The resulting crude protein hydrolysate may undergo fractionation processes to yield an enriched Bleomycin chemical structure bioactive peptide preparation or additional purification steps to isolate single peptides. Following the identification of the sequence of the isolated peptides, bioactivity is validated by testing chemically synthesized pure peptides. The plethora of literature abounding on bioactive peptides derived from proteins notwithstanding, most of these empirical studies have not recognized the importance of using a systematic approach for process development, to optimize the multiple factors that affect production and purification. Hanke and Ottens [4•]

commented that trial-and-error and one-factor-at a time experimentation is largely obsolete, being replaced by systematic design of experiments (DOE) approaches incorporating the ‘science,

process understanding and risk management to design the production process to consistently deliver the pre defined quality objectives’. Knowledge based process development requires an understanding of the critical process parameters GW-572016 (CPP) that affect critical quality attributes (CQA) [4•]. Examples of CPPs for bioactive peptide production are characteristics of the starting source material (e.g. protein content, other major and minor constituents, pH, variability by season) and enzyme preparation (purity, substrate specificity, specific activity, single or multiple enzymatic activity, optimal pH and temperature

conditions for activity and stability), as well as the Dolutegravir clinical trial process conditions (concentrations and relative ratio of enzyme to substrate, pH, temperature, time). Several CQAs may be identified for the protein hydrolysate or peptide fractions, and may require process optimization to obtain products with multiple functions, either within the same peptides (i.e. multifunctional peptides), or in different peptides each contributing to a specific function. Cheung and Li-Chan [5] used a Taguchi’s L16 (45) fractional factorial design to investigate the influence of four CPPs, each tested at four levels, on three CQAs (the extent of hydrolysis, angiotensin-I converting enzyme (ACE)-inhibitory activity and bitterness) of protein hydrolysates produced from shrimp processing by-products. Using this DOE enabled the evaluation of hydrolysates produced under conditions associated with combinations of the four CPPs based on only 16 unique experiments, as opposed to either single-factor-at a time testing (holding three parameters constant while changing the fourth), or a full factorial design (requiring 256 unique experiments). Similarly, Marchetti et al. [6] applied DOE for ‘Quality by Design’ to understand and design the CPPs for peptide separation and recovery by nanofiltration.