Abstract:
A method of extracting and purifying recombinant protein(s) from transgenic plant matter is disclosed. Fractioning of juice that has been extracted from the plant matter is obtained by using a multiple stage filtering process that uses multiple stages of decreasing porosity (preferably screening) followed by preferably membrane type filters, ion exchange, membrane adsorber, and chromatographic processes.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This is a continuation-in-part of co-pending U.S. patent application Ser. No. ______, filed Apr. 10, 2001, entitled “Sugarcane Fractioning System”, which is incorporated herein by reference.  
         [0002]    Priority of U.S. Provisional Patent Application Serial No. 60/196,085, filed Apr. 11, 2000, which is incorporated herein by reference, is hereby claimed. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0003] This work was supported by DOD Grant No. DAAG55-97-1-0096. The government may have rights in this invention. 
     
    
     
       REFERENCE TO A “MICROFICHE APPENDIX” 
         [0004]    Not applicable  
         BACKGROUND OF THE INVENTION  
         [0005]    1. Field of the Invention  
           [0006]    This invention relates to a novel technology for the separation of proteins from transgenic plant matter (eg. stalk, leaf and/or grain) and fractional purification of proteins so that proteins can be separated and further purified from other compounds. The invention allows for the rapid separation and fractional purification of large quantities of proteins. The present invention more particularly relates to a separation and fractional purification process that can be applied with any type of transgenic vegetal plant material (including, eg. plant stalk, leafy material and/or grain) such as but not limited to cane, barley, corn, potatoes, alfalfa, used to produce essentially any category of recombinant protein(s) such as but not limited to monoclonal antibodies (MAB), lectins, collagens, enzymes, or therapeutic proteins. During each step or any of the steps of this novel process, unconventional or conventional laboratory analysis could be performed in order to monitor the streams and the concentration of the protein(s) of interest.  
           [0007]    2. General Background of the Invention  
           [0008]    The extraction of the protein(s) of interest from the feedstock can be performed through different means that will not be destructive to the different proteins. The extraction can be performed through a preparation or comminuting step for size reduction of the feedstock followed by maceration or leaching steps. Comminuting of the feedstock will be done with apparatus such as but not limited to crusher, grinder, and cutting machine. For example, starting with genetically modified sugarcane stalks containing the protein(s) of interest, and passing the stalks through a pressure system such as roller/crusher allows extracting a liquid in this case also called pression juice, which contains the protein(s) of interest.  
           [0009]    The present invention relates to processing of plant matter for the recovery (fractional purification) of high value proteins so that these proteins can be separated and further purified from other compounds.  
           [0010]    The present invention enables the rapid separation and fractional purification of large quantities of proteins. This process is preferably applied to any type of transgenic vegetable plant material such as, for example; cane, sugarcane, barley, corn, potatoes, alfalfa, etc., and can be used to produce essentially any category of recombinant protein(s) such as, but not limited to, monoclonal antibodies (MAB), lectins, collagens, enzymes, or therapeutic proteins. During each step or any of the steps of the process of the present invention, unconventional or conventional laboratory analysis could be performed in order to monitor the streams and the concentration of the protein(s) of interest.  
         BRIEF SUMMARY OF THE INVENTION  
         [0011]    The present invention provides a process for extracting high value protein(s) directly from transgenic plant material.  
           [0012]    The extraction of transgenic plant feedstock containing the protein(s) of interest can be performed through different means that will not be destructive to the different proteins. Genetically modified and/or non-genetically modified plants containing the protein(s) of interest and able to produce a juice when passing through a pressure system can be processed. For example, starting with genetically modified plant material containing the protein(s) of interest, and passing the plant matter through a pressure system such as roller/crusher allows extracting a liquid, in this case also called “pression juice”, which contains the protein(s) of interest.  
           [0013]    The extracting pressure system can for example use: (a) different geometry rollers, (b) a plurality of rollers, (c) water for further extraction of pression juice, (d) series pressure system where the plant matter after the first pressure system will feed a second pressure system in series with the first pressure system, etc. (e) a buffer solution which will avoid partially or entirely, oxidation or degradation of some compounds contained in the pression juice.  
           [0014]    In order to improve the juice extraction of the pressure system, the plant matter can be previously shredded. Both shredder and pressure system are preferably part of the extraction step. The extraction step can work continuously or discontinuously. Extraction could also be performed through a leaching process such as diffusion.  
           [0015]    Following the extraction step, the pression juice is transmitted, either by gravity or by means of pumping to a screening system that is preferably composed of one or several screening steps. For example, a three step screening system can be used that is comprised of: (a) a first screening step that removes selected particulate, eg. matter larger than about 500 microns to 1000 microns, (b) the second screening step can be used for removing particulate size larger than about 150 microns to 250 microns, (c) the third screening step can be used to remove particulate size larger than about 10 to 60 microns.  
           [0016]    The screening system can include, for example, screens that are stationary, vibrating, rotary or any combination of these types of screens. Screens could also be self-cleaning units. The screened juice is recovered for further processing and the reject is discarded or sent to alternate processing. Press filter(s) or other filtering devices such as pressure filters could be used as an option to the screening step.  
           [0017]    The screened juice is transferred to a receiving/mixing tank where its pH is adjusted to a value that is preferably in the range of between about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank could be equipped with a low shear rate-mixing device. The tank is preferably designed to control the temperature of the juice to a value between about 4° Celsius to 70° Celsius.  
           [0018]    The juice from the receiving/mixing tank is transmitted (eg. pumped) at constant flow into a first membrane separation system. This first system performs the separation of suspended solids with a size larger than between about 0.1 to 0.2 microns. The clean juice contains the protein(s) of interest. This clean juice or “first permeate fraction” is sent to a receiving tank before transmission to the next method step.  
           [0019]    The membrane reject or first retentate fraction is discarded or sent to alternate processing. The first retentate fraction contains contaminants such, as but not limited to: dextrans, waxes, bagacillo, bacterias, yeast, and suspended solids that are typically larger than about 0.1 to 0.2 microns. Membranes can be of different types, materials and configurations. As an example, hollow fiber polymeric membranes can be used. However, composite membranes can be used as well as inorganic (for example, ceramic and coated stainless steel tube membranes) and polymeric membranes with different, selected configurations.  
           [0020]    The first membrane separation system can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes obtained are preferably in the range of between about 15 to 160 gfd (gallon per square foot per day) at different trans-membrane pressures. During this step some properties of the membrane such as hydrophilicity can enhance the separation process.  
           [0021]    The permeate (also called clean fraction from the first step membrane) is collected into a tank called first permeate tank.  
           [0022]    The product from the first fraction tank is used to feed at preferably constant flow, the second membrane separation system. This second membrane separation system performs the separation of particulate larger than between about 0.01 to 0.05 microns. The permeate fraction is collected into a tank called the second fraction tank. The retentate fraction is collected into a tank called second retentate tank. According to its (their) molecular size(s), the protein(s) of interest could be in either the second retentate fraction or the second permeate fraction.  
           [0023]    Membranes can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used. However, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with arrangement including hollow fiber, spiral, plate and tubular module configurations.  
           [0024]    The second membrane separation system can be composed of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes can be in the range of between about 5 to 80 gfd (gallon per square foot per day) at different transmembrane pressures. The system can be hydraulically designed in order not to exceed a shear rate of 10,000 sec −1 . During this step some properties of the membrane such as hydrophilicity can enhance the separation process. The discarded fraction is sent to alternate processing.  
           [0025]    The fraction containing the protein(s) of interest, either the second permeate fraction or the second retentate fraction is collected into a second fraction tank. From the second fraction tank, the second fraction is transmitted (eg. pumped) at preferably constant flow into the third membrane separation system, which has cut size of about 5,000 to 80,000 molecular weight.  
           [0026]    The membranes used in the third separation system can be made of different material with different shape and configuration. Membranes can be of different types, materials and configurations. The membrane used can be a flat plate configuration, often referred to as “cassettes”. However, hollow fiber and spiral wound membranes could also be used. Different materials either regenerated cellulose or polyethersulfone membranes can be used. Other materials that could be used such as polymeric membranes with arrangement including hollow fiber, spiral, plate or tubular module configurations.  
           [0027]    The third membrane separation system can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 0.1 to 30 gfd (gallon per square foot per day) at different transmembrane pressures. The system can be hydraulically designed in order not to exceed a shear rate 10,000 sec −1 .  
           [0028]    The third membrane separation system produces two fractions: (a) the third permeate fraction and (b) the third retentate fraction. The protein(s) of interest is (are) in one of these two fractions. The discarded fraction is sent to alternate processing.  
           [0029]    The fraction containing the protein(s) of interest is collected into a third fraction tank prior to any further treatment step during the purification process. The third fraction tank is a receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of between about 5.2 to 8.3, accordingly to the protein(s) of interest. The third fraction tank can be equipped with a low shear rate-mixing device. The third fraction tank can also be temperature controlled to maintain the temperature of the juice to a value between about 4° Celsius to 70° Celsius.  
           [0030]    The protein fraction of interest after pH adjustment is transferred (eg pumped) at a rate of about 0.5 to 3.0 beds volume per hour through an ion exchange column containing a weak anionic resin with higher affinity (at this pH of about 4.5 tp 8.3, preferably 5.2 to 8.3) for colorants than any other compounds. Temperature during this step is maintained at a value between about 4° Celsius to 70° Celsius. Decoloration of the incoming feed is between about 25% and 95%.  
           [0031]    The decolorized fraction containing the protein(s) of interest is collected into a ion product tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The ion product tank could be equipped with a low shear rate-mixing device. The ion product tank could also be designed to control the temperature of the juice to a value between about 4° Celsius to 70° Celsius. The juice from this ion product tank is transferred (eg. pumped) at a rate of about 0.1 to 3.0 beds volume per hour through an ion exchange chromatographic process for further purification. The ion exchange chromatographic process step produces several fractions, one of them with higher concentration of the protein(s) of interest. A membrane adsorber could replace the ion exchange chromatographic step.  
           [0032]    The resulting fraction containing the protein(s) of interest is collected into an ion exchange chromatographic receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The ion exchange chromatographic tank could be equipped with a low shear rate-mixing device. The ion exchange chromatographic tank could also be temperature controlled to maintain the temperature of the juice to a value between about 4° Celsius to 70° Celsius. The fraction of the protein(s) of interest could be sent to a concentration step such as a low temperature evaporating system for further concentration such as flash/freeze dry. The product from the concentration step (eg. evaporation station) contains the fractionated desired protein(s) partially purified and concentrated. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0033]    For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein:  
         [0034]    [0034]FIG. 1 is a schematic flow diagram illustrating the preferred embodiment of the method and apparatus of the present invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0035]    [0035]FIG. 1 is a schematic diagram of the preferred embodiment of the apparatus of the present invention, designated generally by the numeral  10 . FIG. 1 also shows the various method or process steps of the preferred embodiment of the apparatus of the present invention. Transgenic plant processing system  10  receives plant feedstock  11  that are treated preliminarily by extraction step  13 . The feedstock  11  can be any type of transgenic plant material such as eg. cane, sugarcane, barley, corn, potatoes, alfalfa. As used herein, the terms “transgenic material should be construed to include any plant material such as leafy material, stalks, grain, or the like.  
         [0036]    The extraction step  13  can start with genetically modified plant matter  11  containing the protein(s) of interest, and passing the stalks through a pressure system such as roller/crusher allows extracting a liquid in this case also called pression juice  14 , which contains the protein(s) of interest.  
         [0037]    The extracting pressure system or extraction step  13  can use: (a) different geometry rollers, (b) any quantity of rollers, (c) water  12  for further extraction of pression juice  14 , (d) series pressure system where the plant matter  11  after the first pressure system will feed a second pressure system in series with the first pressure system, etc. (e) a water and/or buffer solution  12  which will avoid partially or entirely oxidation or degradation of some compounds contained in the pression juice  14 .  
         [0038]    In order to improve the juice extraction of the pressure system, the plant matter  11  can be previously shredded. Both shredder and pressure system are part of the extraction step  13 . The extraction step  13  can work continuously or discontinuously.  
         [0039]    Extraction  13  could be also performed through a leaching process such as diffusion.  
         [0040]    Following the extraction step  13 , the pression juice  16  feeds, either by gravity or by means of pumping to a screening system composed of one or several screening steps  17 ,  19 ,  21 . For example, a three steps screening system will be composed of: (a) the first screening step  17  could remove particulate matter larger than about 500 microns to 1000 microns, (b) the second screening step  19  used for particulate size larger than about 150 microns to 250 microns, (c) the third screening step  21  removing particulate size larger than about 10 to 60 microns.  
         [0041]    Screens  17 ,  19 ,  21  could be stationary, vibrating, rotary or any combination of these types of screens. Screens  17 ,  19 ,  21  could also be self-cleaning units. The screened juice is recovered at mixing tank  24  for further processing and the reject  18 ,  20 ,  22  is discarded or sent to alternate processing.  
         [0042]    Press filter(s) or other filtering devices such as pressure filters could be used as an option to the screening step comprised of screens  17 ,  19 ,  21 .  
         [0043]    The screened juice is transmitted to receiving/mixing tank  24  where its pH is adjusted to a value preferably in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank  24  can be equipped with a low shear rate-mixing device. The tank  24  can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius.  
         [0044]    The juice from the receiving/mixing tank  24  is transmitted (eg. pumped) at constant flow into a first membrane separation system  25 . This first membrane separation system  25  performs the separation of suspended solids with a size larger than about 0.1 to 0.2 microns. The clean juice contains the protein(s) of interest. This clean juice or first permeate fraction is sent to first fraction tank  27  before going into the next step. The membrane reject or first retentate fraction  26  is discarded or sent to alternate processing.  
         [0045]    The first retentate fraction  26  contains contaminants such as but not limited to: dextrans, waxes, bagacillo, bacterias, yeast, and suspended solids larger than 0.2 microns. Membranes that are used in system  25  can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used; however, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with different configurations. The first membrane separation system  25  can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 15 to 160 gfd (gallon per square foot per day) at different trans-membrane pressure. During this step at first membrane separation system  25  some properties of the membrane such as hydrophilicity can enhance the separation process. As previously indicated, the permeate also called clean fraction from the first step membrane is collected into a tank  27  called first fraction tank.  
         [0046]    The product from the first fraction tank  27  is used to feed (at preferably constant flow) the second membrane separation system  28 . This system  28  performs the separation of particulate larger than about 0.01 to 0.05 microns. The permeate fraction  29  is collected into a tank called second permeate fraction.  
         [0047]    The retentate fraction  30  is collected into a tank called second retentate tank. Accordingly to its (their) molecular size(s), the protein(s) of interest can be either into the second retentate fraction  30  or the second permeate fraction  29 . Membranes in the second membrane system  28  can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used: However, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with arrangement including hollow fiber, spiral, plate and tubular module configurations. The second membrane separation system  28  can be composed of a single or several membranes working in parallel or in series.  
         [0048]    Operating temperature is preferably in the range of a value about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 5 to 80 gfd (gallon per square foot per day) at different transmembrane pressure. The second membrane separation system  28  is hydraulically designed in order not to exceed a shear rate of 10,000 sec −1 . During this step some properties of the membranes  28  such as hydrophilicity can enhance the separation process. Any discarded fraction can be sent to alternate processing.  
         [0049]    The fraction containing the protein(s) of interest can be either the second permeate fraction  29  or the second retentate fraction  30  and is collected into the second fraction tank  31 . From the second fraction tank  31 , the second fraction is transmitted (eg. pumped) at preferably constant flow into the third membrane separation system  32 , which has cut size of about 5,000 to 80,000 molecular weight.  
         [0050]    The membrane(s) used in the third separation system  32  can be made of different material with different shape and configuration. Such membranes can be of different types, materials and configurations. The membrane(s) can be flat plate configuration, often referred as cassettes. However, hollow fiber and spiral wound membranes can be used. Different materials such as either regenerated cellulose or polyethersulfone membranes can be used. Other materials can be used such as eg. polymeric membranes with arrangement including hollow fiber, spiral, plate or tubular module configurations.  
         [0051]    The third membrane separation system  32  can be comprised of a single or several membranes working in parallel or in series. Operating temperature is in the range of a value about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 0.1 to 30 gfd (gallon per square foot per day) at different transmembrane pressure. The third membrane separation system  32  can be hydraulically designed in order no to exceed a shear rate 10,000 sec −1 . The third membrane separation system  32  produces two fractions: (a) the third permeate fraction  33  and (b) the third retentate fraction  34 . The protein(s) of interest is (are) in one of these two fractions  33 ,  34 . Any discarded fraction can be sent to alternate processing.  
         [0052]    The fraction containing the protein(s) of interest is collected into the third fraction tank  35  prior to any further treatment step during the purification process. The third fraction tank  35  is preferably a receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank  35  can be equipped with a low shear rate-mixing device. The tank  35  can also be temperature controlled to maintain the temperature of the processed juice in the tank  35  to a value about 4° Celsius to 70° Celsius. The protein fraction of interest after pH adjustment is transferred (eg. pumped) at a rate of about 0.5 to 3.0 beds volume per hour through an ion exchange column  36  containing a weak anionic resin with higher affinity at this pH of about 5.2 to 8.3 for colorants than any other compounds. Temperature during this step at column  36  is maintained at a value about 4° Celsius to 70° Celsius. Decoloration of the incoming feed is between about 25% and 95%.  
         [0053]    The decolorized fraction containing the protein(s) of interest is collected into an ion product receiving/mixing tank  38  where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank  38  can be equipped with a low shear rate-mixing device. The tank  38  can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius. The juice from tank  38  is transferred (eg. pumped) at a rate of about 0.1 to 3.0 beds per volume through an ion exchange chromatographic process step  39  for further purification. The ion exchange chromatographic process step  39  produces several fractions, one of them with higher concentration of the protein(s) of interest. Membrane adsorber could replace the ion exchange chromatographic step  39 .  
         [0054]    The resulting fraction containing the protein(s) of interest is collected into an ion exchange chromatographic receiving/mixing tank  41  where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank  41  can be equipped with a low shear rate-mixing device. The tank  41  can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius. The fraction of the protein(s) of interest could be sent to a low temperature concentration step  42  (eg. evaporating system) for further concentration. Such a concentration step can be, for example, a flash/freeze dry step. The product from the concentration step  42  (evaporation station) contains the fractionated protein(s)  44  partially purified and concentrated.  
         [0055]    The following is a list of suitable parts and materials for the various elements of the preferred embodiment of the present invention.  
                                                   PARTS LIST            PART NO.   DESCRIPTION                    10   plant matter fractioning system       11   plant matter feedstock       12   water/buffer       13   extraction step       14   pression juice       15   reject (bagasse)       16   flowline       17   first screen       18   reject       19   second screen       20   reject       21   third screen       22   reject       23   ph buffer       24   mixing tank       25   first membrane       26   first retentate fraction       27   first fraction tank       28   second membrane       29   second permeate tank       30   second retentate fraction       31   second fraction tank       32   third membrane       33   third permeate       34   third retentate       35   third fraction tank       36   ion exchange       37   rejects       38   ion product tank       39   ion exchange chrom       40   reject       41   ion exchange chrom tank       42   concentration step       43   condensates       44   partially purified protein                  
 
         [0056]    The foregoing embodiments are presented by way of example only; the scope of the present invention is to be limited only by the following claims.