A single-use purification process for the production of a monoclonal antibody produced in a PER.C6 human cell line

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.     

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg‐based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin‐Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25–30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers ≥8.6 log₁₀ FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn‐around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production. Biotechnol. Bioeng. 2010;106: 906–917. © 2010 Wiley Periodicals, Inc.     

Disposable bioreactors for plant liquid cultures at Litre‐scale

Driven by the demands of the market and the manufacturing industry, disposable bioreactors have gained in importance in cell culture‐based processes during the last 10 years. Today they are widely accepted in R&D and also in manufacturing where process simplicity, safety and flexibility have top priority. Although disposable bioreactors are mainly used for cell expansions, glycoprotein secretions and virus generations realised with mammalian and insect cell lines, there are several reports delineating their suitability for the cultivation of plant cell and tissue cultures. This review describes the current disposable bioreactor types suitable for growing plant cell suspensions and organ cultures (hairy roots, meristematic clusters, somatic embryos) at Litre‐scale. Based on a definition of the term “disposable bioreactor”, a categorisation of the prevalent types for plant liquid cultures is presented. We describe the bioreactor regimes, working principles and bioengineering parameters of mechanically and pneumatically agitated bag bioreactors, which have advantages of process scalability and efficiency. Furthermore, results from the literature and data from our own research (obtained during production of undifferentiated bioactive cells, expressions of secondary metabolites and glycoproteins, and micropropagations of plant tissues) are discussed.     

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 10⁹ virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.     

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality.     

Experimental and computational characterization of mass transfer in high turndown bioreactors

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.     

Fluid mechanics,cell distribution,and environment in CellCube bioreactors

Cultivation of MRC-5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine (1), is performed in the CellCube reactor, a laminar flow fixed-bed bioreactor with an unusual diamond-shaped, diverging-converging flow geometry. These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation (2, 3). Early testing of the CellCube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase. Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained. Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment.     

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.     

Experimental characterization of flow conditions in 2- and 20-L bioreactors with wave-induced motion

Quantifying the influence of flow conditions on cell viability is essential for a successful control of cell growth and cell damage in major biotechnological applications, such as in recombinant protein and antibody production or vaccine manufacturing. In the last decade, new bioreactor types have been developed. In particular, bioreactors with wave‐induced motion show interesting properties (e.g., disposable bags suitable for cGMP manufacturing, no requirement for cleaning and sterilization of cultivation vessels, and fast setup of new production lines) and are considered in this study. As an additional advantage, it is expected that cultivations in such bioreactors result in lower shear stress compared with conventional stirred tanks. As a consequence, cell damage would be reduced as cell viability is highly sensitive to hydrodynamic conditions. To check these assumptions, an experimental setup was developed to measure the most important flow parameters (liquid surface level, liquid velocity, and liquid and wall shear stress) in two cellbag sizes (2 and 20 L) of Wave Bioreactors®. The measurements confirm in particular low shear stress values in both cellbags, indicating favorable hydrodynamic conditions for cell cultivation. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015     

Proliferation of meristematic clusters in disposable presterilized plastic bioreactors for the large-scale micropropagation of plants

Summary Proliferation of meristematic clusters of several plants in an inexpensive airlift bioreactor system, consisting of a disposable presterilized light transmittable plastic film vessel is described. The optimal shape, size, and structural function of the disposable plastic bioreactor are based on the bubble column and airlift glass bioreactors. The disposable bioreactors are designed in a conical configuration with a single inoculation and harvest port and multiple use dispensing and mixing accessories. Shearing damage and foaming problems known to exist in bioreactors due to the plant's rigid cell wall and size were greatly reduced in the disposable plastic bioreactors. The disposable bioreactors were used for propagule proliferation and growth, using meristem and bud clusters of potato, fern, banana, and gladiolus. The clusters' biomass increased five-to eightfold over a period of 26–30 d, depending on the species. The clusters were separated mechanically by a chopper made of a grid of knives. The chopped propagules were inoculated to agar medium for further growth and developed into transplantable plants. In the case of gladiolus and potato, corms and tubers developed in a sucrose-elevated storage organ induction medium, respectively, after the initial formation of small shoots. The plantlets and storage organs were transplanted to an acclimation greenhouse and continued to grow with a 95–100% survival, depending on the species. Plant development was followed for a period of 16 wk in fern and 12–14 wk in potato, banana, and gladiolus and normal shoot and leaf growth was observed. The feasibility of large-scale liquid cultures for plant micropropagation is discussed.     

Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.KEY WORDS: biologics, manufacturing technology, quality risk management, regulatory science, single-use system     

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer^® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors.Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer^® and the BIOSTAT^® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer^® bioreactor were chosen based on comparable mixing times.Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer^® and in the BIOSTAT^® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

Disposable bioreactor for cell culture using wave-induced agitation

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of mass transfer and mixing in rigid lab‐scale disposable bioreactors at low power input levels

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied. These settings are generally used for cultivation of mammalian cells growing adherent to microcarriers. The rigid disposable vessels showed similar engineering characteristics compared to a traditional glass bioreactor. On the basis of the presented results appropriate settings for adherent cell culture, normally operated at a maximum power input level of 5 W m^?3, can be selected. Depending on the disposable bioreactor used, a stirrer speed ranging from 38 to 147 rpm will result in such a power input of 5 W m^?3. This power input will mix the fluid to a degree of 95% in 22 ± 1 s and produce a volumetric mass transfer coefficient of 0.46 ± 0.07 h^?1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1269–1276, 2014     

Beyond heuristics: CFD-based novel multiparameter scale-up for geometrically disparate bioreactors demonstrated at industrial 2kL–10kL scales

The timely delivery of the most up-to-date medicines and drug products is essential for patients throughout the world. Successful scaling of the bioreactors used within the biopharmaceutical industry plays a large part in the quality and time to market of these products. Scale and topology differences between vessels add a large degree of complication and uncertainty within the scaling process. Currently, this approach is primarily achieved through extensive experimentation and facile empirical correlations, which can be costly and time consuming while providing limited information. The work undertaken in the current study demonstrates a more robust and complete approach using computational fluid dynamics (CFD) to provide potent multiparameter scalability, which only requires geometric and material properties before a comprehensive and detailed solution can be generated. The CFD model output parameters that can be applied in the scale-up include mass transfer rates, mixing times, shear rates, gas hold-up values, and bubble residence times. The authors examined three bioreactors with variable geometries and were able to validate them based on single-phase and multiphase experiments. Furthermore, leveraging the resulting CFD output information enabled the authors to successfully scale-up from a known 2kL to a novel and disparate 5kL single-use bioreactor in the first attempted cell culture. This multiparameter scaling approach promises to ultimately lead to a reduction in the time to market providing patients with earlier access to the most groundbreaking medicines.     

A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine

Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (>5 l) than that described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two-liquid-phase bioreactors for enhanced degradation of hydrophobic/toxic compounds

Two-liquid-phase culture systems involve the addition of a water-immiscible, biocompatible and non-biodegradable solvent to enhance a biocatalytic process. Two-liquid-phase bioreactors have been used since the mid-seventies for the microbial and enzymatic bioconversion of hydrophobic/toxic substrates into products of commercial interest. The increasing popularity of bioremediation technologies suggests a new area of application for this type of bioreactor. The toxicity and the limited bioavailability of many pollutants are important obstacles that must first be overcome in order to improve biodegradation processes. Two-liquid-phase bioreactors have the potential to resolve both limitations of biotreatment technologies by the enhancement of the mass-transfer rate of compounds with low bioavailability, and by the controlled delivery of apolar toxic compounds. This technology can also be useful in accelerating the enrichment of microorganisms degrading problematic pollutants. In this paper, we discuss the application of two-liquid-phase bioreactors to enhance the biodegradation of toxic/poorly bioavailable contaminants. Important microbial mechanisms involved in this type of system are described. Uptake of the substrates can be achieved by microorganisms freely dispersed in the aqueous phase and/or bound at the interface between the aqueous and the immiscible phases. Production of surface-active compounds and adhesion abilities are microbial features involved in the process. General guidelines for the design of two-liquid-phase bioreactors for biodegradation purposes are presented. Solvent selection should be established on specific criteria, which depend on the characteristics of target compound(s) and the microorganism(s) implicated in the biodegradation process. The central importance of maximizing the interfacial surface area is highlighted. The potential of this approach as an alternative to current biotreatment technologies is also discussed.