Patent ID: 12221603

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is particularly suited for the on-demand manufacturing of therapeutic proteins (either cell-based or cell-free) that are suitable for direct delivery to a patient. Therefore, the present invention will be primarily described and illustrated in connection with the manufacturing of therapeutic proteins. However, the present invention can also be used to manufacture any type of protein, including toxic proteins, proteins with radiolabeled amino acids, unnatural amino acids, etc. Further, the present invention is particularly suited for the on-demand manufacturing of proteins using cell-free expression, and thus the present invention will be described primarily in the context of cell-free protein expression. However, the present invention can also be used in connection with cell-based protein expression.

FIG.2is a block diagram that illustrates the principles of operation of one preferred embodiment of the present invention. The bioprocessing system100includes a production module200, a purification module300and a fluid storage/dispensing module400that are fluidly coupled via coupling components500. A processor600may be in electrical communication with one or more of the production module200, purification module300, coupling components500and fluid storage/dispensing module400for controlling and monitoring the operation of the system100.

The fluid storage/dispensing module400is adapted to store the solutions needed for the production of a protein. The fluid storage/dispensing module400may also include containers for storing any waste product produced during the production of the protein. The fluid storage/dispensing module400may be temperature controlled, if needed, to maintain the solutions at a required temperature.

The production module200is adapted to receive the solutions required for production of a protein, such as a therapeutic protein, from the fluid storage/dispensing chamber via coupling components500. The production module200may suitably include a bioreactor adapted for maintaining living cells that incorporates non-invasive optical chemical sensing technology for monitoring culture parameters (e.g., pH, oxygen, optical density, fluorescence, absorbance, redox, temperature, etc.), such as the bioreactors and optical chemical sensing technology illustrated and described in commonly assigned and related U.S. Pat. Nos. 6,673,532 and 7,041,493, as well as co-pending commonly assigned and related patent application Ser. No. 12/991,947, whose disclosures are incorporated by reference herein in their entirety. These types of bioreactors are particularly suited for cell-based production of therapeutic proteins. Alternatively, the production module200may suitably include a stirred mini-reactor such as, for example, the BioGenie Minibioreactor sold by Scientific Bioprocessing, Inc., that is adapted for the cell-free production of a protein, and that are also equipped with sensors for monitoring reaction parameters (e.g., pH, oxygen, optical density, fluorescence, absorbance, redox, temperature, etc.).

The production module200as illustrated inFIG.2is designed for batch mode protein production where all the components for protein production (DNA, cell-free lysate, reaction buffer, etc.) are combined in a single step and then delivered to the purification module via coupling components500at the end of the reaction (3-6 hours). The production module200may also be a cup with a dialysis membrane bottom or a dialysis cassette with dialysis membrane on both sides. The cup or cassette will be surrounded by a dialysis buffer to remove reaction waste products such as inorganic phosphate, but also to maintain the concentration of nutrients such as amino acids and creatine phosphate. The solutions for protein production are delivered to the dialysis cup or the dialysis cassette and the surrounding dialysis buffer from the fluid storage/dispensing chamber400via coupling components500.

After the reaction is complete, the raw product is then transferred to the purification module300via coupling components500. The purification module300contains the necessary purification components for purifying the protein from the reagents. The purification module300can include, for example, chromatography components and dialyses components for purifying the biologic.

The production module200and the purification module300may each include sensors for monitoring reaction parameters and/or product quality parameters. The parameters monitored can include, but are not limited to, conductivity, temperature, pH, oxygen and CO2. The sensors may be any type of invasive sensor known in the art for monitoring these parameters, where the sensors are in contact with the process fluid. In addition, the sensors may be non-invasive optical chemical sensors, such as those described in U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S. patent application Ser. No. 12/991,947. In addition, spectrometers known in the art can be used in the production module200and/or the purification module300to monitor the product stream and/or the inputs to each module. The parameters measured by such spectrometers can include, but are not limited to, absorbance, fluorescence, Raman scattering, circular dichroism and infrared spectral characteristics.

FIG.3is a schematic diagram of a bioprocessing system700, in accordance with another preferred embodiment of the present invention. The system700is particularly suited for the cell-free production of proteins and will be described in this context.

The system700includes a reactor210, in which protein expression takes place, a membrane chromatography component310, a diafiltration component320and a fluid storage/dispensing module400. The reactor210preferably includes a heating and cooling element220, suitably a thermoelectric cooler, for controlling the temperature of the solution230inside the reactor210. The reactor also preferably includes sensors240and250for monitoring parameters in the reactor solution230, such as pH, oxygen, redox, conductivity or any other parameter that can be measured with existing sensors. The sensors240and250can be implemented with any type of sensor known in the art for measuring the desired parameters. However, the sensors240and250are preferably non-invasive optical chemical sensors.

The system700also includes a processor600that is in communication with one or more of the reactor210, optoelectronics270, membrane chromatography component310, diafiltration component320, fluid storage/dispensing module400and pumps520A and520B for controlling and/or monitoring the operation of the system700.

Optoelectronics270are provided for exciting the optical chemical sensors240and250with excitation light242and244, respectively, and for receiving and detecting emission light246and248from the optical chemical sensors240and250, respectively. As discussed above, commonly assigned and related U.S. Pat. Nos. 6,673,532 and 7,041,493, as well as co-pending commonly assigned and related U.S. patent application Ser. No. 12/991,947 describe in more detail how non-invasive optical chemical sensing technology can be used to monitor parameters.

InFIG.3, two optical chemical sensors240and250are shown, and are preferably adapted to measure pH and dissolved oxygen, respectively. However any number of optical chemical sensors (including only one) may be used depending on the number and type of parameters being measured. Optoelectronics270include optical excitation sources (not shown) for generating the excitation light242and244, as well as photodetectors (not shown) for detecting the emission light246and248from the optical chemical sensors240and250. The type of optical excitation source or sources are the types used in optoelectronics. Any combination of optical excitation sources and optical chemical sensors may be used, depending on the number and types of parameters being measured. Examples of optical excitation sources that can be used included in optoelectronics270include, but are not limited to, light emitting diodes and laser diodes. Alternatively, the optoelectronics270may just be used to measure optical properties of the reactor contents in their entirety absent any sensors.

Further, for each optical chemical sensor240and250, two possible placements on the reactor210are shown. The two possible placements for optical chemical sensor240are shown as240A and240B. The two possible placements for optical chemical sensor250are shown as250A and250B. The use of other non-contact sensors (i.e. Raman, contact free conductivity sensors etc) is also possible in this context.

In the “A” placement (240A and250A), the optical chemical sensors240A and250A are positioned inside the reactor210on a reactor wall260. With this placement, the optical chemical sensors240A and250A are in physical contact with the solution230, and the reactor wall260on which the optical chemical sensors240A and250A are placed is optically transparent to the excitation light242and244, so that the excitation light can reach the optical chemical sensors240A and250A.

In the “B” placement (240B and250B), the optical chemical sensors240B and250B are positioned outside the reactor210on reactor wall260. With this placement, the thickness of the reactor wall260is sufficiently small so as to allow the analytes that are being measured to diffuse through the reactor wall260and contact the optical chemical sensors240B and250B. Alternatively, the portions of the reactor wall260on which the optical chemical sensors240B and250B are attached can be replaced with barrier membranes249A and249B that are adapted to allow the analytes being measured to diffuse through so that they come in contact with optical chemical sensors240B and250B. The use of barrier membranes and thin reactor walls to effectuate diffusion of the analytes of interest through a container wall to optical chemical sensors is described in more detail in commonly assigned and related U.S. patent application Ser. No. 13/378,033, which is incorporated herein by reference in its entirety.

In theFIG.3embodiment, the fluid storage/dispensing module400preferably includes a buffer solution container410for holding buffer solution, an mRNA/DNA solution container420for holding mRNA/DNA solution, a reaction solution container430for holding reaction solution, a waste storage container440for holding waste solution and a product storage container450for holding the purified protein. In operation, reaction solution, mRNA/DNA solution and buffer solution are directed to reactor210via conduits510A,510B,510C and pump520A.

After the reaction in the reactor210, the raw product is directed to membrane chromatography component310via conduit510E and pump520B for purification of the protein from the reagents. Membrane chromatography component310may suitably include a cylindrically shaped housing which contains porous membrane layers (preferably at least 10 porous membrane layers), where the individual membranes consist of an appropriate polymer, such as polymethacrylate, that has been chemically functionalized with a ligand, such as a diethylaminoethyl (DEAE), a quaternary amine (Q), or a carboxymethyl (CM) ligand for the case of ion-exchange chromatography, or a phenyl or butyl ligand for the case of hydrophobic interaction chromatography, or a mercaptoethylpyridine (MEP) ligand for the case of mixed mode chromatography. One preferred embodiment of the membrane chromatography component310will be discussed in more detail below in connection withFIG.5. Waste from the membrane chromatography process is directed to waste storage container440via conduit510F. The purified product is directed to diafiltration component320for dialysis via conduit510G and pump520C.

Membrane chromatography component310may also include one or more sensors312for monitoring product quality parameters, such as conductivity, temperature, pH, oxygen, CO2, absorbance, fluorescence, Raman, circular dichroism and infrared spectral characteristics. The sensors312may be any type of invasive or noninvasive sensor known in the art for measuring these parameters including, but not limited to, spectrometers. In addition, the sensors may be non-invasive optical chemical sensors, such as those described in U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S. patent application Ser. No. 12/991,947. In addition, membrane chromatography component310preferably includes a heating and cooling element314, suitably a thermoelectric cooler, for controlling the temperature of the solution (raw product) inside the membrane chromatography component310.

The diafiltration component320may suitably include a hydrophilic polymeric membrane for use as a separation mode for dialysis for separating proteins in a diluent liquid on the basis of differences in their ability to pass through a membrane or in the alternative for diafiltration to simply exchange the buffer solutions. Such hydrophilic polymeric membrane may include, but not limited to, polyethersulfone, a cellulosic, or a polyvinylidene fluoride (PVDF) membrane with a well-defined pore structure that yields a desired molecular weight cut-off (MWCO) value in the range of 10 k to 200 k Da as appropriate for a given application. The final protein that comes out of the diafiltration component320is directed to product storage container450via conduit510H. The waste product produced from the dialysis process in the diafiltration component320is directed to waste storage container440via conduit510I.

Diafiltration component320may also include one or more sensors322for monitoring product quality parameters, such as conductivity, temperature, pH, oxygen, CO2, absorbance, fluorescence, Raman, circular dichroism and infrared spectral characteristics. The sensors322may be any type of invasive or noninvasive sensor known in the art for measuring these parameters including, but not limited to, spectrometers. In addition, the sensors may be non-invasive optical chemical sensors, such as those described in U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S. patent application Ser. No. 12/991,947.

In addition, diafiltration component320preferably includes a heating and cooling element316, suitably a thermoelectric cooler, for controlling the temperature of the solution (raw product) inside the membrane chromatography component320.

In addition to the pumps520A,520B and520C, any number of valves or other hydraulic components, such as additional pumps, may be used throughout the system700to assist in controlling the flow of solution/product between the various components of the system700.

The present invention is particularly suited to miniaturization by using micropumps and microfluidic technology.FIG.4is a schematic diagram of a microscale bioprocessing system800, in accordance with another embodiment of the present invention. The system800includes many of the same components of the system700ofFIG.3, and common elements are labeled with common element numbers.

The system800contains a fluid storage/dispensing module400that includes a buffer solution container410for holding buffer solution, an mRNA/DNA solution container420for holding mRNA/DNA solution, a reaction solution container430for holding reaction solution, a waste storage container440for holding waste solution and a product storage container450for holding the purified protein. The system800also includes a reactor210, a membrane chromatography component310, a diafiltration component820, a processor600, optical chemical sensors840chosen and positioned to monitor finished product quality parameters, such as, for example, conductivity, redox, pH, UV spectrum and protein concentration, and optoelectronics830for providing optical excitation light and for detecting emission light from the optical chemical sensors840. The optoelectronics830may also just be used to measure the optical properties of the finished product absent any sensors.

The reactor210can be of any size, but in the microscale embodiment ofFIG.4, it preferably has a volume capacity of less than approximately 50 milliliters, and more preferably approximately 20 milliliters or less, in order to keep the system800relatively compact. The reactor210may be implemented, for example, with the BioGenie minibioreactor system manufactured by Scientific Bioprocessing, Inc.

Micropumps850A and850B and conduits510A-510I direct solution to the various components in a manner similar to pumps520A,520B and conduits510A-510I in the system700ofFIG.3. Although not shown inFIG.4, the reactor210contains optical chemical sensors and optoelectronics for monitoring parameters in the reactor solution230in a manner similar to system700ofFIG.3. The micropumps850A and850B may be implemented with any type of micropump known in the art such as, for example, the mp5 micropump or the mp6 micropump manufactured by Bartels Mikrotechnik.

The housing lid850may contain a display, such as an LCD display860, that connects to the processor600and that can provide information about the system800, such as, for example, diagnostic information, reaction parameters and/or finished product quality parameters, such as, for example, conductivity, redox, pH, UV spectrum and protein concentration.

The processor600inFIGS.2,3and4may be implemented with a general purpose desktop computer or a general purpose laptop computer. In addition, the processor may be implemented with a tablet computer or smartphone, such as iOS or Android-based tablets and smartphones. However, processor600can also be implemented with a special purpose computer, programmed microprocessor or microcontroller and peripheral integrated circuit elements, ASICs or other integrated circuits, hardwired electronic or logic circuits such as discrete element circuits, programmable logic devices such as FPGA, PLD, PLA or PAL or the like. In general, any device on which a finite state machine capable of executing code for implementing the functionality described herein can be used to implement the processor600.

FIG.5shows a membrane chromatography component310that can be used in systems700and800, in accordance with one preferred embodiment of the present invention. The membrane chromatography component310includes a housing2000and porous membrane layers2010(preferably at least 10 porous membrane layers). As discussed above, the individual porous membrane layers2010preferably consist of an appropriate polymer, such as polymethacrylate, that has been chemically functionalized with a ligand, such as a diethylaminoethyl (DEAE), a quaternary amine (Q), or a carboxymethyl (CM) ligand for the case of ion-exchange chromatography, or a phenyl or butyl ligand for the case of hydrophobic interaction chromatography, or a mercaptoethylpyridine (MEP) ligand for the case of mixed mode chromatography.

The membrane chromatography component310can be of any size, but in the microscale embodiment ofFIG.4, it preferably has a volume capacity of less than approximately 100 milliliters, and more preferably less than approximately 5 milliliters, in order to keep the system800relatively compact. The membrane chromatography component310may be implemented, for example, with a Sartobind® Q SingelSep Nano manufactured by Sartorius Stedim Biotech, which has a bed volume of 1 ml and a membrane area of 36 cm2.

Raw product from reactor210is mixed with elution buffer solution via three-way valve2015, and the mixture enters the membrane chromatography component310via inlet2020. Purified product and waste exits via the outlet2030. Three-way valve2040directs the purified product to the diafiltration component320/900/1100and directs the waste to waste storage440.

FIGS.6A-6Cshow a diafiltration component900that can be used in systems700and800, in accordance with one preferred embodiment of the present invention. The diafiltration component900includes serpentine-shaped product and buffer sections910and920, respectively. The diafiltration component900ofFIGS.6A-6Cinclude a product section910that is a serpentine-shaped channel formed on a first substrate1000. Similarly, the buffer section920is a channel formed on a second substrate1010with the same serpentine shape as the product section910. A diafiltration membrane930is sandwiched between the first and second substrates1000and1010, such that the serpentine-shaped channels that form the product and buffer sections910and910substantially overlap each other. The substrates1000and1010are attached to each other, with the diafiltration membrane930sandwiched between them, with any adhesive known in the art.

In the diafiltration component900ofFIGS.6A-6C, a diafiltration buffer solution flows through the serpentine-shaped product section920and purified product from the membrane chromatography component310flows through the serpentine-shaped product section910. Diffusion takes place from the product section910to the counterpart, similarly shaped buffer section920via the diafiltration membrane930.

The purified product from the membrane chromatography component310enters the product section910via inlet buffer reservoir1020and inlet1030. The diafiltered product exits the product section910via outlet1040and outlet buffer reservoir1050. Diafiltration buffer enters the buffer section920via inlet1060and exits the buffer section via outlet1070. The diafiltration buffer is chosen to facilitate the transfer of components through the diafiltration membrane930, and could be, for example, 25 millimolar phosphoric acid titrated to pH 7 with sodium hydroxide, or 25 millimolar citric acid tritrated to pH 5 with sodium hydroxide.

The inlet and outlet buffer reservoirs1020and1050are optionally used in order to dampen the back-and-forth oscillating flow, if needed. A makeup buffer solution is preferably added to the diafiltered product via the outlet buffer reservoir1050in order to replace the fluid that was that passed through the diafiltration membrane930with an equivalent volume of a different type of buffer, thereby transferring the protein of interest to the makeup buffer. Alternatively, the volume of the makeup buffer added via the outlet buffer reservoir1050can be less than the volume of fluid that has passed through the diafiltration membrane930, in which case the diafiltration component900accomplishes both buffer exchange and protein concentration.

As discussed above, diafiltration membrane930may suitably be a hydrophilic polymeric membrane, such as a polyethersulfone, a cellulosic, or a polyvinylidene fluoride (PVDF) membrane with a well-defined pore structure that yields a desired molecular weight cut-off (MWCO) value in the range of 10 k to 200 kDa as appropriate for a given application.

FIG.7shows a diafiltration component1100in accordance with another embodiment of the present invention. The diafiltration component1100may be used in system700or system800ofFIGS.3and4, respectively. The diafiltration component1100includes a buffer section1120, and a product section1110that comprises tubing1112that is passed through the buffer section1120. The tubing1112that makes up the product section1110can be any type of tubing known in the art that can function as the dialysis membrane1140between the product1115in the product section1110and the buffer1130in the buffer section1120.

The tubing1112is preferably flexible so that a larger amount of tubing can be placed inside the solvent section1120. The more tubing1112is present in the buffer section1120, the more diffusion can take place between the tubing1112and the buffer1130due to the larger tubing surface area in contact with the buffer1130. End portions1140and1150of the diafiltration component1100contain openings1160for the tubing1112to enter and exit the diafiltration component1100. The end portions1140and1150also contain an inlet1170for receiving diafiltration buffer solution, and an outlet1180for expelling used diafiltration buffer solution (waste). Although the diafiltration component1100is shown as rectangularly-shaped, it can be any other shape, such as cylindrically-shaped. Further, the diafiltration component1100can suitably be a flow cell that has been modified to pass the tubing1112through the buffer section1120.

Protein Expression in In Vivo and Cell-Free Systems

A protein is expressed in three main steps: replication, transcription and translation, as shown inFIG.8. DNA multiplies to make multiple copies by a process called replication. Transcription occurs when the double-stranded DNA is unwound to allow the binding of RNA polymerase producing messenger RNA (mRNA). Transcription is regulated at various levels by activators and repressors, and also by chromatin structure in eukaryotes. In prokaryotes, no special post-transcriptional modification of mRNA is required. However, in eukaryotes, mRNA is further processed to remove introns (splicing), to add a ‘cap’ (M7 methyl-guanosine) at the 5′ end and to add multiple adenosine ribonucleotides at the 3′ end of mRNA to generate a poly(A) tail. The modified mRNA is then translated.

The translation or protein synthesis is also a multi-step process with Initiation, Elongation and Termination steps and is similar in both prokaryotes and eukaryotes. The difference is that in eukaryotes, proteins may undergo post-translational modifications, such as phosphorylation or glycosylation. The translation process requires cellular components such as ribosomes, transfer RNAs (tRNA), mRNA and protein factors as well as small molecules like amino acids, ATP, GTP and other cofactors.

The difference between in vivo and in vitro (cell-free) protein expression is that in cell-free expression, the cell wall and the nuclei are no longer present.

Cell-Free Protein Expression from an Engineer's Perspective

To obtain the cell extract for cell-free protein expression, cells (E. coli, wheat germ, mammalian cells) are subjected to cell lysis followed by separation of the cell wall and nuclear DNA. The desired protein is synthesized by adding a DNA or mRNA template into the cell extract together with a reaction mix comprising of biological extracts and/or defined reagents. The reaction mix is comprised of amino acids, nucleotides, co-factors, enzymes and other reagents that are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc. When DNA is used as template (i.e. linked reaction), it is first transcribed to mRNA. Alternatively mRNA could also be used directly for translation.

The template for cell-free protein synthesis can be either mRNA or DNA. Translation of stabilized mRNA or combined transcription and translation converts stored information into a desired protein. The combined system, generally utilized inE. colisystems, continuously generates mRNA from a DNA template with a recognizable promoter. Either endogenous RNA polymerase is used, or an exogenous phage RNA polymerase, typically T7 or SP6, is added directly to the reaction mixture. Alternatively, mRNA can be continually amplified by inserting the message into a template for QB replicase, an RNA dependent RNA polymerase. Purified mRNA is generally stabilized by chemical modification before it is added to the reaction mixture. Nucleases can be removed from extracts to help stabilize mRNA levels. The template can encode for any particular gene of interest.

Salts, particularly those that are biologically relevant, such as manganese, potassium or ammonium, may also be added. The pH of the reaction is generally run between pH 6-9. The temperature of the reaction is generally between 20° C. and 40° C. These ranges may be extended.

In addition to the above components such as cell-free extract, genetic template, and amino acids, other materials specifically required for protein synthesis may be added to the reaction. These materials may include salts, polymeric compounds, cyclic AMP, inhibitors for protein or nucleic acid degrading enzymes, inhibitors or regulators of protein synthesis, oxidation/reduction adjusters, non-denaturing surfactants, buffer components, spermine, spermidine, etc.

The salts preferably include potassium, magnesium, ammonium and manganese salts of acetic acid or sulfuric acid, and some of these may have amino acids as a counter anion. The polymeric compounds may be polyethylene glycol, dextran, diethyl aminoethyl dextran, quaternary aminoethyl and aminoethyl dextran, etc. The oxidation/reduction adjuster may be dithiothreitol (DTT), ascorbic acid, glutathione and/or their oxides. Further DTT may be used as a stabilizer to stabilize enzymes and other proteins, especially if some enzymes and proteins possess free sulfhydryl groups. Also, a non-denaturing surfactant such as Triton X-100 may be used at a concentration of 0-0.5 M. Spermine and spermidine may be used for improving protein synthetic ability, and cAMP may be used as a gene expression regulator.

Synthesized product is usually accumulated in the reactor within the production module, and then is isolated and purified according to the methods of the present invention for protein purification after completion of the system operation. The amount of protein produced in a translation reaction can be measured in various fashions. One method relies on the availability of an assay that measures the activity of the particular protein being translated. Examples of assays for measuring protein activity are a luciferase assay system and a chloramphenicol acetyl transferase assay system. These assays measure the amount of functionally active protein produced from the translation reaction. Importantly, activity assays will not measure full length protein that is inactive due to improper protein folding or lack of other post translational modifications necessary for protein activity. As used herein, the term “activity” refers to a functional activity or activities of a peptide or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide. Preferably, the activity of produced proteins retain at least 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or more of the initial activity for at least 3 days at a temperature from about 0° C. to 30° C.

Another method of measuring the amount of protein produced in a combined in vitro transcription and translation reactions is to perform the reactions using a known quantity of radiolabeled amino acid such as35S-methionine or14C-leucine and subsequently measuring the amount of radiolabeled amino acid incorporated into the newly translated protein. Incorporation assays will measure the amount of radiolabeled amino acids in all proteins produced in an in vitro translation reaction including truncated protein products.

In another study, expression of a fusion protein consisting of murine GM-CSF (granulocyte macrophage colony stimulating factor) and a scFv antibody, in reactor systems such as thin film, bubble column and Eppendorf tube without membrane, were studied, producing protein up to >500 μg/ml protein with significant amount of precipitated protein (apprx eq.50%). Recently, rhGM-CF was expressed in a 100 L stirred tank reactor expressing protein up to 700 mg/L which was subsequently purified with DEAE resin, tangential flow filtration membrane (3 kD cut off) and Sephacryl S-100 size exclusion chromatography with 99% purity and 65% recovery. Cell-free expression has not only been successful in the expression of bacterial proteins, but also successfully produced glycoproteins like human choriogonadotropin (hCG) and envelope glycoprotein (gp120) of human immunodeficiency virus type-1 (HIV-1) in hybridoma cell extract (HF10B4).

For protein purification, people have relied on column chromatography traditionally, but in recent years membrane chromatography has emerged as an additional aid in this field, eliminating column chromatography at specific steps like capture and polishing of protein at final step with overall cost reduction up to 65%. Column chromatography is still useful for gradient purification of proteins, but membrane chromatography could also be studied by relying on the fact that step elution of protein and removal of the impurities could be done at different buffer conditions.

The chart below compares cell-free and in vivo protein expression systems.

In vivoCell freeBiological cell requiredNo cell, but cellular machinery is requiredTime consuming processTime effective processToxic protein may be difficult to expressToxic protein could be expressedMultiple steps in purification requiredRelatively less number of steps requiredHigher fraction of misfolded protein alongReduced levels of misfolded protein reported,with folded proteinalong with folded protein but precipitatedHigher endotoxins challengeRelatively less endotoxins challengeHigher amount of impurities in crude proteinRelatively less impurities, enhancing capturecausing challenges in capture stepand increasing yield of the proteinEstablished scale upHas significant potential to scale upProtein expression up to g/lProtein expression up to mg/l
Biomolecules for Protein Expression

The following biomolecules are preferably used for protein expression. To carry out a protein expression reaction, energy components and amino acids are supplied externally and may include, but not limited to the following components:a. A genetic template for the target protein (mRNA or DNA) expression;b. T7 RNA polymerases for mRNA transcription;c. 9 Translation factors (initiation, elongation and termination);d. 20 aminoacyl-tRNA synthetases (ARSes) for esterification of a specific amino acid to form an aminoacyl-tRNA;e. Methionyl-tRNA transformylase transfers hydroxymethyl-, formyl-groups;f. Creatine kinase converts ATP to ADP;g. Myokinase catalyzes the inter conversion of adenine nucleotides;h. Pyrophosphatase are acid anhydride hydrolases that act upon diphosphate bonds;i. 4 nucleoside triphosphates (ATP, GTP, CTP, TTP) for DNA formation;j. Creatine phosphate which serves as a reserve of high-energy phosphates for rapid mobilization;k. 10-formyl-5,6,7,8-tetrahydrofolate for the formylation of the methionyl initiator tRNA (fMet-tRNA);l. 20 amino acids for protein synthesis;m. Ribosomes for polypeptide translation;n. 46 tRNAs in protein synthesis; ando. Cellular components which assist in proper protein folding.

Some of the proteins that may be expressed by the present invention for on-demand production may include, but not limited to, adrenocorticotropic hormone peptides, adrenomedullin peptides, allatostatin peptides, amylin peptides, amyloid beta-protein fragment peptides, angiotensin peptides, antibiotic peptides, antigenic polypeptides, anti-microbial peptides, apoptosis related peptides, atrial natriuretic peptides, bag cell peptides, bombesin peptides, bone GLA peptides, bradykinin peptides, brain natriuretic peptides, C-peptides, C-type natriuretic peptides, calcitonin peptides, calcitonin gene related peptides, CART peptides, casomorphin peptides, chemotactic peptides, cholecystokinin peptides, colony-stimulating factor peptides, corticortropin releasing factor peptides, cortistatin peptides, cytokine peptides, dermorphin peptides, dynorphin peptides, endorphin peptides, endothelin peptides, ETa receptor antagonist peptides, ETh receptor antagonist peptides, enkephalin peptides, fibronectin peptides, galanin peptides, gastrin peptides, glucagon peptides, Gn-RH associated peptides, growth factor peptides, growth hormone peptides, GTP-binding protein fragment peptides, guanylin peptides, inhibin peptides, insulin peptides, interleukin peptides, laminin peptides, leptin peptides, leucokinin peptides, luteinizing hormone-releasing hormone peptides, mastoparan peptides, mast cell degranulating peptides, melanocyte stimulating hormone peptides, morphiceptin peptides, motilin peptides, neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides, orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides, pancreastatin peptides, pancreatic polypeptides, parathyroid hormone peptides, parathyroid hormone-related peptides, peptide T peptides, prolactin-releasing peptides, peptide YY peptides, renin substrate peptides, secretin peptides, somatostatin peptides, substance P peptides, tachykinin peptides, thyrotropin-releasing hormone peptides, toxin peptides, vasoactive intestinal peptides, vasopressin peptides, and virus related peptides.

Conventional and Non-Conventional Method of GBP Production

The systems and methods of the present invention can be used, for example, for the cell-free expression and purification of glucose binding protein (GBP). Glucose is a major carbon and energy source in cellular metabolism of animal body and in bioprocess industry. Glucose is not always beneficial in bioprocesses, it could also be detrimental in bacterial culture leading to self-lysis of cells by formation of acetate in Krebs cycle and reducing the pH of the culture. Thus, fast and efficient concentration detection of glucose is desired.

Glucose binding protein is a protein which could bind to glucose and serve this purpose by acting as a biosensor. A biosensor is an analytical device used for the detection of an analyte that combines a biological component with a physicochemical detector component. GBP is such a biosensor, where GBP binds with glucose and binding is analyzed using fluorescence intensity and the corresponding signal is compared with standard glucose signal to estimate concentration of unknown sample. GBP is a monomeric periplasmic protein with molecular weight of 34 kD (kilo Dalton) and is synthesized in the cytoplasm ofE. coli.

In the conventional method, GBP (L225C mutant) is produced in multiple steps, pre-inoculation ofE. colimutants in Luria Bertani (LB) broth, culturing, harvesting, cell washing, osmotic shock, labeling, liquid chromatography and dialysis. All these steps are time consuming (around 4 days) and cumbersome. The present invention enables a non-conventional cell free expression of GBP where expression is faster and the resulting protein is relatively pure. This protein would preferably be labeled using a fluorophore called acrylodan (6-Acryloyl-2-dimethylaminonaphthalene) and purified by D15 (DEAE) chromatography membrane. The protein would preferably further be concentrated and dialyzed against 5 mM tris-HCl, pH 7.5.

Cell Free Method of Production of Granulocyte Colony Stimulating Factor (G-CSF) and Pharmaceutical Analog Fligrastim

For proof of principle, G-CSF also known as the pharmaceutical analog Filgrastim is used as a model therapeutic protein. Notably, the same method holds for any therapeutic protein for administration at the point-of-care. Filgrastim is used to stimulate the production of granulocytes (a type of white blood cell) in patients undergoing cancer therapy with specific drugs that are known to cause low white blood cell counts.

Cell-free protein synthesis system was tested for G-CSF protein expression, the DNA used as the template in the system was G-CSF plasmid: 80 μg (concentration: 0.47 μg/μL) in combination with lyophilized CHO lysate in an amount of 1 mL, Gadd34-Myc plasmid 8 ug (@ 0.4 ug/uL)=20 uL)), Thermo Reaction Mix (5×): 400 uL, nuclease free water: balance to 2 mL, and 1×CHO dialysis buffer at 25 mL. Six batches were run with a total batch volume for each batch of about 2 mL and the process took about 6 hours at a temperature of about 30° C. The bioreactor used was SLIDE-A-LYZER™ dialysis cassette (10 kDa cutoff: 3 mL).

About 1.98 mL of harvested product was subjected to purification in an IMAC spin column with loading buffer: 10 mM imidazole, wash buffer 1: 10 mM imidazole, wash buffer 2: 30 mM imiadazole and elution buffer: 150 mM imidazole. Notably PBS buffer, without DTT, was used in the purification process. The fractions were collected from triplicate runs where G-CSF was expressed over a six hour period in the presently claimed cell-free system. The G-CSF was purified using a His-tagged affinity column. The data show the remarkable consistency of the expression and purification of the target product.

FIGS.9A and9Bshow Western Blot results with an Anti-G-CSF antibody. The pellets discussed in both figures were washed once with 500 μL of PBS and solubilized in 1500 μL of PBS with 1% Tween-20 and 1.5% Triton X-100. The “H” represents the Harvest, the “P” is the Pellet and the “E” is the Elute. The elution column shows a clear band representing G-CSF for all three runs showing the remarkable consistency of the expression and purification of the target product. The far right column shows the G-CSF standard that was also run.FIG.9Ashows the results of Run #3 andFIG.9Bshows the results of Run #4. Clearly the results are consistent and provide evidence that the process of the present invention is reproducible.

FIG.10Ashows the quantified values of the harvested proteins and purified protein of Run #3 ofFIG.9AandFIG.10Bshow the values of Run #4 ofFIG.9B.

Further it was shown that proteins produced in the on-demand system of the present invention provide for improved and increased potency relative to freeze/thaw data of the prior art method and it is evident that using a freeze/thaw cycle impacts activity. As shown inFIG.11, in the results of separate runs of 3 and 4, it can be seen that the freshly made G-CSF has potency twice that of the reconstituted lyophilized standard. The same molecule loses its potency after just one freeze-thaw cycle approaching that of a boiled control. These data prove our assertion that administering a freshly made therapeutic protein (at most, refrigerated for a few days) provides maximal potency and has the additional advantage of no additives. This is a significant and surprising improvement over the current paradigm of biologics production and delivery.

In another example, the therapeutic protein Erythropoetin (EPO, used to stimulate red blood cell production in the human body) was produced in the cell free system.FIG.12shows the same consistency of expression in 4 separate batches as evidenced by the bands on the Western blot.

As the cell proliferation-based activity assay shows in the top panel ofFIG.13A, the EPO in the extract is more active compared to controls. It should be noted that the EPO tested from the cell free process was diluted 100-fold, so the activity in the extract was in excess of 100,000 units/mL. Given the typical EPO dosage is between 50-100 units/mL/kg, it appears that 1 mL product contains sufficient EPO to dose10adults. These data show the remarkable potential of point-of-care manufacturing as the freshly expressed protein shows very high activity. This is likely due to virtually no “aging” of the protein that normally takes place in conventionally manufactured proteins (i.e. deamidation, oxidation, aggregation etc.).

Streptokinase was also produced successfully in a cell free stirred tank bioreactor. A representative sample of activity at two harvest times is shown inFIG.13B, lower panel. As can be seen, the harvest time may be used to pick a desired activity in the case of Streptokinase, several dosing regimens are in use clinically and the harvest can be timed to conform to the desired dose, eliminating any dilution for the final delivery to obtain the correct dose. As can be seen, active EPO was produced and had extremely high activity (samples 32K14 and 36K14 were diluted 100 fold for the assay). For Streptokinase, lysate was harvested at two time points and activity measured. This approach may be used to determine the dose needed for delivery to the patient.