Oxidative protein refolding on size exclusion chromatography: From batch single-column to multi-column counter-current continuous processing

Challenges in Integrative Research in Therapeutic and Diagnostic Proteins: Translation and Conformational Engineering

“…The inherent process complexity offers substantial advantages in terms of simultaneous increases in productivity, high product purity and reduced solvent requirements. Taking these advantages for bioprocessing requires fundamental understanding of continuous bio-chromatography using protein-based therapeutic biomolecules, which is the one of the key objectives applicant's research program [24][25][26][27] The limitation of SMB is the inability to achieve linear solvent gradients, or gradients other than step gradient as required in protein refolding and purification of products from a mixture of many compounds with very similar adsorptive properties.…”

“…In addition, the classical 4 zone SMB is restricted to binary separation. The new recent development in SMB technology is the use of gradients in solvent strength that alters the affinity of proteins towards the ion-exchange resins and the elution sequence36, and the concept of MCC to permit solvent gradients in each column paving for purification of more complex multi-component mixtures [26]. Different salt concentrations in the feed and eluent create regions of high and low affinity.…”

“…Due to mass transfer limitations and selectivity's among the antibody variants close to 1, the realized yield is below 10% for a desired purity of above 80% unless solvent gradient is used. Our recent work [24][25][26] on lysozyme protein refolding and solvent gradients showed improved performance (high productivity and low buffer consumption). Based on recent contemporary literature, researchers need to generate new knowledge through fundamental studies to facilitate rational process design and scale-up methodologies of MCC-SMB aimed in enhancing productivity and purity, reducing solvent consumption, improving process economics and time to market.…”

Mechanistic Study of Protein Adsorption on Mesoporous TiO2 in Aqueous Buffer Solutions

“…In summary, both the ionic strength and surface geometry have significant effects on protein adsorption. − However, there is no consensus, which factor dominates the adsorption of biomolecules on nanomaterials . Meanwhile, most of the previous study has illustrated the apparent phenomena, and it is still unclear what is the intrinsic mechanism for interpreting the experimental findings.…”

β-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on Sepharose Surface Provide an Effective Matrix for Protein Refolding

“…The efficacy of this step can be relatively high, and then the solubilized inactive protein is refolded into its active form with use of diverse approaches. − One of the refolding strategies is the dilution, which reduces denaturant concentration. , The second one is free solution in the presence of several species (e.g., additives, micelles, nanoparticles, − and nanotubes). Another method is solid-state or on-column refolding − (e.g., size-exclusion chromatography (SEC), − ion exchange chromatography (IEC), , immobilized metal affinity chromatography (IMAC), , and hydrophobic interaction chromatography (HIC) , ), and the most well-known and advanced method, which is an imitation of the in vivo folding called “artificial chaperone-assisted refolding”. ,, Rozema and Gellman developed this method, which has two steps, capturing and stripping . First, hydrophobic patches of denatured protein are shielded by the detergent, and a protein-detergent complex is formed.…”