Multi-variable operational characteristic studies of on-column oxidative protein refolding at high loading concentrations

Oxidative protein refolding on size exclusion chromatography at high loading concentrations: Fundamental studies and mathematical modeling

“…This may be due to increased non-specific interaction at higher concentration of early intermediates with the gel in quenched experiments compared to short-lived early intermediates in reactive experiments. However, this is in contrast with that presented in our previous work [31] where the same elution volumes were observed and suggest that Superdex75pg surface properties changes over time well before expiry of the gel as indicated by manufacturer (∼2 years). Binding of blue dextran to the gel during void volume measurements (as mentioned before) was another indication of such change over time.…”

“…The above loading concentration range was selected based on previous studies which demonstrated no aggregation formation within this range [31]. Fractions of 1 mL were collected and analysed using RP-HPLC to determine the concentration of correctly refolded protein.…”

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.…”

“…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.…”

“…The common approach used to retrieve active protein from inclusion bodies involves three steps: inclusion body isolation, solubilization of the aggregated protein, and refolding of the solubilized protein. The efficiency of the first two steps is relatively high but renaturation yield is limited by the accumulation of inactive misfolded species and aggregates [24]. Critical to the development of an efficient and economic denaturant based solubilization steps for full bioactivity recovery in the refolding are: a) Determination of the minimum amount of denaturant (e.g., urea) needed.…”