Statistical tools for setting in-process acceptance criteria.

Establishing meaningful and reasonable acceptance criteria for process validation or continual monitoring is crucial to approval and successful manufacturing. The limits should be based on statistical analysis of historical data when possible. The control limits of "mean +/- 3 standard deviations" is one industry standard. However, the limits may be artificially constraining if the standard deviation does not reflect the true variance of the process. Under-estimation of process variance is common with small data sets. This paper presents three methods for correcting underestimated variance, allowing the setting of acceptance criteria that are slightly larger than +/- 3 standard deviations. These limits are more meaningful in that they account for true process variability and will signal process deviations due only to a specific cause.

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor.

A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

The occurrence of cyclic AMP in archaebacteria.

Cyclic AMP was found in species representative of the three major groups of the archaebacteria. In Methanobacterium thermoautotrophicum starvation for H2 led to a significant increase in cellular cAMP. The findings suggest that the occurrence of cAMP antedates the divergence of the major kingdoms of biology; the observations also imply that cAMP constitutes a very early regulatory molecule.

Levels of cyclic-2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum during phosphate limitation.

Batch-grown Methanobacterium thermoautotrophicum cells grew nonexponentially in the absence of exogenous Pi until intracellular cyclic-2,3-diphosphoglycerate (cyclic DPG) had fallen below 2 mumol/g (dry weight), the limit of detection. Growth resumed immediately upon transfer to medium containing Pi Cyclic DPG levels were also below detection in Pi-limited chemostat cultures operating at a dilution rate of 0.173 h-1 (4-h doubling time), with reservoir Pi concentrations below 200 microM. At this dilution rate, the Pi concentration in the culture was 4 microM. An H2-limited steady state was achieved with 400 microM Pi in the inflowing medium (67 microM in the culture). The cyclic DPG content of these cells was 72 to 74 mumol/g, about one-third the amount in batch-grown cells. The specific growth rate accelerated immediately to 0.36 h-1 (1.9-h doubling time) under washout conditions at high dilution rate. The cellular content of cyclic DPG declined over a 2-h period, and then increased rapidly as the Pi level in the medium approached 200 microM. Expansion of the cyclic DPG pool coincided with a marked increase in Pi assimilation. These results indicated that M. thermoautotrophicum accumulated cyclic DPG only when Pi and H2 were readily available.

Cyclic-2,3-diphosphoglycerate levels in Methanobacterium thermoautotrophicum reflect inorganic phosphate availability.

Methanobacterium thermoautotrophicum was grown in phosphate-limited chemostat cultures at a dilution rate corresponding to a doubling time of 13.2 h. The cyclic-2,3-diphospho-D-glycerate content of these cells was 8 to 10-fold lower than that of cells grown in batch cultures having a doubling time of 11.5 h. This metabolite accounted for 5% of cell dry weight during batch growth on 2 mM phosphate. In the chemostat the steady-state concentration of phosphate was 4 microM, showing that this methanogen is adapted to highly efficient growth at low phosphate concentrations. Since growth rates were similar in both cultures, the growth rate clearly does not depend on intracellular levels of cyclic-2,3-diphosphoglycerate.

A novel diphospho-P,P'-diester from Methanobacterium thermoautotrophicum.

A phosphorus-containing metabolite that accounts for up to 80% of the total phosphate in perchloric acid extracts of Methanobacterium thermoautotrophicum ATCC 29183 was purified by chromatography on an anion exchange column. The proton-decoupled 31P NMR spectrum of the sodium salt in D2O exhibits an AB quartet with lines at -9.48, -9.78, -10.78, and -11.08 ppm upfield from 85% phosphoric acid. The phosphorus-phosphorus coupling constant is 18.5 Hz, a value characteristic of a pyrophosphate linkage. The structure of this compound has been tentatively established as cyclic-2,3-diphosphoglycerate (Kanodia, S., and Roberts, M. F. (1983) Proc. Natl. Acad. Sci. U.S.A., in press). Although rapidly degraded in neutral cell extracts, the diphosphodiester was stable for hours in cells whose metabolism had been arrested by depletion of H2 from the medium. If the cells were transferred to phosphate-free medium in the presence of H2 and CO2, the diester disappeared in about one cell generation. These findings point to a possible role as a phosphate storage compound, but the real function(s) of this unusual diphosphodiester must await a complete analysis of its structure and further metabolic studies.