Continuous Crystallization of Proteins in a Tubular Plug-Flow Crystallizer

Abstract

Protein crystals have many important applications in many fields, including pharmaceutics. Being more stable than other formulations, and having a high degree of purity and bioavailability, they are especially promising in the area of drug delivery. In this contribution, the development of a continuously operated tubular crystallizer for the production of protein crystals has been described. Using the model enzyme lysozyme, we successfully generated product particles ranging between 15 and 40 μm in size. At the reactor inlet, a protein solution was mixed with a crystallization agent solution to create high supersaturations required for nucleation. Along the tube, supersaturation was controlled using water baths that divided the crystallizer into a nucleation zone and a growth zone. Low flow rates minimized the effect of shear forces that may impede crystal growth. Simultaneously, a slug flow was implemented to ensure crystal transport through the reactor and to reduce the residence time distribution.

Figure 1

Set-up of the tubular crystallizer (schematic), T = temperature, L = length of tube in the respective water bath, RT = room temperature.

Figure 2

Implementation of the slug flow. Gas bubbles separate the liquid flow into segments to achieve the optimal transport of crystals along the reactor and a narrow residence time distribution (adapted from Besenhard et al.).

Figure 3

Saturation and concentration gradient along the tubular reactor. Lysozyme concentrations were determined experimentally. Supersaturation was estimated based on the solubility data shown in Table 1.

Figure 4

Microscopic picture of crystals produced in the continuous crystallizer.

Figure 5

Number density distribution of product crystals.

Figure 6

Low flow rates (v less than 1.9 mm/s) regularly led to crystal aggregation.