key: cord-0747266-520wq23x authors: De Luca, Chiara; Felletti, Simona; Lievore, Giulio; Chenet, Tatiana; Morbidelli, Massimo; Sponchioni, Mattia; Cavazzini, Alberto; Catani, Martina title: Modern trends in downstream processing of biotherapeutics through continuous chromatography: the potential of Multicolumn Countercurrent Solvent Gradient Purification date: 2020-09-24 journal: Trends Analyt Chem DOI: 10.1016/j.trac.2020.116051 sha: 279611ea5bd5217eaa91e97cf2b6f8f9ec0668cb doc_id: 747266 cord_uid: 520wq23x 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. Since the 1980s, biopharmaceuticals have emerged as an innovative class of therapeutics, due to their highly specific activity, a feature that cannot be imitated by traditional drugs. [6]. They usually include nucleic acids, host cell proteins, lipids, components of the cell culture media, salts, etc. which derive from the manufacturing process. Very often, affinity into the column until its breakthrough. The product specifically binds to the stationary 74 phase, whereas all the other different species flow through the column and can be discarded. For instance, Staphylococcus Protein A-based stationary phase is largely employed for the chromatography leads to several advantages, especially increased recovery and better resin utilization, but this comes at the expense of the hardware complexity [23] . specifically designed for challenging separations where many product-related impurities are 105 present. Its operating principles will be discussed and its advantages over traditional single-106 column techniques will be presented. Method transfer from batch to continuous will be also 107 illustrated, together with a synthetic overview of most interesting applications of MCSGP. In doing this, an effort has been done to describe the process from the viewpoint of 109 analytical chemists (more than that of chemical engineers) in order to make the technology 110 more familiar to this community. Purity is the first parameter that is essential for pharmaceutical scopes. It is defined as the 117 ratio between the area of the product peak and the total area of the HPLC chromatogram: 118 purity is calculated as the mean of the purities of the pools at the steady state. The outcome of the separation (i.e. resolution of the main peak from the impurities) has a 145 high impact on the performance of the whole process. As mentioned before, it frequently happens that batch purifications, especially when many 147 product-related impurities are present, are affected by a yield-purity trade-off. This situation is schematically represented in Fig. 1 . If the overlapping regions are completely discarded, An appealing possibility to overcome the limit of batch chromatography described above is to replace the single column process with a continuous (or semi-continuous) countercurrent chromatographic process, where the chromatographic system is continuously fed with the 165 crude mixture. To realize the continuous (or semi-continuous) mode, the instrument must be equipped with two or more (identical) columns connected through a series of valves. The The use of continuous chromatography operations has considerable advantages not only in 172 terms of recovery of the product (as it will be illustrated in the following) but also in terms 173 of automatization of the purification process. The interactions. For the sake of space, this technique will not be described in this paper, therefore the interested reader is addressed to other recent papers on the subject [6,23,30- • Zone 2: weakly adsorbing impurities (from now on called W), which are less retained 212 than the target product, start eluting from the column. • Zone 3: product (P) starts eluting from the column, but the weakly adsorbing impurities and thus it is discarded. At this stage, the columns are disconnected. • Then, valves switch position and the columns get interconnected. This means that W/P, 262 the overlapping region of W and P, is directly loaded from column-1 (zone 5) into column-2 (zone 1). Inline dilution is applied to ensure that W/P is re-adsorbed on 264 column-2. • The columns work again in batch mode and a window where product purity satisfies the 266 requirements imposed is recovered from column-1 (zone 6). At the same time, column-2 is loaded with some fresh feed (zone 2). • After that, the columns get interconnected again to allow the recycling of P/S region from 269 column-1 (zone 7) into column-2 (zone 3). Inline dilution is applied to ensure that P/S is The characteristic times of the design batch chromatogram in Fig. 1 correspond to the 292 switching of the inlet and outlet valves of the columns in MCSGP (see Fig. 2 ), that regulates 293 the path accessible to the eluent stream. Fig. 3 shows in detail the path followed by the An important aspect to be considered is that the overlapping regions contain a higher 299 percentage of modifier than at the beginning of the gradient. Therefore, when they are recycled, they need to be diluted with an inline dilution stream, so that the product can be 301 adsorbed on the stationary phase. The fraction containing W/P is diluted to reach the 302 modifier concentration that can be found at t B , so that the product adsorbs on the stationary 303 phase while the weak impurities start moving along the column. The window containing P/S 304 is diluted to reach the percentage of organic modifier at the beginning of the gradient (t A ), because both the product and the strong impurities must be retained. The amount of fresh feed which is injected switch after switch (zone 2 of Fig. 2 ) is 307 calculated in order to maintain the mass of target compound constant into the system. Therefore, the mass of P to be loaded at every switch is the difference between the quantity 309 of target product loaded in the batch run and the amount of target product which is recycled 310 within the overlapping regions (zone 1 and 3). The first thing to do in order to transfer a batch method to MCSGP is to calculate a Pareto 314 curve reporting purity as a function of yield for the batch method (see Fig. 5 ). This is The first trial is usually done by choosing the values of t C and t D corresponding to a certain 335 hypothetical pool in which purity fulfills the requirements and recovery is sufficiently high. t B and t E must be set in order to minimize the amount of product eluting in the waste using a 2-column MCSGP equipment. In that case, the yield was 23% higher than the batch, purification strategy is that of oligonucleotides. This technique applied to a mixture of 386 oligonucleotides allowed to increase the mass recovered by 50% at a target purity of 92% [50]. MCSGP can be applied also in the case of cannabinoids identification and purification. benefit also with respect to the economics of production [41] . potential to act as driver for the shift to precision medicine [52] . relies essentially on the well-known theory of nonlinear chromatography, robust, validated Although, as seen above, MCSGP can be applied very conveniently down-stream to batch 428 or fed-batch bioreactors, we believe that it will play a major role also in the establishment of The authors declare that they have no conflict of interest. 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