Enrichment and recovery of oligonucleotide impurities by N-Rich twin-column continuous chromatography.

N-Rich is a twin-column continuous chromatography technology well suited for small-scale isolation and the enrichment of product related impurities. For the first time, N-Rich was used for impurity isolation from a double-stranded RNA (dsRNA) therapeutic synthetic oligonucleotide (ON), produced by solid-phase synthesis. By employing the N-Rich process, where the desired impurities are recycled and selectively enriched, and interfering substances are depleted, it was possible to obtain substantial amounts of high purity marginal impurities with a reproducible, automatized, and productive method. The productivity-purity tradeoff inherent to traditional impurity isolation methods, i.e., analytical chromatography, was effectively alleviated. Using N-Rich, satisfactory purity values and mass recoveries of several low-concentrated impurities could be obtained simultaneously. A performance comparison demonstrated an up to 15-fold increase for purity values and up to 20-fold mass impurity isolation and concentration with the N-Rich technology in comparison to conventional isolation procedures, drastically reducing processing times, manual handling, and waste production.

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography.

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.

Mass transfer kinetics on modern Whelk-O1 chiral stationary phases made on fully- and superficially-porous particles.

In this work, a detailed study of mass transfer properties of trans-stilbene oxide (TSO) enantiomers on two Whelk-O1 chiral stationary phases (CSPs) has been performed. The CSPs were prepared by using both fully-porous silica particles of 2.5 µm particle diameter and superficially-porous ones of 2.6 µm particle diameter as base materials. By combining stop-flow and dynamic measurements in normal-phase conditions, the different contributions to mass transfer have been estimated. The study of intraparticle diffusion has revealed that the adsorption of both enantiomers is localized (i.e., characterized by absence of surface diffusion). The determination of thermodynamic binding constants (measured through adsorption isotherms) supports this finding.

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.

Shedding light on mechanisms leading to convex-upward van Deemter curves on a cellulose tris(4-chloro-3-methylphenylcarbamate)-based chiral stationary phase.

An unusual convex-upward van Deemter curve was observed for the more retained enantiomer of a chiral sulfoxide (2-(benzylsulfinyl)benzamide) on a cellulose tris(4-chloro-3-methylphenylcarbamate)-based chiral stationary phase (CSP), prepared on silica particles of 1000 Å pore size. In contrast, the firstly eluted enantiomer of the same molecule exhibited the traditional convex-downward van Deemter curve. A detailed kinetic and thermodynamic investigation has revealed that this unusual phenomenon, which however has already been observed in chiral chromatography, originates when the adsorption of the compound is very strong and the solid-phase diffusion negligible. Experimentally, the intraparticle diffusion of the more retained enantiomer of the sulfoxide was found to be one order of magnitude smaller than that of the first eluted one. Overall, this translates into very little longitudinal diffusion (b-term of van Deemter curve) accompanied by high solid-liquid mass transfer resistance (c-term). Finally the comparison with another, differently-substituted chiral sulfoxide (whose enantiomers both exhibit traditional van Deemter curve behavior) has allowed to correlate these findings to the specific characteristics of the molecule.

From batch to continuous chromatographic purification of a therapeutic peptide through multicolumn countercurrent solvent gradient purification.

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized. Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%. A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity.

Investigation of mass transfer properties and kinetic performance of high-efficiency columns packed with C18 sub-2 µm fully and superficially porous particles.

Three columns packed with 2.0 µm superficially porous particles, 1.7 µm fully porous particles, and monodisperse 1.9 µm fully porous particles with narrow particle size distribution have been deeply characterized from a kinetic point of view. The 1.9 µm column showed excellent kinetic performance, comparable to that of the superficially porous one. These two columns also exhibit flatter c-branches of the van Deemter curve compared to the 1.7 µm fully porous particles column, resulting in smaller loss of efficiency when they are operated at higher flow rates than the optimal ones. The independent evaluation of each contribution to band broadening has revealed that the difference in kinetic performance comes from the very small eddy dispersion contribution on the 1.9 µm column, surprisingly even lower than that of the superficially porous one. This finding suggests a very good packing of the monodisperse 1.9 µm column. On the other hand, the potential of 1.7 µm fully porous particles is completely broken down by the strong frictional heating effect already arising at relatively low flow rates.

Modeling the nonlinear behavior of a bioactive peptide in reversed-phase gradient elution chromatography.

The thermodynamic behavior of octreotide, a cyclic octapeptide with important pharmaceutical functions, has been simulated under reversed-phase gradient elution conditions. To this end, adsorption behavior was firstly investigated in isocratic conditions, under a variety of water/acetonitrile + 0.02% (v/v) trifluoroacetic acid (TFA) mixtures as mobile phase by using a Langmuir isotherm. Organic modifier was varied in the range between 23 and 28% (v/v). Adsorption isotherms were determined by means of the so-called Inverse Method (IM) with a minimum amount of peptide. The linear solvent strength (LSS) model was used to find the correlation between isotherm parameters and mobile phase composition. This study contributes to enlarge our knowledge on the chromatographic behavior under nonlinear gradient conditions of peptides. In particular, it focuses on a cyclic octapeptide.