CHES is a zwitterionic buffer used for alkaline biochemical work, typically within pH 8.6–10.0. Also known as 2-(cyclohexylamino)ethanesulfonic acid (CAS 103-47-9), it has a pKa near 9.4 at 25°C.[1] Published applications include enzyme stability studies, monoclonal antibody chromatography and protein crystallization.
For biologics scientists, process engineers and academic researchers, CHES offers an option when screening alkaline conditions. Biobrightech offers low-sodium CHES with sodium content below 10 ppm (10 mg/kg), helping teams control unintended sodium introduced with the buffer raw material.
Applications of CHES
Enzyme Research and Biocatalysis
CHES has been investigated in alkaline enzyme systems where buffer identity, cofactors and handling conditions influence performance.
Merz and colleagues studied the transaminase from Silicibacter pomeroyi in 100 mM CHES or Tris at pH 9.0. With 10 mM pyridoxal 5′-phosphate (PLP), the CHES system achieved an enzyme half-life of approximately 720 hours in darkness. Under the corresponding light-exposed conditions, the half-life fell to approximately eight hours.[2]
This makes CHES worth considering in buffer screens for alkaline biocatalysis, while showing why light exposure and cofactor chemistry must be evaluated together. The result concerns one enzyme under defined conditions; it does not establish universal superiority over Tris.
Monoclonal Antibody Chromatography
CHES also has a documented role in analytical method development for therapeutic antibodies.
Farsang and colleagues evaluated alternative mobile phases for cation-exchange chromatography (CEX). A citric acid/CHES system was promising for monoclonal antibody separations, particularly when combined with a moderate NaCl gradient. The study demonstrated that mobile-phase composition and salt concentration could be adjusted to tune selectivity.[3]
For biologics analytical teams, this supports evaluating CHES-containing systems during CEX method development. It demonstrates an analytical application rather than proof of routine manufacturing-scale use.
Protein Solubility and Crystallization
CHES can also be valuable when screening buffer conditions for difficult proteins.
Collins and colleagues identified 100 mM CHES at pH 9.25 as a favorable buffer for the archaeal protein AF2059. Following buffer optimization, rescreening against 192 crystallization conditions produced 24 crystal leads and ultimately a crystal suitable for 2.2 Å structure determination.[4]
The optimized protein buffer also contained 100 mM NaCl. This is evidence for a particular CHES-containing formulation, not evidence that removing sodium improves crystallization. Researchers should screen buffer identity, pH and salt composition as separate variables.
Value of Low Sodium CHES
Biobrightech’s low-sodium CHES is particularly relevant when sodium background is a controlled experimental or process variable. Its potential advantages follow from reduced raw-material sodium input; the studies above did not test Biobrightech material or establish a low-sodium performance benefit.
Development need | Value of a low-sodium starting material |
|---|---|
Sodium-sensitive experiments | Reduces unintended sodium background before deliberate ion additions |
Potassium-based buffer development | Gives greater control over sodium carryover when CHES is titrated with a suitable potassium-based reagent |
Matched formulation comparisons | Helps reduce raw-material sodium as a confounding variable |
Raw-material qualification | Allows a specific sodium acceptance limit to be linked to the formulation’s impurity budget |
The magnitude of the benefit depends on concentration. With Biobrightech CHES containing less than 10 ppm sodium, a 10 g/L CHES solution receives less than 0.10 mg/L sodium, equivalent to less than 4.35 µM, from the CHES raw material. This calculated upper bound helps researchers plan a sodium budget before adding titrants, salts or other ingredients.
Low-sodium CHES does not automatically produce a sodium-free buffer. NaOH used for pH adjustment, added NaCl, other ingredients and water can dominate the final sodium concentration. In salt-rich formulations, reducing trace sodium from CHES may have little practical effect. Likewise, replacing sodium with potassium changes ion identity without automatically lowering ionic strength.
Selecting CHES for Your Application
Evaluate CHES at the intended working pH and temperature, with the actual protein, cofactors and salts. Compare activity, solubility, chromatographic behavior or stability against the current formulation.
For low-sodium work, specify the sodium limit in mg/kg, the reporting basis and the analytical method, such as validated ICP-OES or ICP-MS with a suitable quantification limit. A low-sodium designation alone does not establish suitability for a regulated manufacturing process.
Contact Biobrightech to discuss CHES with sodium below 10 ppm, application-specific requirements and batch documentation for your development project.
References
1. Merck/Sigma-Aldrich. Biological Buffers Selection Guide. Buffer properties and temperature dependence.
2. Merz LM, van Langen LM, Berglund P. The Role of Buffer, Pyridoxal 5′-Phosphate and Light on the Stability of the Silicibacter Pomeroyi Transaminase. ChemCatChem. 2023;15:e202201174. doi:10.1002/cctc.202201174.
3. Farsang E, et al. Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation. J Pharm Biomed Anal. 2019;168:138–147. doi:10.1016/j.jpba.2019.02.024.
4. Collins B, Stevens RC, Page R. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Acta Crystallogr F. 2005;61:1035–1038. doi:10.1107/S1744309105035244.