Peptide Optimization at the Drug Discovery-Development Interface: Tailoring of Physicochemical Properties Toward Specific Formulation Requirements.

Physicochemical properties of peptides need to be compatible with the manufacturing process and formulation requirements to ensure developability toward the commercial drug product. This aspect is often disregarded and only evaluated late in discovery, imposing a high risk for delays in development, increased costs, and finally for the project in general. Here, we report a case study of early physicochemical peptide characterization and optimization of dual glucagon-like peptide 1/glucagon receptor agonists toward specific formulation requirements. Aggregation issues which were observed at acidic pH in the presence of phenolic preservatives could be eliminated by modification of the peptide sequence, and chemical stability issues were significantly improved by addition of stabilizing formulation excipients. We describe structural, analytical, and biophysical characterization in different compositions to analyze the effect of pH and formulation excipients on physical and chemical stability. Molecular models have been generated to rationalize peptide stability behavior based on computed physicochemical descriptors and interactions with excipients. To conclude these studies, a general roadmap is proposed how to assess and optimize early physicochemical peptide properties in a sophisticated way by combining experimental and in silico profiling to provide stable peptide drugs under relevant formulation conditions at the end of discovery.

Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA.

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from (14)C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD(+), indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD(+)/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

Assay of poly(3-hydroxybutyrate) depolymerase activity and product determination.

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.