- Email: [email protected]
Electrokinetic bioprocessing under microgravity in France as illustrated by space bioseparation: A programme initiated in France and in cooperation with Belgium and Spain
0273—1177189 $0.00 ± .50 Copyright © 1989 COSPAR
Adv. Space Res. Vol.9, No. 11, pp. (11)105—(11)109, 1989 Printed in Great Britain. All rights reserved.
flECTROKINETIC BIOPROCESSING UNDER MICROGRAVITY IN FRANCE AS ILLUSTRATED BY SPACE BIOSEPARATION: A PROGRAMME INITIATED IN FRANCE AND IN COOPERATION WITH BELGIUM AND SPAIN H. Bozouklian,* V. Sanchez,5~M. Clifton,** 0. Marsal*** and A. Esterle* *
Centre National d’Etudes Spatiales, 2 place Maurice Quentin, 75039 Paris
Cedex 01, France * ~Laboratoire CNRS de Genie Chimique, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France ***Centre National d’Etudes Spatiales, 18 avenue Edouard Belin, 31055 Toulouse Cedex, France ABSTRACT
The use of electrokinetic phenomena should prove to be a promising bioseparation technique especially as an application of bioprocessing under microgravity. Therefore, a bioseparation Research and Development programme involving several research teams has been under way for three years in France. Based upon the results of this fundamental research, a programme is now proposed for the development of an automated process chain making it possible to obtain biological macromolecules of high purity under microgravity. This Space Bio Separation programme received the Eureka label at the 6th Ministerial Eureka conference. The project will last six years. It will involve the cooperation of several scientific and industrial partners in France, in Spain and in Belgium. The programme includes the development and validation of three different bioseparation facilities for space. INTRODUCTION As part of its general mission, the Centre National d’Etudes Spatiales (CNES) fosters the utilization of microgravity environment as a tool for either the improvement of existing industrial processes or the development of new techniques. Msong promising areas for the application of microgravity, an important one is bioprocessing in space, a major aspect of which is biopurification. It is generally assumed that the potential usefulness of electrokinetic phenomena in biopurification should be better assessed in weightlessness. It is even considered that weightlessness offers unique advantages for the development of electrokinetic purification methods. In fact, several electrophoretic devices have already been constructed in different countries and tested in space. Several other attempts are planned in the future. This paper describes the basis and the general organization of a new programme which is aimed at — the unambiguous assessment of microgravity in facilitating the purification of biological macromolecules at high resolution, — the development of the tools necessary for the subsequent use of the microgravity environment in biopurification. This Space Bio Separation (SBS) programme involves the cooperation of several industrial partners in three European countries. A major emphasis will be placed on the scientific groundwork required as programme basic support. SBS ORGANIZATION 585 is a six year development.~programne. Its final objective is the building of an automated purification chain able to operate in space. This device will be used for the separation of biological substances, especially for the purification of specific biological macromolecules at high resolution. Such a purification step is an essential link in the whole process leading to the definition of the three dimensional structure of biological macromolecules of interest. Progress in this field will undoubtedly open the door to the creation of new biological effectors in the future. The SBS programme includes the participation of industrial partners in France, in Spain and in Belgium. SBS has been proposed as a European advanced technology development programme. This was formalized on 15 and 16 Jun 1988 at the 6th Ministerial Eureka Conference, where SBS obtained the Eureka label. JASR 9:11—H
(11)105
H. Bozouklian et a!.
(11)106
As far as France is concerned, three organizations are involved — Roussel—Uclaf, as a biological and pharmaceutical specialist interested in biopurification, — Matra, as prime technical coordinator of the project, — CNES, as coordinator of the scientific research effort which coordinates each technical project, through ground—based and space experiments. The emergence of this “joint venture” has been made possible by several factors. The first element is the unique research and industrial perspectives offered by new improvements in biopurification at high resolution. Another factor originates from the complexity of such a project which requires many different coinpetences as well as an important budgetary effort. Since 1984, a Research and Development programme on electrokinetie separation has been directed by the three SBS partners. As far as CNES is concerned, this scientific effort has involved three laboratories from universities and from the Centre National de is Recherche Scientifique (CNRS) — two research teams from the CNRS “Laboratoire de G~nieChimique” in Toulouse .at the “Laboratoire de Chimie Physique et Electrochimie” of the Universit~ Paul Sabatier, a group directed by Dr. Sanchez, .at the “Ins titut du G&iie Chimique”, a group directed by Dr. Bertrand, — the “Centre d’H~motypologie” from the CNRS in Toulouse, a group directed by Dr. Constans. SBS SCIENTIFIC BACKGROUND So far, research has been essentially focused on continuous—flow zone e].ectrophoresis, a separation method which should take particular advantage of the microgravity environment. Results obtained in this field include a theoretical approach to the phenomenon as well as some experimental work. Modelling of the flow structure has been undertaken /1/. The established model enables a hydrodynamic stability criterion of the process to be defined. A heat generation and transfer model has also been proposed /2/. This model takes into account the operating mode of the process and is applicable to a given continuous—flow electrophoresis instrument. The whole hydrodynamic model has been used for the calculation of the trajectory of a given particle from the beginning to the end of the separation process. This result is used in turn to determine which operating conditions should be optimally chosen to avoid signal deformations during the separation process leading to the purification of a given protein species.
II
-
Injection
..
~
a0
0..-~
Fig. 1. Predicted outflow concentration of three model proteins
J~O
Electrokinetic Bioseparation
(11)107
Figure 1 gives an example of the outflow concentrations of three model proteins, corresponding to the properties of serum albumine, hemoglobin and cytochrome C. The plot predicts that the central sample will show highest resolution, while the other two samples show the classical crescent deformation. The model teaches how to adjust sample mobility (through ph control) to maximize resolution. A separation chamber has been built to allow a comparison of theoretical predictions with experimental results. Several protein separation experiments have been performed /3/. In each case, the results of the comparison were found satisfactory. Some other in—orbit experiments are required to further qualify the model. This scientific background constitutes a reliable basis for the start of the first phase of SBS (see SBS programme). Some new scientific developments are also planned in the future — a progressive improvement of existing mathematical models, — an optimization study of the process, based on a resolution or yield criterion and taking into account process constraints, as a first step towards the determination of the best combination of operation parameters, — a study of the effects observed when using extreme values of some operating parameters, — new validation experiments on simple cases and some more purification tests. Some scientific work has also started on another electrokinetic process, namely the isoelectric focusing of proteins. Such a process can be coupled with continuous flow electrophoresis. SBS PROGRAhME The aim of the programme is to develop an automated process chain for the purification of biological molecules both in space and on earth. The bBS programme should last six or seven years starting Jun 1988 as illustrated in Figure 2. 88
89
f
A
A
90
A
91
A
f
92
A
93
A
94
A
A
SBS 1
SBS 2 SBS 3
Fig. 2 ~-:
.
-ci-:
SBS instrument and flight programme beginning of SBS 1, 2 and 3 phases flights
This represents three distinct but partially overlapping phases. Each phase corresponds to a new facility development and includes one flight for concept validation (see Figure 2) — SBS 1 : a first—generation instrument performing exclusively continuous—flow zone electrophoresis, which will allow confirmation of the performances predicted by numerical modelling. This instrument will include a cooling system for isothermal operation and a UV photometer for checking protein concentrations in outlet fractions — SBS 2 : a second—generation instrument using a modular design which will allow three different aspects of electrophoresis to be studied : continuous—flow electrophoresis, isoelectric focusing and electrohydrodynamic effects /4,5/. This instrument will contain one or more separation chambers depending on whether or not a satisfactory phase separator can be developed (see below). Cross Section illumination will be provided to facilitate the observation of electrodynamic effects. An overall diagram of this facility is shown in Figure 3.
(11)108
H.
et a!.
Bozouklian
SES 3 : a complete facility for automated purification of biological molecules. The processes employed will depend on the substance to be treated, but will include continuous—flow zone electrophoresis, isoelectric focusing, membrane processes and chromatography. —
SEPARATION
PHASES
UNIT
SEPARATOR
r1
j
____________________________________________________
PHAS ES SEPARATOR
1’
__
SUNSDRS
I I
1 I
CARRIER ~.ZCTRO0, I ,uPPtR *UFFESSJ
IREPaINC
________
a2crRQIwIETIa --
___
COLLECTOR
LASER
®~
cOtl1~ri~4lu5
I
L
CHAMSER
J
.
IPHorocRspHy~ 1
____
I
FLUID STORACE
{ 1_~~_PU~PS
~j
—~
UNIT
—
ThERMAl. CONTSCL UNIT HEAT
EXC}LANGER COOLING LOOP
________________
r
SPOrES CONTROL
~ REF IGERAT0R~ SAMPLE
H
PRETREATMENT
H
DATA
WIlT
~
CQWSITIO
TEI.EMETRY
AND
PORES SUPPLY
Fig. 3. The second—generation facility (SBS 2) SPACE FLIGHT PLANS Prior to the implementation of this flight programme, a first technological flight should occur by the end of 1989 to test a fluid management concept including a new phase separator using hydrophobic and hydrophilie membranes. As well as the technological cooperation between several partners within Europe, which is the basis of SBS, new scientific cooperations can also be stimulated by such a programme. For instance, in the course of SBS, a flight should take place in 1993. This objective is compatible with the Spacelab IML 2 mission scheduled for May 1993. Thus, it is possible to take advantage of SBS implementation for the pursuit of international scientific cooperation. For this reason, a facility called “Recherches Appliqu~es sur les M~thodesde Separation Electrophorétique Spatiales” (RAI’lSES) has been proposed by CNES as a payload candidate for IML2. RAMSES will be developed within SBS. The modular concept of this proposed facility enables the combination of many scientific objectives regarding the study of electrokinetic phenomena in space. CONCLUSION SBS is a technological development programme which is under the responsability of industrial partners. In spite of this particular context, the pursuit of SBS, like any other programme of this kind, largely depends on the progress of fundamental research. All S3S partners are aware of the essential role played by this basic scientific support. Therefore, this programme is supported by CNES in the same way as other scientific projects. For instance, the R.AMSES facility which is one of the SBS instruments, will be offered to the international scientific community as a Spacelab IFsL 2 payload. REFERENCES 1. 8. Biscans, P. A].inat, J. Bertrand, and V. Sanchez, Influence of flow and diffusion on protein separation in a continuous flow electrophoresis cell : computation procedure, Electrophoresis 9, 84—89 (1988) 2. M.J. Clifton and 0. harsal, heat transfer design of an electrophoresis experiment, Ante Astronautica, in press (1988)
Electrokinetic Bioseparation
(11)109
3. F. Escale, Etude du fonctionnement d’uae cellule d’electrophorese de zone a écoulement continu, au sol, en vue d’experimentations en microgravit&, These de Docteur—Ingenieur, Universit6 Paul Sabatier, Toulouse, France (Jun 1987) 4. R.S. Snyder, P.H. Rhodes, and T.Y. Miller, Continuous Flow Electrophoresis System Experiments on Shuttle Flights STS—6 and STS—7, NASA TP 2778, Marshall Space Flight Center, Alabama, USA (Oct 1987) 5. P.R. Rhodes and R.S. Snyder, Preparative Electrophoresis for Space, NASA TP 2777, Marshall Space Flight Center, Alabama, USA (Oct 1987)
Adv. Space Res. Vol.9, No. 11, pp. (11)105—(11)109, 1989 Printed in Great Britain. All rights reserved.
flECTROKINETIC BIOPROCESSING UNDER MICROGRAVITY IN FRANCE AS ILLUSTRATED BY SPACE BIOSEPARATION: A PROGRAMME INITIATED IN FRANCE AND IN COOPERATION WITH BELGIUM AND SPAIN H. Bozouklian,* V. Sanchez,5~M. Clifton,** 0. Marsal*** and A. Esterle* *
Centre National d’Etudes Spatiales, 2 place Maurice Quentin, 75039 Paris
Cedex 01, France * ~Laboratoire CNRS de Genie Chimique, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France ***Centre National d’Etudes Spatiales, 18 avenue Edouard Belin, 31055 Toulouse Cedex, France ABSTRACT
The use of electrokinetic phenomena should prove to be a promising bioseparation technique especially as an application of bioprocessing under microgravity. Therefore, a bioseparation Research and Development programme involving several research teams has been under way for three years in France. Based upon the results of this fundamental research, a programme is now proposed for the development of an automated process chain making it possible to obtain biological macromolecules of high purity under microgravity. This Space Bio Separation programme received the Eureka label at the 6th Ministerial Eureka conference. The project will last six years. It will involve the cooperation of several scientific and industrial partners in France, in Spain and in Belgium. The programme includes the development and validation of three different bioseparation facilities for space. INTRODUCTION As part of its general mission, the Centre National d’Etudes Spatiales (CNES) fosters the utilization of microgravity environment as a tool for either the improvement of existing industrial processes or the development of new techniques. Msong promising areas for the application of microgravity, an important one is bioprocessing in space, a major aspect of which is biopurification. It is generally assumed that the potential usefulness of electrokinetic phenomena in biopurification should be better assessed in weightlessness. It is even considered that weightlessness offers unique advantages for the development of electrokinetic purification methods. In fact, several electrophoretic devices have already been constructed in different countries and tested in space. Several other attempts are planned in the future. This paper describes the basis and the general organization of a new programme which is aimed at — the unambiguous assessment of microgravity in facilitating the purification of biological macromolecules at high resolution, — the development of the tools necessary for the subsequent use of the microgravity environment in biopurification. This Space Bio Separation (SBS) programme involves the cooperation of several industrial partners in three European countries. A major emphasis will be placed on the scientific groundwork required as programme basic support. SBS ORGANIZATION 585 is a six year development.~programne. Its final objective is the building of an automated purification chain able to operate in space. This device will be used for the separation of biological substances, especially for the purification of specific biological macromolecules at high resolution. Such a purification step is an essential link in the whole process leading to the definition of the three dimensional structure of biological macromolecules of interest. Progress in this field will undoubtedly open the door to the creation of new biological effectors in the future. The SBS programme includes the participation of industrial partners in France, in Spain and in Belgium. SBS has been proposed as a European advanced technology development programme. This was formalized on 15 and 16 Jun 1988 at the 6th Ministerial Eureka Conference, where SBS obtained the Eureka label. JASR 9:11—H
(11)105
H. Bozouklian et a!.
(11)106
As far as France is concerned, three organizations are involved — Roussel—Uclaf, as a biological and pharmaceutical specialist interested in biopurification, — Matra, as prime technical coordinator of the project, — CNES, as coordinator of the scientific research effort which coordinates each technical project, through ground—based and space experiments. The emergence of this “joint venture” has been made possible by several factors. The first element is the unique research and industrial perspectives offered by new improvements in biopurification at high resolution. Another factor originates from the complexity of such a project which requires many different coinpetences as well as an important budgetary effort. Since 1984, a Research and Development programme on electrokinetie separation has been directed by the three SBS partners. As far as CNES is concerned, this scientific effort has involved three laboratories from universities and from the Centre National de is Recherche Scientifique (CNRS) — two research teams from the CNRS “Laboratoire de G~nieChimique” in Toulouse .at the “Laboratoire de Chimie Physique et Electrochimie” of the Universit~ Paul Sabatier, a group directed by Dr. Sanchez, .at the “Ins titut du G&iie Chimique”, a group directed by Dr. Bertrand, — the “Centre d’H~motypologie” from the CNRS in Toulouse, a group directed by Dr. Constans. SBS SCIENTIFIC BACKGROUND So far, research has been essentially focused on continuous—flow zone e].ectrophoresis, a separation method which should take particular advantage of the microgravity environment. Results obtained in this field include a theoretical approach to the phenomenon as well as some experimental work. Modelling of the flow structure has been undertaken /1/. The established model enables a hydrodynamic stability criterion of the process to be defined. A heat generation and transfer model has also been proposed /2/. This model takes into account the operating mode of the process and is applicable to a given continuous—flow electrophoresis instrument. The whole hydrodynamic model has been used for the calculation of the trajectory of a given particle from the beginning to the end of the separation process. This result is used in turn to determine which operating conditions should be optimally chosen to avoid signal deformations during the separation process leading to the purification of a given protein species.
II
-
Injection
..
~
a0
0..-~
Fig. 1. Predicted outflow concentration of three model proteins
J~O
Electrokinetic Bioseparation
(11)107
Figure 1 gives an example of the outflow concentrations of three model proteins, corresponding to the properties of serum albumine, hemoglobin and cytochrome C. The plot predicts that the central sample will show highest resolution, while the other two samples show the classical crescent deformation. The model teaches how to adjust sample mobility (through ph control) to maximize resolution. A separation chamber has been built to allow a comparison of theoretical predictions with experimental results. Several protein separation experiments have been performed /3/. In each case, the results of the comparison were found satisfactory. Some other in—orbit experiments are required to further qualify the model. This scientific background constitutes a reliable basis for the start of the first phase of SBS (see SBS programme). Some new scientific developments are also planned in the future — a progressive improvement of existing mathematical models, — an optimization study of the process, based on a resolution or yield criterion and taking into account process constraints, as a first step towards the determination of the best combination of operation parameters, — a study of the effects observed when using extreme values of some operating parameters, — new validation experiments on simple cases and some more purification tests. Some scientific work has also started on another electrokinetic process, namely the isoelectric focusing of proteins. Such a process can be coupled with continuous flow electrophoresis. SBS PROGRAhME The aim of the programme is to develop an automated process chain for the purification of biological molecules both in space and on earth. The bBS programme should last six or seven years starting Jun 1988 as illustrated in Figure 2. 88
89
f
A
A
90
A
91
A
f
92
A
93
A
94
A
A
SBS 1
SBS 2 SBS 3
Fig. 2 ~-:
.
-ci-:
SBS instrument and flight programme beginning of SBS 1, 2 and 3 phases flights
This represents three distinct but partially overlapping phases. Each phase corresponds to a new facility development and includes one flight for concept validation (see Figure 2) — SBS 1 : a first—generation instrument performing exclusively continuous—flow zone electrophoresis, which will allow confirmation of the performances predicted by numerical modelling. This instrument will include a cooling system for isothermal operation and a UV photometer for checking protein concentrations in outlet fractions — SBS 2 : a second—generation instrument using a modular design which will allow three different aspects of electrophoresis to be studied : continuous—flow electrophoresis, isoelectric focusing and electrohydrodynamic effects /4,5/. This instrument will contain one or more separation chambers depending on whether or not a satisfactory phase separator can be developed (see below). Cross Section illumination will be provided to facilitate the observation of electrodynamic effects. An overall diagram of this facility is shown in Figure 3.
(11)108
H.
et a!.
Bozouklian
SES 3 : a complete facility for automated purification of biological molecules. The processes employed will depend on the substance to be treated, but will include continuous—flow zone electrophoresis, isoelectric focusing, membrane processes and chromatography. —
SEPARATION
PHASES
UNIT
SEPARATOR
r1
j
____________________________________________________
PHAS ES SEPARATOR
1’
__
SUNSDRS
I I
1 I
CARRIER ~.ZCTRO0, I ,uPPtR *UFFESSJ
IREPaINC
________
a2crRQIwIETIa --
___
COLLECTOR
LASER
®~
cOtl1~ri~4lu5
I
L
CHAMSER
J
.
IPHorocRspHy~ 1
____
I
FLUID STORACE
{ 1_~~_PU~PS
~j
—~
UNIT
—
ThERMAl. CONTSCL UNIT HEAT
EXC}LANGER COOLING LOOP
________________
r
SPOrES CONTROL
~ REF IGERAT0R~ SAMPLE
H
PRETREATMENT
H
DATA
WIlT
~
CQWSITIO
TEI.EMETRY
AND
PORES SUPPLY
Fig. 3. The second—generation facility (SBS 2) SPACE FLIGHT PLANS Prior to the implementation of this flight programme, a first technological flight should occur by the end of 1989 to test a fluid management concept including a new phase separator using hydrophobic and hydrophilie membranes. As well as the technological cooperation between several partners within Europe, which is the basis of SBS, new scientific cooperations can also be stimulated by such a programme. For instance, in the course of SBS, a flight should take place in 1993. This objective is compatible with the Spacelab IML 2 mission scheduled for May 1993. Thus, it is possible to take advantage of SBS implementation for the pursuit of international scientific cooperation. For this reason, a facility called “Recherches Appliqu~es sur les M~thodesde Separation Electrophorétique Spatiales” (RAI’lSES) has been proposed by CNES as a payload candidate for IML2. RAMSES will be developed within SBS. The modular concept of this proposed facility enables the combination of many scientific objectives regarding the study of electrokinetic phenomena in space. CONCLUSION SBS is a technological development programme which is under the responsability of industrial partners. In spite of this particular context, the pursuit of SBS, like any other programme of this kind, largely depends on the progress of fundamental research. All S3S partners are aware of the essential role played by this basic scientific support. Therefore, this programme is supported by CNES in the same way as other scientific projects. For instance, the R.AMSES facility which is one of the SBS instruments, will be offered to the international scientific community as a Spacelab IFsL 2 payload. REFERENCES 1. 8. Biscans, P. A].inat, J. Bertrand, and V. Sanchez, Influence of flow and diffusion on protein separation in a continuous flow electrophoresis cell : computation procedure, Electrophoresis 9, 84—89 (1988) 2. M.J. Clifton and 0. harsal, heat transfer design of an electrophoresis experiment, Ante Astronautica, in press (1988)
Electrokinetic Bioseparation
(11)109
3. F. Escale, Etude du fonctionnement d’uae cellule d’electrophorese de zone a écoulement continu, au sol, en vue d’experimentations en microgravit&, These de Docteur—Ingenieur, Universit6 Paul Sabatier, Toulouse, France (Jun 1987) 4. R.S. Snyder, P.H. Rhodes, and T.Y. Miller, Continuous Flow Electrophoresis System Experiments on Shuttle Flights STS—6 and STS—7, NASA TP 2778, Marshall Space Flight Center, Alabama, USA (Oct 1987) 5. P.R. Rhodes and R.S. Snyder, Preparative Electrophoresis for Space, NASA TP 2777, Marshall Space Flight Center, Alabama, USA (Oct 1987)