Technical refolding of proteins: Do we have freedom to operate?
Résumé
Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins, that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features in the industrial scale. However, the value of an inclusion body based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies, economically and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein in its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤ 0.1 mg/mL). However, that requires large buffer tanks and time-consuming concentration steps. We attempt to give an answer to which extent refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in the solution and shielding hydrophobic patches. Progresses were established in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer were reduced by newly developed devices and the introduction of specific mixers. Concepts of continuous refolding were introduced in order to reduce tank sizes and increase yields. A few patents covering refolding of proteins will expire soon or have expired. This gives more freedom to operate.
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