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FEATURED CASE STUDY
Enabling Ultra-rapid Development and Scaled-up Production of Critical Reagent Preparation for Pandemic Diagnostics and Clinical Applications
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A plethora of example suggests, overproduction of recombinant proteins in E. coli can form insoluble aggregates, misfolded conformations resulting functionally inactive product. Moreover, scaling up those proteins commonly leads to aggregation followed by precipitation resulting loss of productivity always been a painstaking experience.

At Premas Biotech, we provide technology to solve the problem during the course of process development followed by successful scale-up. An in-house case study exemplified the development from cloning to large scale high yield production of functionally active protein X at reduced cost for the diagnostics tests within short time span.

Challenges:

Protein X, the most critical reagent for the development of the rapid diagnostic tests, comprises of more than 1600 amino acids and largest among orthologs. Moreover, it was having unanticipated structural differences due to presence of distinct motifs with different structural domain orientation and inter-domain interactions resulting unique bifunctional activity. The protein was highly sensitive to temperature, pH, redox-potential, viscosity of the solution, concentration, mechanical shear and macromolecular surroundings. These biophysical parameters tempting the biochemical functionality of the protein instantly and also in the long-time storage.

Premas, Breaking the Ground:

To solve this puzzle, we planned strategy for each unit operations. At the cloning level, where we incorporated a small overhang to the original sequence without hampering functional domain to help in the appropriate folding of the protein. Interestingly, during assessing the activity post-purification at small scale, we observed that sequence having overhang was functionally active on the contrary other one was not.

After large number of colony screening using high yield expression system, we took forward the clone for further purification at R & D scale with challenges of instant aggregation. We screened wide range of buffer composition and additives for proper cushioning; developed mechanical setup for ultra-slow mixing resulting significantly less aggregation while doing buffer exchange. During at-scale (50L) chromatographic run we found dendritic thread like aggregates were appearing in elution fractions. We scouted resins with different properties, binding capacities, played with bead volume and retention time to get rid of the aggregated fibrils. At larger scale, final formulation was the most difficult step to optimize due to high shear sensitivity of the protein X. So, we optimized the step with diverse lumen size of the diafiltration unit, different material of construct and suitable transmembrane pressure. This step highly contributes to the critical quality attributes (CQA) of the product because during this step, genesis of any aggregated nanofibrils could act as nucleus of higher order aggregates resulting loss of activity in the long-time line.

Thus, we successfully produced multiple grams of the product at 5L and 50 L fermentation scale, with high yields of protein expression/litre. This material was tested for its activity and other release parameters and used for field trials and commercial kit manufacturing. And remarkably the entire development process was less than a quarter of a year. This product was successfully used for screening large number of populations on-field with higher sensitivity and specificity.

Measurable Impact:

1) Successfully expression and purification of protein X with high yield.
2) In short time frame, it was scaled up-to 50L fermentation batch producing more than 6gm of material per batch for preclinical and clinical studies.
3) Reduction of cost of goods (COGs) by scale-up, process development using high expressing clone and higher recoveries during purification. This was highly impactful in reducing the manufacturing cost of the kit.


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