**5. Preformulation studies for biopharmaceuticals development: proteins, peptides, and vaccines**

The concept of recombinant DNA technology and clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 is flourishing for biopharmaceutical product development. These technologies when coupled with artificial intelligence gel large genome data for the proper designing of peptide, protein, and vaccine products [70]. Proteins are classified into secondary, tertiary, and quaternary structures based on their kinds, which in turn affects each structure's molecular size. With the help of the prodrug approach, the vulnerable peptide backbones undergone proteolytic cleavage can be mitigated. Therefore, peptides can be chemically altered to create more stable and high plasma-concentration prodrugs. The prodrug can be produced by chemical modification and substitution reactions like dehydroamino acid substitution, D-amino acid substitution, thio-methylene modification, carboxyl reduction, and PEG-amino acid joining [71].

Preformulation studies are facilitated the right path of selecting the appropriate adjuvants and other formulation conditions required during manufacture. For example, the stability profile of a live attenuated Ty21a bacterial typhoid vaccine performed by spectroscopic techniques provides time-dependent real-time high throughput information at a broad range of temperatures (10–85°C) and pH (4–8). The above information is useful during preformulation studies of other similar type of peptide drugs. An empirical phase diagram, which was created using data from circular dichroism and fluorescence techniques suggests Ty21a cells exist in various physical states and the most stable state occurs at pH 6–7 below 30°C. Among other potential stabilisers, 10% sucrose and 0.15 M glutamic acid show the strongest protective properties, increasing Ty21a cells' transition temperature by roughly 10°C each. Foam-dried formulations have also been the subject of preliminary research as a potential alternative strategy for further stabilising Ty21a cells. Additionally, in-process stability can be improved by 10% sucrose and trehalose solutions [20, 72].
