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    • 专利摘要:
    X Large-scale downstream processing of secreted recombinant proteins is provided in a single device, wherein the contents of a plurality of bioreactors are combined simultaneous to their harvesting and purification resulting in significant savings of time and the cost of manufacturing. It may sometimes be necessary to divide a batch into a number of sub-batches, which are later brought together to form a final homogeneous batch. In the case of terminal sterilization, the batch size is determined by the capacity of the autoclave. In continuous manufacture, the batch must correspond to a defined fraction of the production, characterized by its intended homogeneity.
    公开号:AU2013222455A1
    申请号:U2013222455
    申请日:2013-02-21
    公开日:2014-09-11
    发明作者:Sarfaraz Niazi
    申请人:Therapeutic Proteins International LLC；
    IPC主号:C07K1-16
    专利说明:
    WO 2013/126533 PCT/US2013/027077 DOWNSTREAM BIOPROCESSING DEVICE FIELD OF THE INVENTION [00011 The instant invention relates generally to the field of large-scale manufacturing of target proteins using biological cultures that secrete target proteins wherein the contents of a plurality of bioreactors are combined simultaneous to their harvesting and purification in a downstream bioprocessing device resulting in significant savings of time and the cost of manufacturing. BACKGROUND OF THE INVENTION [00021 Large scale manufacturing of target proteins using mammalian cells such as Chinese Hamster Ovary Cells (CHO) or other similar cells currently constitutes about three-fourth of all recombinant manufacturing methods used today. As more target proteins, particularly the monoclonal antibodies (MABs), come off patent, there is a rising unmet need for manufacturing systems that will be affordable, easily installed and operated with fewer regulatory challenges. None of the currently used systems, regardless of their cost, offer these advantages. As an example, a mammalian cell manufacturing facility to provide at least 20% of the world market for a single MAB could cost over $100 Million for cGMP production. The requirement for such a large investment has kept many companies outside of this field of manufacturing, resulting in a worldwide monopolization and price controls for these products. [00031 There is a large unmet need to develop methods for manufacturing target protein at the lowest possible cost and this can be achieved by a novel combination of various concepts including: WO 2013/126533 PCT/US2013/027077 a. Use of smaller bioreactors to create large batches by combining the output in compliance with CFR 21 requirement of the definition of a cGMP batch, to reduce the cost of scaling up and validation, reduced cost of contamination failures and using smaller manufacturing facilities; b. Eliminating the costly steps of cell separation, nutrient media volume reduction and lengthy chromatography column loading; c. Allowing purification using either step or gradient elution; d. Performing all of the above operations in a single container under fully automated conditions to allow for unattended operations. [0004] The instant invention provides a novel solution for cost-containment in target protein manufacturing by combining all of the above key elements in a novel system that can be used to manufacture mainly the type of target proteins which are secreted in the nutrient media, more particularly the large dose products like monoclonal antibodies, with a significantly lower capital cost requirement and with the lowest possible operating cost, and the shortest turn-around time for development and manufacturing of new products. More generally, the instant invention can be used to pool, harvest and purify any recombinant substance either as expressed or in any stage of purification. Representative examples will be the pooling and concentration of target proteins at the stage of refolding of proteins. [00051 The novel downstream processing system claimed is not an obvious outcome of the known art; several novel steps, hardware components and methods had to be created to make this system function optimally. 2 WO 2013/126533 PCT/US2013/027077 SUMMARY OF THE INVENTION [00061 A pharmaceutical production batch size is defined in accordance with CFR 21 (Code of Federal Register) as a homogenous mixture of ingredients. A "batch" or "lot" as defined in the WHO GMP guideline (TRS 908 Annex 4) as "a defined quantity of starting material, packaging material, or product processed in a single process or series of processes so that it is expected to be homogeneous. It may sometimes be necessary to divide a batch into a number of sub-batches, which are later brought together to form a final homogeneous batch. In the case of terminal sterilization, the batch size is determined by the capacity of the autoclave. In continuous manufacture, the batch must correspond to a defined fraction of the production, characterized by its intended homogeneity. The batch size can be defined either as a fixed quantity or as the amount produced in a fixed time interval." [00071 In those instances where smaller sub-batches are manufactured and pooled together, it requires combining them in a larger container wherein the sub-batches can be mixed to a homogenous mixture. However, in many situations, a larger container use can be prohibitive, such as in clean rooms, and thus there is an unmet need to invent systems that will allow mixing between containers without the need to mix the entire content in a larger container. [0008] The idea of mixing contents of multiple containers also offers many significant financial and regulatory advantages. 100091 There are additional advantages in combining smaller sub-batches to produce a larger batch. The science of pharmaceutical manufacturing teaches us that changing the size of a batch is not a simple exercise. As the size of a batch changes, the dynamics of mixing also changes along with the dynamics of any reactions taking place in the manufacturing process and, as a result, a manufacturer is required to conduct studies to validate the conditions of manufacturing to assure that a specific size of 3 1 batch would consistently result in the same WO 2013/126533 PCT/US2013/027077 product. Therefore manufacturers are required to invest substantial time and money in validating different batch sizes to meet their need for specific quantities of the product. [0010] Biological manufacturing of products such as proteins using bioreactors even faces greater challenges as changes in the volume of liquid (nutrient media and biological culture) in the bioreactor container significantly changes the conditions required to produce a product consistently. The factors that are of significant importance include the geometry of the container, the amount of gasification, the amount and the nature of agitation of the liquid and as a result it is not possible to predict the behavior of manufacturing process unless it is practiced and appropriate corrections made to various parameters of the manufacturing process. 100111 Since the manufacturers of products are often faced with a choice of making a larger or a smaller batch at a time, the most obvious exercise conducted is to validate several batch sizes and use a specific batch size based on the current need of manufacturing. The use of different batch sizes also require making available different size of vessels, and other technical attachments to a bioreactor, making the cost of maintaining several validated batch sizes very high. However, as target proteins are the most expensive to manufacture and often have a shorter shelf life, it is inevitable for the manufacturers not to maintain several validated batch sizes. 100121 Since bioreactors mainly employ liquid contents, they are easier to mix and finding a solution to mix the contents of several bioreactors in a manner that it would meet the requirement of the FDA in accordance with CFR 21 for a single batch would reduce the cost of manufacturing significantly by reducing the number of batches that need to be validated and affording the flexibility to manufacturers to produce different sizes of batches at will using fewer variations in the manufacturing equipment. 4 WO 2013/126533 PCT/US2013/027077 [00131 There is no prior art that teaches on combining the contents of several bioreactors in a continuous manner using much smaller mixing vessels to constitute a single batch. The instant invention not only resolves this critical hurdle in reducing the cost of production but also teaches a commercial level applications where hundreds and thousands of liters of liquid can be processed using low-cost solution to mix liquids. The instant invention offers a two step method for uniting very large volumes of nutrient media from several bioreactors; first, all bioreactors pour into a small mixing plenum, which then introduces the liquid into a much smaller container compared to the size of the bioreactor. The intent is not to hold but to continuously mix and drain out the nutrient media and the biological culture and only keep the active target protein by binding it to a resin capable of binding it. [0014] The instant invention provides a continuous mixing capturing of target protein. Traditionally, once a target protein has been expressed in a bioreactor, the process of harvesting and purification currently requires separation of cells, reduction in the volume of nutrient media and loading of chromatography columns. All of these are extremely time consuming steps, cause substantial degradation of the expressed target protein and require very large capital investment. [0015] The instant invention combines all processes, more particularly for target proteins like monoclonal antibodies, by first capturing the expressed target proteins using a chromatography media capable of binding the target protein and then discarding the nutrient media and the biological culture; the complex of target protein and chromatography media is then washed and finally eluted to obtain a highly purified form of target protein using a unitary downstream bioprocessing into which the bioreactors drain. [00161 The present invention capitalizes on the recent availability of many resins that are capable of binding target proteins in large quantities. Most modem resins will bind between 20-125 mg of target protein per mL of resin. Many of these resins are highly specific to the 5 WO 2013/126533 PCT/US2013/027077 target proteins and many of them can be combined to remove any type and quantity of a target protein from a solution by a simple process of physicochemical binding that is strong enough to retain the target proteins attached to the resin while the nutrient medium is removed from the bioreactor. The art has also advanced significantly in the field of target protein purification wherein we now have a much better ability to elute these bound target proteins from resins by adjusting the pH, the ionic strength or other characteristics of the eluting buffer to break the binding between the resin and the target protein. This allows removal of target proteins from a bioreactor as a highly concentrated solution that is ready for further purification and in some instances it can even be the final product for use. [0017] Affinity chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces, which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and its target compound makes it highly specific. [0018] Many membrane products are glycoprotein and can be purified by lectin affinity chromatography. Detergent-solubilized products can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. [0019] Immunoaffinity chromatography resin employs the specific binding of an antibody to the target protein to selectively purify the target protein. The procedure involves immobilizing an antibody to a column material, which then selectively binds the target protein, while everything else flows through. [0020] Some of the state of the art resins binding technologies include: a. Novozymes's newly patented Dual Affinity Polypeptide technology platform replaces Protein A process steps with similar, but disposable, technology. 6 WO 2013/126533 PCT/US2013/027077 b. Stimuli responsive polymers enable complexing and manipulation of target proteins and allow for control of polymer and target protein complex solubility, which results in the direct capture of the product without centrifuges or Protein A media, from Millipore Corp. c. Mixed mode sorbents to replace traditional Protein A and ion exchange, for improved selectivity and capacity with shorter residence times. These media, with novel chemistries, include hydrophobic charge induction chromatography, such as MEP, and Q and S HyperCel from Pall Corp. d. Monoliths, involving chromatography medium as a single-piece homogeneous column, such as Convective Interaction Media monolithic columns from BIA Separations. e. Simulated moving beds, involving multicolumn countercurrent chromatography, such as BioSMB from Tarpon Biosystems. f. Protein G (multiple vendors). g. Single domain camel-derived (camelid) antibodies to IgG, such as CaptureSelect from BAC. h. New inorganic ligands, including synthetic dyes, such as Mabsorbent AlP and A2P from Prometic Biosciences. i. Expanded bed adsorption chromatography systems, such as the Rhobust platform from Upfront Chromatography. j. Ultra-durable zirconia oxide-bound affinity ligand chromatography media from ZirChrom Separations. k. Fc-receptor mimetic ligand from Tecnoge. 1. ADSEPT (ADvanced SEParation Technology) from Nysa Membrane Technologies. m. Membrane affinity purification system from PurePharm Technologies. n. Custom-designed peptidic ligands for affinity chromatography from Prometic Biosciences, Dyax, and others. o. Protein A- and G-coated magnetic beads, such as from Invitrogen/Dynal. 7 WO 2013/126533 PCT/US2013/027077 p. New affinity purification methods based on expression of target proteins or MAbs as fusion target proteins with removable portion (tag) having affinity for chromatography media, such as histidine) tags licensed by Roche (Genentech). q. Protein A alternatives in development, including reverse micelles (liposomes), liquid-nutrient medium extraction systems, crystallization, immobilized metal affinity chromatography, and novel membrane chromatography systems. r. Plug-and-play solutions with disposable components (e.g., ReadyToProcess), process development AKTA with design of experiments capability, and multicolumn continuous capture, from GE Healthcare. [00211 It is surprising that while great advances have been made in the design of resins available to capture target proteins, these have been only used in the downstream processing of purification. Adding resins to a crude mixture of target proteins and host cells will be no different than the current practice of art that teaches first concentrating the nutrient media and then loading it onto columns with all of the impurities in it. [0022] Aiming at a cell line that produces 1 mg/mL of target protein and that the binding capacity of the resin used is 50 mg/mL, this will require 20 L of resin when operating a 1000 L bioreactor. The cost of resins suitable for the manufacturing of monoclonal antibodies can range from $15-$20,000 per liter, such as Protein A. As a result, most manufacturers would rather run several sub-batches of purification using a smaller quantity of the resin. However, given that these can be used for hundreds of times, the cost is readily amortized for use and avoids the tediousness and regulatory hurdles in preparing sub-batches. [00231 The biological components that may be processed in accordance with the invention are described in the paragraphs which follow and include, but are not limited to, cell cultures derived from sources such as animals (e.g., hamsters, mice, pigs, rabbits, dogs, fish, shrimp, nematodes, and humans), insects (e.g., moths and butterflies), plants (e.g., algae, corn, 8 WO 2013/126533 PCT/US2013/027077 tomato, rice, wheat, barley, alfalfa, sugarcane, soybean, potato, lettuce, lupine, tobacco, rapeseed (canola), sunflower, turnip, beet cane molasses, seeds, safflower, and peanuts) and human. The only requirement is that the biological culture used should express the target protein by secreting it in the nutrient media as opposed to the formation of inclusion bodies in some instances. DETAILED DESCRIPTION OF THE INVENTION [00241 The instant invention provides a means of connecting a plurality of pre-validated, smaller-scale bioreactors to a downstream bioprocessing device holding a chromatography media capable of binding the target protein in the nutrient media of the bioreactors ready for harvesting. The nutrient media and the biological culture are allowed to enter the downstream bioprocessing device causing the chromatography media to float upwards and thus creating and expanded bed chromatography system. A significant modification to the classical expanded bed chromatography is provided in the instant invention wherein the chromatography resin is kept in a continuous state of uniform distribution throughout the container, a container, e.g., a cylinder that holds the nutrient media, the biological culture and the chromatography media. This modification is crucial to the success of the downstream bioprocessing device and also to provide it to operate unattended and automatically. 100251 As the nutrient media and the biological culture rise to the top of the downstream bioprocessing device, these flow out while the chromatography media is held in the downstream bioprocessing device as a filter is installed in the downstream bioprocessing device; the porosity of the filter is smaller than the size of chromatography resin (generally 50-300 microns). By calculating the quantity of chromatography media such as Protein A resin accurately through prior experimentation of the binding capacity of the target protein, one can assure that the entire quantity of target protein is bound to chromatography media. 9 WO 2013/126533 PCT/US2013/027077 However, it is well-realized that a certain reaction time is required for the binding to take place, so the flow rate of nutrient media from the bioreactors into the downstream bioprocessing device must be carefully controlled; one test for the correctness of the flow rate is the measure of recombinant product concentration in the nutrient media that reaches the top of the downstream bioprocessing device and is decanted. A continuous monitoring of the concentration of target protein will allow adjusting the flow rates. Generally, the outflowing nutrient media should not contain more than 1% of the incoming concentration of the target protein; sampling the incoming nutrient media and using it as a reference while the nutrient media exiting the device is treated as a test item determine this. Only when the ratio of the concentration between the incoming and outgoing media is 1:100 that the nutrient media is allowed to flow through; generally a cut off range will be 1:100 to 2:100 to conserve the maximum quantity of target protein and provide the highest efficiency of the device. [0026] Once the entire nutrient media and the biological culture is removed from a plurality of the bioreactors and passed through the downstream bioprocessing device, the bioreactors are sealed at the bottom outlet. The target proteins-media complex in the downstream bioprocessing device is washed by adding a washing buffer through an inlet at the bottom of the device and allowed to flow out from the top port until a pre-determined level of debris is removed. It is then followed by introducing an elution buffer to break the bonding between the media and the target proteins, either by passing the elution buffer through the bottom port and collecting pure target protein solution through the upper port or by allowing the chromatography media to settle down in the downstream bioprocessing device and allowing the elution buffer to pass through the compacted bed of the chromatography media and collecting the pure solution of target protein through the bottom port. 10 WO 2013/126533 PCT/US2013/027077 [00271 The above invention is operated in a manner where only gravity flow is used to transfer the nutrient media from the bioreactors to the downstream bioprocessing device by placing the inlet port of the downstream bioprocessing device below the level of the outlet port of the bioreactors. Placing the bioreactors around the downstream bioprocessing device and using the same length of connecter tubes can uniformly maintain the flow rates across many bioreactors. Obviously, there are many mechanical means of transporting the nutrient media to the downstream bioprocessing device and these include use of various pumps that may equally be useful. However, the use of gravity assures that the degradation of target protein is minimized during the transfer process. BRIEF DESCRIPTION OF THE DRAWINGS [0028] Figure 1 shows a cross-sectional side view of the claimed downstream processing device. [00291 The claimed device has many unique and specific features that are required for its optimal operation. It comprises a cylinder 1, a cylindrical, hard-walled container that serves as the main processing space for the nutrient media and biological culture 2 entering the cylinder 1 from a plurality of bioreactors. The volume of the cylinder 1 is important in some instances for one reason and is important for another reason in another instance. When operating it to perform as an expanded-bed chromatography system to capture and to purify the target protein, the height of the cylinder 1 is important to allow it enough dwell time as nutrient media flows upwards through a column chromatography resin 3. The volume of cylinder 1 is optimally at least 1.5-3 times the volume of chromatography resin 3 in the cylinder 1. Generally, an optimal relationship of the diameter of the cylinder 1 and its height would also be established from simple studies of the efficiency of binding of a target protein to a chromatography resin; these are specific binding reactions and whereas the capacity of binding of the resin may be known, the rate of binding will depend on many factors including 11 WO 2013/126533 PCT/US2013/027077 the time allowed for contact, temperature of nutrient media and the agitation of the nutrient media. It should be realized that the claimed device provides an expanded chromatography column to bind the target protein to the chromatography resin; the longer is the time allowed to bind, the higher will be the binding; however, physical limitations of the height of the column and other considerations as discussed below pertaining to purification of the target proteins will limit on the height of the cylinder 1 used. [00301 The chromatography resin 3 is retained in the cylinder 1 by providing a bottom filter 4 that has a porosity smaller than the diameter of the chromatography resin 3; this would generally be 50-300 microns; note that in typical chromatography preparative column a much finer filter is used requiring often application of pressure or requiring very long times for a buffer to pass through a bed of chromatography resin. The bottom filter 4 is kept in place by a bottom cap 5 that can be removed for a complete cGMP cleaning of the device and also to remove the chromatography media 3, which is reused. The bottom cap 5 has a bottom liquid port 6, which would generally be in the center of the cap; the bottom liquid port 6 has a bottom sampling port 7, which in turn has a bottom sampling port control valve 8 to start and stop flow of nutrient media and a filter 9 to remove biologivcal culture prior to sampling. The bottom liquid port 6 has a bottom flow control valve 10, and connected to a plenum 11 with a plurality of ports 12 and control valves 13 attached to each of the plenum ports 11; the plenum 11 is capable of being connected to other bioreactors and sources of a washing or elution buffer and also be used to allow a washing buffer or elution buffer to exit the device. [00311 The top side of the cylinder 1 is also supplied with a top filter 14 to retain the chromatography resin 3; the top filter 14 is kept in place by a top cap 15 that has at least one upper liquid port 16 to which is connected an upper sampling port 17 and a upper sampling port valve 18 to start and stop sampling of the nutrient media and a filter 19 to remove biological culture prior to sampling. The upper liquid port 16 is connected to an upper flow 12 WO 2013/126533 PCT/US2013/027077 control valve 20, which then discharges the nutrient media out and is also used to introduce an eluting buffer in a packed column mode purification protocol. 10032] Inside the cylinder 1 is installed an auger 21 connected through an auger shaft 22 to a motor 23 placed outside of the cylinder 1; the shaft 22 passes through a sealed ball-bearing 24 installed in the center of the top filter 14 and through a hole in the center of the top cap 15; the bottom of the auger shaft 22 rests in an auger shaft socket 25 that is embedded in the bottom filter 4; this helps prevent wobbling of the auger when it is rotated. [00331 The auger 18 is a critical element of the claimed invention; it is conical in shape wherein the larger blades are in the bottom and smaller blades in the top. Since the goal of using the auger is to provide a laminar movement of liquid upwards, the ratio of the diameter of the blade at the bottom and at the top is critical. Optimally, the bottom blade would cover approximately 80% of the diameter of the cylinder 1 and the top blade would approximately be 20% of the diameter of the cylinder 1. When rotated at slow speeds ranging from 1-20 rpm, it creates a gentle movement of the nutrient media from the bottom to the top in a sweeping motion that keeps the flow of the nutrient media laminar. This creates a novel fluid bed of chromatography resin by keeping it uniformly suspended throughout the cylinder. Without this feature, the contact efficiency between the resin and the target proteins target proteins is minimized and the efficiency of capturing the target protein reduced. This becomes more important as the chromatography resin becomes saturated with the target protein. This element of innovation provides a method of completely saturating the chromatography resin while a fast flow of nutrient media is maintained. [0034] It is important to maintain a Reynolds number (Re) of less than 10,000 in the cylinder; this number is determined by five factors: the density, the viscosity, the diameter of the agitator, the rotational speed of the agitator (rpm) by the following equation in a stirred vessel: 13 WO 2013/126533 PCT/US2013/027077 Re = [(pND 2 )/p], where p the density is and R is the viscosity; ND is velocity as D is the diameter and N is the rotational speed (RPM) [R. K. Sinnott Coulson & Richardson's Chemical Engineering, Volume 6: Chemical Engineering Design, 4th Ed (Butterworth Heinemann) page 473]. [00351 The cylinder 1 has additional means of heating or cooling 26 to keep the nutrient media at an optimal temperature; it is noteworthy that the claimed device provide a continuous flow of the nutrient media but the flow rate is at a slower speed and so it is possible to maintain a certain temperature given the standard coefficients of heat exchange with the wall of the cylinder 1. A variety of methods are described and include using a jacketed cylinder 1, wrapping the cylinder 1 with a heating or cooling blanket, exposing the cylinder to an infrared lamp when the goal is heat the contents and many other devices widely available in the art. [0036] The claimed device is vertically placed on a support 27 that is generally a ring stand but any other means suitable to firmly place the claimed device in a vertical position would work. It is noteworthy that the claimed invention utilized a gravitational flow method of transferring nutrient media from the bioreactors to the claimed device; for this reason, the means of supporting 26 should be of such mechanical nature that it will allow the claimed invention to rest at a level lower than the lowest part of the bioreactor from which the nutrient media is removed and transferred to the claimed invention. [00371 The target protein-harvesting and purification device described in the figure above is generally used at the end of the bioreactor cycle wherein a plurality of bioreactors is simultaneously connected to the claimed device. The amount of chromatography media contained in each device would be easily calculated by the binding efficiency of the target protein For example, Protein A chromatography media shows a binding of 30-50 mg/mL of the chromatography media. Assuming a 1,000 L nutrient media is used in a 2,000 L 14 WO 2013/126533 PCT/US2013/027077 bioreactor and the production cycle has come to an end, a point where the CHO are no longer producing sufficient quantity of target protein Further assuming that the productivity of the recombinant cell line is about 1 G/L; thus, in this case, there is about 1000 G of target protein in solution in the nutrient media that is to be removed and purified. [00381 On a theoretical basis, assuming a lower end of binding of 30 mg/mL, it will take about 33 L of chromatography media to bind substantially all of target protein in the solution. It should be noted that while Protein A is rather specific to monoclonal antibodies, it is likely that the binding capacity of the chromatographic media will be compromised because of binding of other components in the nutrient media. This can be readily studied by withdrawing a small volume of the nutrient media and adding to it incremental amounts of the chromatography media used until the concentration of the target protein in solution reaches to a pre-determined low value. This would be called titrating the nutrient media. [0039] The instant invention combines the processing of a plurality of bioreactors, for example, as described above, each requiring a 33L volume of Protein A to purify the target protein. Assuming that the contents of five bioreactors are combined, it will require 165 L of chromatography resin and given that there should be at least twice the volume of the main holding container, it will take a 230L to process 5000L of nutrient media; a traditional system of combining 5000L media would be to combine the individual volumes in a 5000L container, which is an expensive exercise. Instead, a container less than 10% of the size is all that is required in the instant invention. [0040] Using the instant invention in the example above, nutrient media from several bioreactors will be allowed to enter the main container of the claimed device where the target protein would bind to the chromatography resin as the nutrient media rises from the bottom through a filter disk that retains the chromatography media from leaving the container; another filter at the top retains the media at the top end. The key to a successful capture step 15 WO 2013/126533 PCT/US2013/027077 is allowing flow nutrient media at a rate that allows optimal binding. Gentle mixing is critical to this invention and this provided by a novel design of an auger blade that pushes the nutrient media upwards. [0041] An optimal process would remove substantially all of target protein from nutrient media; to assure this, the instant invention introduces an automated system of controlling the flow control valves installed at both end of the device; a dual sampling method where the incoming nutrient media and the outgoing nutrient media are continuously sampled and using the incoming nutrient media as the standard, it is easy to calculate the quantity of target protein in the nutrient media flowing out of the device and discarded. Should there be a rise in the concentration of target protein in the drained liquid, the valves close down and retain the nutrient media in the main container until complete binding is reached. Given the large quantity of nutrient media flowing out, an automated system described in the instant invention allows automated operations, a key requirement to large-scale commercial manufacturing. [0042] The instant invention introduces a system of automatically processing the batch wherein the concentration of target protein is measured continuously in two points, the point of entry and the point of exit for the nutrient media; the samples are first filtered through a filter that retains any biological culture to reduce interference from suspended particles. The nutrient media from the entry point is used as a reference sample and the nutrient media from the exit point serves as a sample in a spectrophotometric detection device. The technique of detection is widely available in the prior art and is not claimed. (M. H. Simonian, Spectrophotometric determination of protein concentration, Curr. Protocol. Cell Biol, Appendix 3b, 2002). Absorbance measured at 280 nm A(280) and 205 nm A(205) is used to calculate protein concentration by comparison with a reference but in the instant invention the purpose is not to measure the concentration but a relative concentration between the 16 WO 2013/126533 PCT/US2013/027077 reference and the standard. Given that there are going to be dissolved impurities and lysates and impure protein in the media, the A(280) and A(205) methods can be used. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from reference solutions to determine the relative concentration. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample. However, the instant invention does not limit the type of detection method used; with evolving science of detection of proteins, it may be possible to device a battery of tests including spectrophotometric, fluorometric, infrared or lasers to provide a relative measure of the concentration of the target protein in the nutrient media. [0043] Once the entire nutrient media has passed through the container, a washing buffer is used to replace the nutrient media to wash off the chromatography resin on a continuous basis, following which an elution buffer is introduced in a similar manner to collect purified target protein solution in the outflow through the top liquid port. However, several other methods of purification are available for use within the same device design. One of them requires allowing the elution buffer to stay in the container for a length of time to allow complete breakdown of the binding between the chromatography media and the target protein and then removing the elution buffer through the bottom liquid port. Alternately, the washing buffer can be drained out of the bottom liquid port and the chromatography resin allowed to settle down as a packed column when the elution buffer can be introduced through the top liquid port and the elution buffer either as a single buffer or in a gradient elution buffer allowed to pass through the chromatography resin bed to collect various fractions of the elution buffer and the fractions containing the highest concentration and least impurities are combined to provide the final processed product. 17 WO 2013/126533 PCT/US2013/027077 [0044] In a first embodiment, the instant invention provides a method of combining several sub-batches to produce a large single batch in compliance with the U.S. FDA CFR 21 definitions reducing the cost of production by allowing use of smaller bioreactors, reducing the risk of a larger batch going bad and eliminating the capital cost of bioreactors of several sizes and also of a larger container to mix the contents of several bioreactors. This novel approach makes it possible for even small size companies to develop and manufacture large commercial scale batches at the lowest capital and running cost. [0045] The instant invention obviates the need for installing larger bioreactors when larger quantities of target proteins are produced. The regulatory compliance of mixing smaller sub batches to make a larger batch is met by allowing the contents of a plurality of bioreactors to mix in a plenum prior to entering the claimed device; secondly, as the entire contents of a plurality of bioreactors is passed through the claimed device containing a fixed quantity of a chromatography resin, the captured target proteins constitutes a single batch in a smaller quantity of volume and thus reducing the problems related to handling large containers. [0046] In a second embodiment, the present invention provides a method of avoiding several steps in the harvesting of target proteins including separation of cells, reducing the volume of nutrient media and loading of chromatography column, all of which add substantially to capital cost of equipment, cost of running the equipment, lengthy times required to complete these steps and causing degradation of target proteins. In the present invention, a nutrient media containing host cells and target protein are subjected to a non-specific or specific treatment with chromatography media or a combination of chromatography media contained in the device that would bind all or substantially all of charged or uncharged molecular species, this step is followed by removing the debris of cells and other components from chromatography media-target protein complex by simply washing it with a washing buffer. 18 WO 2013/126533 PCT/US2013/027077 [0047] The present invention thus obviates a major hurdle in the harvesting of products that involves filtering out host cells using a fine filter, not larger than 5p, to retain host cells such as Chinese Hamster Ovary Cells. When large volume of media is used, this process takes a very long time, adds substantial cost of filters, pumps, containers and space management. This step is then generally followed by a concentrating step wherein the volume of nutrient media is reduced most to one-tenth its volume using a cross-flow or micro filtration process, which takes a very long time to complete and again adds substantial cost of equipment and manpower and in some instances causes degradation of target protein. [0048] The instant invention combines these two steps into one simple step. The argument that if it is the intent to harvest and concentrate target protein from a complex mixture containing host cells, why would it not be more efficient to remove the target protein from the mixture instead of removing other components that are present in much larger quantities. This is what would be considered a contrarian teaching. [0049] In the instant invention, those peculiar characteristics of target proteins are exploited to separate them from the rest of the mixture by a non-specific binding to a chromatography media or a mixture of chromatography medias. Obviously, such non-specific capture of target proteins would also capture other components of the mixture and that only requires using a much larger quantity of chromatography media or a specific type of chromatography media that might have specific affinity for the target protein The removal of target protein chromatography media complex is a much simpler process than the removal of host cells or reduction in the volume of mixture; any mechanical process such as decanting, centrifugation or even filtration would work. It is noteworthy that the slowest of all processes would be filtration but even the much larger pore size filter can be used and since the purpose is to collect the filtrate, not the eluate, the cost of manufacturing is lowered substantially. 19 WO 2013/126533 PCT/US2013/027077 [00501 In a third embodiment, the instant invention allows for the use of a expanded-bed chromatography system to purify a target protein in the same container wherein the target protein is captured. An expanded-bed chromatography system allows for continuous operation of the claimed device, provide automated controls to achieve the highest level of capture and purification in an unattended operation. [00511 The instant invention can also use a mixed-bed chromatography media that may contain an ionic chromatography media, a hydrophobic chromatography media and an affinity chromatography media all used together to optimize the efficiency of harvesting. It is well established that the use of ionic chromatography medias does not allow complete capture of products because of the logarithmic nature of ionization; a combination of chromatography medias used in the instant invention allows for a more complete recovery of target proteins. [00521 In a fourth embodiment, the present invention allows purification of a target protein using standard column purification and a gradient elution profile as well as step elution profile; in this comparison, the claimed invention acts similar to a conventional chromatography column with all of its limitations yet without the lengthy steps of loading the column. [00531 In a fifth embodiment, the present invention teaches methods of gravity flow to transfer the contents of a bioreactor to the claimed device; this reduces considerable strain on the target protein is peristaltic pumps are used as it is customary and thus increases the yield of production. [00541 In an sixth embodiment, the present invention teaches the use of a novel mixing component in the device comprising preferably a conical auger that pushes the liquid contents up while maintaining a laminar flow of the liquids reducing strain on the target proteins target 20 WO 2013/126533 PCT/US2013/027077 proteins and also helping maintain the integrity of the chromatography media; no prior disclosure the use of a mixing system for a chromatography system. [0055] The embodiments described above do not in any way comprise all embodiments that are possible using the instant invention and one with ordinary skills in the art would find many more applications specific to a complex process or even in those processes where such needs might not be immediately apparent. [0056] Prior art on using chromatography media to harvest target protein is non-existent; the US patent 7306934 issued on 11 December 2007 to Arora et al., teaches the use of porous solid ion exchange wafer for immobilizing biomolecules, said wafer comprising a combination of an biomolecule capture-chromatography media containing a transition metal cation of +2 valence; it also teaches a separative bioreactor, comprising an anode and a cathode, a plurality of reaction chambers at least some being formed from a porous solid ion exchange wafers (above) having a combination of art biomolecule capture-chromatography media and an ion-exchange chromatography media and having a genetically engineered tagged biomolecule immobilized on said biomolecule capture chromatography media, each of said porous solid ion exchange wafers being interleaved between a cation exchange membrane and an anion exchange membrane, and mechanism for supplying an electric potential between the anode and the cathode. The instant invention is significantly different from the separative bioreactor taught by Arora. First, the instant invention does not require use of electrodes, chromatography medias with a transition cation of +2 valence or immobilized metal ion affinity chromatography. The use of EDI (electrodeionization) and specific use of tags and limited nature of solvents to remove the captured products mainly enzymes makes this patent teachings distinctly different from the instant invention. Moreover, the Arora patent adds a hardware that adds to the cost of processing purification of 21 WO 2013/126533 PCT/US2013/027077 products while the instant invention combines several processes into one without adding any new cost element. [0057] The idea of using expanded bed chromatography to purify target protein is know in the prior art such as the US patent 7,608,583 that teaches purification of insulin using an expanded bed chromatography. The novel aspect of the instant invention is to combine the expanded bed chromatography principles to a process that combines the contents of a plurality of bioreactors, captures and purifies the target protein. However, providing the multi-functionality to a traditional chromatography requires several innovative modifications including a method retaining the chromatography resin in the column at both ends, a means of mixing the contents in the chromatography column and a set of ports and controls that allow use of both step and gradient elution. [00581 All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0059] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods 22 WO 2013/126533 PCT/US2013/027077 described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [00601 Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 23
    权利要求:
    Claims (31)
    [1] 1. A downstream processing device for pooling, harvesting and purifying a target protein comprising: a. a container having a top opening, a bottom opening, wherein the container is suitable for holding a nutrient media, a biological culture and a chromatography media; b. a top filter covering the top opening and a bottom filter covering the bottom opening of the container capable of retaining the chromatography media; c. a removable top cap to hold the top filter in place and further comprising at least one top liquid port and a top flow control valve; d. a top sampling port connected to the top liquid port further comprising a top sampling port valve, and filter capable of removing the biological culture; e. a removable bottom cap to hold the bottom filter disk in place and further comprising a bottom liquid port, a bottom flow control valve, and a plenum having a plurality of liquid ports with means of closing and opening them; f. a bottom sampling port connected to the bottom liquid port, a bottom sampling port valve and a filter to remove the biological culture; g. a mixing apparatus capable of keeping the chromatography media uniformly suspended throughout the container; and h. a vertical support for the container.
    [2] 2. The downstream processing device of claim 1, further comprising a heating element, a cooling element, at least one device to measure the turbidity of the contents of the container, and/or a sensor for measuring the temperature of the contents of the container.
    [3] 3. The downstream processing device of claim a or claim 2, further comprising an electronic means of automatically controlling: 24 WO 2013/126533 PCT/US2013/027077 a. the opening and closing of the bottom flow control valve in response to the ratio of the concentration of the target protein in the top and the liquid ports; b. the heating and cooling elements; c. the contents of the container in response to the temperature of the nutrient media; and/or d. controlling the intensity the mixing means in response to the differences in the measurement of turbidity in the container.
    [4] 4. The downstream processing device according to claims 1-3, wherein the container is a cylinder.
    [5] 5. The downstream processing device according to claims 1-4, wherein the container comprises a plastic, a metal or glass.
    [6] 6. The downstream processing device according to claim 1, wherein the container is disposable.
    [7] 7. The downstream processing device according to claim 1, wherein the container is flexible and housed inside a rigid container.
    [8] 8. The downstream processing device according to claim 1, wherein the container has an inner volume ranging from 1.5 to 3 times the volume of the chromatography media held in the container.
    [9] 9. The downstream processing device according to claim 1,wherein both the top and the bottom filters comprise a flexible plastic or metal mesh supported by a rigid porous structure, a rigid ceramic plate or a porous plastic septum.
    [10] 10. The downstream processing device according to claim 1, wherein both the top and the bottom filters have a porosity ranging from 50 to 300 microns.
    [11] 11. The downstream processing device according to claim 1, wherein the mixing apparatus comprises a horizontally disposed magnetic stirrer operated by a magnetic field source external to the container. 25 WO 2013/126533 PCT/US2013/027077
    [12] 12. The downstream processing device according to claim 1, wherein the mixing apparatus is a central rotating pedal, turbine or propeller driven by a motor external to the container.
    [13] 13. The downstream processing device according to claim 1, wherein the mixing apparatus comprises a conical auger blade vertically disposed in the container with its tip pointing upwards, and the lower end of the shaft supported in a socket in the center of the bottom filter and the upper end of the shaft connected to an external motor after passing through sealed ball bearings in the center of the top filter and the top cap.
    [14] 14. The downstream processing device according to claim 13, wherein the diameter of the base of the auger blade ranges from 80-95% of the diameter of the container and the diameter of the tip of the auger blade ranges from 10-25% of the diameter of the container.
    [15] 15. The downstream processing device according to claim 13, wherein the auger is rotated at a speed of 1-20 rpm and operated in a direction to move the contents of the container from bottom to the top in a laminar flow pattern.
    [16] 16. The downstream processing device according to claim 2, wherein the sensors for measuring the turbidity in the nutrient media in the container are disposed at least one below the top filter and one above the bottom filter and the intensity of the means of mixing adjusted to achieve uniform turbidity in the container.
    [17] 17. The downstream processing device according to claim 1, wherein the vertical support comprises a ring stand, a hook, a hanger or a flat surface.
    [18] 18. The downstream processing device according to claim 1, wherein the chromatography media comprises a resin capable of binding a target protein.
    [19] 19. The downstream processing device according to claim 18, wherein the resin comprises an ionic-exchange resin, a hydrophobic resin, an affinity resin or a mixture thereof.
    [20] 20. The downstream processing device according to claim 18, wherein the resin comprises a protein or a peptide as a ligand.
    [21] 21. The downstream processing device according to claim 18, wherein the resin has a specific affinity towards a target protein. 26 WO 2013/126533 PCT/US2013/027077
    [22] 22. The downstream processing device according to claim 1, wherein the chromatography media comprises a plurality of resins.
    [23] 23. The downstream processing device according to claim 1, wherein the plenum is capable of receiving the nutrient media and buffers from a plurality of sources by gravitational flow.
    [24] 24. The downstream processing device according to claim 2, wherein the heating and/or cooling elements comprise a resistant heating element wrapped around the container, a liquid cooled or heated jacket wrapped around the container, a heated or cooled cabinet placed to surround the container, an infrared lamp placed in close proximity to the container, or a fan blowing heated or cooled air over the container.
    [25] 25. The downstream processing device according to claim 2, wherein the ratio of the concentration of the target protein in the top sampling port and the bottom sampling port ranges between 1:100 to 2:100.
    [26] 26. A method for pooling, harvesting and purifying a target protein expressed in the nutrient media of a plurality of bioreactors comprising: a. providing the downstream processing device according to claims 1-25; b. locating the downstream processing device at such height that the top liquid port is below the lowest level of the plurality of sources of nutrient media or buffers; c. closing the bottom flow control valve; d. connecting the liquid ports of the plenum to a plurality of bioreactors containing the nutrient media in need for pooling, harvesting and purifying a recombinant protein already expressed in the nutrient media; e. removing the top cap and adding to the container a quantity of chromatography media sufficient to bind essentially all of target protein present in the nutrient media ready for processing; f. replacing the top cap and opening the top flow control valve; g. opening the bottom flow control valve; 27 WO 2013/126533 PCT/US2013/027077 h. starting a flow of the nutrient media under gravity pressure from a plurality of bioreactors into the container through the plenum and allowing the nutrient media to fill the container ; i. closing the bottom flow control valve; j. starting the mixing apparatus to achieve a uniform distribution of the chromatography media in the container as indicated by the measurement of turbidity by the sensors in the top, middle and bottom of the container and automatically adjusting the speed of the mixing means by the electronic means to achieve a uniform turbidity; k. heating or cooling the contents of the container to a pre-determined level if required and automatically maintaining the pre-determined temperature in the nutrient media by the electronic means of control; 1. connecting the top sampling port to a test continuous flow cell and the bottom sampling port to a control continuous flow cell of a spectrophotometer capable of measuring the concentration of the target protein and turning on the bottom flow control valve when the ratio of the concentration in the top sampling port and the bottom sampling port reaches 1:100 and closing the bottom flow control valve when the ratio reaches 2:100; m. allowing the electronic means of control to open and close the bottom flow control valve and maintaining the flow of nutrient media from a plurality of bioreactors into the container and allowing the nutrient media to flow out of the top liquid port and discarding it until the contents of all bioreactors have passed through the container ; n. closing the bottom flow control valve and the liquid ports of the plenum and disconnecting the bioreactors from the plenum; o. opening the bottom flow control valve to allow the nutrient media in the container to drain out through one of the unoccupied ports in the plenum and then closing that port; p. connecting to the liquid ports of the plenum to at least one source of washing buffer suitable for washing off any debris in the container and at least one source of elution buffer capable of breaking the binding of the target protein to the chromatography media; 28 WO 2013/126533 PCT/US2013/027077 q. starting the flow of the washing buffer into the container through the plenum and allowing the washing buffer to drain out of the top liquid port until a pre-determined minimum level of debris is reached in the drained wash buffer; r. stopping the flow of the washing buffer; s. stopping the mixing means; t. opening one of the unoccupied liquid ports in the plenum to allow the washing buffer to drain out of the container and closing that liquid port of the plenum; u. starting the flow of an eluting buffer through the plenum to fill the container and closing the flow of elution buffer to the plenum; v. closing the bottom flow control valve; w. starting the mixing means; x. continue mixing the contents of the container for a pre-determined time to allow complete breaking of the binding between the target protein and the chromatography media; y. stopping the mixing means; and z. opening the bottom flow control valve and one of the unoccupied liquid ports in the plenum and collecting the elution buffer in a pre-sterilized container as a purified concentrated solution of the target protein.
    [27] 27. The method according to claim 26, wherein the steps (p) to (t) are repeated when using more than one washing buffer successively.
    [28] 28. The method according to claim 26, wherein the steps (u) to (z) are repeated when using more than eluting buffer successively in a step elution method.
    [29] 29. The method according to claim 26, wherein the steps (u) to (z) are replaced by following steps: opening the bottom flow control valve; adding the elution buffer through the top liquid port at a pre-determined rate; allowing the eluting buffer to pass through the chromatography media in the container under gravity flow; collecting the elution buffer as a plurality of timed fractions as it appears through the plenum liquid port; selecting the 29 WO 2013/126533 PCT/US2013/027077 fractions containing the highest concentration of the target protein and pooling them in a sterilized container as a purified concentrated solution of the target protein.
    [30] 30. The method according to claim 29, where the elution buffer is introduced as a gradient elution buffer.
    [31] 31. The method according to claim 27, wherein the steps (u) to (z) are replaced by the following steps: opening the top and bottom control valves; starting the flow of an elution buffer through the plenum into the container; starting the mixing means; allowing the eluting buffer to fill the container and pass through the top filter and collecting a purified form of target protein in the outflow of the eluting buffer through the top liquid port; continuing the flow of the eluting buffer and collection of a purified form of target protein until the concentration in the collected eluting buffer reaches a pre-determined minimum level. 30
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    同族专利:
    公开号 | 公开日
    HK1205140A1|2015-12-11|
    EP2817321A4|2015-12-09|
    US20160237111A1|2016-08-18|
    US8506797B2|2013-08-13|
    CA2865016A1|2013-08-29|
    US20120149885A1|2012-06-14|
    EP2817321A1|2014-12-31|
    US20130296538A1|2013-11-07|
    JP2015507024A|2015-03-05|
    US9321805B2|2016-04-26|
    WO2013126533A1|2013-08-29|
    CN104395334A|2015-03-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3615222A|1968-09-04|1971-10-26|New England Nuclear Corp|Method and apparatus for measuring the amount of a component in a biological fluid|
    US3721528A|1970-06-04|1973-03-20|L Mead|Method and apparatus for measuring the amount of a component in a biological fluid|
    DE2964379D1|1978-11-25|1983-01-27|Knut Stache|Apparatus for producing a potable liquid from sea-water, polluted water and the like by osmosis|
    US4384957A|1980-09-08|1983-05-24|Amf Incorporated|Molecular separation column and use thereof|
    SE8205033L|1982-09-03|1984-03-04|Nils M L Engstrom|DIVIDABLE CHROMATOGRAPHIPALS WITH DETECTIVE AND SIGNAL DEVICE AND ELUTION SYSTEM - IN COMBINATION OR TEMPORARY|
    US4816161A|1987-11-27|1989-03-28|The United States Of America As Represented By The Secretary Of Agriculture|Isopotential available ion extractor|
    EP0446285A4|1988-12-05|1993-03-10|Primus Corporation|Method for determination of glycated proteinaceous species|
    US4976866A|1989-04-03|1990-12-11|The Dow Chemical Company|Concentric tubular membrane device and process useful in ion exchange or absorbent processes|
    US5221483A|1990-06-29|1993-06-22|Coulter Corporation|Process and apparatus for removal of DNA, viruses and endotoxins|
    US5423982A|1994-05-31|1995-06-13|Biosepra Inc.|Liquid chromatography column adapted for in situ chemical sterilization|
    JP3737516B2|1995-06-26|2006-01-18|パーセプティブバイオシステムズ，インコーポレーテッド|Fast automated continuous flow, multidimensional molecular sorting and analysis|
    US5698004A|1996-10-21|1997-12-16|Hartmann; Richard O. W.|Method and packaging utilizing calcium cyanamide for soil treatment|
    US5855789A|1997-04-01|1999-01-05|Ntec Solutions, Inc.|Process for removing selenium from selenium-containing waste streams|
    US6048374A|1997-08-18|2000-04-11|Green; Alex E. S.|Process and device for pyrolysis of feedstock|
    US6610528B1|1997-08-26|2003-08-26|Diversa Corporation|Microbial enrichment using a container having a plurality of solid support particles|
    ES2223017T5|1997-09-23|2010-10-15|Rentschler Biotechnologie Gmbh|LIQUID FORMULATIONS OF A BETA INTERFERON.|
    US6241980B1|1997-11-04|2001-06-05|Becton, Dickinson And Company|Sample processing method using ion exchange resin|
    JP4317957B2|1998-01-16|2009-08-19|ソニー株式会社|Speaker device and electronic device incorporating speaker device|
    US5948998A|1998-02-09|1999-09-07|Alberta Research Council|Sampling device for taking sterile samples|
    JPH11309333A|1998-04-30|1999-11-09|Toyo Dynam Kk|Water supply and discharge type microbiological deodorizing apparatus|
    GB2344543B|1998-12-10|2002-11-27|Millipore Corp|Chromatography column system and method of packing a chromatography system|
    US7311880B2|1999-12-23|2007-12-25|3M Innovative Properties Company|Well-less filtration device|
    CA2397797A1|2000-01-20|2001-07-26|Free-Flow Packaging International, Inc.|System, method and material for making pneumatically filled packing cushions|
    AU2001258238B2|2000-05-12|2005-06-23|Upfront Chromatography A/S|A bed adsorption system|
    DE10034076C1|2000-07-13|2002-01-24|Merck Patent Gmbh|Connection system for plastic columns|
    SE0100714D0|2000-07-13|2001-02-28|Ap Biotech Ab|Reaction vessel and method for distributing fluid in such a vessel|
    US6727104B2|2001-02-05|2004-04-27|The Board Of Regents Of The University Of Nebraska|Microcolumn chromatographic immunoassays|
    WO2003000853A2|2001-06-20|2003-01-03|Caprion Pharmaceuticals Inc.|Protein aggregation assays and uses thereof|
    US20030091976A1|2001-11-14|2003-05-15|Ciphergen Biosystems, Inc.|Methods for monitoring polypeptide production and purification using surface enhanced laser desorption/ionization mass spectrometry|
    WO2003068402A1|2002-02-13|2003-08-21|Nanostream, Inc.|Microfluidic separation column devices and fabrication methods|
    GB0211805D0|2002-05-22|2002-07-03|Prometic Biosciences Ltd|Endotoxin-binding ligands and their use|
    GB0212513D0|2002-05-31|2002-07-10|Amersham Biosciences Ab|Packing device and method for chromatography columns|
    US7306934B2|2002-11-05|2007-12-11|Uchicago Argonne, Llc|Porous solid ion exchange wafer for immobilizing biomolecules|
    AU2003278550A1|2002-12-05|2004-06-23|Edupuganti B. Raju|Process for the extraction and isolation of insulin from recombinant sources|
    US6987263B2|2002-12-13|2006-01-17|Nanostream, Inc.|High throughput systems and methods for parallel sample analysis|
    US7270784B2|2003-04-30|2007-09-18|Aurora Discovery, Incorporated|Automated laboratory for high-throughput biological assays and RNA interference|
    GB0311854D0|2003-05-23|2003-06-25|Amersham Biosciences Ab|Chromatography column with movable adapter|
    US7003899B1|2004-09-30|2006-02-28|Lam Research Corporation|System and method for modulating flow through multiple ports in a proximity head|
    EP1685852A1|2005-02-01|2006-08-02|Fondation pour la Recherche Diagnostique|Set of disposable bags for viral inactivation of biological fluids|
    US7435350B2|2005-04-04|2008-10-14|Millipore Corporation|Intelligent system and method for automated packing of chromatography columns|
    US20070221557A1|2006-03-23|2007-09-27|Agilent Technologies, Inc.|Chromatography column having flexible substrate|
    MY145048A|2006-05-09|2011-12-15|Porex Corp|Porous composite membrane materials and applications thereof|
    US8362217B2|2006-12-21|2013-01-29|Emd Millipore Corporation|Purification of proteins|
    EP2522730A1|2007-03-02|2012-11-14|Boehringer Ingelheim Pharma GmbH & Co. KG|Improvement of protein production|
    US20100310548A1|2007-10-15|2010-12-09|Biogen Idec Ma Inc.|Methods of Manufacturing a Biologic Using a Stable Storage Intermediate|
    JP2011504596A|2007-11-26|2011-02-10|ウオーターズ・テクノロジーズ・コーポレイシヨン|Internal standards and methods for use in quantitative determination of analytes in samples|
    US20090188211A1|2008-01-25|2009-07-30|Xcellerex, Inc.|Bag wrinkle remover, leak detection systems, and electromagnetic agitation for liquid containment systems|
    US8293100B2|2009-03-13|2012-10-23|Terrasep, Llc|Methods and apparatus for centrifugal liquid chromatography|
    US8945933B2|2009-10-12|2015-02-03|The Board Of Trustees Of The Leland Stanford Junior University|Liquid chromatography-mass spectrometry methods for multiplexed detection and quantitation of free amino acids|
    US20110117538A1|2009-11-13|2011-05-19|Niazi Sarfaraz K|Bioreactors for fermentation and related methods|
    LT2501822T|2009-11-17|2017-10-25|E. R. Squibb & Sons, L.L.C.|Methods for enhanced protein production|
    US8506797B2|2011-04-10|2013-08-13|Therapeutic Proteins International, LLC|Downstream bioprocessing device|
    US8668886B2|2011-04-24|2014-03-11|Therapeutic Proteins International, LLC|Separative bioreactor|
    US8932843B2|2011-09-07|2015-01-13|Therapeutic Proteins International, LLC|Buoyant protein harvesting method|
    US8852435B2|2011-11-29|2014-10-07|Therapeutics Proteins International, LLC|Purification and separation treatment assembly for biological products|
    JP2016504024A|2012-12-11|2016-02-12|ポール テクノロジー ユーケイ リミテッドＰａｌｌ Ｔｅｃｈｎｏｌｏｇｙ Ｕｋ Ｌｉｍｉｔｅｄ|Receptors for cell culture|US8506797B2|2011-04-10|2013-08-13|Therapeutic Proteins International, LLC|Downstream bioprocessing device|
    US8852435B2|2011-11-29|2014-10-07|Therapeutics Proteins International, LLC|Purification and separation treatment assemblyfor biological products|
    WO2015008302A1|2013-07-19|2015-01-22|Biogenomics Limited|Apparatus for refolding of recombinant proteins|
    CN103756901B|2014-01-02|2015-04-29|广州中国科学院先进技术研究所|Multifunctional mini biological cell culture device|
    CN104923094A|2015-05-29|2015-09-23|安徽育安实验室装备有限公司|Solution blending device|
    CN105123270A|2015-08-31|2015-12-09|佛山市高明区更合镇鹏鹄食用菌专业合作社|Basswood ganoderma lucidum planting method|
    US20170101435A1|2015-10-13|2017-04-13|Therapeutic Proteins International, LLC|Harvesting and perfusion apparatus|
    EP3717624A1|2017-11-30|2020-10-07|Corning Incorporated|Package for batch chromatography|
    US10390551B1|2018-05-16|2019-08-27|Black Acom Ventures|Processing lupines|
    CN110694321A|2018-07-10|2020-01-17|苏州真蒂壁纸有限公司|Wallpaper slurry inlet device with filtering device|
    CN110756089A|2019-11-26|2020-02-07|湖南湘衡彩印有限公司|Raw material stirring device for paper product processing|
    CN111364734B|2020-03-11|2021-06-08|江苏天沃新能源科技有限公司|Epoxy color sand rapid extrusion stirring device|
    法律状态:
    2017-09-14| MK4| Application lapsed section 142(2)(d) - no continuation fee paid for the application|
    优先权:
    申请号 | 申请日 | 专利标题
    US13/400,627|US8506797B2|2011-04-10|2012-02-21|Downstream bioprocessing device|
    US13/400,627||2012-02-21||
    PCT/US2013/027077|WO2013126533A1|2012-02-21|2013-02-21|Downstream bioprocessing device|
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