The way to ultrafast, high-throughput enantioseparations of bioactive compounds in liquid and supercritical fluid chromatography

Until less than 10 years ago, chiral separations were carried out with columns packed with 5 or 3 μm fully porous particles (FPPs). Times to resolve enantiomeric mixtures were easily larger than 30 min, or so. Pushed especially by stringent requirements from medicinal and pharmaceutical industries, during the last years the field of chiral separations by liquid chromatography has undergone what can be defined a “true revolution”. With the purpose of developing ever faster and efficient method of separations, indeed, very efficient particle formats, such as superficially porous particles (SPPs) or sub-2 μm FPPs, have been functionalized with chiral selectors and employed in ultrafast applications. Thanks to the use of short column (1–2 cm long), packed with these extremely efficient chiral stationary phases (CSPs), operated at very high flow rates (5–8 mL/min), resolution of racemates could be accomplished in very short time, in many cases less than 1 s in normal-, reversed-phase and HILIC conditions. These CSPs have been found to be particularly promising also to carry out high-throughput separations under supercritical fluid chromatography (SFC) conditions. The most important results that have been recently achieved in terms of ultrafast, high-throughput enantioseparations both in liquid and supercritical fluid chromatography with particular attention to the very important field of bioactive chiral compounds will be reviewed in this manuscript. Attention will be focused not only on the latest introduced CSPs and their applications, but also on instrumental modifications which are required in some cases in order to fully exploit the intrinsic potential of new generation chiral columns

The Role of Adsorption and pH of the Mobile Phase on the Chromatographic Behavior of a Therapeutic Peptide

The impact of two different stationary phases and ion-pair reagents on the retention behavior of glucagon, a therapeutic peptide consisting of 29 amino acidic residues, has been investigated under reversed-phase elution conditions. Retention of glucagon was investigated under isocratic conditions by varying the fraction of the organic modifier in the range of 28–38% (v/v). The two stationary phases have been characterized in terms of excess adsorption isotherms to understand the preferential adsorption of eluent components on them. Results suggest that the ligand characteristics and the pH of the mobile phase play a pivotal role on retention

Sustainability in peptide chemistry: current synthesis and purification technologies and future challenges

Developing greener synthesis processes is an inescapable necessity to transform the industrial landscape, mainly in the pharmaceutical sector, into a long-term, sustainable reality. In this context, the renaissance of peptides as medical treatments, and the enforcement of more stringent sustainability requirements by regulatory agencies, pushed chemists toward the introduction of sustainable processes to prepare highly pure, active pharmaceutical ingredients (APIs). Innovative upstream (synthesis) and downstream (purification) methodologies have been developed during the last 5 years with the introduction and optimization of several technologies in solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS), chemo-enzymatic peptide synthesis (CEPS), and chromatographic procedures. These innovations are also moving toward the introduction of continuous processes that represent one of the most important targets for iterative processes. This overview discusses the most recent efforts in making peptide chemistry greener. The extensive studies that were carried out on green solvents, reaction conditions, auxiliary reagents and purification technologies in the peptide segment can be useful to other fields of organic synthesis

Modern trends in downstream processing of biotherapeutics through continuous chromatography: The potential of Multicolumn Countercurrent Solvent Gradient Purification

Single-column (batch) preparative chromatography is the technique of choice for purification of biotherapeutics but it is often characterized by an intrinsic limitation in terms of yield-purity trade-off, especially for separations containing a larger number of product-related impurities. This drawback can be alleviated by employing multicolumn continuous chromatography. Among the different methods working in continuous mode, in this paper we will focus in particular on Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) which has been specifically designed for challenging separations of target biomolecules from their product-related impurities. The improvements come from the automatic internal recycling of the impure fractions inside the chromatographic system, which results in an increased yield without compromising the purity of the pool. In this article, steps of the manufacturing process of biopharmaceuticals will be described, as well as the advantages of continuous chromatography over batch processes, by particularly focusing on MCSGP

Liquid Chromatography for Cannabinoids Separations: State of the Art and Future Perspectives

In the last years, several countries have legalized cannabis for medical purposes and/or for recrea5onal use. Therefore, the demand for accurate analy5cal methods, able to provide a detailed characteriza5on of cannabis samples and extracts, and fast, efficient, and cost-effec5ve methods for the isola5on of cannabinoids has increased. In this context, HPLC is emerging as the golden standard for rou5ne potency tes5ng analysis and for prepara5ve purposes. However, method development for the separa5on of cannabinoids is s5ll in its early stages. Indeed, almost the totality of the available LC methods is based on the classical achiral reversed-phase liquid chromatography (with C18 sta5onary phases and acetonitrile as organic modifier), where the resolu5on of species with very similar chemical structures (such as the Δ9-THC/Δ8-THC pair) can be par5cularly challenging. Moreover, only a few fundamental studies about reten5on and adsorp5on of cannabinoids have been performed. As a consequence, the development of separa5on processes is s5ll based on trial-and-error strategy, cons5tu5ng an important limita5on, especially from the industrial viewpoint. Thus, the need of exploring novel adsorbents, different elu5on modes, various experimental condi5ons, and possibly to exploit fundamental informa5on for the successful method development. These studies will contribute to obtain different selec5vity and more and more efficient runs, if compared to the classical methods, being able to overcome (almost) all the observed limita5ons

Perspectives and Pitfalls in Potency Testing of Cannabinoids by High Performance Liquid Chromatography (HPLC)

Cannabis products have been recently legalized in many countries for recreational or medicinal use. Therefore, rigorous analytical methods to test the potency of samples is required prior to commercialization. In addition, growing interest in the properties of minor cannabinoids has increased the demand for high-throughput methods that can separate the largest number of compounds in the shortest amount of time. High performance liquid chromatography (HPLC) is emerging as the preferred analytical method for potency testing of cannabinoids, but more fundamental work is needed to solve critical issues and contribute to advancing knowledge

Split-Intein Mediated Affinity Chromatography for the Purification of a C-intein Tagged Protein

The purification of biomolecules is a very challenging process due to the presence of a very complex mixture of other cell culture components. In order to achieve high protein yield and purity, a so-called affinity chromatography step is widely used [1]. In this study, affinity chromatography has been performed through the use of split-inteins. The stationary phase resin is functionalized with a N-intein affinity tag, while the Protein of Interest (POI) has a C-intein tag. The capture of the POI from the crude lysate occurs due to the high affinity between these two tags [2]. The subsequent cleavage causes the release of the purified protein. After the elution, a regeneration step is required in order to get rid of the C-intein tag (see Figure). This stationary affinity phase was tested and characterized. The pore size distribution of the resin was measured by inverse size-exclusion chromatography (iSEC). The binding and saturation capacities were determined through breakthrough curve (BTC) fitting. In the initial purification processes, proteins pre-purified by IMAC (Immobilized Metal Affinity Chromatography) as well as cell lysate were utilized. Then the results were compared and the purification protocol has been optimized for the crude lysate. Afterwards, using a model based optimization [3], both a batch capture process and a two-column CaptureSMB process were simulated in order to predict the best conditions in terms of productivity and column capacity utilization

Impact Of High Porosity Silica on Zwitterionic Teicoplanin-based Columns For Ultra-High Performance Chromatography

The research in the area of enantioselective Ultra High Performance Chromatography (eUHPC) is continuously focused on achieving higher efficiencies and, at the same time, faster analyses. In this work, a novel Chiral Stationary Phase (CSP) was prepared by covalently bonding the teicoplanin selector (TE_A2-2) on Halo 2.7μm 160Å Superficially Porous silica Particles (SPPs) by employing an already known synthetic procedure allowing to obtain a zwitterionic teicoplanin based CSP, which was used to produce the UHPC-FPP-Titan- Tzwitt CSP based on 1.9 μm 120Å Fully Porous monodispersed silica Particles (FPP) and UHPC-SPP-Halo90-Tzwitt CSP 2.0 μm [1-3]. These CSPs were packed into columns (L.: 50 and 100 mm, I.D.: 4.6 mm) and were characterized in terms of permeability, efficiency and thermodynamic under HILIC condition. van Deemter curves were used as main instrument for the kinetic performance evaluation. The UHPC-SPP-Halo160-Tzwitt 2.7 μm showed excellent efficiencies on both achiral (>323,000 theoretical plates/meter, N/m; hr: 1.14) and chiral analytes (>240,000 N/m; hr: 1.53), proving the high potential of this CSP from the kinetic point of view also in comparison to the UHPC-SPP-Halo90-Tzwitt CSP 2.0 μm and UHPC-FPP-Titan120-Tzwitt CSP 1.9 μm. Furthermore, taking into account the thermodynamic viewpoint, on the one hand, the UHPC-SPP-Halo160-Tzwitt 2.7 μm exhibited significantly smaller retention factors (k’) in comparison to those observed on the two sub- 2μm UHPLC columns (as a consequence of the lower selector loading on the silica). On the other hand, the SPP-Halo 160Å column showed the best resolution power (Rs/tr,2) thanks to its enantioselectivity values because of the larger selector density on the silica matrix. In conclusion, in this study we present the potential of the use of high-porosity SPP silica particles in the UHPLC chiral field opening an interesting scenario in this area

Thermodynamic comparison of zwitterionic teicoplanin-based 2μm superficially and sub-2μm fully porous particles for ultrafast chiral liquid chromatography

The study and understanding of adsorption and enantiorecognition mechanisms between chiral analytes and stationary phases is essential for different reasons: pushing forward the limits of high efficient and ultrafast chiral liquid chromatography and understanding the basis of thermodynamics of adsorption [1]. In this work, thermodynamic performances of two zwitterionic teicoplanin-based chiral stationary phases (CSPs) prepared respectively on 2.0 μm superficially porous particles (SPPs) and on 1.9 μm fully porous particles (FPPs) of narrow particle size distribution (nPSD) have been analyzed and compared [2]. More in details, the adsorption isotherms of Z-D,L-Methionine enantiomers have been studied under Hydrophilic Interaction Liquid Chromatography (HILIC) mode by using the so-called Equilibrium Dispersive (ED) model [3]. The Inverse Method (IM) has been used for isotherm determination. Results have shown the competitive Bi-lanmguir isotherm model to be suitable for the description of the separation of Z-D,L-Methionine enantiomers on the two columns, indicating the presence of two different types of adsorption sites: one selective, responsible for the chiral recognition mechanism, and one nonselective, in which the two enantiomers behave the same. On the one hand, FPPs show higher selectivity values in comparison to SPPs, highlighting the greater enantioselective potential of these particles. On the other hand, SPPs are characterized by slightly larger selective and nonselective binding than FPPs. These data correlate with the specific loading of chiral selector, which was found to be larger on SPPs than on FPPs. This could negatively impact when moving to ultrafast separations