During large-scale manufacturing of protein by E. coli fermentation, cells are typically lysed by
pressure homogenization to release the protein for further purification. To evaluate the efficiency of
protein recovery by this method, a sample of cells is collected for analysis before homogenization
and lysed by applying Bugbuster reagent and Lysonase in combination with sonication. This
chemical treatment is assumed to cause 100% lysis and release all protein for quantitation.
However, it has been observed that the relative amount of protein recovered by pressure
homogenization of the bulk material was greater than that from the chemically lysed samples. We
hypothesize that differences in lysis efficiency between pressure homogenization and chemical
treatment causes this discrepancy. We conducted a study of different chemical lysis parameters with
the goal of optimizing those conditions to achieve total protein recovery from analytical samples. This
was evaluated by comparing the titer results from the tested sample processing conditions to titer
values from lysis via bulk-scale pressure homogenization. No significant improvement in recovery
using increased amounts of Bugbuster and Lysonase was observed. However, a comparison of
recovery from whole cell extract analytical samples to such samples clarified by centrifugation
demonstrated that this step causes protein loss that is exacerbated by increased Bugbuster
volumes. Protein stability could be impacted by Bugbuster detergents, resulting in insoluble
misfolded protein that pellets during centrifugation. Such species were not included in previous titer
determinations which accounts for the lower recovery by chemical lysis. We also demonstrated that
higher concentrations of lysate may have protective effect on the stability of the protein. These
resultsshowtheimportanceofoptimizinglysisprotocolsforanalyticalsamplesforaspecificprotein
of interest.