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F&T -70° Why deep freeze? The storage of high-quality biopharmaceuticals is a major challenge for the industry. Proteins are easily affected by changes in ambient conditions and react sensitively to changes in their environment. The complex threedimensional protein structure is held together by weak, intra-molecular interactions and is therefore particularly delicate. The native folding of protein substances is essential for their efficacy: only if a protein is correctly folded will the right molecules be bound. Changes in the structure will have fatal consequences; in certain cases, they do not only result in a loss of efficacy, but may even cause pathological effects.
This means the process of storing an active protein substance must be the result of an exceptionally careful and sophisticated design process and all influencing factors must be known since the substance will often need to be stored for years.
All Photographs: F&T Services ŠZETA.
The pharmaceutical industry has found deep-freezing to be a stable method of storing protein solutions. The active ingredient is cooled in freezing chambers or in controlled freezing containers until the whole bulk solution has reached the required
temperature level. However, we know from practice that rearrangements and structural effects will occur in proteins even in the frozen state, which can also result in changes to their native structure. Damaging alterations may, for example, lead to increased aggregate formation or flocculation. In order to preserve the product quality, freezing at very low temperatures is required. A change in the protein structure is to be prevented by choosing temperatures below the glass transition temperature. The change in product quality and the various freezing effects depend on the one hand on the process parameters selected, and on the other hand they are strongly influenced by the composition of the protein solution. In most cases, quality changes caused by the effects described above will entail additional process steps and drug losses. Product loss must in any case be reduced to a minimum. Identifying the relationships between the composition of the protein solution, concentration, pH value, additives, cryo-protectors or additives and the behaviour of the quality of the protein solution after one or more freeze and thaw cycles is the basis for advanced understanding of the F&T process. More risk management, less product loss The frozen protein solution becomes increasingly concentrated as ice crystals form during freezing. The auxiliary substances, such as buffer salts, gradually lose their effect depending on their solubility. The pH value of a sodium phosphate buffer can, for example, drop from 7.0 to below 4.0 during freezing. The ionic strength can also increase significantly. In addition to that, the formation of ice crystals leads to an ice-liquid interface which can impact protein folding. The influencing
parameters and their effects differ widely for each substance. This is why they are examined for each product individually in the lab, using tailor-made F&T equipment. The aim is to understand the product behaviour in one freeze and thaw cycle in order to be able to derive optimum process understanding from this knowledge. Once the basic behaviour of the protein solution during freezing has been understood, the F&T processes developed in the lab will be scaled to pilot scale. The freezing process is analysed, optimised further and precisely defined. The specified process is the basis for industrial processing and thus an important element in the development of optimum storage of the protein substance. The pilot scale allows for various analytical methods and represents a valuable intermediary scale. The most suitable process parameters for the product need to be determined and optimised. Contrary to initial, business driven assumptions this does not necessarily have to be the speed of freezing, because one thing is for sure in the F&T process: quality takes precedence over efficiency. As explained above, the PilotFreeze examines the processes that were previously developed in the laboratory to prepare them on a larger scale for industrial processing. Practical applications have shown that the PilotFreeze can also be used to verify existing industrial F&T processes. The processes are then checked again and optimised in the course of optimisation or retrospective process validation before they are fed back to industrial production by means of a technology transfer. In summary we can say the PilotFreeze analysis is as important for the process development of new protein solutions as it is for the optimisation of current processes.