The microenvironment of frozen biotherapeutics and its implications for the cryopreservation of proteins and cells

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The microenvironment of frozen biotherapeutics and its implications for the cryopreservation of proteins and cells

Prof. Miguel Rodrigues

Departamento de Engenharia Química
Instituto Superior Técnico
Universidade de Lisboa

Decreasing temperature is generally expected to decrease degradation kinetics. However, the complexity of biological products (as proteins or cells), associated to multiple intricate phenomena that are triggered by cooling, often brings unanticipated results. For example, when a solution is freezing, the growth of ice crystals causes all the solutes to concentrate, typically by one or two orders of magnitude, potentiating interactions, crystallization, aggregation, pH shifts, osmotic and hydrostatic pressure, ionic force increase, protein unfolding by cold or extensive ice interfacial area. Moroer, oxygen and other gases reach saturation levels, forming air interfaces (bubbles) and dehydration is generalised. The mechanistic contribution of each of these variables cannot be clearly deconvoluted and it is also not easily correlated with product quality because of the stochastic nature of nucleation, the spatial anisotropy that is generated by freezing (and thawing or drying) and the amplification of the previous factors by limited (or deficient) process control. Experimental and modelling approaches were developed to understand and anticipate some of these mechanisms within the ice structure. Computational Fluid Dynamics was used to access the microscale and to virtually display local transient stresses, which was complemented by kinetic aggregation studies under ice-growth inhibition by isochoric cooling. This strategy has already contributed to clarify important mechanisms for biotherapeutic degradation induced by freezing and is used to assist the development of more rational formulations and optimised freeze-thaw systems.