Reversed-phase high-performance liquid chromatography (HPLC) is the most versatile and widely used technique peptide purification. With its proven efficiency, this technique is a reliable separation tool for a wide range of peptide active pharmaceutical ingredients (APIs) and intermediate building blocks [1].
When purifying peptides, it is convenient to directly load the crude feed solution containing the peptide product and other impurities for HPLC purification. The crude feed solution, which consists only of water (under 100% aqueous conditions), can be introduced directly into the HPLC purification system without the need to mix it with organic solvents or carry out additional steps such as desalting, dilution, or concentration.
Peptides are most often separated using stationary phases based on alkyl-bonded spherical silica
microbead. An alkyl or carbon chains comprising 4, 8, or 18 carbon atoms are chemically attached to the surface of silica microbeads. These alkyl-bonded spherical silica are known as C4, C8, and C18 silica (Figure 1). Exposing this reversed-phase material to 100% aqueous conditions leads to a problem called dewetting during the loading step.
The consequence of dewetting is that water, the mobile phase carrying the analyte (peptides), does not facilitate sufficient interaction between the analyte and the stationary phase. Thus, the analyte does not separate efficiently after passing through the stationary phase [2, 3].
What is dewetting?
To sufficiently understand dewetting, we need to revisit the reverse phase chromatography separation mechanism to understand dewetting. In a previous blog, we provided a brief primer on this mechanism. In reverse phase chromatography, “like dissolves like,” i.e., non-polar analytes and solvents (hydrophobic or water-repelling possessing neither positive nor negative charge) interact with non-polar stationary phases (hydrophobic surfaces such as C18). Water is a polar molecule and does not interact efficiently with these stationary phases. So, it did not wet the surface of these stationary phases very well. This phenomenon is called dewetting.
How is this dewetting problem currently solved?
1) Adding an organic modifier. Adding a non-polar solvent that interacts efficiently with these stationary phases with their surfaces very well and reverses the dewetting phenomena. This non-polar solvent (also known as organic modifier) is typically a organic solvent used in reverse phase chromatography. Acetonitrile is the widely used organic modifiers in peptide purification. A thumb rule to prevent dewetting is to add the organic modifier at equal to or more than 5% of the mobile phase volume. Otherwise, if dewetting has already occurred, at least 50% organic modifier must be passed through the column for 30 to 40 minutes to restore the stationary phase.
2) Changes to stationary phase material chemistry. Manufacturers of alkyl-bonded silica stationary phase material also make chemical modifications [2] of this material to make it more “wettable.” The three main strategies are:
• Reducing the density of the alkyl groups on the surface. By reducing the no of alkyl chains on the surface of silica, the stationary phase is rendered less hydrophobic, and more hydrophilic amenable to wetting.
• No end capping. A long chain alkyl group is chemically attached onto silica microbeads via a
reaction with alcohol (-OH) group present on the surface of silica microbeads. However, not all available -OH groups are attached with alkyl groups (see Figure 2). The free -OH groups are chemically capped with small hydrophobic alkyl groups. This process is called end-capping. If this end capping is not done, then the water molecules interact with the hydrophilic -OH groups and wet the silica surface.
• Polar end capping the stationery. Another option is to use a polar end cap group that will interact with water (a polar molecule) rather than alkyl groups (non-polar molecules).
What are the effects of dewetting issues on costs and sustainability?
Although the problem is well described and products that circumvent the dewetting risk are available in the market, commercial purification of peptides use standard alkyl-bonded spherical silica that exhibit this dewetting effect. Thus, peptide manufacturers first pass at least 5% organic modifier (typically acetonitrile) to thoroughly wet the stationary phase before loading the crude feed solution. Additionally, this organic modifier is mixed with crude feed solution and maintained at or above the 5% threshold during the purification step.
For example, a standard industrial chromatography column (Length of 40 cm and internal diameter of 40 cm) with a volume of 50 liters requires a minimum of 25 liters (50% of column volume) of acetonitrile for each cycle. Considering that hundreds of cycles are typically needed to purify 1 kg of peptide, this results in the usage of thousands of liters of acetonitrile. Additional acetonitrile is also needed for optimal purification and to equilibrate the columns. The cumulative volume of organic solvents used can end up being tens of thousands of liters.
Broader Implications.
The demand for peptide-based drugs, particularly GLP-1 analogs for treating diabetes and promoting weight loss, has led to a significant increase in peptide manufacturing. However, this increase in production has also caused a rise in industrial waste. Roughly a decade ago, only three peptides were being manufactured at 100 kg per year. Today, the demand is for multi-metric-ton quantities of peptides, leading to an unprecedented increase in waste [4].
A report by the American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable estimates that manufacturing 1 kg of GLP-1 analog peptide requires up to 14 metric tons of solvent, resulting in high amounts of waste [5]. Peptide manufacturing is reported to have the highest waste generation compared to other active pharmaceutical ingredients.
The chromatographic purification step, a crucial part of the synthetic peptide production process, is a significant contributor to waste generation, with acetonitrile accounting for most of the waste in the purification step. "Waste prevention" remains a challenge, and addressing issues such as the use of acetonitrile to prevent dewetting issue in the purification step will be an essential step in waste prevention efforts.
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.
Millennial Scientific' NanoPak-C All Carbon microbeads as reverse phase chromatography media allows 100% crude feed solution without loss of retention reducing acetonitrile use and promoting sustainability.
Contact us to discuss how we can support your GLP-1 analog and other peptide purification challenges. Please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
References
[1] S.W. Pettersson, B.S. Persson, M. Nyström, General Method Allowing The Use Of 100% Aqueous Loading Conditions In Reversed-Phase Liquid Chromatography, Journal of Chromatography B 803(1) (2004) 159-165.
[2] M. Przybyciel, R.E. Majors, Phase Collapse in Reversed-Phase LC, LCGC Europe (2002) 780.
[3] F. Gritti, T. Walter, Retention Loss of Reversed-Phase Columns Using Highly Aqueous Mobile Phases: Fundamentals, Mechanism, and Practical Solutions, LCGC North America 39(1) (2021) 33-40.
[4] A. Pratap, What making weight-loss drugs means for the environment, Chemical & Engineering News 102(13) (2024) 18.
[5] I. Kekessie, K. Wegner, I. Martinez, M.E. Kopach, T.D. White, J.K. Tom, M.N. Kenworthy, F. Gallou, J. Lopez, S.G. Koenig, P.R. Payne, S. Eissler, B. Arumugam, C. Li, S. Mukherjee, A. Isidro-Llobet, O. Ludemann-Hombourger, P. Richardson, J. Kittelmann, D. Sejer Pedersen, L.J. van den Bos, Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis, The Journal of Organic Chemistry 89(7) (2024) 4261-4282.
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