Good Practices for the Laboratory Performance Testing of Aqueous Oral Inhaled Products (OIPs): an Assessment from the International Pharmaceutical Aerosol Consortium on Regulation and Science (IPAC-RS).

Multiple sources must be consulted to determine the most appropriate procedures for the laboratory-based performance evaluation of aqueous oral inhaled products (OIPs) for the primary measures, dose uniformity/delivery, and aerodynamic particle (droplet) size distribution (APSD). These sources have been developed at different times, mainly in Europe and North America, during the past 25 years by diverse organizations, including pharmacopeial chapter/monograph development committees, regulatory agencies, and national and international standards bodies. As a result, there is a lack of consistency across all the recommendations, with the potential to cause confusion to those developing performance test methods. We have reviewed key methodological aspects of source guidance documents identified by a survey of the pertinent literature and evaluated the underlying evidence supporting their recommendations for the evaluation of these performance measures. We have also subsequently developed a consistent series of solutions to guide those faced with the various associated challenges when developing OIP performance testing methods for oral aqueous inhaled products.

Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities.

The development of a chip-based sensor array composed of individually addressable polystyrene-poly(ethylene glycol) and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. A single drop of fluid provides sufficient analysis media to complete approximately 100 assays in these microetch pits. The cavities possess pyramidal pit shapes with trans-wafer openings that allows for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microspheres. Identification and quantitation of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data are extracted from the array efficiently using a charge-coupled device allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, metabolic cofactors, and antibody reagents.

Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array.

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify beta-D-glucose and beta-D-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.

The production of heterologous plasma proteins.

Plasma proteins have been early commercial targets for heterologous expression in cell systems. Much of the impetus for the design and implementation of such processes has come from the desire to supply a market with products that do not rely on the fractionation of donated plasma. The production of recombinant plasma proteins would avoid the hazards of blood-derived products, the most notable of which is viral contamination. Although, when processed correctly, blood-derived products are virtually free from transmitting viral infections, a perceived risk of contamination exists for the manufacturer, user and patient. These risks have been well-documented in the press over the past decade. In this article, the production of human serum albumin (HSA) is used to illustrate the challenge of manufacturing a nature-identical plasma protein from a heterologous source.

Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations.

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.

A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.

Expression of recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae.

A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.

Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter.

The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment. Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters. Chromosomal mapping indicates that this genomic fragment is located on chromosome XII. The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.

Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae.

Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.

Saccharomyces cerevisiae strains that overexpress heterologous proteins.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.

5'-secondary structure formation, in contrast to a short string of non-preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast.

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5'-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5'-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5'-end of the mRNA (overall delta G = -36.6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae.

A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined. "High-copy" number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.

The secretion and post translational modification of interferons from Saccharomyces cerevisiae.

Studies with three interferon molecules, IFN-alpha 2, IFN-beta 1, and a "hybrid" interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves. Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast alpha factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site. Secretion studies with heterologous proteins in S. cerevisiae are aimed at maximising yield, the percentage of extracellular product and correct amino terminus sequence. The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence. This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process.

Secretion of mammalian polypeptides from yeast.

Yeast cells are capable of expressing and secreting foreign polypeptides into the medium. Mammalian glycoproteins are glycosylated when secreted from yeast although the exact oligosaccharide sequence is not reproduced.

Production and genetic analysis of yeast cybrids.

Data presented here demonstrate that fusion of protoplasts of two different haploid strains of Saccharomyces cerevisiae having the same mating type leads to the formation of "fusants" and "cytoplasmic hybrids". The nuclear and cytoplasmic genome of a "fusant" combine those of the parent haploid strains. The "cytoplasmic hybrid" possesses the haploid genome of one parent and the combined cytoplasmic genomes of both. In mouse cells lines such products have been termed "cybrids" and this term has therefore been adopted here (Bunn and Wallace 1974).

Genes which control cell proliferation in the yeast Saccharomyces cerevisiae.

In many eukaryotes it is thought that cell proliferation is regulated at a point in G1 close to the initiation of DNA synthesis. Hartwell and his colleagues have shown such a point in G1 phase in the budding yeast, Saccharomyces cerevisiae, defined by the cdc 28 mutation. He has termed this point 'start' and showed that for cells to proceed beyond start, initiate DNA synthesis and produce a bud, various conditions must be met. Two of these conditions are the presence of adequate nutrients in the medium and attainment of a critical size. We identify here some of the genes controlling start by isolating mutants which are altered with respect to the conditions in which start occurs. Two types of mutant have been isolated. One results in bud initiation when the parent cell is only half the size at which bud initiation occurs in wild-type cells. Such mutants define a single gene, whi-1, and they are apparently analogous to the size mutants isolated by Nurse and his colleagues in Schizosaccharomyces pombe. A second type of mutation affects a second gene, whi-2, which is involved in the mechanism whereby cells arrest in G1 in stationary phase. whi-2- cells growing exponentially initiate buds at the same size as wild-type cells. In stationary phase, however, whi-2- cells growing exponentially initiate buds at the same size as wild-type cells. In stationary phase, however, whi-2- cells, unlike wild-type cells, are predominantly budded and are smaller than wild-type cells.