What We Do

Purification

Small Molecule Purification

Hypha are able to purify metabolites, natural products and other small molecules from a variety of different matrices, including from clinical samples and complex microbial fermentation extracts.

Our extensive purification capabilities are based on a wealth of small molecule purification expertise, mostly originating in natural product discovery operations. This has expanded through application to products generated by biotransformation and chemical synthesis, as well as challenging isolations of minor metabolites from other complex biological matrices, including from human and other mammalian excreta.

Our laboratories are well equipped with analytical UPLC-MS and HPLC-MS systems for high-throughput analyses and chromatographic method development. Our preparative HPLC systems include mass-directed instrumentation and a number of Waters auto-purification systems, which enable purification of products ranging from sub milligram to multigram quantities.

Hydroxylated or conjugated drug metabolites tend to be polar molecules and as such are suited to purification by reversed phase HPLC methods. Generally, two or three purification stages using different column/mobile phase chemistries may be needed to purify a metabolite to the degree of purity required. For more lipophilic compounds, normal phase flash chromatography often proves to be convenient and economical for large scale separations.

Client case studies

Purification of multiple late-stage oxidised derivatives

A late-stage lead diversification project used PolyCYPs enzymes to generate multiple oxidised products using Hypha’s PolarExplorer process. One of the reactions was conducted at a scale of 1.7 litres. Preparative HPLC purification using two different column chemistries afforded six pure products with yields between 2 and 9 mg, along with a number of semi-pure fractions, all of which were provided for bioactivity testing. The analytical UPLC-UV chromatograms below provide a comparison of the crude extract against four of the purified products.

Purification of metabolites from a microbial biotransformation reaction

A glucuronide metabolite of a drug in clinical development was produced by conducting a microbial biotransformation at a scale of 16 litres. The metabolite was extracted and purified by successive preparative reversed phase HPLC purification steps employing acidic and then basic mobile phase gradients and the final yield comfortably exceeded the target quantity of 5 g. The final product purity was 96% by LC-UV, >99% by LC-ELSD and 90% by qNMR. A higher-level Certificate of Analysis was provided for this material.

purification oxidised derivatives case study image

Purification of metabolites from urine

A sub milligram amount of a metabolite of a drug in clinical development was isolated from over 20 litres of human urine. The purification involved five successive reversed phase preparative HPLC purification steps and enabled structure elucidation by NMR for definitive metID.

In another purification project, tens of milligrams of the R– and S-O-glucuronides of carisbamate, a neuro modulator developed by SK Life Science, were purified to > 95% purity from 150 ml of urine using a three-step purification method. Confirmation of the structures were obtained by NMR spectroscopy, enabling their use as analytic standards for bioanalysis.

The papers below highlight published examples of microbial natural product compounds produced and purified at Hypha.

Kebede, B.; Wrigley, S.K.; Prashar, A.; Rahlff, J.; Wolf, M.; Reinshagen, J.; Gribbon, P.; Imhoff, J.F.; Silber, J.; Labes, A.; Ellinger, B. Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production. Marine Drugs, 2017, 15, 84.

Mbekeani, A.J., Jones, R.S., Bassas Llorens, M., Elliot, J., Regnault, C., Barrett, M.P., Steele, J.,Kebede, B., Wrigley, S.K., Evans, L., Denny, P.W. Mining for natural product antileishmanials in a fungal extract library. IJP: Drugs and Drug Resistance, 2019, 11, 118-128.

Moraes, C. B., Witt, G., Kuzikov, M., Ellinger, B., Calogeropoulou, T., Prousis, K. C., … Gul, S. Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform. SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2019, 24(3), 346–361.

Resources

Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.

If clearance mechanisms of the test drug results in sufficient quantities of the major metabolites in biological material such as faeces or urine, purification and subsequent identification of metabolites from such matrices is possible. One such project undertaken at Hypha resulted in tens of milligrams of the R- and S-O-glucuronides of carisbamate, a neuromodulator developed by SK Life Science, which were purified to >95% purity from 150 ml of urine using a three-step purification method.

In Chapter 4 of the book on “Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters”, Hypha authors summarise the different methods employed for producing metabolites of drugs, illustrated with representative examples from the literature and work undertaken at Hypha. The chapter also includes a discussion and examples of the use of NMR spectroscopy for structure elucidation of metabolites.

Related articles

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Hypha Discovery came highly recommended and we subsequently contracted with Hypha to extract and purify metabolites out of rabbit urine. As they were so successful with that extraction, we again requested help for a second program to extract metabolites from human urine and/or synthesize/biosynthesize 3 metabolites. As expected, Hypha has been successful preparing these metabolite reference standards along with structural elucidation and certificates of analysis. In addition, synthesis of one of the metabolites had been attempted at 2 other labs without success; however, Hypha was able to synthesize this difficult metabolite which allowed us to do further evaluations on the metabolite.

Head of Toxicology/DMPK

US Pharma company

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