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.

Nowadays, environmental problems are drawing the attention of governments and international organisations, which are therefore encouraging the transition to green industrial processes and approaches. In this context, chemists can help indicate a suitable direction. Beside the efforts focused on greening synthetic approaches, currently also analytical techniques and separations are under observation, especially those employing large volumes of organic solvents, such as reversed-phase liquid chromatography (RPLC). Acetonitrile has always been considered the best performing organic modifier for RPLC applications, due to its chemical features (complete miscibility in water, UV transparency, low viscosity etc); nevertheless, it suffers of severe shortcomings, and most importantly, it does not fully comply with Environmental, Health and Safety (EHS) requirements. For these reasons, alternative greener solvents are being investigated, especially easily available alcohols. In this work, chromatographic performance of the most common solvents used in reversed-phase chromatography, i.e., acetonitrile, ethanol and isopropanol, have been compared to a scarcely used solvent, dimethyl carbonate (DMC). The analytes of interest were two small molecules, caffeine and paracetamol, whose kinetics and retention behaviour obtained with the four solvents have been compared, and all contributions to band broadening have been assessed. Results about kinetic performance are very promising, indicating that a small amount (7 % v/v) of DMC is able to produce the same efficiency as a 2.5-times larger ACN volume (18 % v/v), and larger efficiency than alcohols. This paper reports, for the first time, fundamental studies concerning the mass transfer phenomena when DMC is used as an organic solvent in RPLC, and, together with the companion paper, represents the results of a research whose final aim was to discover whether DMC is suitable for chromatographic applications both in linear and preparative conditions.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps. For oligonucleotides, and biomolecules in general, preparative liquid chromatography is the technique of choice to perform large scale purifications, typically in batch mode, i.e. using a single column. Specifically, since ONs are mainly large, hydrophilic and charged molecules, Anion Exchange chromatography (AEX) and Ion Pair Reversed Phase chromatography (IP-RP) are the preferred chromatographic modes for their downstream processing. Nevertheless, these approaches suffer from a purity-yield trade-off, and for this reason, more than one purification step is usually required. The two chromatographic modes can therefore be used consequently to remove different groups of impurities, thanks to their orthogonality. In this work, a multidimensional and orthogonal approach on a (semi)preparative scale, namely "Integrated Batch process", was applied for the purification of a single-stranded DNA oligonucleotide. This process combines two chromatographic steps without any hold step, operator intervention or sampling of the first step. The performance parameters of the Integrated Batch were compared to those obtained in the single batch runs under different experimental conditions (chromatographic mode, eluent systems), showing the potential of this integrated approach. This proof-of-concept study illustrates how this technique can considerably reduce overall production time and how it allows to increase the robustness and reproducibility of the method, since the process is highly automated.

Peptides are a class of biomolecules with a great potential from the therapeutic point of view, because of their unique biological properties. Industrially, the production stategies adopted produce both the target peptide and a series of impurities that must be removed. Preparative chromatography is the technique of choice for the large-scale purification of biomolecules, generally performed in reversed-phase mode, using hydrophobic adsorbents (e.g., C8 stationary phases). A promising and innovative alternative is represented by mixed-mode columns, which bear two different ligands on the particle surface, exploiting two different retention mechanisms to improve the separation. This work represents a proof-of-concept study focused on the comparison of a hydrophobic adsorbent and a mixed-mode one (bearing both hydrophobic groups and charged ones) for the purification of a crude peptide mixture. Thanks to more-favourable thermodynamics, it was found that, when collecting the whole peak excluding fractions of the peak tail, the mixed-mode column led to an increase in the recovery of roughly +15%, together with a slight improvement in purity at the same time, with respect to the traditional hydrophobic column. In addition, if the whole peak, including the tail, is collected, the performance of the two columns are similar in terms of purity and recovery, but the pepetide elutes as a narrower peak with the mixed mode. This leads to a collection pool showing a much-higher peptide concentration and to lower solvent volumes needed, which is a beneficial achievement when targeting more sustainable processes. These results are very advantageous from the industrial viewpoint, because they also involve a decrease in the peptide amount contained in the peak tail, which must be reprocessed again to satisfy purity requirements.