High cell density suspension culture of mammalian anchorage independent cells: oxygen transfer by gas sparging and defoaming with a hydrophobic net.

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs. During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 l reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l.h equivalent to a consumption rate of 1 X 10(8) cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net. Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 5 l scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7 X 10(7) cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4 X 10(7) cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.


Strategies for the recovery of chemicals from fermentation: a review of the use of polymeric adsorbents.

Many different compounds can be produced by using microorganisms or enzymes. An important element in the design of a viable biotechnological process is the selection of an economical and efficient separations train. Production of chemicals via biotechnology generally requires isolation and purification from dilute, aqueous solution. A general framework for separation process design relies on exploiting a unique molecular physicochemical property (or properties) for separating the molecule of interest from water and the other species in solution. Important properties that can be utilized for the recovery of low molecular weight polar compounds are molecular charge, hydrophobicity, Lewis acidity or basicity, volatility, and limited solubility. In turn, it can be useful to characterize molecular properties by using separation processes, such as, for example, hydrophobicity by measuring octanol/water partition coefficients. This paper reviews the use of adsorption onto hydrophobic, nonpolar macroreticular polymers and Lewis acid-base complexation by using functionalized polymers for the recovery of amino acids, carboxylic acids, alcohols, and ketones from dilute aqueous solution. The focus will be on utilizing physical and chemical properties to predict uptake capacity. This information will be relevant to separation process development and will help to characterize molecular properties in aqueous solution.


Application of population balance model to the loss of hybridoma antibody productivity.

A simple dynamic model has been applied to explain the population dynamics of monoclonal antibody (MAb) producing (producer) and nonproducing hybridoma cells (nonproducer) coexisting in culture. The events of mutation or loss of genes associated with antibody synthesis have been incorporated into the model to account for the conversion of a producer to a nonproducer. The model shows that the cell population is not necessarily dominated by the nonproducer, and a steady balance of producer and nonproducer populations can be achieved. A nonproducer population is undesirable, and cultivation strategies to maximize MAb production are suggested, taking into account the dynamics of a nonproducer population.


Regulation and expression of transforming growth factor type-beta during early mammalian development.

We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-beta) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-beta activity increases approximately 25-100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-beta activity reflects the induction of TGF-beta 2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-beta 2 as well as a distant member of the TGF-beta family, Vgr-1. Transcripts for TGF-beta 2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC-13 but not in their undifferentiated counterparts. Moreover, TGF-beta 2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-beta 2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-beta 2 and Vgr-1 is closely associated with the induction of differentiation during early development.


Membrane modifications in the course of hepatocyte isolation.

A transmission E/M, scanning E/M and freeze fracture ultrastructural study has been performed on the rat hepatocyte in the course of isolation from the liver parenchyma. The cell submicroscopic aspect indicates a good morpho-functional preservation from the liver perfusion to the final stages of cell isolation. The freeze fracture membrane analysis evidentiates the constant presence of gap junctions and tight junctions, characterized by particular structural alterations, probably due to progressive functional uncoupling. The persistence of these cell differentiations until complete cell isolation may be considered a further morphological expression of the maintenance of the differentiated stage of the hepatocyte. Fragments of membranes from adjacent cells, still adherent to isolated hepatocyte surfaces, can also be occasionally detected by freeze-fracture techniques.


Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures.

The aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.


Antibody production in packed bed reactors using serum-free and protein-free medium.

The present work demonstrates the utility of packed bed reactors for the production of monoclonal antibody. We present data from a continuous process run for the production of over 100 grams of antibody, using serum-free medium. An additional pilot run also demonstrates the potential for continued antibody production under protein-free conditions, using a standard basal medium.


Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants.

The causal agent of heartwater disease of domestic ruminants, Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0-6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.


Bioconversion of avermectin into 27-OH avermectin.

The bioconversion of avermectin to its 27-hydroxy derivative is achieved with Nocardia autotrophica subsp. canberrica. The approach of increasing bioconversion productivity rather than efficiency was adopted in these studies. Process improvement studies focused on the physico-chemical conditions of the fermentation, examined initially at the shake-flask scale. Bioconversion yields were affected by pH, substrate concentration, time of substrate addition, substrate solubilization, carbon to nitrogen ratio, and medium strength. Optimization of these parameters resulted in a 8-fold process improvement. During pre scale-up studies, the sensitivity of this bioconversion to the antifoam employed was demonstrated and lard oil was selected as giving the best results. Additional process changes were required during scale-up efforts in larger vessels, including replacement of the original substrate solvent with dimethylsulfoxide.


DNA sequence analysis of endoglucanase genes from Pseudomonas fluorescens subsp. cellulosa and Pseudomonas sp. NCIB 8634.

The DNA of two previously isolated recombinant clones, one from Pseudomonas sp. NCIB 8634 (= Cellvibrio mixtus) (pPC71) and another from Pseudomonas fluorescens subsp. cellulosa (pPFC4) that express endoglucanase activity in E. coli was sequenced. Plasmid pPC71 had three open reading frames, two of which include portions of plasmid pBR322. The third open reading frame occurs entirely within the Pseudomonas DNA insert and encodes a protein with a molecular mass of 5845 Da. The DNA insert in pPFC4 was found to contain an open reading frame (PFC-ORF) that encodes a protein of 32189 Da. The major endoglucanase produced in E. coli cells carrying pPFC4 is about 30,000 Da. It is concluded that PFC-ORF encodes this endoglucanase. Both ribosome and catabolite gene activator protein binding sites lie upstream from the initiating codon of PFC-ORF. An interesting feature of the PFC-ORF protein is the presence of amino acid motifs Val-Ser-Ser-Ser-Ser and Val-Val-Ser-Ser-Ser-Ser-Ser that occur within a 25 amino acid span.


Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis.

Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-14C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin by Saccharothrix aerocolonigenes.


Biochemical fingerprints of Legionella spp. by the BIOLOG system: presumptive identification of clinical and environmental isolates.

Six Legionella strains were characterized with the BIOLOG identification system. A specific metabolic pattern for the six Legionella strains was observed after 24 h. Several positive reactions were detected intermittently in all six strains tested. The possible application of the BIOLOG system for identification of Legionella spp. from environmental or clinical samples is discussed.


Isolation and characterization of a cryptic plasmid from mesophilic aeromonads: potential as a cloning vector.

A 2.6 kb covalently closed circular plasmid has been isolated from clinical and environmental isolates of Aeromonas sobria and A. hydrophila. The possibility that the plasmid carries genetic determinants that mediate resistance to a variety of anti-microbial agents has been eliminated. The plasmid is stable at approximately 20-25 copies per chromosome equivalent which, together with its relatively small size and the presence of unique restriction sites, makes it a good candidate for development as a cloning vector.


Electroporation-induced transformation of Escherichia coli: evaluation of a square waveform pulse.

Four strains of Escherichia coli were tested for electroporation-induced plasmid transformation using a high voltage pulse in a square waveform. Conditions for optimal transformation were determined for each strain and transformation frequencies found to be comparable to those reported previously for E. coli with a sinusoidal waveform of exponential decaying pulse.


Generation of deletions in pTV1ts following transformation of Staphylococcus aureus protoplasts.

Plasmid pTV1ts was introduced into Staphylococcus aureus by transformation of protoplasts at frequencies ranging from 6.6 x 10(1) to 2.8 x 10(5) per micrograms of DNA when selection was made for resistance to chloramphenicol and erythromycin, respectively. Phenotypic analysis of regenerated transformants demonstrated three distinct classes. Analysis of the plasmid profiles of several isolates revealed that two classes harboured deleted pTV1ts derivatives.


Conjugal transfer of recombinant plasmids into gellan gum-producing and non-producing variants of Pseudomonas elodea ATCC 31461.

The conjugal transfer of recombinant plasmids into a gellan gum-producing, rifampicin-resistant strain of Pseudomonas elodea and into one of its non-producing variants, was studied in order to facilitate the cloning of gellan genes. The mobilization frequency of recombinant plasmids derived from pKT240, and of cosmid pJRD215 from Escherichia coli HB101 (hsdM), into the mucoid strain was below the values for the non-mucoid variant and decreased exponentially with plasmid size. Reducing the mating time on solid surfaces led to higher mobilization frequencies. Under optimal conditions, a gene bank (40-50 kb) constructed in the cosmid pJRD215 was efficiently mobilized into Gel- mutants during complementation experiments.


Oligonucleoside methylphosphonates as antisense reagents.

Oligonucleoside methylphosphonates contain nonionic internucleotide bonds that resist degradation by cellular nucleases and allow the oligomers to be taken up intact by mammalian cells in culture. Antisense methylphosphonate oligomers targeted against cellular or viral mRNA initiation codon or coding regions or against precursor mRNA splice sites effectively and specifically inhibit mRNA expression in cells. The efficacy of antisense methylphosphonate oligomers can be enhanced by derivatization with functional groups that allow the oligomer to covalently cross-link with its targeted mRNA. These oligonucleotide analogs will be useful tools for studying and controlling gene expression and are also promising candidates for development as therapeutic agents.


Facilitated in vitro refolding of human recombinant insulin-like growth factor I using a solubilizing fusion partner.

We describe a new approach to refolding recombinant proteins in which an affinity fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A, is used to solubilize misfolded molecules before, during and after reduction and reoxidation. We show that human insulin-like growth factor I (IGF-I) can be refolded as a fusion protein at a concentration as high as 1-2 mg/ml without the use of denaturing agents. A process scheme suitable for large scale application is described in which the yield of correctly folded human IGF-I with full biological activity is substantially increased.


Bioconversion of n-octane to octanoic acid by a recombinant Escherichia coli cultured in a two-liquid phase bioreactor.

The alk genes from the catabolic OCT plasmid of Pseudomonas oleovorans, which encode the enzymes involved in the oxidation of n-alkanes to carboxylic acids, were introduced into E. coli W3110. The resulting recombinant converts n-octane in a two-liquid phase medium into the corresponding alkanoate and excretes this compound into the aqueous phase. The rate of octanoic acid production by the recombinant E. coli is equal to or better than the alkane oxidation rate of P. oleovorans, suggesting that two-liquid phase fermentations with E. coli might have future industrial applications.


The development of virus-resistant alfalfa, Medicago sativa L.

We have generated more than 100 transgenic alfalfa plants, via Agrobacterium-mediated gene transfer, from genotypes selected from five alfalfa cultivars. These plants express the genes for kanamycin resistance and for the coat protein of alfalfa mosaic virus (AMV). The strongest expressers accumulated nearly 500 ng coat protein per mg soluble leaf protein. AMV inoculation of protoplasts from these strong expressers indicated that they were resistant to infection by AMV, while protoplasts from plants containing about a hundred-fold less coat protein and from control untransformed plants were not. Transgenic alfalfa plants containing large amounts of coat protein were, likewise, resistant to AMV. These plants did not develop systemic infections following inoculation with up to 50 micrograms/ml AMV, while inoculated control plants developed systemic infections following inoculation with as little as 10 micrograms/ml AMV. These results demonstrate that expression of the AMV coat protein gene confers resistance to AMV infection in transgenic alfalfa plants.


Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6.

We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.


Expression of heterologous proteins in Aspergillus.

Filamentous fungi, in particular those of the genus Aspergillus have been well exploited for their ability to produce high levels of extracellular proteins in an inexpensive manner. Since many human proteins with the potential to be used therapeutically are secreted and require post-translational modification for biological activity, eukaryotic expression-secretion systems have been targeted for development. Recent developments in DNA-mediated transformation systems have allowed the utilization of Aspergillus as a host for the production of recombinant proteins. Several features such as well-characterized genetics and the availability of many mutants make Aspergillus nidulans the organism of choice for development of expression secretion systems. Recombinant strains contain integrated expression cassettes often in multiple copy, which are mitotically stable. In this review, we discuss the recent progress made in the use of Aspergillus as expression secretion hosts for the production of proteins of therapeutic significance.


Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.


Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.


Identification and expression of the ACV synthetase gene.

The gene coding for ACV synthetase has recently been identified and cloned. Analysis of its structure and expression, along with similar studies of other genes involved in beta-lactam biosynthesis, should lead to a better understanding of the molecular basis of regulation of the pathway and the possibility of modifying yield and diversity of fungal antibiotics.


Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.


Stabilizing effect of penicillin G sulfoxide, a competitive inhibitor of penicillin G acylase: its practical applications.

We have found that penicillin G sulfoxide (pen G SO) behaves as a general stabilizing agent of two bacterial penicillin G acylases (PGAs) from E. coli and from K. citrophila), and this role is related to a strong inhibitory effect on the enzymes. The stabilizing effect has been observed during two different inactivation processes: (i) thermal inactivation of soluble enzymes at alkaline pH, and (ii) inactivation of immobilized enzymes as a consequence of covalent multiinteraction with highly activated agarose aldehyde gels. At the same time, pen G SO behaves as a strong competitive inhibitor of these two enzymes. The inhibition constant is more than 10-fold lower than the one corresponding to another smaller competitive inhibitor, phenylacetic acid (PAA), the structure of which is exactly the acyl donor moiety corresponding to pen G SO. In turn, PAA hardly exerts any stabilizing effect on PGAs. The stabilizing effect of pen G SO allowed the preparation of derivatives of these PGAs preserving full catalytic activity in spite of being 1,400- and 650-fold more stable than the corresponding soluble or one-point attached immobilized enzymes.


Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.


Immobilization of invertase by encapsulation in polyelectrolyte complexes.

Free and polystyrene-bound invertase from Saccharomyces cerevisiae were encapsulated within symplex membranes which were composed of cellulose sulfate as the polymeric anion and poly(dimethyldiallylammonium chloride) as the polymeric cation. The kinetics and the performance of the encapsulated enzyme preparations have been compared to the free enzyme employing the hydrolysis of sucrose. The pH and temperature optima were only slightly affected by the encapsulation. The kinetic constants, however, were changed by the encapsulation as a result of diffusional limitation. Encapsulated invertase showed a high storage stability and a high operational stability if low substrate concentrations were applied. The coimmobilization of invertase with living cells, which are not capable of utilizing sucrose, in the described capsules, opens many possibilities in fermentation technology.


Formation of peptide bonds by carboxypeptidase c from orange leaves.

Carboxypeptidase c partially purified from orange leaves was studied as a catalyst for enzymatic peptide synthesis. Various N-protected ester- and nucleophile compounds were evaluated in order to determine the substrate specificity. For further characterization of the synthetic reaction, optimum pH and the influence of the N-terminal protecting group were studied. Kinetic investigations revealed considerable differences in Km and Vmax for the nucleophile when the N-terminal protecting group of the substrate was varied.


Diffusion in immobilized-cell gels.

The relationship between the diffusion coefficient, the effective diffusion coefficient and the partition coefficient for a solute in a cell-containing gel is discussed. The use of correlation equations that are based on some kind of physical model is recommended when the effect of cell concentration on diffusion is interpreted.


Synthesis and secretion of hirudin by Streptomyces lividans.

To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the alpha-amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.


Overproduction of an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" in Escherichia coli.

The xynC gene coding for an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli strain RR28 by cloning the gene downstream from the lacZ promoter region of pUC18 (pNZ1447) or downstream from the temperature-inducible lambda pRpL promoters of pJLA602 (pNZ1600). The protein formed high molecular weight aggregates in induced cells of RR28/pNZ1600 but not in RR28/pNZ1447. The enzyme constituted up to 10% of the total cell protein and was located in the cytoplasmic fraction of RR28/pNZ1447. The acetyl esterase was most active at pH 6.0 and 70-75 degrees C with a half-life of 64 h at 70 degrees C and 30 h at 80 degrees C, respectively.


A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods.

A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 10(4) cfu/ml, and at ca. 10(5) cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.


Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells.

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3-0.5 ng/10(5) cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10(5) cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.


Purification and some properties of proliferation suppressing factor from human lung adenocarcinoma PC-8.

Extracts by 3 M KCl from a human lung adenocarcinoma cell line, PC-8, suppressed the proliferation of phytohemagglutinin activated lymphocytes. The proliferation suppressing factor (PSF) was purified from the extracts by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The purified PSF had a molecular weight of 50 kDa by SDS-PAGE and was acid- and heat-labile. Since the PSF also suppressed the proliferation of three established human cell lines, it is unlikely that the PSF hinders the interaction between lymphocytes and mitogens.


A two-compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds.

A new bioreactor for animal cell cultivation employs two compartments for cells and medium respectively. The two chambers are separated by an ultrafiltration membrane. Cells and solution of collagen or collagen/chitosan mixture were loaded to the cell chamber and were allowed to form gel inside. Contraction of the cell-laden gel occurred subsequently to create a new zone in the cell chamber. In such a bioreactor cells are retained in the reactor, the high molecular product(s) accumulate in the cell chamber, while the small molecular weight nutrients and metabolites are replenished and removed from the medium chamber. By adjusting the flow rates for cell and medium chambers, the resident time for cells, high and low molecular weight components of the system can be manipulated separately. The new bioreactor, in both flat-bed and hollow-fiber configurations, was used to cultivate recombinant human cell, 293, for Protein C production over 60 to 90 days.


A new human fusion partner, HK-128, for making human-human hybridomas producing monoclonal IgG antibodies.

A hypoxanthine-aminopterin-thymidine (HAT) sensitive human fusion partner cell line, HK-128 was established from a human plasmacytoma line, LICR-LON-HMy2 (HMy2). The HK-128 cells showed a 100% cloning efficiency. Fusion efficiency of HK-128 was so high that one hybridoma cell was produced by fusion of 10(5) cells of HK-128 with lymphocytes, obtained from lymph nodes of breast cancer patients. About 90% of the resulted hybridomas were IgG producers. The remainder revealed IgM producing activity, which was lost by long term culture. This result indicates that the HK-128 cell line has an advantage for making hybridoma cells producing IgG. Among ca. 7,000 hybridomas obtained by fusion of HK-128 with lymphocytes of a breast cancer patient, we could establish a hybridoma cell line which produced IgG specifically reacting to a human breast cancer cell line, MCF-7.


The effect of bioreactor configuration on production of HIV and cell-virus interaction.

In an attempt to establish a bioreactor system for generation of HIV that is practicable, efficient, biologically contained, and capable of scale up, the production of two strains of this virus was examined in suspension culture and the 'Porosphere' fixed bed system. HIV 1 and HIV 2 were grown successfully in both these types of reactor. The porosphere reactor theoretically appears to offer a better environment for HIV production, but evidence for significantly improved yields from this system, compared to suspension, was equivocal. However, this configuration facilitated media changes during culture. The data clearly showed that the culture system and cell environment significantly affected cell-virus interrelationships. Switches between lytic--and persistent--type infections, and changes in the virus population were observed.


Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method.

We previously reported the expression of human beta-interferon (beta-IFN) (Miyaji et al., 1989) and human lymphotoxin (Miyaji et al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A beta-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of beta-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved beta-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted beta-IFN at a level as high as 5 micrograms/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, beta-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.


A compatible support system for cell culture in biomedical research.

The lack of a suitable system to culture epithelial cells for a long period under a luminal-antiluminal medium gradient, was the reason to develop a new system. It consists of an interchangeable sheet of permeable support material, which is set in place by two tight fitting holding rings. For special demands the supports can be coated with extracellular matrix proteins improving cellular attachment and terminal differentiation. The handling of the sheets by forceps proceeds easily and quickly, thus fastening the transfer of cultured cells without additional manipulations. The sheets can be transferred to a newly developed microperfusion chamber on which an apical and a basal perfusion over a long culture period parallel to a transepithelial electrophysiological registration becomes possible. The chamber has an extremely low amount of fluid dead space. The separate perfusion of cultured cells under isotonic, hypotonic or hypertonic conditions opens new possibilities. Thus, culture can be performed under most natural conditions e.g., that found within the kidney.


3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: a new fluorescent dye to detect Mycoplasma contamination in cell cultures.

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.


The advantage of the aggregate culture of isolated ovarian cell types over the monolayer culture.

Ovarian cells such as theca interna, granulosa and corpus luteum cells were isolated from pig ovaries and cultured in Erlenmayer flasks (25 ml) containing 3.5 ml of culture medium. The media were replaced every second day and frozen to -20 degrees C for later steroid analysis. The reaggregation of cells was completed within 2-3 days and this was then followed by a period of cell migration. During the subsequent 5-6 day period the reaggregates became larger. The best results were obtained in cultures of isolated theca alone and in combination with granulosa cells, as well as of early corpus luteum cells. Granulosa cells did not aggregate as easily or as completely as the corpus luteum cells. All types of cells investigated were able to secrete progesterone into the culture medium. They secreted more progesterone and for a longer time than cells cultured as monolayers. The aggregate culture seems to be a good model to study the secretion of ovarian cells, by creating the tri-dimensional, and thus more physiological, culture system.


Production of recombinant proteins in serum-free media.

The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can be carried out without the use of any serum.


Optimization of serum-free fermentation processes for antibody production.

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.


Serum-free medium for fermentor cultures of hybridomas.

Hybridomas lend themselves particularly well to large scale cultivation techniques since they grow as single cells in suspension without requiring attachment to a substrate. Furthermore, many cell strains have been adapted to grow in serum-free (SF) media to a similar cell density and antibody production as in serum containing media. This review will concern itself mainly with the cultivation of hybridomas in SF-media in bioreactors of various types with the ultimate goal of producing large quantities of monoclonal antibodies (mAb).


Cell banking.

The use of cultured cells (diploid strains or continuous cell lines) to produce biopharmaceuticals provides a level of standardization to the manufacturing process that cannot be attained by the more traditional methods of biological extraction from animal or human fluids, tissues, or primary cells. The key to this advantage is the ability to cryopreserve the production cell line as a master cell bank. This bank serves as the common starting source of a given product for the lifetime of the manufacture of that product. Since the MCB is the common and only starting source, it can be exhaustively characterized with regard to contamination by adventitious and endogenous agents. Assays can be developed for cellular components that are potential contaminants of the product. The removal of these components during product purification can be validated and their removal confirmed by rigorous quality control. In conclusion, characterized cell banks are central to the standardization of biopharmaceutical manufacturing processes and give rise to the production of high-quality products not attainable by the traditional methods of extraction of product from sources which are continually changing.


Use of recombinant DNA technology for engineering mammalian cells to produce proteins.

The recent advances in molecular biology have merged with somatic cell genetics and cell biology to allow mammalian cells to be extremely useful for the expression of foreign genes. This chapter has focused primarily on the approaches and potential limitations to high-level expression of proteins in mammalian cells. Future developments will involve the modification of mammalian cells in order to increase the efficiency of the various steps in protein processing and secretion. The ability to genetically engineer mammalian cells to produce high levels of desired proteins is presently complemented by advances in biochemical engineering which involve the ability to grow mammalian cells in very large volumes or at very high densities with reduced serum requirements. As a result, the cost for production of gram quantities of a protein from a mammalian host cell are approaching the cost of proteins from a mammalian host cell are approaching the cost of proteins similarly derived from microbial systems with all the advantages that mammalian systems afford.


Assay requirements for cell culture process development.

Because the structural parameters which influence product "quality" will vary from protein to protein, the screening methods used during development of each production process will also differ. Thus, not all of the methods described above will be needed for each product. One of the challenges of cell culture process development is choosing the correct structural features to monitor. The next level of process development may involve on-line control of many of the parameters discussed above. This would allow product quality-driven manipulations of culture conditions. The major factor limiting this advance may not be the techniques for real-time analysis, but rather a more thorough understanding of the structural features which describe a high-quality pharmaceutical protein.


Nonperfused attachment systems for cell cultivation.

The pressing need for large-scale culture methods has prompted efforts to develop vessels which will accommodate the growth of large numbers of anchorage-dependent cells. The large-scale "cell factories" have been used to grow viruses for vaccine production, nucleic acid studies, and various cancer research projects. Two particular types of culture vessels used for large-scale production of anchorage-dependent cells were discussed and examples of their use in vaccine production given. Development of these large-scale culture systems has enabled the pharmaceutical companies to (1) meet the ever-increasing demands worldwide for vaccine, (2) employ a production process that produces a cost-efficient vaccine product in cell culture, and (3) produce large volumes of bulk vaccine at a single campaign, thus allowing multiple usage of a single production unit over the course of a year. The type of large-scale culture vessel used depends on the purpose of the culture and the type of cells that are to be grown in the vessel. The unit process vessel, with its multidisks, provides superior surface area, but because of the stainless steel housing, the cells cannot be monitored microscopically. Additionally, the unit process vessel can be equipped with a jacket for precise temperature regulation and thus eliminate the need for incubators. The roller bottles, on the other hand, allow microscopic monitoring of the growing cells but are limited in surface area available for growth, and the volume of harvest fluid obtainable from a bottle is very limited. It should be noted that handling of roller bottles is labor-intensive and requires many more manipulations and hence more personnel than employing unit process-type vessels for vaccine-manufacturing operations. The use of mass cultivation techniques in cancer research has also been of immense benefit. The production of large numbers of neoplastic cells has provided researchers with the raw materials to perform molecular biology experiments such as DNA sequencing, which required milligram quantities of cells before sufficient quantities of DNA could be isolated to allow sequencing studies. Additionally, the technique of mass cultivation also provided sufficient quantities of cells for inoculation into either nude mice or immunosuppressed hamsters in an attempt to determine the tumor induction capacity of the cultured cells. Additional characterization studies which require large numbers of cell, enzyme, and isoenzyme profiles can also be performed on cells grown in roller bottle cultures. There are numerous factors which limit the productivity of mass cultivation systems and the scientist should be aware of their existence.(ABSTRACT TRUNCATED AT 400 WORDS)


Suspension culture of mammalian cells.

Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales.


Downstream processing of proteins from mammalian cells.

Less than a decade ago, the use of continuous mammalian cell lines for the production of cloned proteins was considered strictly a research tool. At that time, few thought it possible to allay the many safety concerns associated with transformed cells. It soon became clear that mammalian expression systems had numerous advantages over bacteria for production of therapeutic proteins, initiating a multidisciplinary effort to address these concerns in a thorough and reliable manner. The success of these efforts is exemplified by the emergence of product molecules into the market. Today, there are seven recombinant human therapeutics that have received FDA approval. Almost half of them (OKT3, t-PA, and EPO) are produced in mammalian cells, with the remainder produced in bacteria (insulin, growth hormone, and alpha-interferon) or yeast (hepatitis vaccine). At least a dozen more recombinant cell culture products are in advanced human clinical trials. With the accumulation of data and experience, continuous mammalian cell lines will no doubt be the preferred hosts for many future products of biotechnology.


Management of process technology.

A successful manufacturing scale cell culture process has as its basis a well-designed process. However, even the best designed large-scale process can become unreliable if not managed properly. The primary element to successful management of all large-scale processes is the careful attention to the details, especially the most important ones. The second element that requires serious consideration is the operation of that process within the confines of current regulatory environments. The major considerations are: 1. Adequate raw material specifications and testing. 2. Facility and equipment design that maximizes operational success and meets current regulatory requirements. 3. Thoroughly written procedures covering all aspects of operation and maintenance, and supported by a thorough documentation and review system. 4. A dedicated staff with a thorough understanding of systems and the importance of operational details. 5. Continual training and retraining programs. 6. Sound process and facility validation programs. 7. Comprehensive preventive maintenance programs. 8. Thorough understanding of process and facility trends, and the expected and acceptable ranges of results. 9. Development of sound procedures and practices that minimize batch losses and permit operation within current regulatory requirements. 10. Thorough and continual monitoring of all critical process parameters. 11. Process scheduling to minimize batch jeopardy or loss while maximizing throughout. 12. Maintenance of the appropriate environmental conditions of the plant and general cleanliness of plant and equipment. 13. Adequate batch loss prevention programs. 14. Adequate troubleshooting programs. 15. Adequate support organization and facilities both internally and externally to plant operations. With the appropriate application of sound process design and management principles, and with operational experience, large-scale cell culture process plants can be operated at success rates that exceed 95%.


Methods for the detection of adventitious viruses in cell cultures used in the production of biotechnology products.

The possibility for viral contamination exists in established cultures as well as in primary cultures. The use of established genetically engineered cultures in the production of biologicals for human use requires that these cultures be monitored for adventitious viral agents. Among the methods used for this purpose are animal inoculation and in vitro assays which provide a broad-spectrum screen for viral agents.


Process validation for cell culture-derived pharmaceutical proteins.

Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.


Large-scale propagation of insect cells.

Cultured insect cells have many uses in agriculture and medicine. They can be used in the diagnosis and isolation of a number of viruses infecting both animals and plants and for the laboratory study of these viruses. Large-volume culture of insect cells has been envisioned as a way of producing viruses for use in controlling insect pests and for the production of viral antigens for vaccine preparations. Recently they have become a potentially valuable way of producing a variety of proteins for human and veterinary medicine using the genetically engineered baculovirus expression vectors. The development of satisfactory cell lines and culture methods has proceeded at a slow, irregular pace, inhibited by the lack of knowledge of the physiology of the insect, its small size, and often by the lack of consistent, adequate support for the necessary developmental research. However, now that the basic culture systems are available, cell lines have been developed or can easily be developed for most needs. Suitable media are available and recent developments in refining existing media formations have resulted in low-cost media containing little protein to interfere with down-stream processing of cellular metabolites. Future developments are likely to further improve the media formulations and lower the cost. Technical problems relating to oxygen demand and cell fragility that inhibited the continued development of large-volume culture systems beyond the laboratory a few years ago now appear to be solved or at least are solvable. The successful culture of the Spodoptera cells in bioreactors of 40-liter capacity indicates that means of producing insect cells or their metabolic products on a commercial scale can be made economically feasible.


Large-scale animal cell culture: a biological perspective.

It is generally recognized that no one cell culture system can be universally applied to all cell types commonly used for biopharmaceutical manufacture. The analogous concept that no single cell type may be useful for the expression of all biopharmaceutical products may also gain credence in the biotechnology community. It may be that like specialized bioreactors, there will come to exist a variety of cell types that will be used for the production of different types of biopharmaceutical products. In addition, it may not be enough in the future just to demonstrate the stability of expression of the amino acid backbone of the protein only; the carbohydrate portion of the molecule may become the subject of real scrutiny. Questions such as how the carbohydrate side chain affects the performance of the molecule in vivo are being asked of more DNA constructs. The next question becomes, how can we control the expression of carbohydrate moieties on the molecule? Such questions are in the future of the biotech manufacturing field. Aside from those examples mentioned above dealing with the insertion of receptors, other more subtle attempts at modifying cellular metabolism are taking place. It was reported at a recent meeting that the sialyltransferase gene was inserted into a CHO line which did not normally express this enzyme (116). The transfected line was capable of expressing the transferase and, more importantly, the enzyme functioned correctly in sialylating glycoproteins. Other very complex relationships exist between the substratum and the cell that could have very direct consequences on culture maintenance. For example, researchers recently published results indicating that collagenase synthesis and secretion is stimulated in rabbit fibroblasts by autocrine factors. They determined that these autocrine proteins had sequence homology to serum amyloid-A and beta-2-microglobulin. It may be that using serum supplements in the medium in those systems that couple fibroblast and collagen substratum may not be prudent, especially for long-term culture. The traditional selection of a cell type for expressing heterologous proteins has generally been limited to the more "common" cell types such as CHO cells, C127 cells, and myeloma cells. In many cases these cell types were selected because there was a great deal of preexisting literature on the cell type (i.e., "cookbook" methods of transfection for the cell) or the cell was simply being carried in the lab at the time the effort was made to express a biopharmaceutical product.(ABSTRACT TRUNCATED AT 400 WORDS)


Purification of secreted recombinant proteins from Escherichia coli.

Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic compartments. Thus, hGH and muIL-2 extracted from the cytoplasm of E. coli have been characterized as high molecular weight disulfide-bonded oligomers. It is likely that oligomerization occurs as the polypeptides are released from the reducing environment of the cytoplasm. In contrast, secreted hGH and muIL-2 extracted from the periplasm of E. coli by osmotic shock displayed the properties of a property folded native protein with correct disulfide pairing. In the case of muIL-2 only a small residual fraction (approximately 15%) of the purified secreted protein exhibited incomplete oxidation of cysteine (35). Secretion of heterologous proteins into the periplasm prevents their exposure to the action of proteases located in the cytoplasm of E. coli (58). The smaller polypeptides such as somatostatin (59), IGF-1 (46), and hEGF (54) are known to be particularly susceptible to intracellular degradation.(ABSTRACT TRUNCATED AT 400 WORDS)


Physical and chemical cell disruption for the recovery of intracellular proteins.

There are many ways to disrupt microorganisms and plant and animal tissue. Selecting the best cell disruption method depends on the factors listed in Table 6. The kind or type of cells is an important consideration. For example, some disruption methods which work well for animal tissue do not work at all for microorganisms. A guideline for the suitabiity of a given disruption method for some cell types is given in Table 7. The ratings in this table are not incontestable and, as mentioned earlier, combinations of methods can sometimes produce satisfactory results whereas one method alone fails. The disruptibility of cells can be influenced by their growth and storage history. For microorganisms, cells in log phase growth tend to produce thinner cell walls which are more easy to disrupt. This and other conditions which can influence microbial cell disruptiability are listed in Table 8. The cell disruption method selected will depend on its capability to process samples of a certain size or to be able to process multiple samples in a reasonable period of time. Other considerations are the availability, cost, and general utility of the disruption equipment. Thus, in a research environment the purchase of an expensive cell disrupter which processes a wide variety of cell types may be more easy to justify than a specialized disrupter. And if the long-term goal is to scale up, the choice of disruption methods narrow considerably. Indeed, several of the most successful laboratory cell disruption methods have no possibility of being scaled up. Despite possible scale-up difficulties, in the case of many bioactive recombinant products expressed at high levels in microorganisms, this concern may be irrelevant. Few of these products are likely to be manufactured in really large amounts and current laboratory scale or pilot plant scale production equipment may be entirely adequate. For instance, active human TNF (tissue necrosis factor) can be expressed in Pichia pastoris yeast at levels of 100 g/kg of yeast (dry weight). At this level of expression, only a few kilograms of r-DNA yeast needs be disrupted to meet the worldwide demand for this research material. Finally, the operating and energy requirements which affect the economics of the disruption process (batch versus continuous, disruption yield, cell fragment size, effect of added enzymes on downstream separation, etc.) are important considerations in the selection of production equipment.


The competitive market for antiplatelets and thrombolytics: can a new entrant succeed?

The current lack of available antiplatelet and thrombolytic agents, the limitations of existing products, the possibility of value-pricing, and the sheer number of potential candidates for therapy all combine to produce a market with tremendous potential. Given these very significant incentives, it is no wonder that so many pharmaceutical companies have chosen to pursue research in the antithrombotic field.


Applications of bacterial magnets.

Magnetic bacteria migrate along the lines of the earth's magnetic field, and synthesize intracellular particles of magnetite which are aligned in chains and enveloped by a membrane, thus forming biological magnets. Recent progress in techniques for isolating and culturing one strain offers hope for the large-scale production of bacterial magnets. Potential biotechnological and medical applications will exploit the ability to manipulate conjugates or cells incorporating the particles, whose usefulness depends on their small size and membrane coating.


Studying DNA-protein interactions using NMR.

NMR spectroscopy is emerging as a powerful tool in molecular biology and biotechnology; one aspect of which is the use of one- and two-dimensional NMR methodologies to investigate the interactions of proteins with DNA. The dynamic and structural information which NMR can provide, on the changes in conformation and molecular flexibility, complements X-ray crystallography data and enables mechanistic models of DNA-protein interactions to be formulated.


Bio-techniques for the detection of genetic toxicity in the aquatic environment.

This article reviews the application of sensitive biotechniques in the detection of DNA damage within the aquatic environment as "early warning" indicators of pollution-related genetic toxicity. Bacterial mutagenicity assays have played an important role both in the discovery of mutagens and in the analysis of the ability of aquatic organisms to convert inert substances to reactive genotoxic products. Genetic damage in aquatic species treated with carcinogens has been detected by analysis of DNA adducts, induction of DNA repair, DNA strand breakage and formation of chromosomal aberrations and sister chromatid exchanges. Little information is available on pollution-related DNA damage resulting from field exposure. A 32P-postlabelling technique for the sensitive detection of DNA adducts is a promising development towards this aim and the analysis of changes in oncogene nucleotide sequence in tumour tissue is considered an important future direction.


Electrically controlled proliferation of human carcinoma cells cultured on the surface of an electrode.

Human carcinoma cells, MKN45, were cultured on the surface of a metal-coated plastic plate electrode the potential of which was controlled. The proliferation rate and cell morphology were altered depending on the applied potential. Cell proliferation was halted in the potential range above 0.4 V vs. Ag/AgCl, although cells started to proliferate again when the applied potential was shifted from 0.4 V to 0.1 V vs. Ag/AgCl. Fluorescence probe studies indicated that the fluidity of plasma membrane decreased in association with halting of cell proliferation. These results suggest that electrical stimulation causes cells to temporarily halt proliferation, and that cell proliferation was reversibly controlled by electrode potential. The mechanism is interpreted in relation to the change of plasma membrane structure represented by membrane fluidity.


Examination of serum and bovine serum albumin as shear protective agents in agitated cultures of hybridoma cells.

A murine hybridoma cell line (167.4G5.3) was cultured in batch mode using IMDM containing different serum concentrations and bovine serum albumin (BSA). Cell growth and death, metabolism and antibody production were studied in these cultures. The cells were more susceptible to shear in the stationary and in the decline phase of growth as evidenced by higher death rates. Cell growth was best at high serum concentrations with high specific growth and low specific death rates. When BSA was used instead of serum in IMDM, no protective effect was observed. Cell metabolism and monoclonal antibody production rates were not influenced by the level of serum or by BSA. The use of serum in commercial serum-free media (OPTI-MEM) also resulted in no change in both growth and death rates.


The effect of the partition locus on plasmid stability and expression of a prolonged chemostat culture.

The stability, copy number, and gene expression of the pBR322 plasmid containing the par-locus under prolonged cultivation were studied. In the initial stage of the experiment it was observed that the par-locus had a stabilization effect on plasmid maintenance. This observation was consistent with previously reported results. However, after approximately 225 h, a mixed population of plasmid-containing and plasmid-free cells appeared. The mixed culture was stably maintained for approximately 200 h. In addition, the relative plasmid copy number showed an increase as compared to the par- culture. After 100 h the copy number decreased, reached a minimum, then stabilized. The beta-lactamase activity, was not significantly affected by the par-locus.


On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode.

Two different analysis techniques for on-line monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA). The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae.


On-line determination of glucose concentration throughout animal cell cultures based on chemiluminescent detection of hydrogen peroxide coupled with flow-injection analysis.

A flow-injection analysis (FIA) system for the on-line determination of glucose in animal cell cultures is described. The system is based on immobilized glucose oxidase (GOD). The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol. Based on the measurement of the maximum emitted light intensity, the system was able to analyse hydrogen peroxide over the concentration range of 10(-7) to 10(-2) M. For glucose determination, the system has a linear range of 10(-5) to 5 x 10(-2) M glucose, with an r.s.d. of 3% at the 1 mM level (5 measurements). The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated. The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per h. The system has been successfully applied to on-line monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor. Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).


Development of guidelines for small scale genetic manipulation work.

The Genetic Manipulation Advisory Committee (GMAC) is the national body which reviews research and development work in Australia using genetic manipulation technologies. It supersedes and continues the work of the Recombinant DNA Monitoring Committee (RDMC). GMAC has recently drafted new guidelines for small scale laboratory work. The difference between these guidelines and the ones they have replaced is the broadening of the scope of work to be monitored, from recombinant DNA work to other genetic technologies which may also produce new combinations of inheritable genetic material. Experience from assessing proposals over the life of the RDMC and a previous Academy of Science committee enabled the GMAC Scientific Sub-committee to be more specific about the experiments it wanted to assess before they can begin, and those which could be safely exempted from the guidelines.


International trends in biotechnology regulation: the implications for developing countries.

Evolutionary processes have demonstrated that genetic mutations can, over time, produce a complete restructuring of the previous ecological order. The capacity of biotechnology to manipulate and transform genetic material at a far greater rate than nature is capable of suggests that careful consideration should be given to effectively controlling these technologies. This paper examines the underlying technical and philosophical concerns and arguments which underpin the development of appropriate regulations for biotechnology. The regulatory frameworks which have been set in place around the world are reviewed. Modifications which are being introduced in the light of increasing practical experience and in response to changing societal pressures are discussed. Potential negative impacts of biotechnology on developing countries are specifically addressed. A case is made for co-ordinated international efforts to develop consistent and uniform legislation for the safe and environmentally beneficial application of biotechnology on a more equitable global basis.


Opportunistic infections in AIDS and their diagnosis.

AIDS patients are susceptible to a variety of infections from microorganisms as a result of their immunosuppressed condition. These infections are mainly responsible for the morbidity and mortality in these patients. The organisms responsible include protozoa, bacteria, fungi and viruses. Their presence may be a result of reactivation of latent or previous infection, or exposure to opportunistic agents. The seriousness of the patients' condition makes rapid and early diagnosis imperative so that appropriate treatment can be instituted. Methods of diagnosis including more recent technology are discussed.


Production of recombinant products in yeasts: a review.

This paper reviews the use of yeasts to produce heterologous proteins via the development of transformation systems. The ability to genetically engineer yeast cells has many advantages over prokaryotic systems. Yeasts are already well established in fermentation procedures, are able to secrete glycosylated and modified proteins to render the proteins biologically active, and yeasts do not secrete toxic chemicals. With the techniques available it is possible to explore yeasts as hosts for the expression and secretion of commercially-useful proteins.


Cell cycle analysis in flow cytometry: use of BrdU labelling and side scatter for the detection of the different cell cycle phases.

Cell cycle analysis in flow cytometry is based on the incorporation of labelled precursors in DNA. The use of BrdU versus SSC, in which side scatter substitutes PI fluorescence, has proved to be useful also for the distinction between G2 and Mitotic cells. Mitoses often produce an SSC decrease due to the morphological changes that happen in the nucleus during this phase of cell cycle. Moreover, DNA accessibility to PI varies during mitosis, as well. However, most of these variations, detectable by flow cytometry appear to be basically dependent on the cell line used.


Alternative methods in toxicology tests: in vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develop alternative methods to reduce the required number of animals. The new in vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by the in vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity with in vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems for in vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mitochondrial activity, intracellular content and leakage of enzymes into the medium.


Cell metabolism measurements in culture via microelectronic biosensors.

Silicon-based H(+)-sensitive biosensors in proximity to a cell population detect variations in cell metabolism via local measurements of changes in pH. The feasibility of this approach is shown in the case of ISFET devices.


Characterization of a subfamily of zinc finger genes expressed in human hematopoietic cell lines.

We isolated by low stringency screening of a human erythroleukemia cDNA library (K562) 45 independent clones hybridizing to a Krüppel-like (HF.10) zinc finger cDNA. The expression of 15 such cDNAs in human hematopoietic cell lines was investigated. Preliminary sequence analysis of the zinc finger motifs in these cDNAs indicate that they belong to a subclass of the Cys-Cys/His-His motif, showing the highest homology to the Wilm's tumor and EGR1, EGR2 cDNAs.


Fluorimetric approaches to the study of calcium transients in living cells.

This paper is a short review of the fluorimetric methods used to measure intracellular free Ca++ concentration in living cells. The availability of fluorescent probes has greatly contributed to the understanding of the mechanisms responsible for the cellular homeostasis of this second messenger. Data can be collected from populations of cells by spectrofluorimetry or from small groups or single cells by spectromicroscopy. Finally the fluorescent images can be captured by a high sensitivity camera, digitally processed and convert in Ca++ images of the cell. The technique allows recognition of differences in [Ca++]i transients among adjacent cells in a same field or in different regions of a cell and greatly contributes to the identification of the cellular mechanisms modulating [Ca++]i.


Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes.

There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning the actI, actIII, actIV, and actVII genes (pANT12) of actinorhodin biosynthesis pathway from Streptomyces coelicolor in anthracycline producing streptomycetes. Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kb XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus 31 133. When S. galilaeus 31 671 was transformed with the actI, actVII, and actIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin. When S. galilaeus 31 671 was transformed with only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci, dauG, dnrR1, and asaA, on the production of natural and hybrid polyketides were also shown.


Biological activity of a transforming growth factor-alpha--Pseudomonas exotoxin fusion protein in vitro and in vivo.

Transforming growth factor-alpha (TGF alpha)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor binding activity of TGF alpha and the cell killing activity of PE40. In the current study, we report that a modified TGF alpha-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor--toxin fusion proteins as anticancer agents.


Bioreactor and process design for large-scale mammalian cell culture manufacturing.

Good Manufacturing Practice for pharmaceuticals requires that the manufacturing processes be controlled and reproducible. The biological nature of cell culture-based processes lends them an inherently higher variability than is generally found for processes involving production of defined chemical entities. As one regulatory body has pointed out, this places a greater emphasis on in-process controls and adherence to GMPs in the manufacture of cell culture products. Design of bioreactors and associated process control systems is a key element in the successful implementation of a commercial-scale cell culture manufacturing process capable of meeting such high standards. It should be noted that reactor and control system designs for animal cell culture are still very much in a state of evolution from the modified bacterial fermentation systems that currently predominate, and it is difficult to predict how design strategies may change in the coming years. Having said this, however, it should be clear from the information presented here that satisfactory processes can be devised today using proven equipment configurations such as the stirred-tank reactor.


Second-generation products: antibiotics.

There has been outstanding progress in gene cloning from microorganisms producing useful antibiotics. Especially noteworthy is the cloning of a gene for cephalosporin biosynthesis that, with increased copy number, helps to overcome a rate-limiting step in biosynthesis. Although this technology is often fraught with unexpected difficulties, not the least of which is gene expression, yield improvement is a practical application with clear financial benefits to the pharmaceutical industry. Although access to cloned genes for antibiotic biosynthesis is becoming common, expression of the cloned genes in a manner such that yield improvement is achieved is a challenge that will be exciting and beneficial in years to come. Heterologous (interspecies) gene expression in antibiotic-producing microorganisms, particularly the actinomycetes, must be successful if hybrid antibiotics are to be produced. Currently, there are no documented examples of heterologous gene cloning to yield a hybrid antibiotic of utility. Even though a hybrid antibiotic structure has been reported as a result of heterologous gene cloning, the antibiotic is not clinically useful, and the two species used in the experiments are closely related. Although technological advances have enabled successful gene cloning, insufficient attention has been given to the issue of gene expression, particularly heterologous gene expression. The need to develop heterologous gene expression systems is urgent. Until this is done, the inability to express cloned antibiotic biosynthesis genes in heterologous hosts could be a barrier to the generation of useful hybrid antibiotics. The potential for a wide array of new antibiotic compounds that could be generated by gene-cloning technology is vast. At least until the limitations of heterologous gene expression are overcome, the best available screening technology (including molecular and immunological screens) and chemistry might best serve as the most prolific means of discovering useful new antibiotics for agriculture and medicine.


The preparation and validation of stock cultures of mammalian cells.

The utilization of continuous cell substrates is now widely accepted for the production of biologics. As part of the evaluation and licensing process of these products, the regulatory agencies are requiring extensive validation of the production system. That production system begins with the validation or qualification of a defined cell bank. This includes the validation of the stability cells at both the genetic and biochemical levels during cell culture production. Process validation plus cell bank validation provide the necessary assurance that the final product will be free of contaminating viruses and other adventitious agents. A combination of cell bank characterization and product characterization (peptide mapping or amino acid sequencing, or a combination thereof) will also demonstrate the stability of the production process. The validation of a cell bank for adventitious agents and cell line stability will not, in itself, ensure that the product is sterile and stable. However, cell bank validation is critical for demonstrating the safety of a product when combined with both process validation and end-product testing.


Cultivation, characterization and modulation of rat thymic non-lymphoid cells in vitro.

Thymic fragments of young adult rats were cultivated in vitro using an explant technique. Two weeks later, non-lymphoid cells were characterized by enzyme-cytochemistry and immuno-cytochemistry. Based on cytomorphological criteria and cell-specific markers approximately 95% of cells were classified into 3 main types: epithelial cells, macrophages and fibroblasts. The effect of dexamethasone and amphotericin B, known modulators of cell growth in culture was also studied. Quantitative analysis showed that amphotericin B inhibited proliferation of macrophages and epithelial cells, while dexamethasone suppressed proliferation of fibroblasts and promoted growth of epithelial cells.


Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells. Obtention of clones exhibiting different patterns of enterocytic differentiation.

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.


Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining.

Many microcarriers used for the cultivation of animal cells do not allow for convenient microscopic observation of cell morphology and viability due to their optical properties. Using fluorescent viable stain combining fluorescein diacetate and ethidium bromide, we observed the distribution, morphology and viability of cells on various microcarriers.


Characterization of a new continuous cell line from the flood water mosquito, Aedes vexans.

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (= 2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.


Methods for improving tissue culture of human tracheo-bronchial epithelium obtained at autopsy.

Human tracheo-bronchial epithelium obtained from autopsy, surgery, and organ donation will have areas of both viable and non-viable cells. It is important in the initial establishment of epithelial explant and cell cultures that injured, non-viable mucosal epithelium not be used for the cultures. Autopsy cases selected for culture should initially be chosen on the basis of a shorter post mortem interval and cause of death in order to increase the rate of successful culture. Staining the epithelium with the vital dye, trypan blue, in combination with phase contrast microscopy of the bronchial tissues will further identify those areas of the mucosa that are enriched for viable cells. The dead, non-viable areas are trypan blue positive, while the viable areas are clear and have foci of beating, motile cilia. Treatment of the mucosal tissue with mucolytic agents to remove cell debris, dead cells, and microbes trapped in the mucus material will further improve the chances for successful culture. Human tracheo-bronchial epithelium, although non-sterile and often injured at time zero for numerous reasons, can effectively be used in vitro pathophysiology studies.


Influence of inoculum age on hybridoma culture kinetics.

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.


Primary culture and cryopreservation of mouse astrocytes under serum-free conditions.

The methods of primary culture and cryopreservation of mouse astrocytes under serum-free conditions were examined. Cerebra from newborn C3H/He mice were employed as the source of astrocytes. The cultured cells were able to grow in a serum-free, chemically defined medium containing transferrin, hydrocortisone, biotin, sodium selenite, insulin, fibroblast growth factor and epidermal growth factor. After the culture was maintained in the medium for 3 weeks, purity was assessed using immunofluorescence staining. The great majority of the cells (greater than 98%) contained glial fibrillary acidic protein and S-100 protein which are cell markers of astrocytes. To cryopreserve the enriched astrocytes under serum-free conditions, various cryoprotectants were examined. The combination of 10% dimethylsulfoxide and 0.1% methylcellulose gave the highest survival rate. These methods of primary culture and cryopreservation will be useful in physiological and biochemical studies which require mouse astrocytes.


Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2-3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.


Water activity as a key parameter of synthesis reactions: the example of lipase in biphasic (liquid/solid) media.

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (aw = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high aw (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)


Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier--spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.


Concanavalin A: a useful ligand for glycoenzyme immobilization--a review.

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.


Activity and distribution of urease following microencapsulation within polyamide membranes.

Urease was microencapsulated by forming a semipermeable polyamide membrane around aqueous microdroplets (266 microns mean diameter) containing the soluble enzyme. The yield of the interfacial polymerization technique, determined spectrophotometrically, was 83% of the original enzyme on a mass basis, resulting in a final intracapsular urease concentration of 62.3 mg ml-1 or 0.1 mM. Similar absorption spectra of broken and intact microcapsules suggested that spectrophotometry may be applied in performing direct studies on the intact microcapsules. The high activity yield of urease microcapsules relative to the mass of entrapped enzyme (92.5%) indicated minimal effects of mass transfer limitation. The mass of active urease incorporated into the nylon membrane represented 6% of the encapsulated enzyme activity. The soluble intracapsular enzyme fraction (94%) was released into solution upon rupture of the membrane. A complete mass and activity balance of the encapsulated enzyme was achieved.


Aminopropylsilane treatment for the surface of porous glasses suitable for enzyme immobilisation.

During silylation of porous glass with 3-aminopropyltriethoxysilane, the properties of the carrier affected the concentration of bound amino groups, the formation of aminopolysiloxane mono- or multilayer on the surface, and the hydrolytic stability of the layer formed. The influence of the carrier depended on the specific surface area and the size of pores. In contrast to silylation performed in organic solvents, in aqueous solutions a monolayer of aminopolysiloxane of high hydrolytic stability was formed on the surface of porous glass. Tetraethoxysilane modification of porous glass prior to silylation with aminosilane yields carriers of increased hydrolytic stability. Glucoamylase immobilised on carriers, that were modified in aqueous solutions, exhibit higher enzymatic activity.