Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography

Peptides are organic polymers composed of 2–50 amino acids linked to each other by means of covalent amide (=peptide) bonds. The composition, length and sequence of the amino acid chain have a dramatic influence on the activity of the peptide itself, for example in the human body. Peptides are called bioactive if they have a beneficial impact on body functions, on biological processes and, as a consequence, on health [ 1 , 2 , 3 ]. The literature about different kinds of bioactivity that peptides can exert on the human body is broad. They can interact with different organs and systems, such as digestive, cardiovascular, nervous and immune systems. This is the reason why they have been extensively studied for their potential applications in cosmetics, food and pharmaceutical fields. Some peptides derived from milk proteins, for instance, are considered promising alternatives to classical treatments for cancer therapy, targeting cancer cells specifically, without affecting healthy ones [ 1 ]. Other peptides, as well as some proteins, are considered to be “biomarkers”, which means species that can give an indication about the effectiveness of a treatment [ 4 ]. Recently, some peptides have also been tested to treat some symptoms related to COVID-19 disease. For instance, icatibant (a peptidomimetic constituted of 10 amino acids, antagonist of bradykinin B2 receptors and effective against symptoms of hereditary angioedema) has recently shown its potentiality to improve oxygenation in patients affected by COVID-19 at early stage [ 5 , 6 ]. Moreover, some peptidic vaccines have also been designed to provide immunity to SARS-CoV-2 [ 7 ]. This class of molecules represents an effective alternative to small molecule drugs. Peptides, especially if endogenous, exhibit remarkable advantages such as high bioactivity, specificity to the target tissues, broad range of therapeutic effects, no toxicity nor tendency to accumulate in the human body [ 8 , 9 , 10 , 11 , 12 , 13 ]. For instance, classical drugs with anticancer or anti-carcinogenic activity usually cannot recognize cancer cells from normal ones, and this causes an intrinsic toxicity of “traditional” therapies. Peptide-based treatments not showing this negative effect are being considered more and more appealing [ 1 ]. In 2014, about 60 peptide drugs had already been approved and launched on the market. At that time, the peptide market represented about 1.5% of all pharmaceuticals sales [ 14 ]. The increasing number of peptides entering clinical trials year by year indicates that there is a growing interest from the side of pharmaceutical companies in the use of this class of molecules as drugs [ 14 , 15 , 16 ]. To date, the number of therapeutic peptides approved is 70 [ 17 ]. Peptides can be classified as endogenous, if they are synthesized inside the human body, or exogenous if they are introduced into the organism from an external source. For instance, it is well-known that proteins acquired through food are an essential source of amino acids and it has been demonstrated that some specific portions of dietary proteins exert biological functionalities. Very important sources of bioactive peptides include dairy food, fish, eggs, soybean, rice, corn, peanuts etc. In most cases, the amino acid sequence of the bioactive peptide is contained within a parent protein, where it is inactive, and it can be released by enzymes during digestion [ 18 , 19 , 20 ]. In other cases, the peptide of interest can be synthesized. Recently, peptidic analogues possessing remarkable pharmaceutical properties have been developed and obtained by means of chemical synthesis [ 21 ]. The most suitable technology for the production of a given peptide strongly depends on the molecular size of the target molecule [ 22 ]. Even though often highly selective, all production methods lead to mixtures where the target peptide is present together with a series of other molecules (impurities). The target Active Pharmaceutical Ingredient (API) must be separated from all impurities produced during the manufacturing process, because strict regulations are imposed to all kinds of pharmaceuticals, for quality and safety reasons. Particularly, impurities can be divided into two groups [ 23 , 24 ]: Process-related impurities, deriving from the production method employed (salts, pieces of cells or of DNA, …) Product-related impurities, that are species chemically similar to the target product. The first type of impurities can be easily separated from the peptide of interest, e.g., through affinity chromatography, during the so-called capture step [ 25 ]. In this chromatographic technique, the ligand specifically binds to the target molecule, whereas the impurities are flushed through the column. The peptide or protein is then eluted with a suitable buffer. Elimination of product-related impurities (which is technically defined as the polishing step of the purification process) is much more challenging becaus

Despite the success of other production methods, the technique of choice to produce small to medium peptides, especially for pharmaceutical applications, is still chemical synthesis [ 45 ]. The main reasons are two: firstly, a synthesis method is developed starting from standard and well-established procedures, so its development is less complex and less time-consuming. Secondly, in the synthetic approach, differently than recombinant approach (see Section 2.3 ), modified amino acids can also be incorporated in the peptide chain. These characteristics make chemical synthesis the preferred technique for peptide production. Chemical synthesis can be performed either in liquid- or in solid-phase. Both strategies are based on similar reaction mechanisms, where amino acids and/or fragments of the desired peptide are added successively to the mixture, to react with the growing chain. In Solid Phase Peptide Synthesis (SPPS), firstly developed by Merrifield [ 46 ], one N-protected amino acid reacts with the peptide chain, which is anchored to a solid support (a resin) and, after that, the terminal amino acid is deprotected. Then, the following amino acid undergoes the same procedure. Functionalities on the aminoacid side chain need to be protected as well, in order to avoid side reactions. After the procedure is terminated and all the amino acids have reacted, the peptide is released from the resin during the cleavage step. A scheme representing the Solid Phase Peptide Synthesis is depicted in Figure 1 . The presence of the solid support allows one to recover the product simply by filtration: in this lies the reason of the success of synthesis in solid phase. Moreover, the process can be automated [ 14 , 22 , 47 , 48 , 49 ]. Currently, huge efforts are being made in order to make the synthetic processes as green as possible, by introducing the use of protecting groups and alternative solvents more sustainable than the traditional ones [ 50 , 51 , 52 ]. Liquid phase synthesis plays an important role in the manufacturing of short peptides (up to 10 amino acids). Recently, this approach gained importance for the manufacturing of longer peptides through the coupling of its previously synthesized fragments [ 53 ].

None of the aforementioned upstream methods to obtain the peptide of interest leads to a single product. Actually, a series of impurities are produced together with the target. They must be removed during the downstream step of manufacturing. The reason is that every impurity could potentially exert adverse biological activity on the human body. Therefore, very strict purity requirements are applied to pharmaceuticals. Different purification methods have been developed, with their own advantages and disadvantages. The purification strategy must be evaluated for every single case, in light of imposed requirements that, in turn, vary depending on the particular application [ 55 ]. If a peptide is produced through hydrolysis, for instance, it can be separated by means of ultrafiltration from the enzyme employed during the process and from other protein residues with higher molecular mass. Generally, in this case, the membranes of choice have a low molecular mass cut-off and the size of their pores depends on the molecular weight of the desired peptide. Anyway, the main disadvantage of this technique is the poor selectivity of the membrane [ 1 , 56 ]. A technique particularly useful in case of separation of charged peptides and proteins is IsoElectroFocusing (IEF), which is based on the same separation principles as electrophoresis. The sample is injected in a chamber where an electric field is applied, in presence of a pH gradient. Acidic species move towards the anode and basic ones towards the cathode. When a species reaches a zone with pH identical to its isoelectric point, it stops migrating. Then it can be moved to a detection windows to be identified. Therefore, IEF separates analytes depending on their isoelectric point [ 57 ]. Several modes of IEF have been developed, some of which can be used on analytical while others on preparative scale, which is the case of IEF in solution [ 58 ] or in a cellulose-based separation medium [ 59 ]. Anyway, IEF lies outside the main topic of this review and therefore it will not be treated any further. A common purification issue is the separation of a complex mixture of peptides deriving from solid-phase synthesis. The separation can only be done after cleavage and it is frequently challenging because impurities may differ from the target peptide by a single amino acid or a single functional group, resulting in very similar chemical properties [ 60 ]. Typical by-products due to solid phase synthesis include peptides with one amino acid that did not react or reacted in the wrong position, peptides with side-chain modification (oxidation, deamidation, epimerization, alkylation, ring closure or opening, incomplete deprotection) and truncated peptides [ 61 , 62 ]. Chromatography is the most suitable technique for the purification of valuable products, which is the case of pharmaceuticals, where high resolution and selectivity are required [ 63 , 64 ]. This technique allows one to obtain very high efficiency in the separation of complex mixtures, where the components have similar chemical properties. It is flexible and adaptable since a wide selection of stationary and mobile phases to choose from is available. Additionally, a number of well established chromatographic methods have already been developed at industrial level and are available for practical biopharmaceutical applications [ 55 ]. A disadvantage of chromatographic methods is that it is difficult to handle viscous mixtures, which cause increase in the backpressure; moreover, organic solvents are used almost always as mobile phase, and this poses environmental concerns regarding their toxicity and disposal [ 65 ]. Anyway, chromatography remains the technique of choice for the purification of biomolecules at laboratory, preparative and industrial level. Currently, industrial processes for biopharmaceuticals employ almost exclusively chromatography both for capture and for polishing steps, whose difference has already been explained in Section 1 . Due to the complexity of the peptide mixtures, generally a combination of chromatographic techniques based on different separation principles is required to improve the resolution power [ 66 ]. Therefore, at least two different chromatographic modes are applied consecutively, either online or offline, resulting in a multidimensional separation (e.g., two-dimensional liquid chromatography, 2D-LC). A separation performed on the basis of different types of interactions is often referred to with the term “orthogonality” [ 67 ], meaning that the two dimensions of the separation can remove different impurities. For example, ion-exchange, HILIC and reversed-phase chromatography separate the analytes depending on different features (charge, hydrophilicity and hydrophobicity) and therefore they can be used as dimensions orthogonal to each other [ 68 ]. Otherwise, also reversed-phase in acidic conditions and in basic conditions can be considered to be orthogonal separation method

Hydrophilic Interaction Chromatography (HILIC) and can be considered as a variant of normal-phase chromatography. As it is shown in Figure 3 , it is likely that HILIC involves a partitioning mechanism between the more hydrophobic mobile phase and a layer of solvent rich in water adsorbed on the stationary phase, but the phenomena responsible for such a behaviour are still not well understood [ 93 ]. Alpert [ 87 ] demonstrated that, if at low percentage of ACN the separation mechanism is mainly based on ion-exchange, on the other hand retention increases dramatically at high levels of ACN (e.g., greater than 50%). This is due to hydrophilic interactions. At the same time, electrostatic effects diminish in importance. The order of elution is the opposite than in reversed-phase chromatography, meaning that retention increases from least to most hydrophilic component [ 94 ]. HILIC has been employed for different applications. Nine peptides present in an illicit drug mixture, including oxytocin, leuprorelin, sermorelin, epitalon and melanotan II, have been separated within 35 min using a gradient elution method employing LC-DAD-MS. The mobile phases used for the gradient were MP-A: acetonitrile, deionized water and 100 mM ammonium formate pH 3.0 (80:10:10, v / v / v ), and MP-B: acetonitrile/deionized water/100 mM ammonium formate pH 3.0 (40:50:10, v / v / v ). The column was a ZIC HILIC column, made of bare silica modified with sulfobetaine. In addition, other stationary phases were tested, including CORTECS (made of bare silica) and BEH HILIC column (composed of unbound porous particles hybridized via ethylene crosslinking) [ 95 ]. A comparative study between three HILIC columns (XBridge amide from Waters, bare silica Kinetex HILIC from Phenomenex and silica with quaternary ammonium and sulphonic acid Nucleodur HILIC from Macherey Nagel) was accomplished in the context of quantification of proteotypic tryptic peptides, where chromatography was coupled with ESI-MS. It resulted that a higher sensitivity was obtained using the amide column without salt buffer in the mobile phases [ 96 ]. HILIC technique proved to be a good solution also for the separation of immunoglobulin deriving peptides containing modified residues (deamidated asparagine and oxidized methionine) from their native forms, a procedure necessary for the identification and quantification of critical impurities. The method involved the use of a Penta-HILIC column and a gradient elution program where the percentage of strong solvent (water containing 0.1% formic acid and 50 mM ammonium formate) increased from 5 to 70% within 90 min, whereas the content of ACN decreased [ 97 ]. A variation of the classical HILIC technique, developed by Mant et al. envisaged to perform the peptide separation using a salt gradient (sodium perchlorate) in the presence of isocratic high content of organic solvent, specifically ACN around 80–90%. The column employed was a Halo Penta-HILIC column. This technique was applied to different mixtures of synthetic peptides differing in structure, number of amino acids (from 10 to 26) or charge, and in those circumstances it resulted to be more suitable for peptide separations than the traditional version of HILIC in terms of resolving capabilities [ 98 ]. For instance, Figure 4 shows the differences in the separation of a mixture of three synthetic α -helical peptides by using reversed-phase chromatography (panels A and B), HILIC (panel C), isocratic HILIC (panel D) and HILIC/SALT (panel E). The peptides considered have the same structure (Ac-ELEKLL X ELEKLLKELEK-amide) except for the amino acid X in position 7, which in the three cases is either Ser (LS7), Thr (LT7) or Val (LV7). Ser and Thr are much more polar than Val, which is non-polar, and this explains the order of elution.

4.1. The Need to Investigate the Theoretical Basis of Adsorption The goal of purification procedures is to achieve high purity products with elevated recovery, possibly by means of high throughput and economically viable processes. Combining all these requirements is challenging. Moreover, during the development of purification processes, often a “trial-and-error” strategy is followed, an approach which is time-consuming and costly. Indeed, it is estimated that around 50 to 70% of the whole manufacturing cost is attributable to the downstream processing [ 49 , 65 , 104 ]. In this regard, it appears necessary to model the chromatographic behavior of the species of interest through theoretical and computational instruments, in order to define a priori proper experimental conditions for the purification of the target [ 105 , 106 ]. Particularly, knowing the thermodynamic properties (that is, the adsorption isotherms) involved in the adsorption of the target molecule is the basis to predict its behaviour in a wider range of working conditions, which makes trial-and-error optimization superfluous. Additionally, mechanistic models can be a useful tool to predict the impact of process parameters and experimental conditions on product quality [ 107 ]. Computational methods, such as Inverse Method, allow one to obtain thermodynamic data starting from very little amount of compound, which is particularly advantageous when the material of interest is expensive or present in limited amount, and this could promote the need for model building among pharmaceutical companies during process development phases. Some of the authors of this review have published a study regarding the modeling of the chromatographic behaviour of a peptide in non-linear conditions [ 49 ]. The investigation of thermodynamic adsorption equilibria regulating the separation in batch of the compounds contained in the peptide mixture could also be used to scale the process to continuous chromatography, a field where the knowledge of non-linear chromatography has not been deeply investigated yet.

Bioactive peptides show a broad range of activities, that vary from antimicrobial, antihypertensive, immunomodulatory, antioxidant, etc; therefore, they can be employed as food additives or pharmaceutical ingredients for the therapy of some diseases. They can be produced by means of hydrolysis of the parent protein or they can be chemically synthesized, especially through Solid Phase Peptide Synthesis. Their application as biopharmaceuticals requires the peptide to fulfill very strict purity and quality specifications; as a consequence, choosing proper purification processes is one of the most important parts of the manufacturing process design. Liquid chromatography is usually the best technique employed for purification, especially when used in reversed-phase mode. Additionally, ion-exchange chromatography and hydrophilic interaction chromatography are frequently used, depending on the hydrophobic/hydrophilic properties of the peptide and on its charge. Innovative mixed-mode stationary phases, on the other hand, combine reversed-phase and ion-exchange features. So, depending on the chemical characteristics of the peptide, for a large class of compounds a suitable chromatographic technique can be easily found. Thanks to important technological advancements, continuous chromatographic techniques based on the concept of countercurrent chromatography are becoming established. MCSGP represents a remarkable case of process which is sparking interest, because of improvements gained in product quality, economic advantages related to higher productivity and, nonetheless, a simplification of the process due to the automation of operations. Although huge progress has been done in the purification processing, theoretical studies focusing on the modeling of the process should be implemented, to favour a quick optimization of the procedures. Additionally, some obstacles and barriers must still be faced from the point of view of the regulation, in order to assure to meet Good Manufacturing Practise (GMP).