US20100021880A1 - Automated Macromolecule Sample Preparation System - Google Patents
Automated Macromolecule Sample Preparation System Download PDFInfo
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- US20100021880A1 US20100021880A1 US12/573,438 US57343809A US2010021880A1 US 20100021880 A1 US20100021880 A1 US 20100021880A1 US 57343809 A US57343809 A US 57343809A US 2010021880 A1 US2010021880 A1 US 2010021880A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
- G01N35/1097—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers characterised by the valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/145—Ultrafiltration
- B01D61/146—Ultrafiltration comprising multiple ultrafiltration steps
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/16—Feed pretreatment
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/18—Apparatus therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
- G01N1/20—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials
- G01N1/2035—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials by deviating part of a fluid stream, e.g. by drawing-off or tapping
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
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Abstract
An apparatus for preparing a macromolecule sample from a complex liquid mixture includes a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves. The hydraulic system can be controlled by an automated controller to apply the liquid mixture to one or more filters with a pressure differential across each filter. The filters include a rough filter selected to separate, from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule. Further included is a fine filter selected to separate from the macromolecule at least a portion of one or more fine components in the mixture that are smaller than the macromolecule. A method for preparing a macromolecule sample includes automatically acquiring a liquid mixture, the mixture including a macromolecule. At least a portion of the other components in the mixture are automatically separated from the macromolecule by applying the mixture to each of one or more filters, with a pressure differential across each filter.
Description
- This application is a divisional of U.S. application Ser. No. 10/601,277, filed Jun. 20, 2003, now U.S. Pat. No. 7,601,545, which is related to U.S. application Ser. No. 10/601,096 filed on Jun. 20, 2003, now abandoned; U.S. application Ser. No. 10/601,181 filed on Jun. 20, 2003, now U.S. Pat. No. 7,341,652; U.S. application Ser. No. 10/600,177 filed on Jun. 20, 2003; and U.S. application Ser. No. 10/601,083 filed on Jun. 20, 2003, now U.S. Pat. No. 7,169,599.
- The entire teachings of the above applications are incorporated herein by reference.
- Analysis of macromolecules in complex mixtures is challenging in many chemical and biochemical processes. For example, the analysis of a macromolecule product, e.g., a protein, typically involves first preparing a sample of a macromolecule from a complex mixture for analysis.
FIG. 1 depicts an example of amacromolecule preparation process 100, which involves taking a sample from a complex liquid mixture, e.g. a biofluid in abioreactor 102, separating amacromolecule 104 from other components in the mixture, and processing it to deliver a preparedmacromolecule 104′ for analysis atanalyzer 106. - Effective process control generally requires accurate and frequent sampling, yet sampling of an operating bioreactor is associated with numerous problems, particularly contamination from sampling. For example, a bioreactor fluid typically contains, in addition to the macromolecule of interest, components such as salts, nutrients, proteins, peptides, cells, cell components, biopolymers such as polysaccharides, and the like, all of which can confound analysis of the desired products. Sampling can introduce, for example, foreign or wild bacteria into a bioreactor, which can compete with the process bacteria in the bioreactor fluid. Other contaminants, e.g., chemical contaminants, can affect the growth of the process bacteria and can confound the analysis of process components in the bioreactor fluid. Contamination can also affect the sampling and analysis apparatus. For example, wild or process bacteria can colonize the sampling/analysis system, or the system can accumulate other components form the biofluid, e.g., as salts, nutrients, proteins, peptides, cells, cell components, biopolymers such as polysaccharides, all of which can confound analysis of the desired products. Additionally, frequent sampling can lead to build-up of the molecule or molecules being analyzed, which can lead to inaccuracy.
- In particular, the problem of “backflow”, i.e., liquid cross-contamination, is especially difficult when interfacing two fluidic systems. Simple valve interfaces are inadequate because valves typically have crevices, joints, dead volume, and the like, where contaminants can lodge and accumulate, only to be released during another sample cycle. Additionally, valves can fail and allow undesirable contamination to occur before much measurable fluid has leaked. More complex valved interfaces are known, but some are costly and still suffer some of the problems of simple valve systems, while other examples are unsuitable for high pressure systems. Needle/septa interfaces are known to avoid backflow but have issues with septa lifetime, needle contamination during transfer, and are particularly troublesome for frequent, automated sampling of larger volumes. Furthermore, septa replacement itself opens the system for contamination.
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FIG. 2 depicts typical steps that can be included in a macromolecule sample preparation process operating on amixture 202. If the macromolecule is endogenous, i.e., is at least partly contained in cells, anoptional lysing step 204 opens the cells so that themacromolecule 104 can be separated.Separation step 206 separatesmacromolecule 104 fromrough components 207 andfine components 213.Rough components 207 can include, for example,insoluble cells 208,cellular fragments 210,soluble molecules 212 which are larger thanmacromolecule 104, and the like.Fine components 213 can includesalts 214 andsoluble molecules 215 that are smaller thanmacromolecule 104, and the like. The concentration of ions such as salts and hydrogen (i.e., pH) are adjusted instep 216. Instep 218, the molecule can be denatured, i.e., can be heated and/or combined with a denaturingagent 220, producing preparedmacromolecule 104′, which is typically at an increased concentration compared tomacromolecule 104. - The various steps used for protein preparation in the prior art involve separation of components through labor intensive centrifugation or time-intensive matrix chromatography. Matrix chromatography uses expensive columns that can be prone to plugging when used with complex mixtures that include insoluble or precipitation-prone components. Centrifugation can be effective but can cause contamination problems as there is no way to readily isolate a sample from the environment during the various sample transfers typically employed, and the size of the centrifuge limits the amount of macromolecule that can be prepared at one time. Thus both methods are low throughput in terms of amount of macromolecule that can be prepared.
- Additionally, both methods are low throughput in terms of the sampling frequency, as the time from sample extraction from a complex bioreactor mixture to analysis of the macromolecule can easily be four hours or more. Such a slow analysis time leads to poor optimization of reactor processes, resulting in lowered yields, increased costs, increased purification demands, and increased amounts of potentially hazardous biological waste.
FIG. 3 depicts a hypothetical example comparing two sampling frequencies, wherein a lower sampling frequency versus time (squares) can miss details in the level of a desired macromolecule versus time (solid line) in a reaction mixture, compared to a higher sampling frequency (circles). For example, the lower sampling frequency can miss the maximummacromolecular concentration 302 by measuring onlylower concentration 300. - Electrophoresis is an analytical technique commonly used to separate molecular species, e.g., peptides, proteins, oligonucleotides, small organic molecules, and the like. The molecules, in a separation medium, e.g., a solution or a gel matrix, separate under an applied electric field according to their electrophoretic mobility, which is related to the charge on each molecule, its size, and the viscosity of the separation medium.
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FIG. 10 depicts the separation of asmall molecule 1002 and alarge molecule 1004, each with the same net positive charge, and a small negativelycharged molecule 1006. Application ofelectric field 1008 causes differential motion of the charged molecules according to their electrophoretic mobilities, withcations anode 1010. In the ideal case, theanions 1006 move to thecathode 1012, though experimentally a phenomenon known as electroosmotic flow can reduce or reverse the anion to cathode motion. - In capillary electrophoresis (CE), the separation is performed in a capillary tube having an internal diameter on the order of tens to hundreds of micrometers. In such small tubes the heat generated by the electric field is easily dissipated, so that high electrical fields can be used, leading to fast separations.
FIG. 11 depicts a schematic of anelectrophoresis apparatus 1100. Aninlet vessel 1102 and anoutlet vessel 1104 are connected by acapillary column 1106. The vessels and the capillary contain a buffer with an appropriate electrolyte. Upon loading a sample containing the analyte of interest at the inlet vessel, an electric field provided by a highvoltage power supply 1108 causes the various molecules in the sample to separate, whereupon they can be detected by adetector 1110. - While capillary electrophoresis is powerful and versatile, it is sensitive to variations in acidity (pH), ionic strength, temperature, viscosity and other physical characteristics of the mixture, properties intrinsic to the analytes being studied, and contamination issues. Furthermore, small capillaries are physically fragile and are not suited to high-throughput separations, being easily plugged from the many macromolecules and debris in a complex mixture. In particular, rapid separation and analysis of macromolecules from complex liquid mixtures, for example, during the analysis of proteins produced in a bioreactor, is especially challenging.
- In one example of CE technology a fragile, small diameter capillary is repeatedly applied by robotics to a series of distinct inlet vials. The repetitive motion can easily break the CE column. Column replacement requires time-consuming recalibration of the robotic motion. Another example of CE technology employs microchannels etched into a glass chip. While this hardware is durable, the separation efficiency is limited by the length of CE channel that can be fabricated on a chip. Attempts to extend the channel length by increasing channel density on a chip generally restrict high electric fields from use, increasing separation time. Also, the throughput of this technique is limited. Furthermore, sample transfer as practiced in both the robotic capillary technique and the chip technique expose the analytic solution to undesirable environmental contamination.
- A system and method are provided for preparing a sample of a macromolecule from a complex liquid mixture. In particular applications, methods and apparatus are provided for separating a protein from other components in a complex bioreactor liquid mixture, denaturing the protein, and providing a prepared sample of the protein suitable for analysis.
- An apparatus for preparing a macromolecule sample includes a hydraulic system, comprising a pump and one or more valves adapted for control by an automated controller. The hydraulic system can be controlled by an automated controller to apply the liquid mixture to one or more filters with a pressure differential across each filter.
- The filters may include a rough filter selected to separate, from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule. Further included is a fine filter selected to separate from the macromolecule at least a portion of one or more fine components in the mixture that are smaller than the macromolecule.
- The fine filter may be selected to separate, at least in part, a macromolecule in a liquid mixture from one or more salt components in the mixture.
- A lysis unit may be provided to lyse cells in a liquid mixture that includes cells and a macromolecule. A rough filter is selected to separate from the macromolecule, at least a portion of components in the mixture that are larger than the macromolecule, the components including insoluble lysed cell components.
- A method for preparing a macromolecule sample includes automatically acquiring a liquid mixture, the mixture including a macromolecule. At least a portion of the other components in the mixture can be automatically separated from the macromolecule by applying the mixture to each of one or more filters, with a pressure differential across each filter.
- The methods and apparatus disclosed herein provide significant advantages to preparing and analyzing a macromolecule from a complex liquid mixture. The hydraulic system allows convenient exclusion of the macromolecule preparation process from contamination by the external environment, in contrast to centrifugation. The use of filters to separate salts and components different in molecular weight from the macromolecule reduces cost, improves reliability, and increases throughput compared to chromatographic methods. Furthermore, the combination of high throughput with provision of automatic control allows the preparation and analysis of macromolecules at a higher rate, enabling improved analysis and control of time-sensitive bioreactor processes.
- The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
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FIG. 1 depicts an example of amacromolecule preparation apparatus 100. -
FIG. 2 depicts typical steps that can be included in a macromolecule sample preparation process 200 operating on amixture 202. -
FIG. 3 depicts a hypothetical example comparing two sampling frequencies, wherein a lower sampling frequency versus time (squares) can miss details in the level of a desired macromolecule versus time (solid line) in a reaction mixture, compared to a higher sampling frequency (circles). -
FIG. 4 depicts a schematic of steps that can be included in preparing a macromolecule sample. -
FIG. 5 depicts anapparatus 500 that can conduct the steps inFIG. 4 . -
FIG. 6 depicts thelysis module 508. -
FIG. 7A depictsrough separation circuit 700, containingaseptic separation circuit 752. -
FIG. 7B depicts an asepticfluidic interface apparatus 752. -
FIG. 7C depictsapparatus 752 with arelief valve 758,overflow reservoir 760, andfilter 766. -
FIG. 7D depictsapparatus 752 wherein inlet andoutlet valves -
FIG. 8 depicts desalination/fine filtration circuit 800. -
FIG. 9 depictsdenaturation circuit 900. -
FIG. 10 depicts the separation of asmall molecule 1002 and alarge molecule 1004. -
FIG. 11 depicts a schematic of anelectrophoresis apparatus 1100. -
FIG. 12 depicts steps that can be included in analysis by stationary capillary electrophoresis. -
FIG. 13 depicts a stationarycapillary electrophoresis circuit 1300 that can be controlled to conduct the steps inFIG. 12 . -
FIG. 14 depicts a more detailed schematic of the capillary electrophoresis circuit. -
FIG. 15 depicts a block diagram of apreferred apparatus 1500. -
FIG. 16 is a block diagram of a system including the subsystems described above and a local user interface for providing operational input. -
FIG. 17 is a network diagram including multiple systems ofFIG. 16 connected to remote computing devices across a network. -
FIG. 18 is a block diagram of an industry model in which a business may distribute the system ofFIG. 16 . -
FIG. 19 is a generalized flow diagram of a business method used in the industry model ofFIG. 18 . -
FIG. 20 is a flow diagram of a process used by the manufacturer of the system inFIG. 18 . -
FIG. 21 is a flow diagram of a process used by a customer inFIG. 18 . -
FIG. 22 is a detailed flow diagram of process steps in the flow diagram ofFIG. 21 . -
FIG. 23 is a flow diagram of a process also used by the customer inFIG. 18 . - A description of preferred embodiments of the invention follows.
- The methods and apparatus disclosed herein are generally related to analyzing a sample of a molecular analyte, e.g., a macromolecule, from a complex liquid mixture.
- The invention has particular application to automated methods and apparatus for capillary electrophoretic analysis macromolecules, e.g., proteins, from a complex bioreactor liquid mixture.
- Automated Macromolecule Preparation
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FIG. 4 depicts a schematic of steps that can be included in preparing a macromolecule sample. The liquid, typically aqueous,mixture 202 contains themacromolecule 104, and can also containfine components 213, e.g., salts, molecules smaller than the macromolecule, and the like; andrough components 207, e.g., cells, cell fragments, particulate contaminants, molecules larger than the macromolecule, and the like. -
Macromolecule 104 can be dissolved in the liquid mixture, or can be partially contained in cells, as depicted.Optional lysis step 204 lyses at least a portion of the cells to releasemacromolecule 104. Lysing can be conducted using any method of lysing cells well-know to the art, for example, heating, sonic disruption, addition of lysing agents, e.g., detergents, changes in ionic strength, e.g., by dilution with water or combination with a lysis buffer, and the like. - A
rough separation step 410 applies the liquid mixture to arough filter 412, and a pressure differential acrossfilter 412 directs at least a portion of the liquid,macromolecule 104, and thefine components 213 through the filter, separating at least a portion ofrough components 207 atrough filter 412.Rough filter 412 can be selected to remove at least a portion of components that are larger than the macromolecule, e.g., greater in diameter or molecular weight. Preferably,rough filter 412 removes components that are greater in molecular weight than the molecular weight of the macromolecule by about 150%, more preferably about 125%, even more preferably about 110%, and most preferably, about 105%. In other aspects,rough filter 412 can be selected to remove at least a portion of components that are greater in diameter than about 60 μm, more preferably about 30 μm, even more preferably about 10 μm, or most preferably about 5 μm. - A
fine separation step 414 applies the liquid mixture to afine filter 416, and a pressure differential across the filter directs at least a portion of the liquid and thefine components 213 through the filter to waste 418, separating at least a portion ofmacromolecule 104 at the filter.Fine filter 416 can be preferably selected to remove at least a portion of components that are smaller than the macromolecule, e.g., salt components. Preferably, the fine filter removes components that have a molecular weight that is a fraction of the molecular weight ofmacromolecule 104 of preferably about 50%, more preferably about 75%, even more preferably about 90%, and most preferably, about 95%. - One skilled in the art will recognize that the separation steps can be conducted in any order, for example,
fine separation 414 can be conducted beforerough separation 410. Preferably, the steps are conducted in the order depicted inFIG. 4 . - The liquid mixture remaining at the filter now has a greater concentration of
macromolecule 104, and a reduced concentration of solublefine components 213, e.g., salts. Instep 420, the liquid mixture can optionally be combined withadditional buffer 422 to adjust the concentration ofmacromolecule 104 and other components, e.g., ions. Buffer 422 can contain pH buffer, other ionic buffers, filtration aids, denaturation agents, organic solvents, pure water, and the like. - One skilled in the art will recognize that in
step 420,buffer 422 can be added to either side offilter 418. Preferably, buffer 422 can be directed throughfine filter 416 by applying pressure differential acrossfilter 416. This can dislodge portions ofmacromolecule 104 that can become attached tofine filter 416 infine filtration step 414. Also, one skilled in the art will appreciate thatsteps macromolecule 104 fromfine components 213. - The concentration of the salt components is preferably reduced in
steps 414 and/or 420 by at least 50%, or more preferably, by at least 75%, or most preferably, by at least 90%. - The concentration of the macromolecule is preferably increased by
steps 410 and/or 414 by at least 50%, or more preferably, by at least 100%, or most preferably, by at least 200%. -
Optional denaturation step 218 accepts the liquid mixture and at least partially denaturesmacromolecule 104 toprepared macromolecule 104′. The denaturation step can employdenaturing agent 220 and/or a heating step. Thedenaturing step 218 heats the macromolecule withdenaturation agent 220 to, for example, from about 70° C. to about 100° C. for about 60 to about 600 seconds; more preferably, from about 80° C. to about 100° C. for about 120 to about 450 seconds; or even more preferably, from about 85° C. to about 95° C. for about 250 to about 350 seconds. Preferably,denaturation step 218 heats the macromolecule and the denaturation agent to about 90° C. for about 300 seconds. -
FIG. 5 depicts anapparatus 500 that can conduct the steps inFIG. 4 . Samplingvalve 502 opens toreactor site 102 throughrough filter 412. Pump 506 draws the sample throughoptional lysis unit 508, wherelysis step 204 can be performed, and then throughrough filter 412, removing rough components from the liquid mixture, i.e.,step 410.Valve 502 closes to filter 412 andvalves pump 506 to reverse direction and drive the liquid mixture againstfine filter 416, passing fine components throughfine filter 416 andvalve 516 to waste 514, i.e.,step 414. -
Valve 516 can be closed and pumps 506 and 518 can be operated cooperatively, i.e., pump 506 pushing and pump 518 pulling, to direct a portion of the liquid containingfine components 213 throughfine filter 416. When a portion of the liquid mixture has traversedfilter 416,valve 510 closes andvalve 516 opens, and pump 518 reverses to direct that portion towaste site 514, after whichvalve 516 closes. As alternatives, only one ofpumps fine components 213 throughfine filter 416. - To perform
step 420,valve 520 can open and pump 506 can direct the remaining liquidmixture containing macromolecule 104 todenaturation vessel 526. Preferably, however,valve 522 opens and pump 518 draws a portion of buffer fromreservoir 524.Valve 522 closes,valve 510 opens, and pump 518 directs the buffer throughfilter 416. Preferably, pumps 518 and 506 operate cooperatively to direct the buffer throughfilter 416, and pump 506 then directs the mixture throughvalve 520. Addition of the buffer through the filter can dislodge portions ofmacromolecule 104 that may become associated withfine filter 416 instep 414. - Next, pump 506 drives the combination of
macromolecule 104 tooptional denaturation vessel 526, i.e., performingstep 218, whereupon the denaturedmacromolecule 104′ can be then directed toanalysis site 106. - One skilled in the art will recognize that variations are possible in
apparatus 500. For example, one or more of the valves, depicted as two-way valves, could be combined into a single multifunction valve. The placement of various elements can be varied; for example,valve 502 can be placed beforerough filter 412, and the like. -
FIG. 6 depictslysis module 508.Pump 600 operates to drawliquid mixture 202, including cells, fromreactor 102 throughvalve 602.Valve 602 closes,valve 604 opens, and pump 600 draws a lysis buffer fromreservoir 606, lysing at least a portion of the cells inmixture 202. Pump 600 then directs lysedmixture 202′ throughvalve 608 to second stagerough filter 412, preferably through a first stagerough filter 610. - Aseptic Fluidic Interface Coupled to Macromolecule Preparation Apparatus
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FIGS. 7A-C , 8, and 9 depict a more detailed schematic of one embodiment of the invention. Acontroller 701 is coupled to the various pumps, valves, sensors, heating and cooling elements to provide automatic control of the system. The controller may, for example, be a special purpose microprocessor based system or a general purpose computer. -
FIG. 7A depictsrough separation circuit 700. The liquid mixture can be drawn frombioreactor sample site 102 by openingvalves valves site 102 includes a liquid/air trap region 715, includingwaste valve 704,waste site 720, and flowsensor 718. - By operating
rough pump 506, which is preferably a syringe pump, a sample ofliquid mixture 202 can be drawn fromreactor site 102 at a flow rate of about 2 mL/min to reach a volume of about 10 mL. This action draws the liquid mixture through initial filtering steps involving first and second stagerough filters rough filter 610 can be selected to remove rough components 60 μm or larger, and second stagerough filter 412 can be selected to remove rough components 5 μm or larger. By closingvalve 502, the rough-filtered liquid mixture can be isolated in the syringe chamber ofrough pump 506. -
Rough separation circuit 700 includes a number of valved reservoirs to supply various standards, buffers and cleaning agents, including size standard reservoir/valve 728/706, cleaning solution reservoir/valve 730/731, run buffer reservoir/valve 732/733, isopropyl alcohol reservoir/valve 734/735, and clean water reservoir/valve 736/737, wherein the amount of buffer drawn can be measured atflow sensor 738. One skilled in the art will recognize that a range of useful solvents and buffers can be employed for cleaning, for standardization, for storage, for aiding filtration, and the like. For example, a size standard buffer can be used in a calibration run of the apparatus to determine the separation performance of the apparatus. The size standard buffer can contain a range of components of known size, at known concentrations, i.e., where size can include weight, molecular weight or diameter of the components. The apparatus can be controlled to self-clean by employing a cleaning solution, preferably cleaning the apparatus between each run of a sample of the liquid mixture. Organic solvents, e.g., isopropyl alcohol, can be employed as cleaning aids or to fill the fluid-handling elements of the apparatus when the apparatus is inactive for an extended period. Clean water and run buffer can be used to dilute the liquid mixture, to adjust the concentration of ions, to aid fluid flow, and the like. -
Rough separation circuit 700 also includes a number ofpressure transducers steam flow sensor 750; andwaste site 752. The downstream boundary ofrough separation circuit 700 isvalve 714, through which the liquid mixture can be directed to the desalination/fine filtration circuit 800. - Aseptic Fluidic Interface Apparatus
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FIG. 7B depicts in detail an asepticfluidic interface apparatus 752 that can be used to provide a fluidic interface between two bioprocess systems, a biofluid source site and a biofluid process site, e.g.,bioreactor 102 and apparatus 753 (the balance of rough separation circuit 700). -
Inlet valve 703 is coupled to a biofluid source site, e.g.,bioreactor 102. Asampling conduit 754 extends frominlet valve 703 tooutlet valve 702.Outlet valve 702 is coupled to a biofluid process site, e.g., theapparatus 753.Trap 715 is located atsampling conduit 754.Waste valve 704 is located at awaste conduit 756, and extends fromconduit 754 towaste site 720. A wash fluid source is coupled to at least one of the inlet andoutlet valves valve 728/706 is coupled throughoutlet valve 702. The valves are all adapted for automatic control. -
Trap 715 is a portion ofconduit 754 that is lower in height than either end ofconduit 754, e.g., so that fluid inconduit 754 tends to collect there under gravity. The lowest portion of the trap is generally below the lowest end ofconduit 754 by a multiple of the conduit internal diameter (or average internal diameter) of at least about 3 times, more typically, at least about 5 times, even more typically about 10 times and preferably at least about 20 times. The trap is typically a U-shaped portion of conduit, and the ends, e.g., at input andoutput valves Waste valve 704 can be coupled to any point in the trap but is typically coupled to the lowest point of the trap. The volume of the conduits bounded byvalves - Aseptic
fluidic interface apparatus 752 can control fluid transfer between the two systems so that fluid is transferred in a particular direction at particular times, e.g., only frombioreactor 102 toapparatus 753 during sample collection. For example,automated controller 701 can communicate electronically with the valves, collecting fluid sample frombioreactor 102 by openinginlet valve 703, directing the sample toapparatus 752 by openingoutlet valve 702 whilewaste valve 704 is closed.Reactor 102 andapparatus 753 can be isolated by closinginlet valve 703 andoutlet valve 702, andtrap 715 andsampling conduit 754 can be drained towaste site 720 by openingwaste valve 704. Before transferring a sample, preferably as part of each sample cycle,sampling conduit 754 can be cleaned by openingwaste valve 704 and directing a wash fluid through at least one of inlet andoutlet valves waste valve 704 towaste site 720, e.g., fromwash reservoir 728 throughoutlet valve 702. Anoptional flow sensor 718 can be located inapparatus 752, typically atsampling conduit 754 orwaste conduit 756, preferably atwaste conduit 756 betweentrap 715 andwaste site 720.Flow sensor 718 can be employed bycontroller 701 to sense for fluid flow, particularly when the two biofluid sites, e.g.,bioreactor 102 andapparatus 753, are isolated. If flow is sensed during isolation, a possible backflow condition can be indicated. As used herein, “backflow” means undesirable fluid flow in the system, e.g., due to failure ofvalves -
Apparatus 752 preferably controls fluid transfer so the transfer is aseptic. As used herein, aseptic means that the integrity of the sample is maintained. For example, the sample can contain microorganisms, macromolecules, fluids, salts, etc., e.g., those present inbioreactor 102. However, external contaminants, e.g., microorganisms, macromolecules, and other chemical, biological or particulate contaminants from the external environment can be excluded from the apparatus. Furthermore, in the wash process, the residue from each previous sample can be removed from the apparatus. For example, when a process is sampled over time to determine the concentration versus time of a macromolecule, it can be desirable to remove traces of the macromolecule from a previous sample so that the accuracy of a future sample is not affected. Similarly, microorganisms can be removed to avoid a microorganism lodging in the apparatus and excreting amounts of the macromolecule which could affect accurate measurement. -
FIG. 7C depicts an asepticfluidic interface apparatus 752 with arelief valve 758,overflow reservoir 760, and filter 766, all located onrelief conduit 764.Flow sensor 718 can optionally be located onrelief conduit 764 as shown.Relief conduit 764 extends fromwaste conduit 756 at a point betweentrap 715 andwaste valve 704, and ends in fluid communication with the external environment throughfilter 766.Filter 766 excludes at least a portion of external contaminants from at least a portion of the relief conduit. The filter can be located anywhere betweenvalve 758 and the distal end ofconduit 764, preferably at the end as depicted inFIG. 7C . Typically, the filter is selected to exclude microorganisms and particulate contaminants, e.g., the filter excludes contaminants having a diameter greater than about 1 μm, more typically greater than about 0.5 μm, and preferably greater than about 0.2 μm.Overflow reservoir 760 can be located anywhere betweenvalve 758 and the distal end ofconduit 764, preferably betweenflow sensor 718 andvalve 758 as depicted inFIG. 7C .Flow sensor 718, which can be located anywhere inapparatus 752, is typically atwaste conduit 756 orrelief conduit 764. If the overflow elements are employed,flow sensor 718 is typically atconduit 764 as shown, preferably betweenvalve 758 andreservoir 760. Asecond filter 768 can be employed atconduit 764, e.g., betweenvalve 758 andtrap 715.Filter 768 is sized smaller thanfilter 766, i.e., excludes at least a portion of contaminants that pass throughfilter 766. For example,filter 768 is typically sized to exclude particles less than about 75% of the size excluded byfilter 766, more typically, less than about 50% of the size excluded byfilter 766, an preferably, less than about 25% of the size excluded byfilter 766. -
Automated controller 701 directs wash fluid into the sampling conduit through at least one of inlet andoutlet valves outlet valve 702. A wash fluid can be one or more fluids, e.g. a gas, a vapor, a liquid, a supercritical fluid, a combination, and the like. For example, gases can include compressed air, oxygen, nitrogen, noble gases nitrous oxide, ethylene oxide, carbon dioxide, and the like; vapor can include steam or vaporized solvents; liquids can include water, aqueous solutions of buffers, antiseptics, detergents, and the like; solvents, e.g., organic solvents such as alcohols, ethers, ketones, polar aprotic solvents, and the like; and supercritical fluids can include carbon dioxide, water, and the like. Typically, the wash fluid is sterile. More than one fluid can be employed, for example, the apparatus can be flushed with an aqueous cleaning solution, steam, and then dry compressed air. Preferably, at least one wash fluid is antiseptic or sterilizing, i.e., is able to kill microorganisms. - The automated controller can direct the wash fluid along a number of paths. Starting from
reservoir 728, the fluid can be directed throughoutlet valve 702, From there, it can be directed throughvalve 704 to waste 720, or throughvalve 758 to overflowreservoir 760, or throughinlet valve 703 back intoreservoir 102. -
Automated controller 701 is typically employed with the wash fluid to reduce bacterial count, macromolecule contamination, and/or other contamination to acceptable levels. An “acceptable level” of contamination is that level of contaminants that do not have a measurable adverse effect on the bioprocess site. For example, macromolecule contamination is typically reduced below the detection level of an analysis circuit coupled to the system. Contamination of any portion of the system can be measured using rinse water, e.g., by filling that portion with rinse water, letting stand at 20° C. for 1 minute, and then analyzing the rinse water for the concentration of macromolecules or bacteria. Typically, washing can reduce bacterial contamination, e.g., the number of bacterial colony forming units per milliliter of rinse water to less than about 100, more typically, to less than about 50, and preferably, to less than about 10. Generally, washing can reduce macromolecule contamination in rinse water to less than about 10 parts per million (ppm), more typically, to less than about 1 ppm, even more typically, less than about 0.1 ppm, and preferably, to less than about 0.01 ppm. -
FIG. 7D depicts still other options forapparatus 752. One or more valves, e.g., inlet andoutlet valves outlet valve 703. The wash fluid can then be directed out of the remaining output of double isolatedoutlet gate valve 703 towaste site 720, or alternatively, intosampling conduit 754, up to double isolatedinlet gate valve 702, and then to wastesite 720. -
FIG. 8 depicts desalination/fine filtration circuit 800, which followsrough separation circuit 700 fromFIG. 7A . The liquid mixture, now separated from at least a portion of rough components, can be accepted fromrough separation circuit 700.Valves branch valves Rough pump 506 can be emptied at about 1.5 mL/min to a total of about 7.5 mL. At the same time,fine pump 518 can be controlled to draw the plunger back at about 1.5 mL/min. This creates the force to direct the liquid mixture out ofrough pump 506, acrossfine filter 416 and into the syringe chamber offine pump 518.Fine filter 416 andrough pump 506 retain the macromolecule while passing a solution of fine, e.g., salt components through and into the syringe chamber offine pump 518. - Next,
valves valve 516 can be opened.Fine pump 518 can be activated to push the syringe plunger contents (about 7.5 mL) at about 1.5 mL/min and direct at least a portion of the liquid mixture containing fine components, e.g., sodium chloride, to wastesite 514.Flow sensor 515 can be employed to monitor the liquid sent to waste. - Next, a desalination buffer can be loaded by opening sample
buffer feed valve 522, andmanifold feed valve 806 and drawing sample buffer fromreservoir 524 intofine pump 518. About 7.5 mL of sample buffer can be drawn fromreservoir 524, after which samplebuffer feed valve 522 andmanifold valve 806 are closed. The amount of buffer drawn can be measured atflow sensor 808. Other buffers can be provided, for example,valved reservoir 810/811 can provide, e.g., a pH buffer, pure water, etc. - As described in
FIG. 5 , to performstep 420,valve 520 can open and pump 506 can direct the remaining liquidmixture containing macromolecule 104 todenaturation vessel 526. - Next,
valves valves denaturation circuit 900, wherevalves FIG. 9 ).Pumps rough pump 506 will push a total of about 2.5 mL, whilefine pump 518 will push a total of about 7.5 mL. -
Fine separation circuit 800 also includes a number ofpressure transducers Valve 802 can provide compressed air or steam for cleaning or purging the system. -
FIG. 9 depictsdenaturation circuit 900.Denaturation vessel 526 is preferably a 10 mL stainless steel vessel that contains both heating and cooling coils. The mixture can be heated until at least partial denaturation occurs, for example, heating to at least about 70° C. for about 90 seconds, or more preferably, heating to about 90° C. for about 300 seconds. Subsequently, the cooling coil can be operated to cool the sample to about 25° C. - Upon denaturation, denatured
macromolecule 104′ can be removed by activatingdenaturation pump 916, openingvalve 902 and opening eithervalve vessel 526. The concentration of ions in the mixture, for example, the concentration of hydrogen ions, i.e., the pH of the mixture can be monitored atsensor 909. Subsequently, the mixture containing denaturedmacromolecule 104′ can be passed through a precipitatefilter 932 by closingvalves valves 908 and 924.Pumps filter 932. Precipitatefilter 932 can be selected to exclude insoluble components that can precipitate during the denaturation step. Preferably,filter 932 excludes insoluble components greater in diameter than about 1 μm, more preferably about 0.6 μm, and most preferably about 0.45 μm. Once the mixture is filtered of at least a portion of precipitate and is in the syringe chamber ofpump 930, valve 924 can be closed and analysissite feed valve 928 can be opened, and pump 930 can direct the mixture containingprepared macromolecule 104′ toanalysis site 106. -
Denaturation circuit 900 also includesdenaturation vessel valve 934; compressed air orsteam inlet valves sensors waste sites pressure transducers - Stationary Capillary Electrophoresis
-
FIG. 12 depicts steps that can be included in analysis by stationary capillary electrophoresis. Aliquid sample 1202 includes one or moremolecular analytes 1204 andother components 1206. As used herein, a molecular analyte is any molecule that is soluble or suspended in the liquid sample and has an electrophoretic mobility that is different fromother components 1206 in the liquid sample. The molecular analyte can be any molecule, e.g., inorganics, small molecule organics, biomolecules, synthetic polymers, biopolymers, proteins, peptides, amino acids, nucleic acids, and the like. Preferably, the molecular analyte is a macromolecule, i.e.,macromolecule 104, and most preferably, separatedmacromolecule 104′. - The liquid sample is introduced to the end of an
electrophoresis column 1106 by pressure or electro-kinetic injection instep 1208. Abuffer 1210 that contains, for example, electrolytes know to the art to be suitable for capillary electrophoresis can be added. Additional components of the buffer known to the art can include organic solvents, e.g., acetonitrile; additives which can act to reduce electroosmotic flow; electrophoretic flow modifiers, i.e., ionic agents that complex with molecular analytes to change electrophoretic mobility; spectroscopic or radioactive tags; and the like. - A voltage differential is applied across the column in
step 1212, causing the molecular analyte to separate from other components. Inoptional step 1214, pressure differential can be applied to the column to cause liquid in the column to flow. For example, the effective length of the column can be increased, e.g., by conducting a partialelectrophoretic separation step 1212, pausing, performingoptional step 1214 to flow liquid in the column in a direction contrary to the electrophoretic flow, and then resumingelectrophoretic flow step 1212. One skilled in the art will appreciate thatsteps 1212/1214 can be repeated numerous times. Once the molecular analyte is separated, it can be analyzed indetection step 1216, either while still in the column bydetector 1110, as depicted, or after extraction from the column. -
FIG. 13 depicts a stationarycapillary electrophoresis circuit 1300 that can be controlled to conduct the steps inFIG. 12 . Theinlet chamber 1102 is supplied with the liquid sample bypump 1302 throughinlet valve 1304 fromliquid sample source 1301. Optional precipitatefilter 1303 can be employed to separate insoluble precipitates from the liquid sample by employingpump 1302 to apply the liquid sample to filter 1303 with a pressure differential across the filter. - Each chamber can be supplied independently by a
buffer reservoir 1306 throughvalves valves waste sites Pump 1320 can draw filtered air throughair inlet valve 1326 andair source 1328, and independently direct the air tochambers valves electrophoresis power supply 1108, andoptional detector 1110 are adapted to be controlled by anoptional controller 1330. - The
capillary electrophoresis column 1106 is coupled with the interior of each chamber so that liquid in each chamber can be placed in fluid communication with the respective end ofcolumn 1106. Preferably, the column has a length of at least about 20 centimeters, more preferably at least about 30 centimeters, and most preferably, at least about 50 centimeters. - The
optional detector 1110 can be any detection method known to the art for detection of molecular analytes, for example, absorbance/transmission of radiation, e.g. ultraviolet/visible light; fluorescence detection; refractive index detection; electrochemical detection; mass spectrometric detection; detection of electron or nuclear magnetic resonance; flame ionization detection; binding, e.g., in an enzyme or antibody assay; detection of a spectroscopic or radioactive label; and the like. Whenoptional detector 1110 is an optical detector, it can be configured to detect molecular analytes that areinside column 1106. Or, fractions can be collected from the molecularanalytes exiting column 1106, e.g., atoutlet chamber 1104, and the fractions can be analyzed separately from the column. - Additionally, each chamber can be barometrically sealed, i.e., they can be pressurized or depressurized. For example,
valves valve 1322 can be opened, and pump 1320 can pressurizeinlet chamber 1102. If the pressure inchamber 1102 is greater than the pressure inoutlet chamber 1104, a high to low pressure differential results across the length ofcapillary electrophoresis column 1106. Alternatively, pump 1320 can reduce the pressure inchamber 1102 to less than the pressure inchamber 1104, resulting in a low to high pressure differential, which can direct liquid fromchamber 1102 throughcolumn 1106 tochamber 1104. Or, the valves can be configured so thatpump 1320 can pressurize or depressurizechamber 1104. Optionally, separate independent pumps can be coupled with each chamber and the pumps can operate cooperatively, one pulling and the other pushing, to create a pressure differential acrosscolumn 1106. Creation of a pressure differential betweenchamber 1102 tochamber 1104 throughcolumn 1106 can be employed to fill, purge, or clean the column, or to move fluid through the column, e.g., performstep 1214. -
FIG. 14 depicts a more detailed schematic of the capillary electrophoresis circuit. The line betweeninlet valve 1304 andinlet chamber 1102 can be supplied with compressed air byfiltered air supply 1402 throughvalve 1404. An additionaloptional air source 1406 andvalve 1408 is provided that can be employed to purgeinlet chamber 1102 and/or the waste line betweenvalve 1312 andwaste 1316. The waste from both chambers is provided withflow sensors Pressure transducers reservoir 1306 are provided avalve 1418 and additional valved reservoirs 1420/1421, 1422/1423, and 1424/1425. These reservoirs can supply water, buffer, cleaning solution, solvents, electrolytes, and the like, the flow of which can be sensed atflow sensor 1426. Additional buffer can be supplied to theoutlet chamber 1104 byreservoir 1428 throughflow sensor 1430 andvalve 1432. - Additionally, the level of fluid in
chambers level sensors column 1106 by the electrophoresis current can be removed by aheat exchanger 1438, which can be, for example, a cooling element, a thermoelectric element, and the like. Also,optional degas unit 1440 can be employed to remove at least a portion of dissolved gases. - Automated System for On-Line Aseptic Sampling of Bioreactor Fluids, Macromolecule Separation, Denaturation, and Capillary Electrophoretic Analysis
-
FIG. 15 depicts a block diagram of apreferred apparatus 1500, which couplesrough filtration circuit 700, desalination/fine filtration circuit 800,denaturation circuit 900, andcapillary electrophoresis circuit 1300 into an integrated system. That is,rough filtration circuit 700 inputs a complexliquid mixture 202 comprising a macromolecule, and separates the macromolecule from rough components. The mixture is directed to desalination/fine filtration circuit 800, and the macromolecule is separated from at least a portion of fine components, including salt components. The mixture is then directed todenaturation circuit 900, where the macromolecule is denatured and separated from any insoluble precipitates that form during denaturation. This creates a liquid sample, containing denaturedmacromolecule 104′, that can be directed tocapillary electrophoresis circuit 1300. In this view, several elements described in preceding Figs. can be synonymous, e.g., pumps 930 and 1302 can be the same pump;valves filters automated controllers - One skilled in the art will appreciate that the various elements of
apparatus 1500 can be integrated in different combinations. For example, each of the various individual elements can be integrated with a bioreactor, for example,rough filtration circuit 700 can be integrated with a bioreactor, ordenaturation circuit 900 can be integrated with a bioreactor, and the like. Combinations of the various elements van also be employed, for example, for a particular biofluid source that does not require rough filtration or denaturation,aseptic interface 752 can be combined withfine filtration circuit 800 andcapillary electrophoresis circuit 1300. In another example, a system that does not require fine filtration could employrough separation circuit 700,denaturation circuit 900, andcapillary electrophoresis circuit 1300. In other applications, each circuit or apparatus can be used alone, or in other logical combinations. One skilled in the art will know which circuit or apparatus will be useful in any particular application. - In various embodiments, each of the individual elements in
apparatus 1500 and various combinations thereof can be coupled “on-line” to an operating bioreactor. As used herein, “on-line” means that the apparatus can draw samples directly from the reactor into the apparatus, i.e., the sample is drawn directly into the apparatus without exposure to the external environment and without involving a transfer using a discrete sample container, e.g., a sample vial. - Another feature of particular embodiments of combinations of two or more of the elements of
apparatus 1500 is that each combination can be coupled to an operating bioreactor to form an integrated system. This means that the sample is in complete custody of the system, i.e., is controlled to be free from exposure to the external environment, from the bioreactor to the final operation on the sample (e.g., analysis of the prepared macromolecule at capillary electrophoresis circuit 1300). Furthermore, “integrated” can mean that the various circuits and apparatuses are controlled by the automated controller to operate in a coordinated fashion. - Still another feature of various embodiments of the elements of
apparatus 1500 and their combinations is that each can be coupled to an operating bioreactor to handle “raw” fluids, i.e., complex liquid mixtures containing one or more components typically found in a bioreactor, for example, cells, cellular debris, cell organs, cell fragments, salts, macromolecules including proteins, DNA, RNA, and the like. A “raw” fluid is taken directly from a reactor, typically an operating reactor, without any preprocessing. - In particular embodiments, the
capillary electrophoresis circuit 1300 can be controlled to partially or completely exchange the fluid inside the capillary electrophoresis column in place, i.e., the column can remain fixed with respect to one, or preferably both of the inlet and outlet reservoirs. - In various embodiments, the inside diameter of the capillary electrophoresis column is at least about 50 μm, more typically at least about 75 μm, and even more typically at least about 100 μm. One skilled in the art will know that values larger than 1 mm for the inside diameter of the capillary are possible, but can face diminishing returns in terms of efficiency. In a particular embodiment, the inside diameter of the capillary is from about 50 μm to about 150 μm, or more particularly, from about 100 μm to about 125 μm.
- In various embodiments, each system, circuit and apparatus can draw sample volumes from at least about 0.1 mL to at least about 25 mL, and more typically between about 0.5 mL and about 10 mL. In a particular embodiment, the sample volume is between about 0.75 and about 5 mL.
- The inside diameter of the conduits employed in the various circuits in the system, excluding the capillary itself, can be in various ranges. The inside diameters can be different in different portions of the system. The inside diameters are typically in a range of from about 0.5 to about 10 millimeters (mm), more typically between about 0.75 and about 5 mm, even more typically between about 0.75 and about 2 mm, and preferably between about 1 and about 2 mm.
- The “pressure differential” employed to direct components at or through a filter can be estimated by one skilled in the art by considering relevant system characteristics such as filter pore size, fluid viscosity, approximate concentration of material larger than the filter pore size, time to filter a particular volume, flow rate, and the like. One skilled in the art will know how to use such characteristics to choose an appropriate pressure differential based on the desired filter performance and flow rate. Typically, the pressure differential across the filter is between about 500 and about 7000 millibar, more typically between about 1000 and about 5000 millibar, or even more typically between about 1500 and 3000 millibar. A “pressure differential” can be caused by pressurizing one side of the filter, depressurizing on one side of a filter, or a combination of pressurizing one side and depressurizing the other side in a “push-pull” fashion.
- As used herein, the filters are employed as “direct flow” or “dead-end” filters, and filtration methods employed herein are “direct flow” or “dead-end” filtration methods. This means that during filtration, the pressure differential applied causes the liquid mixture being filtered to be applied directly to the filter, i.e., in a direction substantially perpendicular to the face of the filter.
- Another particular embodiment of the filters and filtration methods employed is a “back-flushing” capability. That is, each filter can be cleaned by directing a fluid, e.g., a buffer, a cleaning fluid, water, a solvent, a desalination buffer, a denaturation buffer, combinations thereof, and the like through the filter in a direction opposite to a previous filtration step. For example, a filter which becomes clogged with debris after a filtration step can be cleaned, at least in part, by directing a fluid through the filter in a direction opposite to the direction of the preceding filtration step.
- The
controllers 701/1330 may receiveoperational input 1615 from an external source, such as a local user interface (not shown). The controller(s) 701/1330 process theoperational input 1615 to send commands orqueries 1505 to thecircuits responses 1510 from these circuits. -
FIG. 16 is a block diagram of anoverall system 1600 shown in the context of additional external systems and input/output data related to thesystem 1500. - From a macromolecule processing point of view, this
overall system 1600 refers to thesystem 1500, theliquid mixture 202 that includes a macromolecule of interest, and theprepared macromolecules 104′. - From a controls point of view, the
overall system 1600 includes thesystem 1500, controller(s) 701/1330 in thesystem 1500, andlocal user interface 1605 connected to thesystem 1500 via a bus orlocal area network 1607. - The
controllers 701/1330 may include executable instructions, provided in the form of software or firmware, which is preferably unchangeable by the user of thesystem 1500. Such unchangeable software may be referred to, and is referred to hereafter, as “compiled” software, meaning that source code was compiled (e.g., compiled C code), and the compiled software exists only in a form usable by thecontrollers 701/1330. Source code may be provided to the user for re-compiling to facilitate modification of the configuration or general operation of the system. However, re-compiling may cause a re-validation and/or re-approval of thesystem 1500 to be required before further usage, which is discussed later in reference toFIGS. 18-23 . - The
operational input 1615 can be provided or written by a designer or end user of thesystem 1500 without having to recompile the compiled software. Theoperational input 1615 typically provides specific operational instructions to customize operation of the system, which may be limited by the compiled software according to a predefined set of limits. - The
local user interface 1605 may include a general purpose computer or custom-designed computer specific for operating thesystem 1500. Thelocal user interface 1605 may send theoperational input 1615 to thecontrollers 701/1330, which process theoperational input 1615 using the compiledsoftware 1610. Responsively, the compiledsoftware 1600 may send commands orqueries 1505 to thesystem components - The compiled
software 1610 may be stored locally or downloaded across thenetwork 1607 and is executed by thecontrollers 701/1330. The compiledsoftware 1610 may also be permanently stored in thecontrollers 701/1330 through the use of firmware, Field Programmable Gate Arrays (FPGA's), Read-Only Memory (ROM), and so forth. - The
controllers 701/1330 may also collect data, such as theproduction data 1625 and/orelectrophoresis data 1620, during operation of the system 1550. Thesedata network 1607 to thelocal user interface 1605 for further processing or display to the user via a Graphical User Interface (GUI), other display, such as LED indicators, or output as sound, such as produced by an audio synthesizer. - The
electrophoresis data 1620 may include information regarding theprepared macromolecules 104′. For example, theelectrophoresis data 1620 may include the molecular weight of the sample and time corresponding to how long the sample takes to travel across the length of theelectrophoresis column 1106. - The
production data 1625 may include information regarding thesystem 1500, such as calibration information, such as throughput/recovery and molecular weight calibrations, equipment specifications, such as capillary diameter, voltage levels, capillary length, cleaning solutions, and number of usages since the last replacement of the electrophoresis column. - The
production data 1625 may also include information related to the production of the macromolecules from theliquid mixture 202, such as discussed in reference toFIG. 5 . - The compiled
software 1610 may have knowledge of a mapping, in accordance with a known industry standard between theoperational input 1615 andsystem components software 1610 is preferably tested and integrated by the manufacturer of thesystem 1500 in a manner also consistent with known industry standards, such as American National Standards Institute (ANSI) (e.g., ANSI ‘C’ programming language). - For example, the manufacturer may validate the system by (i) inputting a complete set of test vectors to the
controllers 701/1330 in a testing phase of thesystem 1500 and (ii) observing activation/deactivation of the valves, pumps, etc. in accordance with the test vectors. Prior to release of the compiled software with thesystem 1500, the compiledsoftware 1610 may be tested extensively for failure modes and/or error checking capabilities for detecting programmatic or out-of-range errors identified in theoperational input 1615. Other forms of testing may include providing test vectors with erroneous or harmful information to ensure the compiledsoftware 1610 handles these situations in a manner that protects thesystem 1500 or components therein. - In a preferred embodiment, when sold or released to a customer, the compiled
software 1610 is unchangeable by the customer. In other words, the customer cannot alter the compiledsoftware 1610 and, therefore, thesystem 1500 continues to operate and be controlled in a manner tested and validated by the manufacturer of thesystem 1500. Theoperational input 1615, however, can be modified by the customer independent of the compiledsoftware 1610 to customize the operation of the system. For example, if a particular macromolecule requires additional filtering cycles or denaturation dwell time, the customer may customize theoperational input 1615 to provide such control. - The
operational input 1615 may be declarative software instructions, where declarative software instructions are defined as instructions of a relational language or functional language, as opposed to an imperative language, where imperative (or procedural) languages specify explicit sequences of steps to follow to produce a result. Declarative languages, in contrast, describe relationships between variables in terms of functions or inference rules, and a language executor (i.e., interpreter or compiler) applies some fixed algorithm to these relations to produce a result. - Thus, for example, the
operational input 1615 may be software instructions, such as BASIC software instructions, that are interpreted in a real-time or pseudo-real-time manner by the compiledsoftware 1610. Theoperational input 1615 may also be forms of data streams that are produced by a graphical user interface (GUI) and processed by the compiledsoftware 1610. - An example set of program instructions or portion of operational input is listed below. The program instructions form a representative script for operating an analyzer, such as the
capillary electrophoresis circuit 1300. The representative script may be referred to as a physical layer between the user and the compiledsoftware 1610 to permit a chemist or operator to program the operation in an english-like language that provides an intuitive understanding for the programming. Use of this technique permits the manufacturer of thesystem 1500 to “hard code” the physical operation of thesystem 1500 while permitting the end user to “soft code” theoperational input 1615 for customizing or modifying a process based on empirical or calculated process flows. -
- “%” Denotes comment and is not executed
- % script—flow to Desalting Filter
- %
- % begin Push Sample to Desalt Filter Routine
- echo “Push Sample to Desalt filter.”
- open_valve SV1022 % open ‘Pump—Desalt Isolation Valve—SV1022’ (
FIG. 8 , valve 714) - sleep 1.0 % Pause 1 second
- open_valve SV1026 % ‘open—Pump 2—Desalt Isolation valve—SV1020’ (
FIG. 8 , valve 510) - sleep 1.0 % Pause 1 second
- % watch_pressure_drop <pt1>, <pt2>, <time_in_seconds>, <warning_low>, <warning_high>, <error_low>, <error_high>
- watch_pressure_drop PT4, PT5, 10.0, 0, 10, −5, 20
- % move sample across filter(s) by activating syringes
- start_syringe SY1, PUSH, 7.5, 0.6
- start_syringe SY2, PULL, 7.5, 0.6
- wait_for_syringes
- end pressure_drop PT4, PT5 % quit reading F3 pressure drop
- % Sample to Desalt Filter transfer complete—release valves
- close_valve SV1022 % close ‘Pump 1—Desalt Isolation Valve—SV1022’ (
FIG. 8 , valve 714) - sleep 1.0 % Pause 1 second
- close_valve SV1026 % close ‘Pump 2—Desalt Isolation Valve—SV1026’ (
FIG. 8 , valve 510) - % end Push Sample to Desalt Filter Routine
- The above script may be stored in the
local user interface 1605 and provided as theoperational input 1615 to thecontroller 1330. The compiledsoftware 1610 on thecontroller 1330 interprets the statements in the above script to generate commands orqueries 1505 to/from components in thecapillary electrophoresis circuit 1300 or valves, syringes, etc. in a preceding circuit, such as thedenaturation circuit 900. - As described above, the compiled
software 1610 includes software instructions unchangeable by the user. The compiledsoftware 1610 interprets statements such as “open_valve SV1022” to mean “provide a signal to energize or deenergize the valve corresponding to the variable SV1022 in a manner such that the valve opens to allow a liquid source to flow into an inlet chamber.” Responsively, the compiledsoftware 1610 causes the controller(s) 701/1330 to produce signals that effect this instruction. Since the compiledsoftware 1610 knows of the correspondence between the valve referred to as SV1022 in theoperational input 1615 to correspond with, for example, valve 714 (FIG. 8 ), the user need only specify valve SV1022 to be sure that the correct valve,valve 714, will be opened. Similarly, valve SV1026 corresponds tovalve 510 as shown inFIG. 8 , so the “open valve SV1026” instruction will be interpreted as such by the compiledsoftware 1610, which, in turn, causes the controller(s) 701/1330 to generate an electrical signal that energizes or de-energizes thevalve 510 to produce the desired “open” state of thevalve 510. - Continuing to refer to the script above, pressure sensors, whose addresses are known to the compiled
software 1610 in connection with the variable names PT4 and PT5, are addressed by thecontroller 1330 executing the compiledsoftware 1610 in response to receipt of theoperational input 1615 that includes the ‘watch_pressure_drop’ statement. The addresses corresponding to the syringes SY1 and SY2 are also known to the compiledsoftware 1610 and addressed by the controller(s) 701/1330 to “push” (i.e., deliver volume) and “pull” (i.e., acquire volume) in response to receipt of the ‘start_syringe’ statements listed above. - As should be understood from the above code, a “plain english” language set of programming instructions may be supported for a user of the
system 1500 for customizing the process for collectingelectrophoresis data 1620 from a sample of a processed macromolecule sample. The variable names (e.g., SV1022, SY1, PT4, etc.) may also be or include mnemonics or other forms of descriptors that are identified by the compiledsoftware 1610 and represent corresponding devices or subsystems to be operated in a manner consistent with the command(s) associated therewith. - The correspondence information may be embedded directly in the code, stored as sets of constants or hard coded variables in the software, or stored in look-up table(s), list(s), such as arrays or linked lists, or calculations used by the
controllers 701/1330 to determine the correspondence between the variable names and elements corresponding thereto. - In this way, once testing of the compiled
software 1610 has been completed, where the testing typically includes an exhaustive set of test vectors that is consistent with a full range of possible inputs provided by users of thesystem 1500, the manufacturer, customer, and user of thesystem 1500 are assured that this correspondence is “fixed” such that inadvertent addressing errors by thecontrollers 701/1330 will not be encountered, excluding electronics errors or failures. In other words,operational input 1615 that includes commands listed above in the example script will result in a known and repeatable effect to ensure proper operation of thesystem 1500 for processing or analyzing macromolecules. -
FIG. 17 is a network diagram of anetwork 1700 that includesmultiple macromolecule systems 1500 connected to a central or distributed network 1705. Each of thesystems 1500 has alocal user interface 1605, as described above in reference toFIG. 16 . In this embodiment, however, thesystems 1500 orlocal user interfaces 1605 include interfaces (not shown) to receiveoperational input 1615 from aremote user interface 1710 across the network 1705. - The
remote user interface 1710 can be employed by a “central” operator to control or monitor a distributed network of themacromolecule systems 1500 for high yield production or analysis of macromolecules. For example, a large pharmaceutical company or manufacturer supplying biological product thereto may employ such a network for high volume production. - Beyond the
operational inputs 1615, theremote user interface 1710 may also request data from a remote processing/data stores device 1715, which is also coupled to the network 1705 for interfacing with thesystems 1500. The remote processing/data stores device 1715 may receive theproduction data 1625 orelectrophoresis data 1620 for processing this data “off-line”. For example, the remote processing/data stores device 1795 may determine yields or quality of the macromolecules processed by thesystems 1500 and provide access to this data across the network 1705, for example, to theremote user interface 1710 or any of thelocal user interfaces 1605. Thus, in response to thedata request 1720, the remote processing/data stores device 1715 may provide requestedinformation 1725, including raw or processed data, across the network 1705 in a typical data exchange manner, such as through packetized communications. - It should be understood that the network 1705 may include various forms of communication networks, such as a Public Switched Telephone Network (PSTN), wired or Wireless Local Area Networks (WLAN's), cellular networks, circuit switching networks, Voice-Over-Internet-Protocol (VOIP) networks, and so forth.
- It should be understood that the compiled
software 1610 operating in thecontrollers 701/1330 of thesystems 1500 may be organized into multiple software “units”, such as a system control unit, network interface unit, local interface unit, and so forth. In this way, the compiledsoftware 1610 can be updated with predetermined re-validation requirements to minimize future costs of maintaining thesystem 1500 by the customers. - Business Method
-
FIG. 18 is a schematic diagram of abusiness model 1800 in which a manufacturer of systems subject to approval by aregulatory body 1820 operates. The regulatory body may (i) provide oversight of a system, such as thesystem 1600 discussed above, produced by the manufacturing company, (ii) provide oversight of the usage of the system, or (iii) provide oversight of products produced by the system. The user of the system and end user of products produced by the system may be the same or different companies or even the manufacturer of the system. By oversight, it is meant that the regulatory body may inspect (i) the system, (ii) usage of the system, or (iii) products produced by the system in a manner that protects workers operating the system or end users of the products produced by the system. As part of the oversight, the regulatory body may require validation of operation or end products of the system and, in turn, provide approval of the system based on the validation data provided by the manufacturer, user, or recipients of products of the system. - An example of a business model in which one or more companies operate under the auspices of a regulatory body is in the case of pharmaceutical material production. The regulatory body in this case is the Food and
Drug Administration 1820. In thisbusiness model 1800, the FDA provides oversight to amanufacturer 1805 of a system for producing macromolecules and/or providing electrophoresis analysis of samples of the produced macromolecules. TheFDA 1820 also oversees operation of the systems as used by a macromolecule producer 1810 (hereafter referred to as the producer 1810). Still further, theFDA 1820 oversees use of the macromolecules produced by thesystem 1500 by a pharmaceutical researcher/developer 1815 (hereafter referred to as researcher 1815). - In a typical business cycle, the
manufacturer 1805 distributes systems (step 1830) to theproducer 1810. Theproducer 1810 distributes materials (step 1835) produced through the use of the system to theresearcher 1815. Prior to distribution of the systems and operation of the systems, themanufacturer 1805 may engage in discussions with the researcher 1815 (step 1825) to assess the needs of theresearcher 1815, such as quality of the macromolecules, volume requirements for the macromolecules, and other production needs so as to design the system with those needs in mind to ensure commercialization of the systems. - Also included in the
business model 1800 are validation and approval cycles (steps 1840, 1845) by each of the aforementioned companies. The validation and approval cycles 1840, 1845 may be required of each of thecompanies business model 1800. For example, in the case of themacromolecule system 1600, before a system can be shipped by themanufacturer 1805, theFDA 1820 may require themanufacturer 1805 to participate invalidation 1840 a of the system (e.g., witness and verify data produced by the system, test results, or performance in response to test vectors provided to the system). After evaluation of validation data by theFDA 1820, theFDA 1820 may grantapproval 1845 a of the system. Following approval, the distribution of the system (step 1830) can occur, and shipment of the system may follow from themanufacturer 1805 to theproducer 1810. - Similarly, the
producer 1810 may have to provide data in the form of validation data to theFDA 1820 forapproval 1845 b before the system can be used by theproducer 1810 to generate macromolecules, for example. Theproducer 1810 may have additional requirements for gainingapproval 1845 b from theFDA 1820. For example, theproducer 1810 may have to customize operational input, as discussed above, to operate the system, and test results may have to be shown. In addition, actual macromolecules produced by the system may also have to be validated by the producer and sent to theFDA 1820 for approval to ensure quality of the macromolecules. - Similarly, the
researcher 1815 may also have to send validation data to theFDA 1820 forapproval 1840 c. Typically, this validation andapproval cycle - It should be understood that the
regulatory body 1820 may be a government or non-government agency. For example, in addition to the FDA, the government agency may be the Department of Defense (DOD) that may be involved in the oversight of non-government entities to monitor systems, such as described above, for use in developing vaccines against toxic substances, such as anthrax, smallpox, and so forth. - Continuing to refer to the business model of
FIG. 18 , there may be a business advantage for themanufacturer 1805 to distribute asystem 1500 that has minimal re-validation and re-approval of the system following development of operational input by theproducer 1810 for its particular mode of operation. By limiting the amount of re-validation and re-approval of the system by theproducer 1810, theproducer 1810 is more likely to have shorter re-validation/re-approval cycles by theFDA 1820, which, ultimately, may lead to increased profits for theproducer 1810 due to higher system usage and more distribution of systems by themanufacturer 1805 for this reason. - As discussed above in reference to
FIGS. 16 and 17 , one way to minimize exposure of theproducer 1810 to re-validation/re-approval cycles 1840, 1845 is to provide executable instructions in thesystem 1500 that are unchangeable by theproducer 1810. One way to make the executable instructions unchangeable is to provide it in a compiled form referred to hereafter as “compiled software” and deploy it in thesystem 1500, for example, in the form of software or firmware. The compiled software preferably conforms to a known industry standard, such as ANSI programming languages or standard protocols for interfacing with devices or subsystems used to operate the system 1600 (FIG. 16 ). - A generalized flow diagram of the process just discussed is depicted in
FIG. 19 . InFIG. 19 , aprocess 1900 is performed by the manufacturer of thesystem 1510. Theprocess 1900 begins (step 1905) upon installation of compiled software in thesystem 1500. Themanufacturer 1805 validates the compiled software in the system 1500 (step 1910), which requires approval by a regulatory body, such as theFDA 1820. Themanufacturer 1805 obtains approval of thesystem 1500 by the regulatory body 1820 (step 1915) independent ofoperational input 1615 to the compiledsoftware 1610. Themanufacturer 1805 then distributes the approved system to a customer 1810 (step 1920). The process ends (step 1925) following distribution of the system 1500 (step 1830). - The
process 1900 may involve additional steps for gaining validation 1840 and approval 1845 by theregulatory body 1820. For example, referring toFIG. 20 , themanufacturer 1805 may have aninternal process 2000 for performing the validation (step 1910). Theinternal process 2000 may begin (step 2005) at a point in which an employee of themanufacturer 1805 compiles software conforming to a known industry standard (step 2010). The employee then downloads the compiledsoftware 1610 to the system (step 2015). In parallel, the same or another employee of themanufacturer 1805 may develop an exhaustive set of system-specific test vectors (step 2020) and use these test vectors to test the compiled software 1610 (step 2025). The employee verifies that the compiledsoftware 1610 operates thesystem 1500 as required to receive the approval from the regulatory body 1820 (step 2030). During the verification (step 2030), the employee collects data for submission to the regulatory body 1820 (step 2035). The employee submits the data to theregulatory body 1820 for approval (step 2040), which completes the internal process 2000 (step 2045). - Referring now to
FIG. 21 , from the point of view of theproducer 1810, aseparate process 2100 is conducted in which customization of thesystem 1500 is provided through custom design and use ofoperational input 1615. Theprocess 2100 begins (step 2105) when theproducer 1810 generates the operational input 1615 (e.g., declarative instructions) (step 2110). Theproducer 1810 validates the system (step 2115) with theoperational input 1615 without having to re-validate the compiledsoftware 1610 in thesystem 1500. Theproducer 1810 then seeks to obtain approval of theregulatory body 1820 for thesystem 1500 with the operational input 1615 (step 2120), which ends the process 2100 (step 2125). - The generation (step 2110) and validation (step 2115) of the
operational input 1615 can include several substeps, which are shown in aprocess 2200 depicted inFIG. 22 . Theprocess 2200 begins (step 2205), and theproducer 1810 generates (step 2110) theoperational input 1615. A scientist or other employee of themacromolecule producer 1810 determines desired macromolecule characteristics (step 2210). The scientist or other employee models (step 2215) theliquid mixture 202 containing the macromolecule. The scientist or other employee specifies (step 2220)system 1500 operation to yield the desired macromolecule from theliquid mixture 202. - Based on the system specifications, the scientist or other employee generates at least one operational input file (file 2225). The validation (step 2115) begins following generation of the operational input file (step 2225). The scientist or other employee validates the system with the operational input file(s) and collects data corresponding thereto (step 2230). The
producer 1810 submits the collected data for approval to theregulatory body 1820 absent data specific to the compiledsoftware 1610 in thesystem 1500. In other words, at this time in the development cycle of thesystem 1500, the validation and approval cycle (steps software 1610 because the compiledsoftware 1610 has not changed in form or function since gainingapproval 1845 a by themanufacturer 1805 prior to thedistribution 1830 of thesystem 1500. Theprocess 2200 ends (step 2240), and theproducer 1810 awaitsapproval 1845 b from theregulatory body 1820. - For any number of reasons, the
macromolecule producer 1810 may want to improve or modify the software in some way to improve the process provided by thesystem 1500 for either producing the macromolecules from theliquid mixture 202 or performing the electrophoresis analysis by thesystem 1500. In this case, all that theproducer 1810 need modify is theoperational input 1615 provided to the compiledsoftware 1610 in thecontrollers 701/1330. In such a case, theproducer 1810 can execute a different business process that is a subset of the business processes discussed above in reference toFIGS. 18-22 . - Referring now to
FIG. 23 , abusiness process 2300 executed by theproducer 1810 begins (step 2305) upon a decision to change the process executed by thesystem 1600 for any number of reasons. A scientist or employee of theproducer 1810 modifies the operational input file(s) (step 2310), causing are-validation 1840 b and re-approval 1845 b of thesystem 1600 to be required by theregulatory body 1820. The re-validation is performed with the new file(s) and the employees collect data based on the operation of the system with the new operational input 1615 (step 2315). Theproducer 1810 submits the data to theregulatory body 1820 for re-approval absent data specific to the compiledsoftware 1610 in thesystem 1500. Again, because the compiledsoftware 1610 is unchangeable by theproducer 1810 following its original validation and approval (steps producer 1810 does not need to repeat these steps. - As used herein, a macromolecule can be a large molecule, typically a biological polymer that can be soluble in the liquid mixture. A macromolecule can be a protein or peptide, for example, a peptide hormone, an enzyme, an enzyme with an associated cofactor, an antibody, a glycoprotein, and the like. A macromolecule can be other biological polymers, for example, polysaccharides, e.g., starches or sugars, polynucleic acids, e.g., deoxyribonucleic (DNA) or ribonucleic acid (RNA), lipids, glycolipids, and the like. A macromolecule can also be other large molecules of interest, for example steroids, carbohydrates, organometallic complexes such as metalloporphyrins, and the like. A macromolecule can also be a non-biological molecule or polymer. A macromolecule can be two or more molecules that are associated through noncovalent interactions to form a complex, for example, an antibody-antigen complex, an enzyme-inhibitor complex, a multi-domain protein where the domains are linked by hydrophobic forces, and the like. Preferably, a macromolecule can be a biopolymer or other biological molecule that is the desired product of a particular bioreactor process. For example, in a bioreactor process designed to grow bacteria genetically engineered to express human insulin, the macromolecule is insulin. The macromolecule can also be a molecule that can be indicative of the desired product of a particular bioreactor process. Most preferably, a macromolecule is a protein. A macromolecule is typically between about 1,000 and about 200,000 atomic mass units (AMU) in molecular weight. Macromolecules are typically between about 10,000 and about 160,000 AMU.
- As used herein, components that are smaller or larger than the macromolecule are those that can be separated from the macromolecule by filtration. Components that are smaller or larger than the macromolecule typically have a molecular weight that is greater or lesser than, respectively, the molecular weight of the macromolecule. One skilled in the art will know, however, that the relation of size to molecular weight for macromolecules and similar components is approximate and depends on a number of factors, including the actual molecular weight, the conformation of the molecule, whether the molecule is aggregated or agglomerated with other molecules, solvent conditions, ionic strength, filter composition, and the like.
- As used herein, rough components can include soluble and insoluble components. Insoluble components include cells, fragments of cells, non-cellular tissue fragments, insoluble agglomerations of macromolecules, particulate contaminants, and the like. Soluble rough components include smaller fragments of cells, macromolecules that are larger than the macromolecule or are greater in molecular weight than the molecular weight of the macromolecule, and the like.
- As used herein, fine components include soluble components. Soluble fine components include macromolecules that are smaller than the macromolecule or are lesser in molecular weight than the molecular weight of the macromolecule. Also included are small organic and inorganic molecules, for example, salts, amino acids, nucleic acids, cofactors, nutrients, metabolites, other macromolecules, fragments of the macromolecule, other biomolecules, and the like.
- As used herein, salt components include salts formed from cations such as sodium, potassium, lithium, cesium, magnesium, manganese, copper, zinc, calcium, iron, ammonium, alkylammonium, phosphonium, sulfonium, and the like. Salt components also include anions including halides, sulfates, thiosulfates, sulfonates, sulfites, nitrates, nitrites, carboxylates, phosphates, phosphates, phosphonates, carbonates, hydroxides, and the like.
- The liquid in the liquid mixture containing the macromolecule can be any solvent, for example, water, organic solvents such as alcohols, e.g., methanol, ethanol, isopropanol, t-butanol, and the like; ethers, e.g., dimethyl ether, diethyl ether, tetrahydrofuran, and the like; ketones, e.g., acetone, methyl ethyl ketone, and the like; aromatic solvents, e.g., benzene, toluene, and the like; halogenated solvents, e.g., chloroform, carbon tetrachloride, trichloroethylene, and the like; polar aprotic solvents, e.g., dimethyl sulfoxide, nitrobenzene, dimethyl formamide, n-methylpyrrolidone, acetonitrile, and the like; mixtures thereof, and the like. Typically, the liquid can be water, optionally with small amounts of one or more organic solvents that are miscible with water, e.g., ethanol, isopropanol, acetonitrile, and the like.
- As used herein, denaturation means changing the conformation and/or the solubility of a macromolecule to prepare it for analysis. For example, when
macromolecule 104 inFIG. 4 is a protein, denaturation can include transformation from a packed three-dimensional conformation 104 to alinear conformation 104′. Denaturation can also include solubilizing the macromolecule with the denaturingdetergent 220. Denaturation can be accomplished by techniques well known to one skilled in the art, for example, addition of one or more denaturation agents, application of heat, disulfide bond reduction, or a combination thereof. Denaturation agents for proteins can include, for example, chaotropic agents e.g., urea, guanidine hydrochloride, and the like; detergents, e.g. sodium dodecyl sulfate, potassium laurel sulfate, and the like; disulfide cleavage agents, e.g. dithiothreitol, dithioerythritol, and the like; acids or bases, e.g., trichloroacetic acid, sodium hydroxide, and the like; and other agents known to the art. Denaturation agents for polynucleic acids can include, for example, chelation agents, e.g. ethylenediamine tetraaceticacid and the like. - As used herein, a denaturation vessel can be any chamber or conduit where denaturation takes place, typically a small volume metal vessel, e.g., a stainless steel vessel between about 1 to about 100 mL. A denaturation vessel is typically coupled to a heating element, i.e., any device known to the art that can be used to heat the fluid mixture, for example, a resistive heating coil, a microwave heater, a combustion heater such as a gas flame, a heat pump, and the like. A denaturation vessel can also be coupled with a cooling element, for example, a heat pump, refrigeration unit, thermoelectric cooling element, radiator, water cooling coil, and the like. One skilled in the art will recognize that heating and cooling elements can be part of a single heat exchanger unit.
- As used herein, a hydraulic system can be a collection of hydraulic conduits, one or more valves, and one or more pumps, coupled so that the pumps can be used to generate fluid pressure in the hydraulic lines and the valves can be controlled to direct the pressurized fluid through the lines. A pump can be any device known to the art that can be used to generate fluid flow, for example, an electro-kinetic pump, or a mechanical pump including a peristaltic pump, a syringe pump, an impeller pump, a pneumatic pump, and the like. A valve can be any device known to the art that can be used to control fluid flow, e.g., a needle valve, a gate valve, a butterfly valve, and the like. An automated controller can be a processor, e.g., an embedded processor, a desktop computer, and the like, that can be programmed to control a system adapted for automatic control, e.g., the hydraulic system.
- As used herein, an ion concentration sensor can be any ion concentration sensor known to one skilled in the art, for example a general ion sensor such as a conductance sensor, or a specific ion sensor such as a chloride sensor, a hydrogen ion sensor (i.e., a pH sensor), and the like.
- As used herein, a buffer can be any liquid that can be added to the mixture to maintain or change the concentration of a particular component, or to combine an additive to change the properties of the process. For example, an ionic buffer, e.g., a pH buffer, can change or maintain the pH of the liquid mixture; a denaturation buffer can contain a denaturation agent; a desalination buffer can be a liquid substantially free of salts or substantially free of a particular salt, e.g., sodium chloride; a lysis buffer can be a liquid that contains a lysing agent (e.g., a detergent) or can be sufficiently low in ionic strength to lyse cells by ionic shock; and the like. Lysing agents can include enzymes, e.g., L-lysine decarboxylase, lysostaphin, lysozyme, lyticase, mutanolysin, and the like. Lysing agents can include detergents, e.g. glycocholic acid sodium salt hydrate, lithium dodecyl sulfate, sodium cholate hydrate, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, N-Nonanoyl-N-methylglucamine, octyl-b-D-1-thioglucopyranoside, 3-(N,N-dimethyloctadecylammonio)propanesulfonate, and the like.
- While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
- The following applications, filed on even date herewith, contain related subject matter and are incorporated herein in their entirety; Docket No.: 3551.1001-000, “Method for Detection of Molecular species in a Crude Sample Using Capillary Electrophoresis,” by Shin-Fuw Lin, et al.; Docket No.: 3551.1003-000, “Stationary Capillary Electrophoresis System,” by George E. Barringer, Jr.: Docket No.: 3551.1004-000, “Method and Apparatus for Operating an Automated Biomolecular Preparation System,” by George E. Barringer, Jr., et al., and Docket No.: 3351.1005-000, “Fluid Interface for Bioprocessor Systems,” by George E. Barringer, Jr.
Claims (39)
1. An apparatus for preparing a macromolecule sample, comprising:
a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;
a rough filter selected to separate from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule;
a fine filter selected to separate from the macromolecule, at least a portion of one or more fine components in the mixture that are smaller than the macromolecule; and
the hydraulic system being controlled to apply the liquid mixture to each filter with a pressure differential across each filter.
2. The apparatus of claim 1 , wherein the fine components comprise salt components.
3. The apparatus of claim 2 , further comprising a reservoir supplying a denaturation agent.
4. The apparatus of claim 3 , further comprising a heating element adapted for control by the automated controller.
5. The apparatus of claim 4 , further comprising a cooling element adapted for control by the automated controller.
6. The apparatus of claim 2 , further comprising at least one ionic concentration sensor adapted to communicate with the automated controller.
7. The apparatus of claim 6 , further comprising at least one flow sensor adapted to communicate with the automated controller.
8. The apparatus of claim 7 , wherein the hydraulic system comprises a second pump adapted for control by the automated controller.
9. The apparatus of claim 8 , wherein at least one ionic concentration sensor is a pH sensor.
10. The apparatus of claim 9 , further comprising a reservoir supplying a desalination buffer.
11. The apparatus of claim 10 , further comprising a reservoir supplying a pH buffer.
12. The apparatus of claim 11 , further comprising the automated controller in communication with the valves, the pumps, and the sensors.
13. The apparatus of claim 2 , wherein the molecular weight of the macromolecule is between about 1,000 and about 200,000 AMU.
14. The apparatus of claim 13 , wherein the molecular weight of the macromolecule is between about 10,000 and about 160,000 AMU.
15. The apparatus of claim 14 , wherein the fine filter is selected to separate components of a molecular weight less than about 90% of the molecular weight of the macromolecule.
16. The apparatus of claim 15 , wherein the rough filter is selected to separate components of a molecular weight greater than about 110% of the molecular weight of the macromolecule.
17. The apparatus of claim 2 , further comprising a lysis unit.
18. The apparatus of claim 17 , wherein the lysis unit comprises a sonic disrupter controlled by the automated controller.
19. The apparatus of claim 17 , wherein the lysis unit comprises a pump and a reservoir supplying a lysis buffer.
20. An apparatus for preparing a macromolecule sample, comprising:
a hydraulic system comprising two or more pumps, buffer reservoirs, a flow sensor, a pH sensor, a heating element, and valves;
at least three filters selected to separate from a macromolecule in a liquid mixture, at least a portion of other components in the mixture, the filters including:
a first stage rough filter selected to separate rough components;
a second stage rough filter selected to separate at least a portion of rough components that pass through the first stage rough filter;
a fine filter to separate fine components comprising salt components;
an automated controller in communication with the valves, the pumps, the heating element, and the sensors that controls the hydraulic system to:
apply the liquid mixture to each filter with a pressure differential across each filter;
direct a desalination buffer from a reservoir through the fine filter into the mixture;
combine a denaturation agent from a reservoir with the macromolecule;
control the heating element to heat the combination of the macromolecule and the denaturation agent until at least partial denaturation of the macromolecule occurs; and
combine a pH buffer from a reservoir to maintain a pH value at the pH sensor in a range from 6 to 8.
21. The apparatus of claim 20 , wherein the liquid mixture comprises cells, further comprising a pump that combines a lysis buffer from a reservoir with the cells.
22. An apparatus for preparing a macromolecule sample, comprising:
a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;
a filter selected to separate, at least in part, a macromolecule in a liquid mixture from one or more salt components in the mixture; and
an automated controller that controls the pump and the valves.
23. The apparatus of claim 22 , further comprising a reservoir supplying a desalination buffer.
24. The apparatus of claim 23 , further comprising:
a reservoir supplying a denaturation agent; and
a heating element adapted for control by an automated controller.
25. The apparatus of claim 24 , wherein the filter is selected to separate components that have a molecular weight less than about 90% of the molecular weight of the macromolecule.
26. An apparatus for preparing a macromolecule sample, comprising:
a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;
a lysis unit that is capable of lysing cells in a liquid mixture comprising cells and a macromolecule; and
a filter selected to separate from the macromolecule, at least a portion of components in the mixture that are larger than the macromolecule, the components comprising insoluble lysed cell components; and
an automated controller that controls the pump and the valves.
27. The apparatus of claim 26 , wherein the molecular weight of the macromolecule is between 10,000 and 160,000 AMU.
28. The apparatus of claim 27 , wherein the lysis unit comprises a sonic disrupter.
29. The apparatus of claim 27 , wherein the lysis unit comprises a reservoir supplying a lysis buffer.
30. The apparatus of claim 29 , further comprising:
a reservoir supplying a denaturation agent; and
a heating element adapted for control by an automated controller.
31. The apparatus of claim 30 , wherein the filter is selected to remove insoluble components, further comprising a rough filter selected to separate from the macromolecule, at least in part, soluble components that have a molecular weight greater than about 110% of the molecular weight of the macromolecule.
32. A method for preparing a macromolecule sample, comprising automatically:
acquiring a liquid mixture, the mixture comprising a macromolecule and one or more cells;
lysing at least a portion of the cells; and
separating from the macromolecule at least a portion of components larger than the macromolecule, the components comprising insoluble lysed cell components, by applying the mixture to a filter with a pressure differential across the filter.
33. The method of claim 32 , further comprising lysing at least a portion of the cells by combining a lysis buffer with the cells.
34. The method of claim 33 further comprising increasing the macromolecule concentration by at least 100%.
35. The method of claim 34 , further comprising increasing the macromolecule concentration by at least 200%.
36. The method of claim 35 , further comprising:
combining the macromolecule with a denaturation agent; and
heating the macromolecule and denaturation agent until at least partial denaturation of the macromolecule occurs.
37. The method of claim 36 , wherein the filter separates insoluble components, further comprising applying the mixture, to a rough filter with a pressure differential across the rough filter, the rough filter selected to separate soluble components that have a molecular weight that is greater than about 110% of the molecular weight of the macromolecule.
38. An apparatus for preparing a macromolecule sample, comprising:
means for automatically acquiring a liquid mixture, the mixture comprising a macromolecule and one or more cells;
means for automatically lysing at least a portion of the cells; and
means for automatically separating from the macromolecule at least a portion of components larger than the macromolecule, the components comprising insoluble lysed cell components, by applying the mixture to a filter with a pressure differential across the filter.
39. An apparatus for preparing a macromolecule sample, comprising:
a hydraulic system comprising:
a plurality of valves;
a rough pump that draws a liquid mixture from a sample site through:
a first stage rough filter selected to separate rough components; and
a second stage rough filter selected to separate rough components that pass through the first stage rough filter;
a fine pump that:
draws a desalination buffer from a reservoir;
operates cooperatively with the rough pump to:
draw a portion of the liquid mixture through a fine filter, the fine filter selected to separate from the macromolecule fine components that have a molecular weight less than about 90% of the molecular weight of a macromolecule;
direct the desalination buffer through the fine filter to combine the desalination buffer with the macromolecule;
a denaturation pump that operates in combination with the rough pump to direct the macromolecule and the desalination buffer to a denaturing vessel, the denaturing vessel comprising a heating element and a cooling element;
a reservoir supplying a denaturation buffer to the denaturation vessel;
a reservoir supplying a pH buffer;
a pH sensor located at the denaturization conduit;
an automated controller in electronic communication with the pumps, the denaturation vessel, and the sensor, that controls the apparatus to acquire a liquid mixture from the sampling site, the mixture comprising a macromolecule, rough components, and fine components;
separate at least a portion of rough and fine components from the macromolecule;
combine a denaturation buffer with the macromolecule;
heat the denaturation buffer and the macromolecule in the denaturation vessel to denature the macromolecule;
control the pH of the mixture to between about 6 and about 8 by adding pH buffer to the mixture; and
direct the denatured macromolecule to the analysis site.
Priority Applications (1)
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US12/573,438 US20100021880A1 (en) | 2003-06-20 | 2009-10-05 | Automated Macromolecule Sample Preparation System |
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US10/601,277 US7601545B2 (en) | 2003-06-20 | 2003-06-20 | Automated macromolecule sample preparation system |
US12/573,438 US20100021880A1 (en) | 2003-06-20 | 2009-10-05 | Automated Macromolecule Sample Preparation System |
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US10/601,277 Division US7601545B2 (en) | 2003-06-20 | 2003-06-20 | Automated macromolecule sample preparation system |
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US20100021880A1 true US20100021880A1 (en) | 2010-01-28 |
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US12/573,438 Abandoned US20100021880A1 (en) | 2003-06-20 | 2009-10-05 | Automated Macromolecule Sample Preparation System |
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US (2) | US7601545B2 (en) |
EP (1) | EP1636563B1 (en) |
AT (1) | ATE381006T1 (en) |
DE (1) | DE602004010658D1 (en) |
WO (1) | WO2004113875A1 (en) |
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Also Published As
Publication number | Publication date |
---|---|
WO2004113875A1 (en) | 2004-12-29 |
ATE381006T1 (en) | 2007-12-15 |
US20040259266A1 (en) | 2004-12-23 |
DE602004010658D1 (en) | 2008-01-24 |
US7601545B2 (en) | 2009-10-13 |
EP1636563A1 (en) | 2006-03-22 |
EP1636563B1 (en) | 2007-12-12 |
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