Project Course 2004
Producing a Protein of the blood clotting cascade
Blood clotting is life necessary, if there is some injury in the organs, muscles, bones or bleeding from the inner joint, for example stitches and wound and external bleeding. In order to reduce the bleeding, the blood itself emerges to form clot in the injury place. In the complex process of blood clotting, the so called coagulant factors are involved. The coagulant factors are protein, which accelerates certain chemical reaction in the blood clotting process. Totally there are 13 different coagulation factors. They are designated with Roman numbers I to XII. The factor V protein is a coagulation factor and plays a central role in the blood clotting process.
Factor V protein can be extracted form blood plasma. To avoid the of possible contaminations with HIV and/or Hepatitis pathogens, factor V protein is produced today by means of biotechnological methods. In this project course, a plant for the production of Factor V protein with a capacity of 3000 kg per year has to be designed.
Under the aid of genetically changed E. coli bacteria, it is produced in a fermentation cascade (pre fermentation and fermentation) process. After inactivation of the bacteria, they are opened (Cell disruption).In subsequent Processes, the fusion protein are split, cleaned by means of chromatography and crystallization and finally the Factor V protein is dried. A special attention is given in planning of the hygienic design as well as the CIP/SIP cleaning of the unit.
There are 9 groups in this project course, which have the following tasks to do:
Group 1 (BVT): Design of prefermentation and fermentation
Group 2 (CRT): Reaction technical design of cyan chloride disruption
Group 3 (TVT): Design of a separation process for Factor V protein separation
Group 4 (LSTM): Solid/Liquid separations and pipe layout design
Group 5 (LFG): Cell disruption and solid handling, Coordination and balancing of the whole plant
Group 6 (LTT): Thermal energy optimization of whole plant and drying the product
Group 7 (iPAT): Hygienic construction, plant layout and cost estimation
Group 8 (RT): Planning of the automation arrangements
Group 9 (LUR): Safety and Environmental aspects
Figure 1: Producing a Protein of the blood clotting cascade
In this joint venture, a live exchange of information between the groups and in the group is necessary.
The task of the individual groups is to design the corresponding single apparatus and to estimate the price. The group 7 calculates the whole plant investment cost according the details given by the other groups and the operating cost of the whole plant. The task range of the group 5 covers both the super ordination and coordination of the project. Providing cooperation at the interfaces of the individual groups and collecting data like Flow, concentration, pressure and temperature, etc. It has to collect and evaluates the resulting data and intermediate results. Group 6 carry out the energetic optimization of the total process and a permanent data matching with group 5. The group 9 carries out the environment compatibility test and safety analysis for the whole plant. This group is also responsible for the selection of plant location and to get the legal permission for the plant.
Group 1 (BVT): Design of prefermentation and fermentation process
For the production of the recombinant protein “factor V” in E. coli Pre-/fermentation plays a central role in the entire process.
Major aim is to design three vessels which can perform the tasks of precultivation of the bacteria, as well as their fermentation and inactivation. All vessels have to be dimensioned in such a way that a minimum yield of 20 kg of the product per batch is achievable.
E.coli is a bacterium of the human intestinal tract and is therefore usually cultured under physiological conditions. I.e. 37°C and pH ~ 7.
Sufficient maintenance of oxygen has to be assured at all time to avoid the reduced vitality in the growth of the bacteria. Additionally produced heat has to be removed from the vessels to maintain constant temperature condition of 37°C.
In general, fermentation of E. coli for recombinant protein production can be subdivided into two different, subsequent steps. These are growing the desired amount of biomass and induction of the actual biosynthesis of the recombinant protein.
Classically gene sequences of the bacteria are modified by introducing a so called GAL ( -glycosidase) or LAC promoter. The promoter enforces the bacteria to start their biosynthesis of the fusion-protein in presence of the inducer IPTG (Isopropyl -D-thiogalactoside).
Figure 2: Electron microscope picture of Escherichia Coli Magnification 15.000x
5 “The recombinant Protein Handbook, Protein Amplification and simple purification”,
Group 2 (CRT): Reaction technical design of Cyan chloride disruption
The APC (activated Protein c)-Resistance, a resistance to the activated protein-c, is the most frequent genetically contingent factor of risk for the formation of thromboses. A thrombosis is the occlusion of a vein through a blood clot. The APC-resistance is evoked through a mutation in the gene for the factor-V in the blood clotting cascade. In 95% of cases it concerns a certain mutation that is designated after the place of its discovery in the Dutch city Leiden with Factor V-Leiden.
To produce Factor V protein, Escherichia- coli k12 bacteria is used. The process is comprised of two fermentation steps, a cell breaking and following cyan chloride disruption to get the desired product Factor V protein. In further steps it is isolated and cleaned.
In this project course, a plant for the production of Factor V protein with a capacity of 3 tons per year has to be designed.
Literature survey about the cyan chloride disruption process.
Determination of reaction conditions (Reagents, Temperature, reaction and time, etc)
Selection and interpretation of suitable batch reactors for the cyan chloride disruption process.
It is to be guaranteed that no cyan chloride is released to the atmosphere (personnel protection)
The by products from the cyan chloride disruption process must be detoxified.
Group 3 (TVT): Design of a separation process for the reconditioning of the Factor V protein
The protein existing after the cyan chloride disruption process has to be separated by a chromatographic separation technique and purified. The plant should be designed according to:
The mobile phase should subsequently be recovered. The outlet from the column has to be characterised (composition and quantity).
Solvent reconditioning: (1)
The solvent from the chromatographic column should be purified with an appropriate technique. For that a process has to be chosen and dimensioned.
Product packing: (2)
The highly diluted, purified product separated from the solvent must be transformed into a solid form. The further reconditioning with the aid of the drying is carried out by another group. A suitable technique must be chosen for this process and dimensioned.
To the design of all process steps, a literature survey in the ScienceFinder and Ullmamm´s is carried out.
Trenntechnik II- Skript Universität Erlangen SS 04 Prof. Arlt
Ullmann´s Encyclopaedia of Industrial Chemistry, 6 th edition, Electronic Release, 2003.
Group 4 (LSTM): Solid/Liquid separation and pipe layout design
In the factor V protein producing project course, Solid/Liquid separation results after the cell disruption. In that the surplus water from mixture is separated by centrifuge. Through further cleaning and processing step, the protein dissolved in the fluid phase is extracted. It is the task of this group to investigate the different possible techniques for the separation process and design a plant for the Solid/Liquid separation. In addition to this it supposes to question about the composition, temperature, etc with other groups.
The second task of this group is to design the piping system to the whole plant. In addition, it supposes to ask the other groups about the component diameter, pressure and temperature, etc. This information is put together in a flow sheet to work in, which is to be optimized thereupon with respect to an ideal arrangement of the components regarding the piping length and security of the total system.
In the selection and dimensioning of the tube connections, Fittings and materials, the face points safety reliability and cost has to be taken into account. At the same time, attention is to be paid to the clear arrangements of the plant equipments. The places where the potential to danger are to be analyzed and encountered corresponding measures. The acquired solution is supposed to be equipped in addition with costs estimation.
Group 5 (LFG): Coordination and balancing of the whole plant, Cell disruption and solid handling
Betreuer: C. Binder, P. Toneva
The value protein manufactured by e-Coli during the fermentation has to be isolated from the production organisms. In addition, the cell walls have to be destroyed, in order to reach their contents. This process is well-known in bio process engineering as cell disruption. The process conditions (temperature, pH value etc.) have to be arranged in such a way that a damage of the sensitive value product is avoided.
The task range of this group covers both the super ordinate and co-ordination of project engineering containing the balance of the material and mass flows of the entire plant, as well as the cell disruption and the solid handling in the whole production chain. The following points are to be worked on thereby:
The last two points are in close co-operation with the groups which are responsible for the respective process steps to work on.
 Angewandte Bioverfahrenstechnik, 1991, D. Bryniok, ISBN 3-437-20480-7
 Biological systems engineering ; Mark R. Marten; Washington , DC : American Chemical Society, 2002
 Engineering and manufacturing for biotechnology ; Marcel Hofman; Dordrecht (u.a.): Kluwer Acad. Publ., 2001
 Mechanischer Aufschluss von Mikroorganismen im Apparatevergleich zwischen Rührwerkskugelmühle und Hochdruckhomogenisator, Martin Pittroff, Dissertation, Karlsruhe 1993
 Zerkleinerungs- und Klassiermaschinen, Höffl, K., Springer Verlag, Berlin, 1996
Group 6 (LTT): Energetic optimisation of the total process and drying of the products
In almost all plants of chemical industry there are facilities where large amounts of energy is required as an input whereas in other parts of the plant energy has to be discharged in the form of heat. This is also valid for the dimensioning of the bio-technological factor V-protein-synthesis which has to be performed in this course.
In order to design the total process in a most efficient and environmentally sound way, it is the task of group 6 to develop a heat network which incorporates all heat sinks and sources.
Therefore an economical concept has to be evolved which makes sensible use of the huge energy amounts which emerge on a relatively low temperature level, for example by supplying this kind of energy to other parts of the process.
In this context heat-exchangers and heat-pumps have to be dimensioned. The respective variants of the apparatuses have to be discussed and designed in co-operation with group7 (IPAT).
Not the detailed design of the apparatuses should stay in the foreground (but has to be performed exemplarily for each different type of apparatus), but the basic optimisation and co-ordination of the energetic net-work of the complete plant.
Moreover the step of product drying has to be designed.
As the product is very sensible to high temperatures and the allowed amount of humidity inside the product is limited to a close range, the type of the dryer has to be chosen carefully in order to avoid possible denaturation of the protein.
For this purpose a close collaboration with groups 7 and 8 is commendable in this case.
 Linnhof, B. et al.: A User Guide on Process Integration for Efficient Use of Energy, Pergamon Press, Kronsberg-Taunus, 1982
 Linnhof, B. et al.: Wärme-Integration und Prozeßoptimierung, Chem.-Ing.-Tech. 59 (1987), 851ff
 Körner, H.: Optimaler Energieeinsatz in der Chemischen Industrie, Chem.-Ing.-Tech. 60 (1988), 511ff
 VDI: VDI-Wärmeatlas, 8. Auflage, VDI-Verlag, Düsseldorf, 1998
 Gregorig, R.: Wärmeaustausch und Wärmetauscher, Verlag Sauerländer, Aarau u. Frankfurt a. M., 1973
 Leipertz, A.: Wärme- und Stoffübertragung, Vorlesungsskriptum, FAU Erlangen, 1992
 Chemikalieninformationssystem zu verbraucherrelevanten Stoffen
Group 7 (iPAT): Hygienic construction, Lay out and Cost Estimation
Hygienic construction/ Lay out
Plants for the bioprocess must fulfil strong Hygienic requirements. So all the plant components, for example stirred tank, piping, fittings and the necessary process machineries must be maintained dead spot free and CIP(cleaning in place)/SIP(sterilization in place) are to be carried out Contaminations between the single installation parts must be prevented if necessary by the installation of sterile interfaces.
The tasks for this group is to acquire the Hygienic condition necessary for the production of Factor V protein and the examination /construction of all installations parts with respect to that acquire hygienic aspect in interaction with other Groups. The suggested process components are supposed to be examined on CIP/SIP capacity and a concept for cleaning and sterilisation of plant has to be generated.
At the end a layout diagram has to be prepared which meets the Hygienic technical requirements.
The main equipment cost of every single process step is to be calculated and thereby to calculate the over all investment cost of the plant. In the frame of the costs calculation, hygienic specific materials and fittings are to be considered. Besides, it is necessary to estimate the investment needed for the clean room.
Group 8 (RT): Automation of the Project
Automation arrangements are integral component of each industrial unit. During the total project settlement, they are involving from the preplanning up to the execution of the planned unit. In coordination with different working groups; they are responsible for the automation of that unit. The design of the automation measures begins with the careful study of the single process steps and the formulation of the respective tasks. It would aim for a process just automation that enables a desired operation method of the single installations parts as well as the total concern.
For the procedure described in the general task, the automation process should correspondingly acquire concrete measures of that fair process, near-control, regulation and even supervisions also. At the same time attention is to be paid especially to start-up and shut down strategies to which additional arrangements and measures are needed with different possible techniques.
Group 9 (LUR): Site location, Safety and Environmental aspects
For the project, a plan has to be prepared to the safety and environment for the given location. This plan has to be designed according to the laws of GenTG and BimSchG to fulfil the personal and environment security. The goal of this group is to plane all necessary measures and legal reserves for the personal and environmental safety against raw materials, intermediate and end products. At the same time security risks that exist in regular operation of the unit, should identified and regular precautions, control methods has to be proposed. Possible disturbances and in addition to that appropriate measures like modifications in construction and procedure have to be stated. Disturbances to the equipments and alarm plan as well as maintenance instruction and training of the personal have to be planned. Furthermore, they should propose the methods for the utilization and/or the disposal of the waste and exhaust gas emission with required threshold limit.
1.1 Plant safety
a) Risk assessment and safety categorisation in relation to the GenTechnik safety law (GENTSV) and Hazardous Incident law
b) Protection from potential hazards
1.2 work protection / Work safety
Technical rules for the biologically dangerous substances TRBK, Exposure with dangerous materials, potential of Danger to personal, MAK and TRK values, Protective measures
1.3. Fire protection
Measures to the passive and active fire protection, for example, Fire alarm, Fire extinguishing device, Alarm schemes1
1.4 Handling of substances hazardous to water
Storage quantity, Storage conditions/protective measures, Handling
2. Waste Disposal
Preparing statements for the waste material quantity, composition and the regulations applied to detoxify or disposal
1. Gefahrstoffverordnung, BGBI
2. Störfallverordnung, Bundesumwelt
4. Umwelt Online: http//www.umwelt-online.de
weitere Gesetze, Verordnungen, Richtlinien, DIN-Blätter