Fed-batch operation of an industrial cell culture process in shaken microwells.

Recently we have demonstrated batch suspension culture of mammalian cells in microwell plates. Here we describe a method for fed-batch culture of an industrially relevant GS-CHO (Glutamine Synthetase-Chinese Hamster Ovary) cell line in shaken 24-standard round well (24-SRW) plates. Use of a commercially available 'sandwich lid' and appropriate dilution of the bolus feeds counteracted liquid evaporation from the wells resulting in similar cell growth and antibody formation kinetics in both 24-SRW plates (800 mul) and shaken flasks (50 ml). Peak viable cell densities obtained were 8 +/- 0.5 x 10(6) and 9 +/- 1.3 x 10(6) ml(-1), respectively, while comparable final titres of a whole IgG of approximately 1.5 g l(-1) were recorded. Use of microwells provides at least a 50-fold reduction in medium requirements compared to shake-flask and other culture devices currently used in early stage cell culture process development. The ability to run multiple wells in parallel and to automate culture operation also offers considerable enhancements in experimental throughput.

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale-up.

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.

Systematic functional analysis of the yeast genome.

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.

Engineering characterisation of a shaken, single-use photobioreactor for early stage microalgae cultivation using Chlorella sorokiniana.

This work describes the characterisation and culture performance of a novel, orbitally shaken, single-use photobioreactor (SUPBr) system for microalgae cultivation. The SUPBr mounted on an orbitally shaken platform was illuminated from below. Investigation of fluid hydrodynamics indicated a range of different flow regimes and the existence of 'in-phase' and 'out-of-phase' conditions. Quantification of the fluid mixing time (tm) indicated a decrease in tm values with increasing shaking frequency up to 90 rpm and then approximately constant tm values in the range 15-40 s. For batch cultivation of Chlorella sorokiniana, the highest biomass concentration achieved was 6.6 g L(-1) at light intensity of 180 µmol m2 s(-1). Doubling the total working volume resulted in 35-40% reduction in biomass yield while shaking frequency had little influence on culture kinetics and fatty methyl esters composition. Overall this work demonstrates the utility of the SUPBr for early stage development of algal cultivation processes.

Combined remediation and lipid production using Chlorella sorokiniana grown on wastewater and exhaust gases.

Substitution of conventional feedstock with waste based alternatives is one route towards both remediation and reducing costs associated with production of algal biomass. This work explores whether exhaust gases and wastewater can replace conventional feedstock in the production of biomass from Chlorella sorokiniana. Exhaust gases were used to augment production in final effluent, anaerobic digester centrate or in standard medium. Cultures were grown in 1L bottles under illumination of 80 µmol m(-2) s(-1). The results showed an average µmax ranging between 0.04 and 0.07 h(-1), whilst the final biomass yield in different media ranged between 220 and 330 mg L(-1). Lipid yield was increased over time to 31 mg L(-1). CO2 addition resulted in complete nitrogen removal between 48 and 96 h in both final effluent and centrate. The results also indicated that levels of carbon monoxide, carbon dioxide and nitrogen oxides in the exhaust gases can be reduced by between 20% and 95%.

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His6-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.

Assessment of the manufacturability of Escherichia coli high cell density fermentations.

The physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale-down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large-scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed-batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6-fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality-by-design principles.

Design and characterization of a prototype enzyme microreactor: quantification of immobilized transketolase kinetics.

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-microm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His(6)-tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-catalysed synthesis of L-erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis-Menten model. Results show that the TK kinetic parameters in the IEMR (V(max(app)) = 0.1 +/- 0.02 mmol min(-1), K(m(app)) = 26 +/- 4 mM) are comparable with those measured in free solution. Furthermore, the k(cat) for the microreactor of 4.1 x 10(5) s(-1) was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His(6)-immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization.

Quantification of kinetics for enzyme-catalysed reactions: implications for diffusional limitations at the 10 ml scale.

The effects of different reaction scales [100 microl reactions in 96-standard round well (SRW) plates and 10 ml reactions in 24-square well (SW) plates] have been investigated using, as a model, transketolase (TK)-catalysed reaction producing L-erythrulose. Reactions were carried out under non-shaking, shaking and at 10 ml scale stirring conditions to assess the effect of diffusional limitations. Statistical analysis confirmed the significance of the observed difference in reaction rates under given conditions. Only when the laboratory scale system (10 ml) was well mixed did the reaction rate become comparable to that in the microwells, where there is negligible diffusional limitation. These findings have important implications for the scale-up (or scale-down) of enzyme-catalysed reactions.

Identification of erythrobactin, a hydroxamate-type siderophore produced by Saccharopolyspora erythraea.

AIMS: To investigate the production of siderophores by Saccharopolyspora erythraea SGT2 and how this production is affected by the inoculum. METHODS AND RESULTS: When grown in a low-iron, chemically defined medium (CDM), the soil dwelling actinomycete S. erythraea secretes a substance that is reactive in the nonspecific chrome azurol S (CAS) assay. Importantly, the production of CAS-reactive substance is highly reduced upon the addition of 0.925 micromol l(-1) iron to the cultures and has a peak of production in the late-log to early stationary growth phase. In addition, the culture supernatants tested were negative in the Arnow and Rioux assays but positive in the Csáky procedure. Interestingly, we also found evidence that the production of this CAS-reactive substance in CDM was highly reduced, when inoculated with cells that had been previously grown to late-stationary phase. Conversely, inocula derived from late-log to early stationary cultures presented high levels of CAS activity. CONCLUSIONS: These results indicate that S. erythraea produces a hydroxamate-type siderophore that we have generically designated as erythrobactin. Additionally, the inocula growth stage plays a key role in siderophore production in S. erythraea. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first evidence for siderophore synthesis in S. erythraea and one of the first examples of non-polyketide secondary metabolite production by this organism.

Studies on the interaction of fermentation and microfiltration operations: erythromycin recovery from Saccharopolyspora erythraea fermentation broths.

Changes in fermentation media not only affect the performance of the fermentation itself (with regard to the kinetics of biomass and product formation and the yields obtained) but also the initial product-recovery operations downstream of the fermentor. In this work, microfiltration experiments to remove Saccharopolyspora erythraea biomass from fermentation broth and to recover erythromycin were carried out using two fundamentally different media; a soluble complex medium (SCM) and an oil-based process medium (OBM). Small-scale batch fermentations of 14-L working volume were carried out in triplicate using both media. Broth samples were taken from each fermentation at regular intervals from the end of the exponential-growth phase onwards. These were then processed using a Minitan II (acrylic), tangential crossflow-filtration module, fitted with a single 60 cm(2) Durapore hydrophilic 0.2 microm membrane, operated in concentration mode. The OBM fermentations produced higher titers of erythromycin but required longer fermentation times due to increased lag phases and slower maximum-growth rates. The OBM also increased the loading on the membrane; at maximum product titers residual oil concentrations of 3 g. L(-1), antifoam concentrations of 2 g. L(-1) and flour concentrations estimated at approximately 10 g/L(-1) were typical. It was found that both the permeate flux and erythromycin transmission were affected by the choice of medium. The OBM had significantly lower values for both parameters (12.8 Lm(-2) h(-1) and 89.6% respectively) than the SCM (35.9 Lm(-2) h(-1) and 96.7% respectively) when the fermentations were harvested at maximum erythromycin titers. Transmission of erythromycin stayed approximately constant as a function of fermentation time for both media, however, for the OBM the permeate flux decreased with time which correlated with an increase in broth viscosity. The relatively poor microfiltration performance of the OBM medium was, however, offset by the higher titers of erythromycin that were achieved during the fermentation. The filtration characteristics of the SCM broth did not show any correlation with either broth viscosity or fermentation time. Image-analysis data suggested that there was a correlation between hyphal morphology (main hyphal length) and permeate flux (no such correlation was found for the OBM broth). Moreover, it has been shown for the OBM broth that the residual flour had a profound effect on the microfiltration characteristics. The influence of the residual flour was greater than that imposed by the morphology and concentration of the biomass. The understanding of the factors governing the interaction of the fermentation and microfiltration operations obtained in this work provides a first step towards optimization of the overall process sequence.

Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae.

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (< or = +/- 4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies.

Quantitative analysis of yeast gene function using competition experiments in continuous culture.

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.