabilizing forces, destabilizing the denatured state, and direct binding to the protein. For instance, surfactants, or surface-active agents, play a major role in preventing protein aggregation [35, 36]. During the manufacturing, storage, and delivery processes, therapeutic proteins encounter various surfaces and materials, such as metals, glasses, oils, and polymers. The inherent surface-active nature of proteins can cause them to interact with surfaces, leading to possible denaturation and subsequent aggregation. Excipients help mitigate these interactions and prevent aggregation, thereby ensuring the stability and quality of the protein formulation [35].

It is important to note that the effects of excipients on protein aggregation during agitation can depend on the balance between the excipient's effects on the conformational stability of the native protein in the bulk solution and the extent of protein gelation at air–water interfaces. Therefore, the selection and use of excipients should be carefully considered to achieve the desired stabilization and minimize aggregation [37]. Various excipients can be used to improve the stability of recombinant proteins. To improve the thermal stability of BlaR-CTD, several excipients were analyzed in this research. On this basis, we used US7745193B2 to design our construction. We produced BlaR-CTD in-house to have an access to bulk recombinant BlaR-CTD for investigating the effect of several factors on the stability of this recombinant protein. Therefore, one of the most innovative parts of this study is to assess the optimum concentrations of several excipients to achieve high thermal stability of recombinant BlaR-CTD.

The conventional method to optimize the formulation is to change one parameter at a time while maintain the other constants. However, because of the high number of required experiments due to multiplex parameters, this approach is not feasible. In addition to the inconvenient nature of this approach, if there is an interaction between different variables, it can cause misinterpretation of the results [17, 38]. RSM is a popularly employed alternative procedure to overcome the aforesaid obstacles. It is a mathematic and statistical utilized to design optimization experiments, to create models, and to evaluate the interplays within different bioprocess parameters; whereas reducing the number of possible experiments [39].

To optimize the formulation of recombinant BlaR-CTD, we designed an experimental RSM/CCD methodology that included seven different factors (Sucrose, Glycerol, Tween-20, Triton, Borate, Tris, and PBS) with four concentrations measured at three different wavelengths (280/260 and 350 nm) and a pH range of 7–9. The point prediction tool of the software was utilized to calculate the optimum value of concentration sucrose (152.5 mM), glycerol (15.3%), and pH (7.6). Eventually, a quadratic model (Eq. 1) was proposed by DOE Minitab software based on the experimental results. The developed model showed that among the three mentioned variables, OD recorded for sucrose and pH had a significant effect in 350 nm and 280/260 nm, respectively.

However, it is suggested that not only further excipients but also other parameters influencing protein stability should be investigated to obtain a higher stability of recombinant BlaR-CTD protein. Therefore, in our upcoming experimental studies, we will assess further on the stability of this recombinant protein.