Peptides are considered “medium molecules.” They typically consist of 2-50 amino acids, and their size is measured in molecular weights greater than 1000 daltons (Da). To put size in perspective, only small molecules have molecular weights less than 500 Da and can typically penetrate our skin's outermost layer. Proteins are considered “large molecules,” with molecular weights greater than 5000 Da.
Tens of peptide products are in the market, and hundreds more are in preclinical or clinical development. Among these, GLP-1 peptides, also known as glucagon-like peptide-1, have been making waves. Initially approved for diabetes treatment, these peptides are now being explored for their potential in combating obesity and other health issues, sparking a wave of optimism in the field.
The current industrial-scale peptide synthesis methods involve using substantial quantities of hazardous reagents and solvents. Many peptides are manufactured through a process called solid-state peptide synthesis (SSPS), which chemically links amino acids together to form the peptides. These processes, developed six decades ago, still require substantial quantities of these hazardous materials, raising significant safety concerns.
This synthesis process is followed by the purification and isolation of peptides, which again involves significant amounts of high water and organic solvents as waste.
Thus, therapeutic peptide manufacturing is an excellent example of pharmaceutical manufacturing wherein solvents contribute to approximately 85-90% of the total mass of materials [1]. Consequently, peptide production has a staggeringly high E factor. This term measures the environmental acceptance of chemical synthesis and manufacturing [2]. It accounts for what could be considered waste, including byproducts, unreacted residual chemicals, and solvent losses. Its value will vary depending on what is regarded as waste. In the best case, the E factor will be zero, and higher E factor values indicate more waste. We elaborate on the E factor and other terms used in green chemistry in this blog, underlining the pressing need for more sustainable practices.
Solid-phase peptide synthesis immobilizes the reactants (amino acids) that generate the final peptide product onto a solid support. This solid support is synthesized using polymeric material (e.g., crosslinked polystyrene) known as resin. It is spherical in shape, with size (diameter) ranging from tens to hundreds of microns. The solvent must swell the resin to access reactive sites otherwise blocked within the polymer matrix.
Peptide synthesis involves an excess of the following chemicals at each amino acid addition step:
• Reactants (amino acids)
• Coupling reagents that facilitate the chemical attachment.
• Bases (e.g., piperidine) that catalyze the couple and protect the amino acids from side reactions to
maximize yield.
• Solvents to wash the resin (e.g., N, N-dimethylformamide (DMF) and N- methyl-2-pyrrolidone (NMP))
Hydrophobic and long peptide syntheses pose additional challenges. These peptides exhibit a propensity to aggregate. Thus, specialized resins with lower reactive sites exacerbate sustainability and economic impact.
The pervasive use of chromatography to purify peptide products also contributes to poor sustainability. The impurities generated during the solid-phase peptide synthesis (SPPS) process include unwanted peptide by-products and various chemicals used in SPPS. It's recommended that no single unknown impurity exceeds 0.1% in the final product.
Preparative chromatography is the dominant method for peptide purification, and reversed-phase chromatography is the most widely used technique. It uses:
• Aqueous mobile phase. Typically buffers.
• Organic solvents. Acetonitrile or alcohol is added to the mobile phase.
• Stationary phase material or media. Four (C-4), eight (C-8), and eighteen carbon (C-18) alkyl molecules chemically attached to a spherical silica surface are the most popular.
Preparative chromatography consumes a large volume of buffers and solvents during the purification process. Additionally, wash organic solvents (e.g., alcohols, such as methanol, isopropanol, hexane) are used for cleaning and regeneration procedures. Alkaline aqueous solutions (e.g., sodium hydroxide) are preferable for cleaning and regeneration procedures. But are judiciously used to avoid dissolution and degradation of silica.
For example, chromatography with a column Length of 40 cm and a column internal diameter of 40 cm has approximately 50 liters of volume. This column is packed with 30 kg of alkyl-bound silica media. Assuming the columns are used for 150 cycles before the media is changed, the total solvent volume consumed during purification is 7500 liters. Additionally, assuming a minimum of 2 column volumes for cleaning and regeneration, 15,000 liters of these solvents are consumed. Thus, the cumulative volume of organic solvents, buffers, and wash solvents would be greater than 20,000 liters. Additionally, even though silica is degradable, the alkyl chains and end-capping agents (usually silylating agents such as trimethylchlorosilane) used in media do not degrade. Thus, an alkyl chain with 10% attachment (carbon loading) would release 3 kg of these alkyl molecules and substantially more significant amounts of end-capping agents into the environment.
Future Perspective. Estimates show that peptide production releases tons of waste per kilogram of produced peptide [2]. This leads to very high process mass intensity (PMI: ratio of the total mass of all input material over the mass of isolated product) and cost of goods. Recent efforts, led by the ACS Green Chemistry Institute (GCI) and others, have focused on:
• Exploring and encouraging manufacturers to replace decades-old solid-state peptide synthesis
practices with alternative eco-friendly options.
• Promoting chromatography processes that improve yields, reduce solvent wastage, or introduce
eco-friendly, greener solvent options.
• Developing appropriate environmental metrics for the peptide manufacturing process to identify
additional opportunities to promote sustainable manufacturing.
• Establishing partnerships and collaborations between relevant stakeholders (academia,
pharmaceutical industry, CROs, CMOs).
References
[1] S. Lawrenson, M. North, F. Peigneguy, A. Routledge, Greener Solvents for Solid-Phase Synthesis, Green Chemistry 19(4) (2017) 952-962.
[2] A. Isidro-Llobet, M.N. Kenworthy, S. Mukherjee, M.E. Kopach, K. Wegner, F. Gallou, A.G. Smith, F. Roschangar, Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production, The Journal of Organic Chemistry 84(8) (2019) 4615-4628.
This blog is part of our broader impact series, which provides an easy-to-understand overview of the implications of our technology and products on science, sustainability, and human health.
For more information on how our NanoPak-C All Carbon media can address your peptide purification challenges or or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
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