Adenine quantitation in yeast extracts and fermentation media and its relationship to protein expression and cell growth in adenine auxotrophs of Saccharomyces cerevisiae.

We have described a method to reliably measure the free adenine content of yeast extract powders or the adenine concentrations found in chemically-defined and complex fermentation samples. This method relies on the selective precolumn derivatization of adenine with chloroacetaldehyde to form the fluorescent adenine adduct 1,N6-ethenoadenine. The derivatized adenine can then be resolved from other components found in samples with reverse phase HPLC and selectively monitored with fluorescence. This method was then used to study the adenine nutritional requirements of adenine auxotrophs of recombinant Saccharomyces cerevisiae. The adenine content of individual yeast extract powders was examined in relation to the cell mass (dry cell weight, DCW) achieved in culture media formulated with these powders. A general increase in DCW was observed with increasing adenine concentration in the yeast extract. Conversely, we observed that as adenine concentration increased in complex media the expression levels of a heterologous protein decreased. This method also allowed us to examine the adenine/DCW ratio in both steady-state continuous culture and batch culture. In both cases, the total in vivo adenine content as measured by the amount of adenine utilized from the culture media was estimated to be ca. 25-40 mg/g DCW. However, data suggest that this value is in excess of what is strictly required for cell growth and represents the quantity of adenine required to saturate intracellular pools of adenine or adenine metabolites. A minimum requirement for cell growth is at least as low as 12.5 mg of adenine/g of cells.

Recent advances in the large scale fermentation of Neisseria meningitidis group B for the production of an outer membrane protein complex.

The Outer Membrane Protein Complex (OMPC) of the bacterium Neisseria meningitidis group B has been used successfully as a protein carrier in a Haemophilus influenza type b (Hib) polysaccharide conjugate vaccine and a Streptococcus pneumoniae (Pn) polysaccharide conjugate vaccine to elicit antipolysaccharide immune responses in young infants. The OMPC carrier is derived by detergent extraction of whole cells and, thus, the consistent generation of suitable biomass is central to an effective production process. Therefore, we have developed a large-scale, high-cell density (5 g/L dry cell weight) fermentation process for the cultivation of N. meningitidis B11. Since current requirements for the production of human biologics mandate strict control of all aspects of the manufacturing process, several key features of the process, including a chemically defined medium and a rational event-based harvest criterion, support current good manufacturing practice (cGMP) and increased productivity.

Influence of strain and medium composition on filtration of escherichia coli suspensions.

Cross-flow filtration of Escherichia coli strains was examined at the laboratory and pilot scales using Romicon 500,000 molecular-weight-cutoff hollow fiber membranes. Both the series resistance and macrosolute polarization models were employed to compare performances. Total dissolved solids content above 90 g/L and viscosity above 1.1 x 10(-3) pac s of cell-free culture media were found to decrease average filtration fluxes by over 60% both in the absence and presence of cells. Broth filtration with culture media of dissolved solids levels below 80 g/L were influenced to a greater extent by harvest cell density. The collodial nature of the complex nutrient responsible for the total solids increase affected prediction of filtration performance. Differences in strain filterability were observed with JM109 preferred over DH5 in high solids-containing media and RR1 preferred over JM109 in low dissolved solids-containing media. Their research demonstrates the importance of cell strain and media selection in the performance of early downstream processing steps. (c) 1994 John Wiley & Sons, Inc.

Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae.

Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents. A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin. Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction. rHIR was purified by a process that consisted of two chromatographic steps and decolorization. Total yield for the purification process was 3.6 g, or 41%. This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography. Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA. Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.

A novel process for the large-scale purification of recombinant tick anticoagulant peptide using perfusion chromatography.

Tick anticoagulant peptide (TAP) is a 60 amino acid peptide (Mr = 6977, pI = 4.9) found in the saliva of the soft tick Ornithodorous moubata that specifically inhibits blood coagulation factor Xa (fXa). A recombinant form of TAP (rTAP) secreted by Saccharomyces cerevisiae was purified from 200 liters of fermentation broth with POROS, strong cation exchange (SCX) and reversed-phase high performance liquid perfusion chromatography (RP-HPLC) media (20 microns nominal particle diameter). The higher linear flow rates and dynamic capacities, as well as low back pressures, obtained with perfusion chromatography media permitted 37.5 g of rTAP to be efficiently captured and significantly enriched from 400 liters of diafiltered fermentation broth (24.3 g yield) with 1.25 liter of SCX media using a low pressure column and peristaltic pump in 4.5 hours. Subsequently, 16.7 g of rTAP obtained from the capture step was purified in a high-resolution mode with the same SCX media, after the media was cleaned and repacked into an 800 ml high-pressure column. By doing multiple rapid cycles at high linear flow rates, preparative-scale high-resolution purification of multi-gram amounts of the peptide was done on this relatively small column in only 10.5 hours. Finally, the peptide was desalted and decolorized on a 200 ml RP-HPLC column of perfusion chromatography media by doing multiple rapid cycles. After lyophilization, 12 g of peptide (46.9% yield) was obtained that was > 96% homogeneous by several analytical criteria and fully active in inhibiting blood coagulation factor Xa (fXa).(ABSTRACT TRUNCATED AT 250 WORDS)

The accuracy of self and informant ratings of physical functional capacity in the elderly.

To compare the accuracy of self-ratings with informant-ratings of physical functional capacity in the elderly, 150 elderly patients attending a geriatric day hospital (GDH) and their formal and informal community carers were administered a questionnaire about their ability to perform activities of daily living (ADL). Direct observation of the patients' performance by therapists at the GDH was used as a standard, after the reliability and validity of this approach had been evaluated. Self-ratings were shown to be more accurate and less biased than informant-ratings, both for individual ADL and overall functional capacity. The accuracy of all ratings tended to be greater for less complex or physically demanding ADL, and informants tended to consistently underestimate functional capacity. The concurrent validity of the adapted Barthel Index in a self-report format was also demonstrated. Wherever possible, information concerning the physical functional capacity of an elderly subject should in the first instance be sought from the subject himself, as the quality of such information may be superior to that of his carers.

cDNA cloning of human and rat brain myo-inositol monophosphatase. Expression and characterization of the human recombinant enzyme.

Inositol monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide inositol required for the resynthesis of phosphatidylinositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine inositol monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.

Large-scale purification and characterization of recombinant tick anticoagulant peptide.

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

Production of cytotoxic proteins in Escherichia coli: a fermentation process for producing enzymatically active HIV-1 protease.

Two fermentation processes for the tryptophan-regulated expression of active HIV protease (HIV-1 prt) in Escherichia coli are described. Since overexpression of HIV-1 prt results in cell death, stringent control of product expression was necessary to attain high enzyme levels. Such control was achieved by separation of growth and production phases in a two-step process or by implementation of nutrient feed in a one-step process. When the two-stage process was used, soluble product was detectable only when induction occurred at low culture density (A550 less than 3.5). Short induction periods of 1-2 h and rapid harvesting were necessary to recover active product. Similar results were obtained when the single-stage process was operated at 37 degrees C; however, cultivation and induction at 28 degrees C resulted in active enzyme formation following induction at increased cell density (A550 = 10).

Harvesting recombinant microbial cells using crossflow filtration.

A contained, crossflow filtration (CFF) membrane system is described for harvesting Saccharomyces cerevisiae and Escherichia coli cells. This system is portable and can be cleaned and sanitized in place. Low- and high-cell density (LCD, HCD) fermentations of recombinant cells in 10- to 200-l volumes were used as the starting material. LCD fermentations, up to 8.3 g l-1 dry weight (dcw) of S. cerevisiae, with volumes of 10 to 200 l were harvested and diafiltered in 0.5 and 1.5 h, respectively. HCD 200-l fermentations of S. cerevisiae (47-63 g l-1 dcw) were harvested and diafiltered in approximately 2 h. E. coli fermentations, LCD and HCD (up to 16.2 g l-1 dcw), of 200-l volumes were harvested and diafiltered in 2.3 h while employing 14 and 75 ft2 of membrane area, respectively. Using hollow fiber or flat sheet membranes from different sources, cell harvesting times were less than 2.5 h. These studies demonstrate that CFF is an efficient method for harvesting and diafiltering recombinant S. cerevisiae and E. coli cells from fermentation broth.

The safety of the hepatitis B vaccine. Inactivation of the AIDS virus during routine vaccine manufacture.

In the United States, one hepatitis B vaccine (Heptavax-B) has been licensed for the prevention of hepatitis B virus infections. Even though this vaccine has been shown to be highly effective and well tolerated in controlled trials and has been recommended for use in those at risk for acquiring infection by hepatitis B virus, many individuals have been reluctant to be immunized for fear of contracting acquired immunodeficiency syndrome (AIDS). In this study, we demonstrate that each of the three inactivation steps used in the manufacture of Heptavax-B independently will inactivate the infectivity of high-titered preparations of the AIDS virus; recipients of the hepatitis B vaccine do not develop antibodies to the AIDS virus; the hepatitis B vaccine does not contain detectable levels of nucleic acids related to the AIDS virus. These observations clearly demonstrate that vaccination with the currently available hepatitis B vaccine poses no demonstrable risk for acquiring AIDS.

Production of purified hepatitis B surface antigen from Alexander hepatoma cells grown in artificial capillary units.

An artificial capillary system was devised for growth of hepatoma cells that yields very high titers of hepatitis B surface antigen (HBsAg). High yield of antigen was facilitated by slowing cellular metabolism through reduction of incubation temperature and addition of 0.1 mM caffeine. Deletion of serum from the medium did not reduce the yield of antigen. HBsAg prepared from the culture fluid by affinity chromatography and additional chemical and enzymatic steps was essentially pure and was indistinguishable from HBsAg prepared from infected human plasma. Preparation of HBsAg from the cell culture source presents advantages over that of human plasma and might be a source of HBsAg for vaccine preparation.

Identification of the metabolites of di-(2-ethylhexyl) phthalate in urine from the African green monkey.

The metabolites of di-(2-ethylhexyl) phthalate (DEHP) found in urine from African Green monkeys after intravenous administration of the 14C-labeled parent compound were isolated and identified. Criteria of identification included cochromatography with rat-derived standards on direct-phase HPLC and a variety of gas-chromatographic columns, as well as correspondence of mass spectra (70-eV electron impact and methane positive chemical ionization) with those of known standards. Approximately 80% of the urinary metabolites were excreted in the form of glucuronide conjugates. This is analogous to what has been reported for the urinary metabolites of DEHP from humans, but in clear contrast to the metabolites found in rat urine. Rat urinary metabolites of DEHP are excreted unconjugated, and consist primarily of derivatives more highly oxidized than the major metabolites produced by monkey or human. It is suggested that the African Green monkey may be a better model for human metabolism of DEHP than is the rat.

Enhanced solubility of 2,8 dihydroxyadenine (DOA) in human urine.

Water solubility of the adenine catabolite 2,8 dihydroxyadenine (DOA) frequently forms the basis for predicting potential DOA crystal formation in human urine following infusion of adenine-fortified blood. Measurements relevant to solubility, ionic dissociation, and supersaturability of DOA in aqueous buffers and human urine at 37 C establish striking quantitative differences in the physico-chemical behavior of DOA in the two media. The basal solubility of DOA is 1.53 +/- 0.04 mg/1 (approximately 9 X 10(-6) M) in water (pH 6.5). DOA is an ampholyte characterized by aqueous thermodynamic macrodissociation constants of pKa1 = 2.6, pKa2 = 8.1, and pKa3 = 11.52. This compound displays pH-dependent solubility, although significant solubility increases beyond basal values do not occur within the physiologic pH range for human urine. Supersaturated aqueous solutions (three to sixteen times basal solubility) can be achieved but are unstable. In contrast, human urine at 37 C exhibits enhanced capacity for solubilizing DOA. In vitro basal solubility is 2.68 +/- 0.84 mg/1 at pH 5.0 and 4.97 +/- 1.49 mg/1 at pH 7.8. The apparent pK 2for DOA in urine of 7.9 to 8.1 is dependent upon urine osmolality. Urine can be supersaturated with Doa in vitro to approximately ten times its basal solubility by adding DOA solubilized in weak base, or by evaporation of a urine-DOA mixture. DOA remains supersaturated in urine for at least 16 hours despite gentle agitation. Little variation in in vitro DOA apparent supersaturation was found among urine samples from four normal individuals (40.38 +/- 3.33 mg/1). A patient receiving oral adenine exhibited urinary DOA solubility in considerable excess (96.0 mg/1) of that predicted from water and from in vitro urine solubility studies. Thus, water solubility of DOA is poorly predictive of in vitro and in vivo DOA solubility in human urine. On the basis of these data, estimates of the load of adenine-fortified blood expected to result in urinary DOA crystal formation may be revised upward.