Interaction Packaging Materials & Pharmaceutical Formulation
Project 2.4: "Interaction between Packaging Materials and Pharmaceutical FormulationsProcess Optimization for Liquid Formulations"
| Project Managers: | Dr. Frank Sinner Prof. Johannes Khinast |
| Duration: | 01.11.2008 to 31.12.2011 |
| Business Partners: | Fresenius Kabi Austria GmbH Zeta Biopharma GmbH |
| Scientific Partners: | JOANNEUM RESEARCH Forschungsgesellschaft mbH Institute for Process and Particle Engineering (TUG) |
Abstract
The freezing of biologics is an increasingly common unit operation in the production of protein therapeutics, which is used for storing bulk solutions before they are processed into the final drug product. Proteins are interacting with water in the liquid phase and with the frozen ice matrix and are subject to structural perturbations that can induce freeze damage. The freezing process leads to the cryoconcentration of proteins and solutes, inducing hot spots with higher concentrations than in the bulk solution. The concentration effects may lead to pH shifts of the buffering solutions, which may harm the proteins. A change in ionic strength or in the protein concentration itself may also have adverse effects on the product’s quality. The cryoconcentration can be explained by the dendritic ice formation that moves the solute to front of the ice. The solutes become trapped and form the local maxima since water is their only compound that can change its aggregation state. The solutes and proteins can either crystallize or form a glassy state. It has been proposed that proteins tend to unfold at the hydrophobic ice patches, which induce the unfolding of the native state. This unfolding is not always reversible and may lead to the protein damage that can eventually result in the protein aggregation. Protein aggregates have been addressed by regulatory agencies due to their potential to trigger adverse immune reactions.
During the formulation step of new protein drugs, the freezing and thawing are standard tests during the protein’s investigation. However, due to the lack of small-scale freezing units, these tests are commonly carried out in uncontrolled conditions by placing simple flasks in a freezer, which often makes working under defined boundary conditions impossible. Additionally, it is impossible to transfer the results of such a system to a large-scale unit. Small-scale tests are performed because of the high costs of protein therapeutics.
To date, the influence of the freezing and thawing unit operations on pharmaceutical proteins has remained poorly understood. There is very limited evidence of structural changes occurring in proteins under these conditions. Therefore, a better overall understanding of the freezing process and of its influence on the protein structure and quality in general is of great interest to the pharmaceutical industry. It is crucial to know at each point in time what the quality of the product is, which calls for a system that can transfer the results from a small scale to an industrial scale.
Project Goals
Development of a simulation routine for the prediction of the freezing and thawing processes in a commercial freeze container
Prediction of the solute distribution in a frozen matrix
Investigation of the freezing and thawing effects on model proteins
Development of a lab-scale freeze container
Development of a scale-down model of the commercial freeze container based on the simulation results
Experimental validation of the simulations
Present Project Results
A simulation routine has been developed that can predict the freezing behavior of water in a commercial freeze container. The simulation results have been verified using the published literature data. The multi-phase model involves the liquid and solid phases. Further improvement of the simulation routine made it possible to predict the movement of the proteins, the evolution of the ice front, the convective effects and the zone of the hase transition.
Based on the simulation results, a lab-scale freeze container was built to investigate the freezing process on a smaller scale and, especially, to evaluate the freezing damage to the proteins. It has a working volume of 200 ml and offers a controlled freezing and thawing process, temperature mapping and an easy sample removal.
A set of enzymes was tested experimentally to function as a reference protein. Biochemical and biophysical methods were established to address especially the freezing-induced protein damage. First experiments in the lab-scale freeze container successfully reproduced the expected cryo-concentration of the solutes at the last point of freeze.
Project Challenges
The freezing of protein solutions is a common process for both industry and science. However, this process is typically not considered a key process and, therefore, the research effort in that field has been limited. Scientific publications dealing with the freeze-damage of proteins are very rare. Companies do not pay much attention to this process and accept a certain amount of product loss during the freeze step.
A big challenge to a scientific investigation of the freezing and thawing process is the limited availability of methods to evaluate changes in the protein. Most biophysical methods are aiming at proteins in the solution. For structure characterization of proteins in the solid state (e.g., X-ray crystallography) a well-ordered crystal state is required. It is therefore necessary to establish methods to evaluate changes in the protein structure in the ice state. The next step is the development of tools for process control. Currently, the only parameter that can be followed is the temperature, which only provides limited information.
Project related PublicationsThe freezing of biologics is an increasingly common unit operation in the production of protein therapeutics, which is used for storing bulk solutions before they are processed into the final drug product. Proteins are interacting with water in the liquid phase and with the frozen ice matrix and are subject to structural perturbations that can induce freeze damage. The freezing process leads to the cryoconcentration of proteins and solutes, inducing hot spots with higher concentrations than in the bulk solution. The concentration effects may lead to pH shifts of the buffering solutions, which may harm the proteins. A change in ionic strength or in the protein concentration itself may also have adverse effects on the product’s quality. The cryoconcentration can be explained by the dendritic ice formation that moves the solute to front of the ice. The solutes become trapped and form the local maxima since water is their only compound that can change its aggregation state. The solutes and proteins can either crystallize or form a glassy state. It has been proposed that proteins tend to unfold at the hydrophobic ice patches, which induce the unfolding of the native state. This unfolding is not always reversible and may lead to the protein damage that can eventually result in the protein aggregation. Protein aggregates have been addressed by regulatory agencies due to their potential to trigger adverse immune reactions.
During the formulation step of new protein drugs, the freezing and thawing are standard tests during the protein’s investigation. However, due to the lack of small-scale freezing units, these tests are commonly carried out in uncontrolled conditions by placing simple flasks in a freezer, which often makes working under defined boundary conditions impossible. Additionally, it is impossible to transfer the results of such a system to a large-scale unit. Small-scale tests are performed because of the high costs of protein therapeutics.
To date, the influence of the freezing and thawing unit operations on pharmaceutical proteins has remained poorly understood. There is very limited evidence of structural changes occurring in proteins under these conditions. Therefore, a better overall understanding of the freezing process and of its influence on the protein structure and quality in general is of great interest to the pharmaceutical industry. It is crucial to know at each point in time what the quality of the product is, which calls for a system that can transfer the results from a small scale to an industrial scale.
Project Goals
Development of a simulation routine for the prediction of the freezing and thawing processes in a commercial freeze container
Prediction of the solute distribution in a frozen matrix
Investigation of the freezing and thawing effects on model proteins
Development of a lab-scale freeze container
Development of a scale-down model of the commercial freeze container based on the simulation results
Experimental validation of the simulations
Present Project Results
A simulation routine has been developed that can predict the freezing behavior of water in a commercial freeze container. The simulation results have been verified using the published literature data. The multi-phase model involves the liquid and solid phases. Further improvement of the simulation routine made it possible to predict the movement of the proteins, the evolution of the ice front, the convective effects and the zone of the hase transition.
Based on the simulation results, a lab-scale freeze container was built to investigate the freezing process on a smaller scale and, especially, to evaluate the freezing damage to the proteins. It has a working volume of 200 ml and offers a controlled freezing and thawing process, temperature mapping and an easy sample removal.
A set of enzymes was tested experimentally to function as a reference protein. Biochemical and biophysical methods were established to address especially the freezing-induced protein damage. First experiments in the lab-scale freeze container successfully reproduced the expected cryo-concentration of the solutes at the last point of freeze.
Project Challenges
The freezing of protein solutions is a common process for both industry and science. However, this process is typically not considered a key process and, therefore, the research effort in that field has been limited. Scientific publications dealing with the freeze-damage of proteins are very rare. Companies do not pay much attention to this process and accept a certain amount of product loss during the freeze step.
A big challenge to a scientific investigation of the freezing and thawing process is the limited availability of methods to evaluate changes in the protein. Most biophysical methods are aiming at proteins in the solution. For structure characterization of proteins in the solid state (e.g., X-ray crystallography) a well-ordered crystal state is required. It is therefore necessary to establish methods to evaluate changes in the protein structure in the ice state. The next step is the development of tools for process control. Currently, the only parameter that can be followed is the temperature, which only provides limited information.
M.Iannuccelli, D. Suzzi, B. Sirnik, A. Rinderhofer, J. Khinast,
Numerical Simulation of Freeze-Thaw Biopharmaceutical Processes
Chemical Engineering Transactions, Volume 24, 2011
