Multivariate Analysis and Statistics in Pharmaceutical Process Research and Development.

The application of statistics in pharmaceutical process research and development has evolved significantly over the past decades, motivated in part by the introduction of the Quality by Design paradigm, a landmark change in regulatory expectations for the level of scientific understanding associated with the manufacturing process. Today, statistical methods are increasingly applied to accelerate the characterization and optimization of new drugs created via numerous unit operations well known to the chemical engineering discipline. We offer here a review of the maturity in the implementation of design of experiment techniques, the increased incorporation of latent variable methods in process and material characterization, and the adoption of Bayesian methodology for process risk assessment.

Pyruvate kinase-deficient Escherichia coli exhibits increased plasmid copy number and cyclic AMP levels.

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

Regulating expression of pyruvate kinase in Bacillus subtilis for control of growth rate and formation of acidic byproducts.

Our prior work has shown that a pyk mutant of Bacillus subtilis exhibited diminished acidic byproduct accumulation, dramatically elevated phosphoenolpyruvate (PEP) pool, and reduced growth rate. To determine if a low acetate-producing but fast-growing strain of B. subtilis could be developed, we placed the expression of the pyk gene under the control of an inducible promoter. Enzyme measurements proved that PYK activity of the inducible PYK mutant (iPYK) increases with the isopropyl-beta-d-thiogalactopyranoside concentration. Batch growth experiments showed that growth rate and acid formation are closely related to the induction level of pyk. Measurements of cell growth rate and acetate formation of the iPYK mutant at different induction levels revealed that a PYK activity of about 12% of wild-type allows for good growth rate (0.4 h(-)(1) versus 0.63 h(-)(1) of wild-type) and low acetate production (0.26 g/L versus 1.05 g/L of wild-type). This is the first report to our knowledge of a metabolically engineered B. subtilis strain that allows good growth rate and low acid production in batch cultures. Finally, it was found that, by varying the pyk induction level, intracellular PEP concentration can be controlled over a wide range. The intracellular PEP concentration is intimately connected to the regulation of the transport of phosphotransferase system (PTS) sugars in the presence of glucose. Because there is no other method for modulating intracellular PEP levels, this finding represents a major advance in one's ability to dissect the function of the PTS and sugar metabolism in bacteria.