Cleaning characterization of protein drug products using UV-vis spectroscopy

This study uses on-line absorbance monitoring to evaluate cleanability of protein drug products. Characterization and validation of equipment cleanliness is a key requirement for a biopharmaceutical facility. A manufacturing-scale cleaning cycle has to be developed and validated for its ability to clean all of the equipment parts for a given soil. Cleaning validation in a multiproduct fill-finish facility could benefit from using a worst-case-based approach that involves validating the cleaning process for the most difficult to clean product. Such an approach minimizes the number of required validation runs. Scaled-down cleaning evaluations can provide helpful information for evaluating multiple products and determine the worst case. This study presents a simple and rapid technique for bench-scale characterization of cleanability of protein drug products. On-line A280 (UV absorbance at 280 nm) measurements are performed using a fiber optic probe, and the data are used to establish the dynamics of protein dissolution in cleaning solution. The model not only helps to estimate cleaning time of different formulated proteins (and peptides) but also provides insights into the kinetics of cleaning under different thermal and chemical conditions. Protein product degradation during cleaning is also evaluated through gel electrophoresis. Such information is useful in designing new cleaning cycles. While the study is performed using drug products, the model as well as the findings are also applicable for characterization of final purified bulk soils relevant to bulk drug manufacturing.     

Design methods for tubular fermentation systems

Methods for the design of tubular fermentation systems are summarized. The first part, on tubular system hydrodynamics, is concerned with the design methods for gas hold-up, pressure drop, drop-size distribution and actual interfacial area, and with non-ideal flow conditions. This being a critical review, only the more important methods have been selected. More recent design methods for the prediction of oxygen transfer coefficients in fermentation systems and methods of determining of true kinetic relations are also reviewed, accounting for the inevitable non-ideality of flow. General rules for system optimization are presented.     

Technical evaluation of the potential for streamlining of equipment validation for fermentation applications

The purpose of this article is to provide a few concrete examples of the potential to acceptably reduce the scope of validation and qualification testing based on scientific justification for the specific area of microbial fermentation. The key areas explored include: autoclave operational qualification (OQ) testing, autoclave load pattern testing, vessel sterilize-in-place testing, spore strip use and failure investigation, grouping of D-values for media and concentrated nutrients, influence of temperature on D-values, and equipment clean-in-place cleaning agent/recovery studies. Suggestions are offered based on technical data and engineering analysis of the procedures involved. Methodologies are described for how to evaluate the systems being tested relative to processing requirements to determine which testing might be minimized and which testing might warrant expansion. The ultimate risk to quality then must be evaluated by the designated quality control groups within the organization for the specific process and equipment in use.     

Artificial neural network based experimental design procedure for enhancing fermentation development

Conventional experimental design techniques are available to assist in the optimization of fermentation processes, but due to the nonlinearities in the bioprocess, they are limited in their effectiveness. This problem is further complicated with recombinant systems as a result of the additional complexities of the process. This article describes a general strategy using artificial neural networks as an alternative approach to fermentation process development laboratory are presented for the neural network based procedures. (c) 1994 John Wiley & Sons, Inc.     

Novel bioreactor internals for the cultivation of spore‐forming fungi in pellet form

This study introduced an automated long‐term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed‐batch fermentation, repeated‐batch fermentation was implemented with the help of additional bioreactor internals (“sporulation supports”). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems (e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.     

Mass transfer in fermentation

An essential consideration in the design and operation of commercial fermenters is to ensure adequate mass transfer. The complex composition of fermentation liquids makes it difficult to predict accurately the mass transfer characteristics in large vessels. Here various aspects of mass transfer are discussed and their relationships examined. Strategies for predicting the most important type of mass transfer — between gases and liquids — in large scale fermentations are presented.     

响应面设计优化绿僵菌固体发酵条件

【目的】为了提高绿僵菌分生孢子产量及孢子质量,应用响应面设计对金龟子绿僵菌菌株CY-1(Metarhizium anisopliae)进行固体发酵培养基的优化。【方法】单因素试验基础上,采用响应面试验设计方法优化培养基组分。【结果】添加了碳、氮营养的最佳固体发酵培养基为玉米粉:稻壳=8:2,料水比1:0.8,葡萄糖0.8%,硫酸铵2.5%,磷酸二氢钾0.8%;在固体培养基上的理论产孢量为7.45×10~9个/g;验证后实际为6.94×10~9个/g。【结论】运用响应面法对绿僵菌固体发酵的培养基成分进行优化,得到了绿僵菌孢子粉,为孢子粉进行地下害虫防治和制剂加工的研究奠定了基础。     

Flux enhancement for membrane filtration of bacterial suspensions using high-frequency backpulsing

A promising method for reducing membrane fouling during crossflow microfiltration of biological suspensions is backpulsing. Very short backpulses (0.1-1.0 s) have been used to increase the net flux for washed bacterial suspensions and whole bacterial fermentation broths. The net fluxes under optimum backpulsing conditions for the washed bacteria are approximately 10-fold higher than those obtained during normal crossflow microfiltration operation, whereas only a 2-fold improvement in the net flux is achieved for the fermentation broths. A theory is presented that is based on external fouling during forward filtration and nonuniform cleaning of the membrane during reverse filtration. The model contains an adjustable parameter which is a measure of the cleaning efficiency during backpulsing; the cleaning efficiency found by fitting the model to the experiments increases with increasing frequency and duration of the backpulses. The theory predicts an optimum backpulsing frequency, as was observed experimentally. An economic analysis shows that crossflow microfiltration with backpulsing has lower costs than centrifugation, rotary vacuum filtration, and crossflow microfiltration without backpulsing.     

Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes

A continuous very-high-gravity (VHG) ethanol fermentation process design, consisting of a chemostat vessel connected to several equal-sized ageing vessels configured in parallel, was developed. The objective of the developed process is to have complete glucose utilization during fermentation stage. The process design integrates the conservation of mass principle and the experimental data of collected residual glucose profiles measured under VHG conditions. An ageing vessel involves three consecutive time periods: filling, ageing and operating. The ageing time is biological relevance, and is affected by the initial glucose concentration, the ethanol concentration, and the yeast viability in an ageing vessel. The operating time period is adjustable; a short operating time means a high discharge rate in order to empty an ageing vessel. The filling time links to the selection of the number of equal-sized ageing vessels that are installed downstream to a chemostat device. The developed process features the use of equal sized fermenters for all chemostat and ageing vessels so that the vessel exchangeability and the flexibility of fermentation operation are increased.     

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.     

Current Progress on Butyric Acid Production by Fermentation

Several issues of butyric acid production with bacteria through fermentation are presented in this review. The current progress including the utilization of butyric acid, the production strains, the metabolic pathway, and regulation are presented in the paper. Process operation modes such as batch, fed-batch, and continuous fermentation are being discussed. Genetic engineering technologies for microbial strain improvement are also being discussed and fermentation systems have been recommended.     

Mass transfer effects in fermentations using immobilized whole cells

The immobilization of whole cells for fermentation processes has many potential advantages over fermentation with free cells, including higher cell concentrations, higher productivites and a higher level of operational stability. Most of the research reported in the literature has been directed towards demonstrating the feasibility of using these systems for various fermentations. The ultimate goal of research in this area is to bring the understanding of immobilized whole cells to the level of heterogeneous catalysis. Immobilized whole cell systems are examined from a mass transfer perspective. Evidence for external and internal mass transfer limitations is presented. Procedures for quantifying these effects in terms of effectiveness factors and determining the reaction kinetics in their presence are reviewed. Development of the reactor design equations and the reactor performance results for fermentations with immobilized cells are also discussed.     

Sliding-mode control of fermentation processes

Continuous fermentation processes described by two nonlinear differential equations with uncertain parameters are considered. Sliding mode control design for these processes is proposed. The control design is carried out with direct use of nonlinear model, expert knowledge and on-line measurement of output variable only. Chattering phenomena are avoided by realizing the sliding mode with respect to the control input derivative. The excellent performance of presented control is proved through simulation investigations.     

Large-scale mammalian cell culture: Design and use of an economical batch suspension system

Large-scale mammalian cell culture in the absence of antibiotics requires stringent conditions of sterility for all vessels, procedure, and systems used. Application of existing fermentation technology suffers from the differences between mammalian and bacterial cultures. Relatively simple and inexpensive 100-L vessels have been designed specifically for medium storage and antibiotic-free mammalian cell culture. These vessels are portable and sterilized in a 2 x 3 x 5 ft conventional or VACUMATIC autoclave. They consist of 30-gal 316 stainless-steel sanitary process drums whose heads have been modified to meet the rapid pressure changes that occur during autoclaving. The vessels incorporate systems for aseptic introduction and removal of both liquids and gases required for inoculation, growth, and harvesting of cell suspensions. A two-disk vibromixer is used for agitation with inoculation at a laminar flow hood and incubation in a warm room. These vessels have been used for culture of one rat and eight human tumor lines for over 2 x 10(5) L of suspension.     

Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs

Development of parallel detection tools using microarrays is critically reviewed in view of the need for screening multiple microorganisms in a single test. Potential research needs with respect to probe design and specificity, validation, sample concentration, selective target enrichment and amplification, and data analysis are discussed. Data illustrating selected probe design issues for detecting multiple targets in mixed microbial systems is presented. Challenges with respect to cost, time, and ease of use compared to other methods are also summarized.     

Cyclodextrin glucosyltransferase production by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> NCR: evaluation and optimization of culture conditions using factorial design

Statistically-based experimental designs were used to optimize the production of cyclodextrin glucosyltransferase (CGTase) from a local isolate of Bacillus megaterium using shack culture fermentation. Seven cultural conditions were examined for enzyme production and specific activity using Plackett-Burman factorial design. Fermentation time and K₂HPO₄ level were the crucial for factors improving enzyme production process. The steepest ascent design was adopted-based on the results recorded with Plackett-Burman design. Maximal enzyme estimates (activity 56.1 U/ml, and specific activity 62.7 U/mg protein) were achieved. A verification experiment was carried out to examine model validation of this optimization.     

An integrated microprocessor-based fermenter control system

An integrated microprocessor-based fermenter controller was developed in 1980 for an operational environment at Cetus Corp. The main goals in the design and construction of the system were (1) to facilitate scale-up; (2) to provide flexibility and high performance for optimizing fermentation processes; and (3) to be cost-effective for 15 in-house systems. It was also developed to work in conjunction with a laboratory minicomputer for on-line optimization experiments. The controller controls temperature, agitation, dissolved oxygen, pH, and foam throughout each fermentation run without manual intervention. The feedback control parameters have been optimized to provide very accurate control over a wide range of setpoint conditions and under rapidly changing metabolic conditions such as induced during an Escherichia coli batch run. The controller has also been configured to monitor, display, and record each of the controlled variables; support the interactive operator console; and communicate with the laboratory computer. In over 4 years of operation, these systems have met the design goals and have proven to be very reliable. The controller is described, its operational performance presented, and a typical fermentation run delineated.     

Design of scraped tubular fermentors

Conventional stirred-tank fermentors are inefficient in carrying out certain fermentation processes because of one or more of the following constraints: media backmix–flow, solids wall–deposits, microbial growth–disruption. Two series of novel design of aerated scraped tubular fermentors have been developed to over come the deleterious effects of these constraints. One design is based on a horizontal tube fitted with an internal mechanical wall-scraper that also promotes media segregation; the other design is based on a vertical array of vessels interconnected by small gas-jetting orifices that promote media segregation and clean-surface operation. Tests with cultures of Trichdorma viride (for single-cell protein production) and Candida lipolytica (for lipase production) have been carried out. It is shown that these novel fermentors can minimize the effects of catabolite repression inherent in both cultures and of wall growth in the former.     

Rheometry of fermentation liquids

This article is concerned with the measurements of rheological properties of nonhomogeneous fermentation liquids. In order to determine the flow curves of such liquids, an impeller measuring system of own design was used, and the method of calibration of the system is presented. The experimental verification was carried out using samples of fermentation broth of Aureobasidium pullulans and it has been shown that the applied measuring technique gives internally consistent results. The sensitivity of the technique proves its applicability for monitoring and control purposes in fermentation technology.     

In vitro degradation of the flavonol quercetin and of quercetin glycosides in the porcine hindgut

The present study investigated the microbial degradation of the plant flavonol quercetin and its naturally occurring glycosides isoquercitrin and rutin in the porcine hindgut. The experiments were carried out with the semicontinuous colon-simulation technique. The fluid and particle phase of pig hindgut contents from freshly slaughtered animals were used for the in vitro incubations. Following a five-day equilibration period, quercetin, isoquercitrin or rutin were administered to fermentation vessels and their turnover rate was determined. None of the flavonols affected parameters of microbial fermentation like pH, redox potential or VFA production. The turnover rate for isoquercitrin was seven times higher than the turnover for the fermentation fluid. The turnover rates for quercetin and rutin were four and twofold higher than fluid turnover, respectively. After administration of isoquercitrin or rutin, their aglycone quercetin was detected as an intermediary metabolite. Under sterile conditions using autoclaved incubation fluids and hindgut contents, turnover rates for quercetin and rutin were still higher than the fluid turnover in the fermentation vessels. This indicates a certain chemical instability of the flavonols and/or adsorption to ingesta particles. Thus, flavonols are subjected to microbial metabolism in the porcine hindgut. The glycosidic structure strongly influences the rate of metabolism.