jean-marc Engasser - Academia.edu (original) (raw)

Papers by jean-marc Engasser

Journal of Chromatography A, Sep 1, 1994

Abstract A method based on fast HPLC allowing the rapid and efficient determination of proteins i... more Abstract A method based on fast HPLC allowing the rapid and efficient determination of proteins in mammalian cell cultures is proposed, involving the use of two chromatographic modes, RP-HPLC and high-performance liquid affinity chromatography. These two sequential chromatographic analyses separate the proteins most often used as supplements in serum-free media such as bovine serum albumin, insulin and transferrin, and also monoclonal antibodies secreted by hybridoma in cultures. Rapidity, reliability and flexibility are the main characteristics of the method. The monitoring of proteins in the course of a discontinuous hybridoma culture is presented.

Springer eBooks, 1989

The shortage of direct sensors in bioprocesses is a crucial problem which restrains their automat... more The shortage of direct sensors in bioprocesses is a crucial problem which restrains their automation. It is therefore important to use at the best those which are already available. Among them the pH probe is an essential instrument, despite its somewhat debatable reliability, and pH measurements are a routine task. Two cases should be examined: when pH is not regulated, its variation can be related to biomass or secondary metabolite variation as in acetic fermentation; when pH is regulated, one looks for a global relation between the consumption of the neutralization reagent and the biomass or metabolite production as in the aerobic growth of Saccharomyces cerevisiae. However the interactions between the broth components and the biological reactions influencing pH make necessary a rigorous analysis of these phenomena in order to use correctly pH measurements. This problem has already be pointed out by San [1].

Biotechnology Letters, Jul 1, 1989

Page 1. Biotechnology Letters Vol ii No 7 515-520 (1989) Received 13th June REPRESENTATION OF CHA... more Page 1. Biotechnology Letters Vol ii No 7 515-520 (1989) Received 13th June REPRESENTATION OF CHANGES IN THE METABOLIC PATTERN OF BAKER'S YEAST FROM MEASUREMENTS OF EXTRACELLULAR PYRUVATE, ACETATE, ACETALDEHYDE AND ETHANOL ...

SUMMARY A new configuration of perfused reactor, with cells confined between two flat membranes, ... more SUMMARY A new configuration of perfused reactor, with cells confined between two flat membranes, is presented. The flow rate of the medium forced through the membranes can be controled independently of the oxygen transport assured by a tubular membrane network. Results indicate negligible membrane fouling and culture duration up to 2 months without product retention. Cells protected against shear stress can be homogeneously irrigated and reached an estimated density of 6×107 cells/mL. Compared to the batch mode, the Cellys reactor allows a 3 times higher MAb concentration, a 2 times lower consumption of medium per gramme of MAb and a 100 times higher daily productivity per liter of cell chamber. Made out of medical grade materials, it is sterilizable and extrapolable.

Biotechnology Letters, Feb 1, 1996

Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Applied Microbiology and Biotechnology, Oct 22, 1996

In order to determine the possible effect of nutrient limitations on the response of Corynebacter... more In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower

Chemical Engineering Science, Dec 1, 1974

An isothermal heterogeneous reactor with first order kinetics is characterized by two virtual max... more An isothermal heterogeneous reactor with first order kinetics is characterized by two virtual maximum efficiencies, Nkin and Ntr, which are readily determined from the appropriate kinetic and transport parameters. The actual efficiency of the reactor, which is related to the conversion, X, by X = 1−e−N, can be accurately approximated as N−1 = N−1tr + N−1kin. The overall additivity relation offers a simple approach either to predict the conversion or to extract the kinetic or transport parameters when the reactor behavior is affected by both reaction and diffusion.

Chemical Engineering Science, 1980

Abstract The transport of protons across a dialysis membrane in the presence of acids and bases i... more Abstract The transport of protons across a dialysis membrane in the presence of acids and bases is investigated both experimentally and theoretically. Due to the reversible binding of protons to bases the transport rate of protons can be considerably augmented by buffer shuttles. The extent of transport facilitation depends on the buffer pK and concentration, up to one million fold increase in transport rate at high concentration, as well as on the pH in the liquid phases. The experimental results obtained with acetate, phosphate and ammonia are in good agreement with the theoretical predictions.

Cytotechnology, Feb 1, 1991

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

Cytotechnology, 1992

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamin... more For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be the result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.

Chemical Engineering Science, 1988

Biochemical engineering deals with the processing of biological or chemical materials using enzym... more Biochemical engineering deals with the processing of biological or chemical materials using enzymes or living cells as biological catalysts. At a central position in a biotechnological process is the bioreactor. Its role is frequently dominant on the overall technical and economical performance of the process. The characteristics of the biological reaction can also affect the requirements on the other steps of the process, such as the preparation of the media and the downstream operations for product recovery and purification. For the optimal design and control of biological reactors, the biochemical engineer can make use of the technologies and concepts developed in the field of chemical reactor engineering. Specially with respect to the more physical aspects of mixing, mass and heat transport, and to the general methodologies of reactor modelling and computer control. But when dealing with bioreactors, theories and practice have to be adapted to the peculiarities of the biotogical catalysts. Simplest are the enzymes, the protein catalysts, which, despite generally more complex kinetics, have many common features with chemical catalysts. Much more complex are the living cells. These growing and adaptive catalysts present many new challenges in reactor design and control. During the last twenty years the area of bioreactor engineering has considerably evolved, in some cases with very significant increase in reactor activities and selectivities (1.2). Nevertheless, if compared to chemical reactors, biological reactors still suffer serious weaknesses. First, relatively low concentrations. reaction rates and productivities, as a result of the relatively dilute culture media. Second, problems of stability with continuous processes. The scale-up and the automation of bioreactors is still in its infancy, due to insufficient understanding of the rate limiting steps and a severe lack of on-line sensors. Sterility is an additional constraint on the design of bioreactors. Safety and toxicity problems, and the associate legal complexities and constraints can also be important in some cases. This review deals with recent progress in the design, modelling and control of biological reactors utilizing living cells. After a brief analysts of some of the key features of cells as catalysts, it illustrates modern technologies for increasing the productivity of bioreactors. It also presents new approaches for the modelling of cells and bioreactors and surveys the general trends for the computer control of biotechnological processes. Without being exhaustive, it mainly emphasizes the adaptation of chemical engineering technologies and methodologies to the physiological particularities of living cells. CELLS FOR BIOTECHNOLOGICAL PROCESSES Living cells utilized in reactors can be classified as microorganisms, animal and plant cells (Table 1). They all have the common features of living entities, namely the possibilities to grow, to reproduce, to extract energy from their environment, to perform chemical syntheses, active transport and work. Similarities also exist in their metabolic pathways and intracellular control mechanisms, both at the genetic and enzymatic levels. But there are major differences in structure and composition between the simple bacteria and the highly organized animal cell. As a consequence, there are significant variations in nutritional requirements, growth and synthesis capabilities, and relationships with their chemical and physical environments. Microorganisms The mOSt widely used are the microorganisms. They include the primitive procaryotic bacteria and algae, and the more complex eucaryotic fungi. yeasts and protozoae. Their industrial exploitation oovers the areas of production of food, pharmaceuticals, chemicals, and of waste treatment.

Applied Microbiology and Biotechnology, Nov 1, 1993

The synthesis of ethyl acetate by Kluyveromyces fragil& on diluted whey permeate was studied. Eth... more The synthesis of ethyl acetate by Kluyveromyces fragil& on diluted whey permeate was studied. Ethanol, lactose and 02 are the direct precursors for ethyl acetate synthesis by this yeast. Ethyl acetate production is affected by m a n y parameters, particularly the carbon/nitrogen (C/N) ratio, Tween 80 and iron. Ethyl acetate synthesis is optimum for C/N = 45. Tween 80 lowered slightly the level of ethyl acetate whereas iron completely stopped ethyl acetate production. The level of ethanol in the feed, the dissolved 02 (DO) and dilution rate (D) were also optimised. Thus at D = 0.24 h-l , for 4 g/1 of ethanol in the feed and 40% D O , the productivity of ethyl acetate was optimal (0.7 g/1 per hour).

Biochemical Journal, Mar 1, 1975

The effect of the interaction between the charged matrix and substrate on the kinetic behaviour o... more The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. A simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect if the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menten kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent K1. 6. When both the inhibitor concentration and the ionic strength change there is no apparent K, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest themselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.

Production of Biologicals from Animal Cells in Culture, 1991

ABSTRACT In batch and continuous hybridoma cultures fed with a serum-free medium, insulin is cons... more ABSTRACT In batch and continuous hybridoma cultures fed with a serum-free medium, insulin is consumed, ascorbic acid produced, whereas the level of transferrin remains constant. Insulin is inhibitory to cell growth above 5 mg/L. Continuous cultures can be maintained at a relatively high density (2 x106 cells/mL) in the absence of added insulin.

Process Biochemistry, 1994

Mixed cultures of Saccharomyces cerevisiae CBS 80% and Kluyveromyces fragilis were used for conti... more Mixed cultures of Saccharomyces cerevisiae CBS 80% and Kluyveromyces fragilis were used for continuous biomass production on media containing whey permeate and glucose (WC) or whey permeate, maltose and glucose (WGM). Glucose and maltose were provided Jiom a totally or a partially hydrolysed starch. For each medium the effects of two yeast extract concentrations (0. I and 0.5 g/ litre) were investigated. The yeast extract concentration markedly @ected the equilibrium of the co-culture and the cell yield which was also influenced by the dilution rate. The yield varied from 0.26 to 0.48 g/g. The results obtained were due to the dserent kinetics of glucose transport in Kluyveromyces fra8ihs and Saccharomyces cerevisiae. Kluyveromyces fragilis possessed two glucose carriers, present at all the tested dilution rates, characterised by their af7inity constants which were much higher than those present in Saccharomyces cerevisiae .

Animal Cell Technology, 1992

ABSTRACT Both the rates of appearance of blue cells and of cell lysis have been found to strongly... more ABSTRACT Both the rates of appearance of blue cells and of cell lysis have been found to strongly depend on the medium composition. When growing hybridoma in a serum containing medium at optimal pH, dissolved oxygen and osmotic pressure, cellular death remained negligible. However, the specific rates of cell death and lysis were found to significantly increase when either decreasing the serum level, reducing the dissolved oxygen, lowering the pH or increasing the osmotic pressure of the medium. Under these limiting environmental conditions, cell death can rise to a specific rate of 0.012 h -1 , with cell lysis accounting for 30 % of the total loss of viable cells.

Animal Cell Technology: Developments Towards the 21st Century, 1995

Sodium butyrate (NaBu) addition was found to increase the production of monoclonal antibodies (MA... more Sodium butyrate (NaBu) addition was found to increase the production of monoclonal antibodies (MAbs) by OKT3 hybridoma cells and of human Gamma-Glutamyltransferase (GGT) by recombinant CHO cells. The membrane-bound GGT enzymatic characteristics were not modified by NaBu induction. The GGT induction was tested in different culture modes with cells in aggregates or on microcarriers. Either for GGT or MAb production the cell growth state at the moment of induction appeared to be important.

Animal Cell Technology, 1994

ABSTRACT The evolution of the intracellular pH (pHi) is followed during hybridoma cultures in bat... more ABSTRACT The evolution of the intracellular pH (pHi) is followed during hybridoma cultures in batch bioreactors, with or without control of the medium pH (pHe). The pH sensitive dye BCECF is used as a pH indicator and analysis of the cell populations is done by flow cytometry. The pHi is found to remain constant at its maximal value during the growth phase of both cultures, after an adaptation period. This value is slightly lower when the pHe is not controlled, however the pH barrier between the inside and outside of the cells is unchanged. The evolution of the pHi can be positively correlated to the evolution of the growth rate. The same relation has been observed in continuous culture. Concentrations of some ions in the medium are also followed.

Animal Cell Technology, 1997

The cDNA coding for rat alpha-2,6-sialyltransferase (α2,6-ST) was expressed in a recombinant CHO ... more The cDNA coding for rat alpha-2,6-sialyltransferase (α2,6-ST) was expressed in a recombinant CHO cell line producing interferon-gamma, using a novel in vitro amplification vector. The α2,6-ST was expressed efficiently, 20–30% of the sialic acid pool on interferon-gamma being linked in the α2,6-conformation. Despite the overall level of sialylation was not significantly increased, growth and interferon-gamma production in bioreactor were not compromised by the transfection and expression of the gene encoding for the α2,6-ST.