If continuous processing is to be employed in pharmaceutical production, it is essential that continuous crystallization techniques can meet the purity and yield achievable in current batch crystallization processes. Recycling of mother liquor in steady state MSMPR crystallizations allows the yield in the equivalent equilibrium batch process to be met or exceeded. However, the extent to which yield can be increased is limited by the buildup of impurities within the system. In this study, an organic solvent nanofiltration membrane was used to preferentially concentrate an API (deferasirox, M.W. = 373 Da) and purge the limiting impurity 4-hydrazinobenzoic acid (MW = 152 Da) from the mother liquor recycle stream in a mixed solvent (THF:ethanol) antisolvent (water) system. Incorporation of the membrane recycle allowed yields of 98.0% and 98.7% to be achieved. This compares to the following: a control MSMPR run without a membrane (70.3%), an equivalent batch process (89.2%), and the current commercial batch process (92%). Comparable product impurity levels were measured for the following: the MSMPR membrane recycle experiments (0.15 ppm and 0.22 ppm), the MSMPR control (0.13 ppm), and batch (0.32 ppm) control experiments. All processes met the regulatory specifications of a maximum of 3 ppm of the impurity 4-hydrainobenzoic acid.
The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.
Two-phase flow is investigated in the context of liquid chromatography. In order to describe the column behavior in the presence of multiple convective phases, an equilibrium theory model is established, accounting for both adsorption and multiphase flow. The validity of this model is studied specifically for the experimental system phenetole−methanol−water on a Zorbax 300SB-C18 column. For this purpose, the experimental system is characterized in terms of thermodynamic liquid−liquid equilibria, adsorption behavior, and hydrodynamic properties by independent experimental campaigns. Mathematical relationships are established, describing the investigated effects. Implementation of these relationships in the equilibrium theory model and application of the model to calculate elution profiles provides a quantitative description of experimental data from dynamic column experiments. The good agreement validates the model, its assumptions, and the established relationships. This study provides a thorough insight on the implications of multiple convective phases in the context of liquid chromatography.
This contribution presents the derivation and solution of model equations for a chromatographic system in the presence of multiple fluid phases. While chromatographic models commonly account for adsorption and one single convective phase, macroscopic models for applications in natural reservoirs deal with multiphase flow but usually neglect adsorption effects. This work considers an equilibrium theory model and a lumped kinetic model which account for both multiphase flow and adsorption phenomena. Analytical and numerical solutions of these models are derived and applied to a specific chromatographic system, which was investigated in detail in a companion paper. The analysis of equilibrium theory solutions provides a deep insight in the mathematical and physical implications of two-phase flow and adsorption. The comparison of analytical and numerical solutions validates both models and highlights advantages and drawbacks. The presented model equations and solutions are of considerable interest not only in the context of liquid chromatography but also for several applications involving natural reservoirs.
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