Analysis of short-chain bioactive peptides by unified chromatography-electrospray ionization mass spectrometry. Part I. Method development

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2‐PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.

Section: Methods Development Protocolmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

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Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Neumann

Schmidtsdorff

Schmidt³

et al. 2023

“…Most recently, an optimization approach of a purity method for a set of 76 linear and cyclic short-chain peptides (< 800 Da) was published. The authors utilized Derringer's desirability functions to define the overall best conditions for a single method covering all 76 peptides based on a limited number of experiments [18]. The orthogonality of RP-LC and SFC was studied subsequently for 43 of these peptides by the same group demonstrating an advantage of SFC over RP-LC due to less coelution and stronger retention.…”

Section: Introductionmentioning

confidence: 99%

Application of sub‐/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide

Neumann

Schmidtsdorff

Schmidt³

et al. 2022

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed‐phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design‐of‐Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry‐compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed‐phase liquid chromatography were resolved by applying the final method.

“…The demand for more costeffective and long-term purifying procedures for these biologics is developing. With more environmentally friendly mobile phases such as carbon dioxide and methanol, SFC can provide a greener peptide purification technique with little environmental impact [54][55][56].…”

Section: Supercritical Fluid Chromatographymentioning

confidence: 99%

Synthetic pharmaceutical peptides characterization by chromatography principles and method development

Sharma

Kukreja

Giri

et al. 2022

As per the United States Food and Drug Administration, any polymer/chain composed of 40 or fewer amino acids is called a peptide, where more than 40 amino acids are considered proteins. On many occasions, there is a change in the source of manufacturing of the peptide active pharmaceutical ingredient, where one has to prove the sameness of that product with the existing formulation by considering several aspects like the presence of impurities/degradation products, the extent of aggregations, and so forth. For the same, several chromatographic characterization techniques such as reversed-phase high-performance liquid chromatography-ultraviolet/high-resolution mass spectrometry, supercritical fluid chromatography, size-exclusion chromatography, ion-exchange chromatography, and so forth, are widely used in the pharmaceutical industry. It is well known that the method development of peptide molecules is often challenging as many variables are to be kept in mind which can affect the separation, recovery, and stability of the molecule. The present review focuses on the basics of peptide degradation and method development by using various chromatographic techniques for characterization. It also covers a deep insight of method development parameters and variables to be considered which might directly or indirectly affect the chromatographic separation and recovery and also provides a guide on the selection of chromatographic parameters.