Quantification of l-alanyl-l-glutamine in mammalian cell culture broth: Evaluation of different detectors

l-Alanyl-l-glutamine (also known as Ala-Gln or GlutaMAX) is widely used as a stable l-glutamine source in cell culture for the production of biopharmaceuticals. System approaches for the optimization of production processes require the analysis of all major substrates and products. We have compared four alternative detection systems for l-alanyl-l-glutamine in culture broth. Matrix effects prevented the use of ultraviolet or evaporative light scattering detection. Fluorescence detection used in routine amino acid protocols is compatible with culture broth and has a broad linear dynamic range. Mass spectrometry has superior sensitivity and can be integrated into quantitative metabolomic workflows.     

Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

Cross comparison of rapid mycoplasma detection platforms

The use of animal and plant derived raw materials in mammalian cell culture processes may provide a possible route of entry for adventitious contaminants such as mycoplasma. Mycoplasma contaminations of cell culture represent a serious challenge to the production of biotechnology derived therapeutics. The slow growing nature of mycoplasma can disguise their infection of cultures since cells may continue to proliferate, though at reduced levels and with lesser output of engineered protein. Rapid identification of mycoplasma contaminated cell cultures and materials enables a faster response time to prevent the spread of the contamination. We describe here the comparison of different mycoplasma detection methods: two nucleic acid-based technologies, the standard mycoplasma culture procedure, and a hybrid culture-quantitative PCR assay. In this study, a cell line infected with two species of mycoplasma was used to compare the different detection methods. Our data demonstrates that the two nucleic acid-based techniques are robust methods for detection of mycoplasma and have similar detection capability. In contrast, no mycoplasma was detected in the standard culture assay or in a hybrid culture-quantitative PCR assay. This shows a potential limitation of the culture assay that relies on the ability of mycoplasma to grow in broth media.     

Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection

Cell culture and fermentation broth media are used in the manufacture of biotherapeutics and many other biological materials. Characterizing the amino acid composition in cell culture and fermentation broth media is important because deficiencies in these nutrients can reduce desired yields or alter final product quality. Anion-exchange (AE) chromatography using sodium hydroxide (NaOH) and sodium acetate gradients, coupled with integrated pulsed amperometric detection (IPAD), determines amino acids without sample derivatization. AE-IPAD also detects carbohydrates, glycols, and sugar alcohols. The presence of these compounds, often at high concentrations in cell culture and fermentation broth media, can complicate amino acid determinations. To determine whether these samples can be analyzed without sample preparation, we studied the effects of altering and extending the initial NaOH eluent concentration on the retention of 42 different carbohydrates and related compounds, 30 amino acids and related compounds, and 3 additional compounds. We found that carbohydrate retention is impacted in a manner different from that of amino acid retention by a change in [NaOH]. We used this selectivity difference to design amino acid determinations of diluted cell culture and fermentation broth media, including Bacto yeast extract-peptone-dextrose (yeast culture medium) broth, Luria-Bertani (bacterial culture medium) broth, and minimal essential medium and serum-free protein-free hybridoma medium (mammalian cell culture media). These media were selected as representatives for both prokaryotic and eukaryotic culture systems capable of challenging the analytical technique presented in this paper. Glucose up to 10mM (0.2%, w/w) did not interfere with the chromatography, or decrease recovery greater than 20%, for the common amino acids arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamate, aspartate, cystine, and tyrosine.     

Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production

Corynebacterium glutamicum, well known for the industrial production of amino acids, grows aerobically on a variety of mono- and disaccharides and on alcohols and organic acids as single or combined sources of carbon and energy. Members of the genera Corynebacterium and Brevibacterium were here tested for their ability to use the homopolysaccharide starch as a substrate for growth. None of the 24 type strains tested showed growth on or degradation of this substrate, indicating that none of the strains synthesized and secreted starch-degrading enzymes. Introducing the Streptomyces griseus amy gene on an expression vector into the lysine-producer C. glutamicum DM1730, we constructed a C. glutamicum strain synthesizing and secreting alpha-amylase into the culture broth. Although some high-molecular-weight degradation products remained in the culture broth, this recombinant strain effectively used soluble starch as carbon and energy substrate for growth and also for lysine production. Thus, employment of our construct allows avoidance of the cost-intensive enzymatic hydrolysis of the starch, which commercially is used as a substrate in industrial amino acid fermentations.     

Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins

Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product. The current U.S. Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures. Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1-10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species. The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1-2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection >/=1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting. In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use. This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures. Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438-470 bp. A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events. In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M. hyorhinis, is 1 cfu/ml. The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry.     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production

Fluorescence spectra of a 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) fermentation culture broth were combined with measurable process variables for off-line and on-line process monitoring. Culture broth fluorescence in UV and visible ranges was acquired by a fiber optic LCD array spectrometer. Process dynamics was followed on-line using a fiber optic probe attached to an external recirculation loop of the bioreactor. Partial least squares and stepwise regression methods were used to correlate measurable process parameters with the components of the fluorescence spectra. Both methods provided adequate approximation of yeast density, HEMF, glucose, and ethanol concentrations from fluorescence spectra. HEMF production was observed during the oxido-reductive growth phase when there was a lack of measurable oxygen in the culture broth and an excess of glucose. The addition of glucose resulted in the rapid production of HEMF and other metabolite intermediates such as ethanol, acetate, and glycerol.     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.     

Production of Polyvinyl Alcohol Oxidase by a Symbiotic Mixed Culture

Production of polyvinly alcohol (PVA) oxidase by Pseudomonas sp. strain VM15C, a PVA degrader of a symbiotic PVA-utilizing mixed culture, was examined in various cultures. Despite the absence of PVA in the culture in nutrient broth, VM15C showed approximately the same productivity of PVA oxidase activity as that in the culture with PVA as the sole carbon source, whereas the productivity in the culture with glucose was lower than that of either the nutrient broth or the PVA culture. PVA oxidase activity produced in the nutrient broth culture was predominantly present in the cells, and most of the activity appeared to be in the cytoplasm. In contrast, in the culture with PVA as the sole carbon source, the activity was present in the culture fluid in a larger ratio than in the nutrient broth culture. Thus, production of PVA oxidase activity by this strain was constitutive and repressible, although localization of the produced activity was changed by growth conditions.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.     

Production of superoxide dismutase in Streptococcus lactis by a combination of use of hyperbaric oxygen and fermentation with cross-flow filtration

The production conditions of superoxide dismutase (SOD) in the cells of Streptococcus lactis by using hyperbaric oxygen (O(2)) are described. The SOD activity of anaerobically grown cells was 5-6 U/mg protein. When the culture broth was pressurized by O(2) at 6 atm, the SOD activity was more than twice as high as that under anaerobic culture conditions. However, there is little or no significant increase in SOD activity by exogenous addition of catalase for detoxifying hydrogen peroxide accumulated in the broth and/or controlling the pH of the broth at 6.8 during the pressurization by O(2). The increase in SOD activity by hyperbaric O(2) was possible not only at the late-logarithmic growth phase but also at the initial time for the stationary growth phase. For improvement of SOD productivity, we tried a two-stage culture in which SOD activity in S. lactis cells was enhanced by pressurizing the culture broth using hyperbaric O(2) after achievement of a high-concentration cultivation in the anerobic fermentation system with a microfiltration module.     

Rheological characterization of culture broth containing the exopolysaccharide PS-EDIV from Sphingomonas pituitosa

Sphingomonas pituitosa excretes the capsular exopolysaccharide PS-EDIV into the culture broth augmenting considerably its fluid viscosity. Since this change particularly affects key processes like mixing and transport during the microbial production, this work was aimed at the rheological characterization of the polymer-containing culture broth of S. pituitosa. The study included investigations on basic properties of the culture broth, but also on the dependence of the biomass–polymer-solution properties on different physicochemical post-cultivation treatment steps like variations of temperature, pH-value or concentration of salts. The essential result is the characterization of the viscoelastic behavior of the culture broth, which was more gel-like than sol-like and exhibited slight elastic properties. This rheological behavior showed that the PS-EDIV culture broth formed non-Newtonian fluids, indicating that it is a pseudoplastic biopolymer, with yield stress appearance and exhibits thixotropic properties. Rheograms were fitted to the Herschel–Bulkley model. The amplitude sweep revealed a deformation of 21% as the limiting value of the linear viscoelastic interval. Furthermore, the PS-EDIV culture broth showed a high viscosity which was strongly influenced by salt type and concentration but weakly influenced by temperature and pH-value within the investigated experimental boundaries.     

Campylobacter contamination in French chicken production from farm to consumers. Use of a PCR assay for detection and identification of Campylobacter jejuni and Camp. coli

AIMS: Campylobacter contamination in French chicken production from the farm to the consumer was determined using a PCR assay for bacteria detection and identification. METHODS AND RESULTS: Samples were bird droppings from poultry houses, neck skins, livers, hearts, gizzards, wings, legs and escalopes from slaughterhouses and gizzards, legs, drumstick, breast and escalopes from a supermarket. Bacterial DNA extraction was performed after an enrichment step in a broth and was followed by PCR. An internal control (IC) was used for both DNA extraction and PCR. Campylobacter were detected in 79.2% of poultry houses. Of the 303 samples, 201 were Campylobacter-positive (i.e. 66.3%) including 43.2% faecal samples, 5.6% slaughterhouse samples and 17.5% supermarket samples. There was no significant difference between the molecular method and the conventional culture technique for Campylobacter detection whatever the samples. The sensitivity was 5 UFC g(-1) of samples and 1.5 x 10(3) UFC ml(-1) of enrichment broth. The use of IC revealed PCR inhibition in 13 samples and problems in the DNA extraction in five samples. CONCLUSION: Significant Campylobacter contamination affects all stages of French chicken production. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Campylobacter contamination at different levels of chicken production and the determination of the best place(s) for intervention are important for significantly decreasing Campylobacteriosis. Our technique is rapid and can be used on different chicken samples for Campylobacter detection and identification.     

Estimation of the Growth of the T1 Strain of Mycoplasma mycoides in Tryptose Broth by the Measurement of Lactate Dehydrogenase: II. Application to Vaccine Production

The measurement of lactate dehydrogenase activity has been used to evaluate the growth of the T(1) vaccine strain of Mycoplasma mycoides var. mycoides in broth during the production of vaccine. Estimations were made during incubation at 37 C and storage at 4 C. The results were compared with titers of the vaccine obtained by two conventional methods of assessing viability titers: the counting of colonies formed on solid medium and a 50% end point titer found after dilutions were made in broth. The method of measuring lactate dehydrogenase activity allowed the rapid enumeration of organisms during incubation of the culture, but estimations made on vaccine stored at 4 C did not compare well with the two conventional methods of assessing viability. The lactate dehydrogenase activity of the culture has enabled rapid monitoring of the production stages of this vaccine.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.