Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2001-04-23
2003-02-11
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S068100, C435S069100, C435S297400
Reexamination Certificate
active
06518058
ABSTRACT:
FIELD OF INVENTION
The present invention concerns to molecular biology and biotechnology, namely to the methods and devices for synthesis of polypeptides in cell-free translation system.
BACKGROUND OF THE INVENTION
Several methods of polypeptide synthesis in cell-free translation system are known. For elimination of restrictions connected with a lower output of target polypeptides and short-term operation of cell-free translation systems a method was suggested which is widely used now (Spirin et al., 1988). This method is based on the principle of continuous removal from a reaction mixture of reaction products and continuous restoration of the initial concentration of low molecular weight components during synthesis. This method underlies several inventions connected with its improvement for increasing the synthesized product output (Alkahov et al., 1991; Baranov et al., 1993; Alakhov et al., 1995).
By the input of feeding solutions and removal of products of synthesis the known methods can be divided as follows: (a) methods in which dialysis is used to add feed solution components to the reaction mixture and to remove low molecular weight components from the reaction mixture through the dialysis membrane or to simultaneously remove low and high molecular weight components from the reaction mixture; (b) methods in which continuous ultrafiltration is used for a simultaneous removing of low and high molecular weight components of products through the membrane and a simultaneous input of feeding solutions directly into the reaction mixture volume or through the membrane; c) methods in which periodic input of a feed solution into the reaction mixture and subsequent removing of low and high molecular weight components through the membrane are used. Input and output of the flows is realized by changing the direction of liquid flows at the exposure of consecutive creation of pulses of positive or negative pressure.
The method (Mozayeny, 1995) is known in which the removal of products with large molecular weight is improved by increasing the area of a ultrafiltration membrane in relation to the reaction mixture volume. One of the main disadvantages of the given invention is that during removal of high molecular weight components through the large area of the membrane with the pore size of 70 kD to 100 kD, together with the final product useful working components of molecular weight up to 100 kD are lost. This is a limiting factor for the operating time of the cell-free system. The larger is the membrane area, the greater is the amount of high molecular components of the cell-free system washed-off from the reactor at a high flow rate. Another disadvantage is the necessity to use an external loop for creation of a tangential flow of the reaction mixture along the membrane surface. During passage of the reaction mixture via liquid communications three factors influence the work of the cell-free systems: (1) when the reaction mixture passes via the loop the feed solution is not added in the part of external volume of the reaction mixture, (2) low weight products which inhibit the cell-free system are not removed from the external volume, (3) the liquid communications and pumps are not thermostable and the reaction mixture changes its temperature depending on the environment. This leads to irreproducibility of results and limits the life time of the cell-free system.
The method in which authors offer to apply repeated pulse for input of the feeding solution in the reactor and removal of low and high molecular weight products of synthesis from the reactor via a membrane is known (Fischer et al., 1990). This is realized by changing the direction of the flow through the membrane. One of the main disadvantages of the given invention is that low molecular weight components of synthesis which inhibit operation of the system are not removed from the reactor during a long period. The time during which the feed solution passes repeatedly via the membrane is equal to the period when a total volume of feed solution passage via the membrane is equal to the complete volume of the reaction mixture. For this purpose N cycles are formed to create positive and negative pressure. Due to the pressure modulation the inhibiting products come back in the reactor together with a regular portion of the feed solution. Another disadvantage of this method is that upon formation of N cycles high molecular weight components of the cell-free system required for prolonged synthesis are intensively washed off the reaction mixture. Thus, repeated returning into the reactor of low molecular weight components inhibits operation of the system and removing from the reactor of high molecular weight components providing effective synthesis impose restrictions on operation of system.
The method (Choi. 1997) is known by which synthesis of polypeptides is carried out with removal of a target product in a dialysis mode of operation. For this purpose a membrane divides the reactor in two parts. The reaction mixture is placed on one side of the membrane and the feed solution on the other side. The reaction mixture is fastly circulating along the membrane surface in tangential direction. A disadvantage of the method is that due to a large pore size components of the system are removed together with the target products. Moreover, in spite of the fact that the dialysis process is quite effective because of the large pore size, its extent is not enough for operation of highly efficient cell-free systems.
The method (Alakhov et al., 1991) is known in which amino acids, ATP, GTP in an aqueous buffer are added to reactor during functioning of the system and low weight components such as AMP, GDP, Pi formed during synthesis and inhibit the system are removed through a membrane. To provide a more economical operation of the system, low molecular weight products are regenerated and come back into the reactor via the membrane. However from the description and the given figure it is not quite clear how low and high molecular weight components of the synthesis are removed from the reactor and in what way the buffer solution is regenerated after removal of the polypeptide. Taking into account the description of examples, low and high molecular weight components are removed from the reactor via the ultrafiltration membrane. The use of an ultrafiltration membrane is described in a number of publications (Spirin et al., 1988; Takanori et al., 1991; Spirin, 1992; Erdmann et al., 1994). A disadvantage of this method is the use of large sizes of the membrane cutoff. In this case high molecular weight components of systems necessary for synthesis are removed from the reactor together with target products. The volume of low molecular weight components is equal to that of the removed components which results in fast closing of the ultrafiltration membrane pores.
Methods of adding feed solution to the reaction zone and removing from it of reaction products for different types of membrane reactors are known in which the reaction zone is placed between two membranes (Matson et al., 1988; Wrasidlo et al., 1990; Dziewulski et al., 1992).
The method described in the patent (Alakhov et al., 1995) is the prototype of the method proposed herein. For synthesis of polypeptides in this invention the reaction mixture is placed between two flat membranes. The membranes differentiate flows of low molecular weight and high molecular weight components and divide the reactor into three zones: zone for input of feed solution, reaction zone, zone of product removal. The first rather weak flow is formed in the reaction zone. It ensures the reaction mixture movement along the internal part of porous barriers, through which they molecular weight components (including synthesized polypeptides) are removed. The second fast flow is formed in the zone of feed solution input. It ensures penetration of low molecular components via the membrane in the reaction system. The fast flow of low molecular weight components and the slow flow of high molecular components are achieved by creat
Biryukov Sergey Vladimirovich
Mayorov Sergey Gennadievich
Shirokov Vladimir Anatolievich
Simonenko Peter Nikolayevich
Spirin Alexander Sergeyevich
Redding David A.
Roche Diagnostic Corporation
Roche Diagnostics GmbH
White Kenneth J.
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