Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis.

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production.

In recent years, there has been a growing interest in therapeutic peptides within the pharmaceutical industry with more than 50 peptide drugs on the market, approximately 170 in clinical trials, and >200 in preclinical development. However, the current state of the art in peptide synthesis involves primarily legacy technologies with use of large amounts of highly hazardous reagents and solvents and little focus on green chemistry and engineering. In 2016, the ACS Green Chemistry Institute Pharmaceutical Roundtable identified development of greener processes for peptide API as a critical unmet need, and as a result, a new Roundtable team formed to address this important area. The initial focus of this new team is to highlight best practices in peptide synthesis and encourage much needed innovations. In this Perspective, we aim to summarize the current challenges of peptide synthesis and purification in terms of sustainability, highlight possible solutions, and encourage synergies between academia, the pharmaceutical industry, and contract research organizations/contract manufacturing organizations.

Star polymers with a cationic core prepared by ATRP for cellular nucleic acids delivery.

Poly(ethylene glycol) (PEG)-based star polymers with a cationic core were prepared by atom transfer radical polymerization (ATRP) for in vitro nucleic acid (NA) delivery. The star polymers were synthesized by ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and ethylene glycol dimethacrylate (EGDMA). Star polymers were characterized by gel permeation chromatography, zeta potential, and dynamic light scattering. These star polymers were combined with either plasmid DNA (pDNA) or short interfering RNA (siRNA) duplexes to form polyplexes for intracellular delivery. These polyplexes with either siRNA or pDNA were highly effective in NA delivery, particularly at relatively low star polymer weight or molar ratios, highlighting the importance of NA release in efficient delivery systems.

Specific interactions of metal ions with Cys-Xaa-Cys unit inserted into the peptide sequence.

In this work five peptides with Cys-Xaa-Cys motif were studied including Ac-Cys-Gly-Cys-NH(2), Ac-Cys-Pro-Cys-Pro-NH(2), their N-unprotected analogues and the N-terminal fragment of metallothionein-3, Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-Pro-NH(2). All these peptides were found to be very effective ligands for Ni(2+), Zn(2+) and Cd(2+) ions. Potentiometric and spectroscopic (UV-Vis, CD and MCD) studies have proved that sulfur atoms are critical donors for the metal ions coordination. The amide nitrogen may participate in the metal ion binding only in the case when Gly is adjacent to Cys residues. Ac-Cys-Gly-Cys-NH(2) may serve as a low molecular weight model for cluster A, which is a binding unit of nickel ion in acetyl coenzyme A synthase. This bifunctional enzyme from anaerobic microorganisms catalyzes the formation of acetyl coenzyme A from CO, a methyl group donated by the corrinoid-iron-sulfur protein and coenzyme A. Other peptides studied in this work were Ac-Cys-Pro-Cys-Pro-NH(2) and Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-NH(2) originating from metallothionein sequence. These motifs are characteristic for the sequence of cysteine rich metallothionein-3 (MT-3) called also neuronal growth inhibitory factor (GIF). Cys-Pro-Cys-Pro fragment of protein was demonstrated to be crucial for the inhibitory activity of the protein.

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing.

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.