1. Introduction
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Peptides represent a unique class of pharmaceutical compounds intermediate in molecular weight between small molecules and proteins, with desirable biological activities, including antioxidant, antihypertensive, antithrombotic, antilipogenic, antibacterial, and anti-inflammatory effects. Special properties, including low toxicity and high specificity, make these molecules of particular interest to the food and pharmaceutical industries. (1−3) At present, more than 80 polypeptide drugs such as oxytocin, vasopressin, and cyclosporine are approved in the global market, with more in preclinical and clinical development. (3) With the fast development in upstream processes such as the synthesis of these peptides, fermentation, and cell culture technologies, the production bottlenecks shift to downstream purification and formulation steps. The current mainstream purification method is chromatography, (4) but it has some significant shortcomings, such as low throughput, large water consumption, and high cost. The current purification cost can account for more than half of the total production cost in the biomanufacturing process. In addition, chromatography is difficult for scaling up in the manufacturing of peptides. (5,6)

Crystallization is a widely used separation and purification technology in the pharmaceutical industry, which presents higher titer and better scale-up capability than traditional packed-bed chromatography. (7,8) The application of crystallization as an alternative pathway for the separation and purification of peptide drugs has become attractive for industrial manufacturing. In addition, from the perspective of drug formulation, crystalline products have the advantages of higher purity, physicochemical stability, and dissolution characteristics compared to standard protein formulations such as aqueous solutions and amorphous precipitated lyophilizes. (9) However, it is challenging to form a highly ordered crystal structure with large, complex, and flexible peptides, proteins, and even monoclonal antibodies. (10) The nucleation and growth kinetics are relatively slow. (11) Yang et al. (12) proposed a set of optimized crystallization conditions that still need 24 h to produce monoclonal antibodies CD20 in needle-shaped crystals in a very reproducible manner with high yield. Therefore, how to obtain high yield and high-quality crystalline products in a short residence time becomes a crucial question. Recently, the research on peptide crystallization has focused on crystallography and structural determination to develop an efficient technology for the separation and purification of biomacromolecule crystals from biological mixture materials. (13) In addition, from the perspective of industrial crystallization, the stability, crystallinity, size, particle size distribution, crystal shape, and purity of the crystalline products are also important. Downstream processing of poor crystalline products such as tableting, filtration, and washing are often difficult, resulting in the lack of process robustness and poor final product quality. (14) Therefore, it is extremely important to control and strengthen this crystallization process, especially for the nucleation and growth process. (15)

The Taylor vortex device, which can generate a constant shear environment, has been widely used in aviation, water treatment, pharmaceutical engineering, and chemical industry. (16) The Taylor vortex is a fluid motion existing in the annular gap of two coaxially positioned cylinders, and through the relative rotation of the inner and outer cylinders, a series of secondary vortices that are alternating in the axial forward and negative, axisymmetric, and orderly arrangement superimposed on the shear flow are generated in the annular gap, as shown in Figure 1a. (17) In the Taylor vortex device, the mixing strength is controlled mainly by adjusting the speed and gap width. The Taylor vortex in the crystallizer can improve the mixing behavior and the flow field distribution, which can effectively enhance heat and mass transfer. (18) Moreover, the Taylor vortex can produce a uniform shear force, which can effectively strengthen the crystallization process and obtain crystalline products with a uniform particle size distribution. (19) It is reported that the Taylor vortex has been applied to a variety of processes in industrial crystallization such as reaction crystallization, drowning-out crystallization, and cooling crystallization. (19) Kim et al. (20) demonstrated from both experimental and modeling prospects that Taylor vortex has a better mass transfer effect than random turbulence, thus promoting stable polycrystalline nucleation and phase transition of histidine. Wang et al. (18) used the Taylor vortex to improve the mixing behavior, so that the velocity distribution of the flow field was uniform, and hydroxyapatite with uniform particle size distribution was obtained efficiently, which provided a reference for the synthesis of inorganic micronanomaterials. The Taylor vortex has also been reported to have great advantages in enhancing the crystallization of biomacromolecule drugs. Sun et al. (21) obtained high-quality lysozyme and thaumatin crystals by using Taylor vortex. Tao et al. (19) successfully solved the crystal structure of lysozyme with 1.86 Å diffraction accuracy by combining Taylor vortex with poly(ionic liquids) and obtained 86% yield within 3 h, which greatly improved the production efficiency of lysozyme crystals.

Vancomycin (glycopeptide drug vancomycin hydrochloride) is widely used in the treatment of infections caused by methicillin-resistant Staphylococcus aureus and penicillin-resistant pneumococcus, (22) which is known as the ″last line of defense″ of antibiotics. (23) The chemical structure of vancomycin is shown in Figure 1b. At present, the industrial purification and separation of vancomycin are mainly realized using chromatography technology. (1) There is limited research on the industrial crystallization of vancomycin. In the studies by Kim et al., (24) a combination of cooling, salting-out, and antisolvent crystallization took 24 h to reach 95% yield. Pu et al. (25) investigated the behavior of vancomycin salting-out crystallization and determined the conditions for the formation of octahedral massive crystals in 96-well plates, further transferring the results to batch crystallization (1.5 and 15 mL) in a sealed vibrating vessel. However, incubating at room temperature for 24 and 48 h under gentle shaking, only 50% yield could be obtained. Therefore, it is important to explore alternative strategies to amplify and accelerate the crystallization process of vancomycin octahedral crystals to increase the yield and productivity.

In this study, vancomycin was selected as the model peptide to perform salt-out crystallization in static conditions, the mixing-tank (MT) crystallizer, and the Couette–Taylor (CT) crystallizer under equal crystallization conditions. The influences of thermodynamic (concentration and temperature) and kinetic (shear rate and gap size) parameters on the crystalline products (crystal shape, yield, and crystal size distribution) in the CT crystallizer were investigated.

Figure 1. Flow pattern of the Taylor vortex device (a) and the chemical structure of vancomycin (b)

Figure 2. Schematic diagram of CT crystallizer (a) and MT crystallizer (b).