Residual Solvents in Peptide Manufacturing

Organic solvents are used in peptide synthesis, particularly in the context of solid phase peptide synthesis (SPPS). source These solvents include highly volatile compounds like trifluoroacetic acid (TFA), acetonitrile (ACN), and dichloromethane (DCM), and less volatile ones like dimethylformamide (DMF), dimethylacetamide (DCA), and N-methylpyrrolidone (NMP). source If not removed during manufacturing and lyophilization, they can stick around in the final peptide product. Understanding their characteristics, health risks, and removal processes is important for ensuring the safety and quality of synthetic peptides.

Common Residual Solvents used in SPPS

TFA (Trifluoroacetic Acid):

• Highly volatile with a boiling point of 72°C, so residual presence typically sublimates out during lyophilization, making its presence unlikely as long as lyophilization was done properly with multiple drying cycles.
• Toxicity concerns arise when TFA is present as a counterion when no counterion ion exchange is used to replace TFA with another counterion such as sodium or acetate, or was not removed to form the base form, as it may affect pharmacokinetics and long-term health.
• If TFA is used as a counterion, it does not all sublimate away during lyophilization. source:Janoshik

DCM (Dichloromethane):

• Boiling point of 40°C, highly volatile.
• Sometimes used as a solvent during SPPS; not as a counterion.

Acetonitrile (ACN):

• Boiling point of 82°C, highly volatile.
• Sometimes used as a solvent during SPPS; not as a counterion.

DMF (Dimethylformamide):

• Boiling point of 153°C, making it less volatile than TFA and potentially more likely to remain in the peptide product after lyophilization.
• Sometimes used as a solvent during SPPS; not as a counterion.

DCA (Dimethylacetamide):

• Boiling point of 165°C, making it less volatile than TFA and potentially more likely to remain in the peptide product after lyophilization.
• Sometimes used as a solvent during SPPS; not as a counterion.

NMP (N-Methylpyrrolidone):

• Boiling point of 202°C, making it less volatile than TFA and potentially more likely to remain in the peptide product after lyophilization.
• Sometimes used as a solvent during SPPS; not as a counterion.

Toxicity and Safety Standards

The International Council for Harmonisation (ICH) guidelines categorize these typical SPPS solvents as Class 2 due to their potential organ toxicity. Permitted Daily Exposure (PDE) levels are established to minimize health risks source:

• ACN: 4.1 mg/day
• NMP: 5.3 mg/day
• DCM: 6.0 mg/day
• DMF: 8.8 mg/day
• DCA: 10.9 mg/day
• TFA: ≤0.1% of peptide raw product source: listed on 503a/b compounding manufacturers’ COAs regulated by US FDA

For weekly sub-mL injections of reconstituted peptides like tirzepatide, the levels of these solvents are unlikely to pose a significant health risk. To provide a real-world example, consider acetaldehyde — a carcinogenic organic solvent produced by the body when breaking down alcohol. Its permissible daily exposure (PDE) is 2 mg/day, while wine generates approximately 34 mg/L of acetaldehyde (4.8mg/glass). source

Trifluoroacetic Acid (TFA)

In synthetic peptide synthesis, the trifluoroacetate counterion form will stick around unless it is ion exchanged for another salt form or cleaved off the peptide to create the base form of the peptide. As tirzepatide is a weekly, lifelong injection, TFA is subject to stricter standards followed by brand-name and 503a/b compounding pharmacies. TFA is an ultra-short chain PFAS, and while TFA itself has not yet been extensively studied, there are many studies linking PFAS (aka a “forever chemical”) to endocrine system disruption with links to infertility and obesity. source Pharmaceutical manufacturers have also said TFA is “undesirable in peptides intended for preclinical and clinical studies”. source So we recommend just staying away from weekly use peptides that use the TFA salt form in lieu of another more common salt form such as tirzepatide sodium or tirzepatide acetate.

Class 1 Solvents

There are other solvents NOT known to be used in the SPPS manufacturing process, but which have much higher toxicity concerns. Class 1 Solvents are set by the International Council for Harmonisation (ICH) and do not have safe PDE limits set for them since they should be avoided in pharmaceutical use. The parts per million (ppm) is set to ensure traces greater than these limits are not present source:

• Benzene: 2 ppm
• Carbon tetrachloride: 4 ppm
• 1,2-Dichloroethane: 5 ppm
• 1,1-Dichloroethene: 8 ppm
• 1,1,1-Trichloroethane: 1500 ppm

Testing for Residual Solvents

Each of these Class 1 solvents has a boiling point <85°C like TFA, which means they are highly volatile and so are highly unlikely to stick around after lyophilization — especially if multiple drying cycles are done under vacuum, which further lowers their boiling points. They are also not known to be used in the SPPS process. For these reasons, we don’t actively recommend testing for these residual solvents due to the low risk of presence. Instead, we suggest the community focus on:

• Spot testing for TFA — since TFA is both typically present in the manufacturing process and highly volatile like the class 1 solvents, it’s a good proxy marker for whether proper lyophilization was done. If TFA isn’t present, it’s highly likely other volatile residuals with similar boiling points (like Benzene, etc.) were also removed.

Available Testing Options

If you did choose to test for Class 1 residual solvents, the following tests are available:

• NMR F-19 Nuclear Magnetic Resonance F-19 spectroscopy is specifically suited for detecting TFA due to its fluorine content (peak near -73 to -76 ppm). source

• GC-FID Gas Chromatography with Flame Ionization Detection is the primary test method for detecting and quantifying residual solvents. USP <467> is used for analysis of residual solvents. source

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