Early engineering approaches to improve peptide developability and manufacturability

Abstract

Downstream success in Pharmaceutical Development requires thoughtful molecule design early in the lifetime of any potential therapeutic. Most therapeutic monoclonal antibodies are quite similar with respect to their developability properties. However, the properties of therapeutic peptides tend to be as diverse as the molecules themselves. Analysis of the primary sequence reveals sites of potential adverse posttranslational modifications including asparagine deamidation, aspartic acid isomerization, methionine, tryptophan, and cysteine oxidation and, potentially, chemical and proteolytic degradation liabilities that can impact the developability and manufacturability of a potential therapeutic peptide. Assessing these liabilities, both biophysically and functionally, early in a molecule's lifetime can drive a more effective path forward in the drug discovery process. In addition to these potential liabilities, more complex peptides that contain multiple disulfide bonds can pose particular challenges with respect to production and manufacturability. Approaches to reducing the disulfide bond complexity of these peptides are often explored with mixed success. Proteolytic degradation is a major contributor to decreased half-life and efficacy. Addressing this aspect of peptide stability early in the discovery process increases downstream success. We will address aspects of peptide sequence analysis, molecule complexity, developability analysis, and manufacturing routes that drive the decision making processes during peptide therapeutic development.

Fig. 1

Examples of diversity among peptide tertiary structure. a The head-to-tail bicyclic structure of the 14-amino acid (C_α’s numbered from N-terminus to C-terminus) sunflower trypsin inhibitor (SFTI-1) is formed by a disulfide linkage between Cys3 and Cys11 (PDB 1SFI). b Three disulfide bonds stabilize the globular fold of the 35-amino acid peptide toxin, ShK, from the S. helianthus sea anemone (PDB 1ROO). c The 38-amino acid human urocortin-3 (Ucn-3) peptide is predominantly helical in DMSO solution (PDB 2RMH). Structures were rendered using PyMol, with carbon in green, nitrogen in blue, oxygen in red, and sulfur in yellow

Fig. 2

Cysteine editing to reduce peptide complexity. a The primary sequence of μ-conotoxin KIIIA (μ-KIIIA) from C. kinoshitai is shown with the native cystine bonding pattern. Cysteines at positions 1 and 9 were replaced with alanine, yielding the μ-KIIIA(C1A,C9A) mutant. b The overlay of μ-KIIIA (PDB 2LXG) and μ-KIIIA(C1A,C9A) (BMRB 20049) indicates only a slight perturbation of the structure with a pairwise RMSD of 0.95 Å. The backbone of μ-KIIIA is shown in teal and the backbone of μ-KIIIA(C1A,C9A) is shown in pink. The side chains of key amino acids responsible for interaction with sodium channels are labeled, with nitrogen colored blue, oxygen in red, and sulfur in yellow. The alanine mutations of μ-KIIIA(C1A,C9A) are highlighted in green

Fig. 3

Methods for avoiding proteolytic degradation. a Octreotide contains two D-amino acids, which cannot be recognized by endogenous peptidases: D-Phe at position 1 and D-Trp at position 4 (PDB 1SOC). Carbon is colored pink, nitrogen in blue, oxygen in red, sulfur in yellow, and the C_α hydrogens of the D-amino acids are in white. b Liraglutide contains a C16 fatty acid chain added to Lys26, providing a physical barrier to enzymatic degradation. Additionally, a Lys34Arg mutation removes a protease recognition site (PDB 4APD). Amino acid carbons are colored teal and the C16 fatty acid carbons are colored yellow. Nitrogen is colored in blue and oxygen is red