High-throughput measurement of protein stability in microtiter plates

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C-1/2 +/- 0.05 M, m(G), and DeltaG(H2)O) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability. (C) 2005 Wiley Periodicals, Inc

Integration of host strain bioengineering and bioprocess development using ultra-scale down studies to select the optimum combination: An antibody fragment primary recovery case study.

An ultra scale-down primary recovery sequence was established for a platform E. coli Fab production process. It was used to evaluate the process robustness of various bioengineered strains. Centrifugal discharge in the initial dewatering stage was determined to be the major cause of cell breakage. The ability of cells to resist breakage was dependant on a combination of factors including host strain, vector, and fermentation strategy. Periplasmic extraction studies were conducted in shake flasks and it was demonstrated that key performance parameters such as Fab titre and nucleic acid concentrations were mimicked. The shake flask system also captured particle aggregation effects seen in a large scale stirred vessel, reproducing the fine particle size distribution that impacts the final centrifugal clarification stage. The use of scale-down primary recovery process sequences can be used to screen a larger number of engineered strains. This can lead to closer integration with and better feedback between strain development, fermentation development and primary recovery studies. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc