Quality Assurance and Control
Molecular understanding at the atomic level
Therapeutic peptides are big business! Over 60 USFDA-approved therapeutic peptides are in use. They are quite commonly manufactured at scale using pure, chemical starting materials.
Many peptide products are human consumed. They should, therefore, be characterised and have their quality assured.
The appeal of NMR spectroscopy
NMR spectroscopy is used extensively for many quantitative and structural analyses. NMR methods can potentially be validated for use in regulated environments.
NMR is always a quantitative analytical method that has no need for response curve determination. Quantitative answers can usually be quite simply incorporated into an analysis. It follows that NMR is an excellent tool for peptide concentration and purity determinations.
NMR has been used for decades to study peptides and proteins, and Prof K Wüthrich received the Nobel Prize in 2002 for developing the science. As much as ever, NMR continues to make significant contributions to understanding macromolecules’ behaviour and roles.
NMR provides unique levels of information of molecules, making it an ideal tool for structure elucidation and characterisation. At its heart, NMR reliably reports the structural integrity of a peptide: which amino acids are present and how are they connected. But, beyond this, NMR has the potential to also report correct peptide chemical conjugation, such as acetylation, fatylation, glycoconjugate synthesis, tagging, etc. No sample modification, such as hydrolysis, is required.
Analysis by NMR has the potential to provide information well beyond simple QA/QC requirements. The technique can reveal information about the way a peptide might fold in solution – it’s secondary structure. Physical characteristics such as molecular rigidity are reported. NMR can give insight into the peptide behaviour which can help as a formulation medium is sought: NMR experiments can report aggregation or interactions with other molecular species.
NMR is a superb test for peptide structure, identity, purity, and authenticity
“Fingerprint” comparisons - partial analysis
A full, detailed analysis of a peptide can be a complex task, but may not always be required. When used for final release testing of a peptide, a simple comparison of spectra for the batch can be treated as a fingerprint and compared with spectra of authentic samples. Thus, NMR is the most reliable identity test for peptides.
Larger peptides such as insulin, and even biological drugs/biosimilars can be assessed quite simply for correct structure and folding using NMR. A fingerprint comparison can determine if samples are authentic, correctly declared, or equivalent.
When used for batch release, a “fingerprint” comparison with spectra of authenticated material is a quick and cost effective alternative.
Spectrum analysis - complete
In some cases a full spectrum analysis is indicated. Sample complexity will affect the analysis complexity. Simple peptides up to ca 9 residues can be quickly analysed, but we can characterise peptides up to ca 35 amino acid residues in size. Peptide modification can usually be handled.
For full identity checks we will perform a full assignment of the 1H, 13C, and 15N NMR signals. Several 2D experiments will be performed to facilitate this task. Tables of assignments and figures are prepared semi-automatically. We are flexible in the reporting level that you require.
Our expertise and proprietary software are key to the spectral assignment being done quickly, correctly, and with assignment validation.
Shorter peptides generally do not have a higher order structure. The solvent choice can favour α-helical conformations. We will use the sample’s chemical shifts and J-coupling to assess the adoption of secondary structure, and rigidity. Computer programs and manual analyses are used to predict the secondary structure with a confidence level. The through-space NOE data also help to describe the peptide’s secondary structure through key interactions.
We use a combination of complementary, well-established data analysis methods to evaluate the secondary structure to arrive at a consistent picture of the peptide's conformation and mobility. In the s[pectra, below, we show where i → i+1 and i → i+3 contacts would be revealed by NOE peaks.
We typically require ~5mg of pure compound.
Sample preparation is often very simple once a suitable solvent has been found. A variety of solvents can be used, including aqueous and DMSO.
NMR spectrum acquisition is a specialist task requiring expensive equipment and trained operators. Luckily, these services can be provided. A set of 5 or 6 datasets is typically acquired overnight to allow for complete analysis.
The processing and analysis of the NMR spectral data is, again, a specialist task. And again, we can provide this as a complete service.
In collaboration with Spectral Service contract labs, a modern 600 MHz solution NMR instrument is used to acquire NMR data.
If required, we can acquire data at much higher field.
This can be as simple or detailed as you need. We will always create and share lists of peak assignments and annotated spectral figures. A full report can be created that consists of a detailed discussion of the results.
Dialogue and consultation
Speak to our experts to discuss the experimental findings.
We can address any special concerns/questions about your peptide you want answered. Speak to us to discuss results and to also plan experiments that will assist you with your task. We can provide the up-to-date, specialist expertise to help you.
Please contact us to discuss your peptide analytical requirement. We will be happy to tailor an analysis and report specification for you.
Lau, J. L.; Dunn, M. K. Therapeutic Peptides: Historical Perspectives, Current Development Trends, and Future Directions. Bioorg. Med. Chem. 2018, 26 (10), 2700–2707. https://doi.org/https://doi.org/10.1016/j.bmc.2017.06.052. Download.
Kellenbach, E.; Rundlöf, T. Chapter 11: Determination of the Identity, Content and Purity of Therapeutic Peptides by NMR Spectroscopy; 2019; Vol. 2019-January. Download.