Dynamic monosaccharide control process

Abstract

To provide a method for controlling nutrient supply in a cell culture process.SOLUTION: A sample is received from a bioreactor containing a cell culture. Viable cell density and residual nutrient measurements are determined from the samples received. Daily nutrient supply targets are calculated based on viable cell density and residual nutrient measurements. Nutrients are supplied to the bioreactor according to the calculated daily nutrient supply target.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 923,188, filed October 18, 2019. No. 5, U.S. Provisional Patent Application No. 62 / 923,204, filed October 18, 2019; U.S. Provisional Patent Application No. 62 / 923,217, filed October 18, 2019, 202 U.S. Provisional Patent Application No. 63 / 000,361, filed March 26, 2020 U.S. Provisional Patent Application No. 63 / 000,366, filed on March 26, 2020, and This application claims the benefit of U.S. Provisional Patent Application No. 63 / 000,371, filed on 2006 / 003 / 11. The entire contents of which are incorporated herein by reference. [Background technology]

[0002] The productivity of cell culture depends on optimizing culture medium management to enable high cell densities. Nutrient supply is an important parameter for process optimization. High cell density processes The amount of nutrients required varies depending on the cell type or density. Varies depending on the Summary of the Invention [Means for solving the problem]

[0003] Improved materials and methods for predicting the target daily nutrient requirements of a given cell line There is a need for a method for detecting and treating a microorganism, which the present invention addresses. The present invention relates to a method for controlling nutrient supply in a cell culture process. Viable cell density and residual nutrient measurements can be received from a bioreactor containing Daily nutrient supply targets can be determined from the viable cell density and residual nutrient measurements. Nutrients can be calculated based on the daily nutritional targets calculated by the bioreaction. It may be supplied to a reactor.

[0004] In one embodiment, the daily residual nutrient concentration is maintained within a predetermined range in the bioreactor. obtain.

[0005] In one embodiment, the daily nutrient supply target is determined by the daily viable cell density and residual nutrient. It may be recalculated based on measurements.

[0006] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and and glutamine.

[0007] In one embodiment, the nutrient may include one or more simple sugars.

[0008] In one embodiment, residual nutrient measurements include assaying nutrient concentrations within the bioreactor. It can be seen.

[0009] In one embodiment, the residual nutrient measurement is one of offline nutrient measurement and inline nutrient measurement. The method may include performing one or more of the following:

[0010] In one embodiment, the bioreactor can be any bioreactor known in the art. In some embodiments, the volume of the bioreactor ranges from about 15 L to about 15,000 L. In one embodiment, the bioreactor is a Chinese hamster ovary (CHO) cell bioreactor. The reactor may be one or more of a 5 L reactor and a 5 L bioreactor. Other mammalian cell types may be used for the production of the cells. Non-limiting examples of such mammalian cell types include: Examples include HEK, 293, and PerC6. This process can be performed in yeast and bacteria, for example. It may also be used for other non-mammalian cell types such as:

[0011] In some embodiments, the cells in the bioreactor can be any type of cell known in the art. In one embodiment, the cells in the bioreactor can be mammalian cells. The cells in the bioreactor can be bacteria, yeast, or insect cells.

[0012] In one embodiment, the cells may be CHO cells, recombinant CHO cells, or a mixture thereof. do.

[0013] In one embodiment, the daily nutrient supply target is 100% bioreactions from at least six cell lines. The reactor is run at least according to a predetermined global average consumption value and growth profile. may also be calculated based in part on

[0014] Another aspect of the disclosed technology relates to a method for controlling nutrient supply in a cell culture process. The sample can be received from a vessel containing a cell culture. Nutritional measurements may be determined from the samples received. Daily nutritional goals are based on the amount of nutrient delivered. Nutrients can be calculated based on cell density and residual nutrient measurements. The container may be fed according to a nutrient feeding target.

[0015] In one embodiment, the container may be a flask.

[0016] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and In one embodiment, the nutrient is selected from an amino acid or a vitamin. You can choose.

[0017] A further aspect of the disclosed technology is to balance glucose supply in the cell growth process. The viable cell density may be determined periodically during the cell growth process. The glucose concentration can be measured periodically during the cell growth process. It may be adjusted periodically based on viable cell density and glucose concentration. The cell growth process may be periodically fed according to course feeding targets.

[0018] One aspect of the disclosed technology relates to a system for controlling nutrient supply in a cell culture process. The processor may be in communication with the bioreactor and the nutrient supply system. The process may be carried out in a bioreactor. A nutrient supply system may supply nutrients to the bioreactor. The processor measures viable cell density and residual nutrients from samples withdrawn from the bioreactor. Daily nutrient feeding targets can be determined based on viable cell density and residual nutrient measurements. The processor may calculate the daily nutrient supply target. The nutrient delivery system may be instructed to deliver nutrients to the

[0019] In one embodiment, the nutrient supply system provides a continuous or discontinuous supply of nutrients during the cell culture process. It can be provided.

[0020] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and and glutamine.

[0021] In one embodiment, the nutrient may include one or more simple sugars.

[0022] Another aspect of the disclosed technology is to balance glucose supply in the cell growth process. The system includes a processor that supplies glucose during the cell growth process. The processor may communicate with a cell supply system. The density and glucose concentration may be determined periodically. The processor may periodically adjust the glucose delivery target based on the glucose concentration. A glucose supply system is provided to supply glucose to the cell growth process according to a supply target. The system may be periodically instructed to:

[0023] A further aspect of the disclosed technology relates to a system for preventing glycation in a cell culture process. The processor may be in communication with the bioreactor and the nutrient supply system. The process may be carried out in a bioreactor. A nutrient supply system may supply nutrients to the bioreactor. The processor may determine the residual amount of the nutrient in the sample withdrawn from the bioreactor. The processor determines the amount of nutrients consumed since the last feeding based on the amount of nutrients remaining. The processor may determine the viable cell density in the sample. The processor may determine the consumption of nutrients. Based on the amount consumed and the viable cell density, the predicted amount of nutrients consumed until the next feeding is calculated. The processor compares the predicted nutrient consumption with a predetermined residual nutrient target before the next feeding. The processor may calculate a target amount of the nutrient for the current supply based on the calculated Instruct the nutrient supply system to supply nutrients to the bioreactor according to the target amount of nutrients. possible.

[0024] One aspect of the disclosed technology relates to a method for regulating the amount of glycation of a drug in a cell culture process. The cell culture process may be carried out in a bioreactor. You can receive the sample and measure the residual amount of nutrients from the sample. The amount of nutrients consumed after the supply of the nutrients can be determined based on the remaining amount of nutrients. The predicted consumption of nutrients until the next feeding can be determined from the received sample. The predicted consumption of nutrients and the next supply of nutrients can be calculated based on the consumption of nutrients and the viable cell density. Based on the predetermined residual nutrient target before feeding, the target amount of nutrients for the current feeding can be calculated. Nutrients may be fed to the bioreactor according to calculated nutrient target amounts.

[0025] In one embodiment, the predicted viable cell density between the current feeding and the next feeding is determined by the determined viable cell density. The rate of nutrient consumption may be determined based at least in part on the density of the nutrient. The predicted consumption rate may be determined based, at least in part, on the predicted viable cell density and nutrient consumption rate. It can be calculated based on the following.

[0026] In one embodiment, feeding may occur daily.

[0027] Another aspect of the disclosed technology is a method for controlling glucose supply in a cell culture process. A sample is received from a bioreactor containing a cell culture. Measure the residual glucose level from the sample when receiving it from the bioreactor. The processor compares the remaining amount of glucose to a predetermined glucose target. The processor calculates the amount of glucose consumed. If the target is greater than the target, the processor will Glucose consumption is determined by determining the amount of glucose consumed. If the residual amount of glucose is below the predetermined glucose target, the processor The processor determines the glucose consumption rate based on the difference between the target and the glucose remaining amount. The processor calculates the integrated viable cell density. The processor calculates the predetermined viable cell density for the next day based on the integrated viable cell density. The processor calculates a ratio based on the glucose consumption rate and the integrated viable cell density. The processor calculates the ratio of glucose consumption rate to a given viable cell density on the following day. The processor calculates the predicted glucose consumption by multiplying the predetermined glucose The glucose goal is calculated by adding the glucose consumption and the predetermined minimum amount of glucose. The glucose is fed to the bioreactor according to a glucose target.

[0028] In one embodiment, feeding may occur daily.

[0029] Further features of the present disclosure and advantages offered thereby are set forth in the specific drawings which are shown in the accompanying drawings. The present invention will be described in more detail below with reference to embodiments, in which like elements are designated by like reference numerals. will be done. [Brief explanation of the drawings]

[0030] Reference will now be made to the accompanying drawings, which are not necessarily drawn to scale,

[0013] The present disclosure is incorporated in and constitutes a part of the present disclosure, and illustrates various embodiments and aspects of the techniques of the present disclosure, The principles of the techniques of the present disclosure will be explained together with the description. [Figure 1] FIG. 1 is a schematic diagram of an exemplary environment that may be used to implement one or more embodiments of the present disclosure. [Figure 2] FIG. 2 is a schematic diagram of an exemplary environment that may be used to implement one or more embodiments of the present disclosure. [Figure 3]FIG. 3 is a flowchart of a glucose algorithm according to one aspect of the disclosed technology. [Figure 4] FIG. 4 is an example of an automated process for delivering glucose according to one embodiment of the disclosed technology. [Figure 5] FIG. 5 is a block diagram of a nutrient supply control system according to one embodiment of the disclosed technique. [Figure 6A] FIG. 6A is an exemplary table showing experimentally determined residual glucose targets by culture day according to one embodiment of the disclosed technology. [Figure 6B] FIG. 6B is another example of a table showing experimentally determined residual glucose targets by day of culture according to one embodiment of the disclosed technology. [Figure 6C] FIG. 6C is an exemplary table showing cell line and bioreactor run counts used to calculate predicted cell growth behavior according to one embodiment of the disclosed technology. [Figure 7] FIG. 7 is a chart showing the median and IQR for the fold change in ΔIVCD over time according to one embodiment of the disclosed technology. [Figure 8] FIG. 8 is a chart showing the median and IQR of ΔIVCD / VCD over time according to one embodiment of the disclosed technique. [Figure 9A] 9A-9B show charts of percent error in VCD prediction over time according to one aspect of the disclosed techniques. [Figure 9B] 9A-9B show charts of percent error in VCD prediction over time according to one aspect of the disclosed techniques. [Figure 10A] 10A-10C show charts of residual glucose concentrations measured over time in different cell lines according to one embodiment of the disclosed technology. [Figure 10B] 10A-10C show charts of residual glucose concentrations measured over time in different cell lines according to one embodiment of the disclosed technology. [Figure 10C] 10A-10C show charts of residual glucose concentrations measured over time in different cell lines according to one embodiment of the disclosed technology. [Figure 11A] 11A-11B show additional charts of residual glucose concentrations measured over time in different cell lines according to one embodiment of the disclosed technology. [Figure 11B] 11A-11B show additional charts of residual glucose concentrations measured over time in different cell lines according to one embodiment of the disclosed technology. [Figure 12] FIG. 12 shows a chart of residual glucose concentration measured by RSV over time according to one embodiment of the disclosed technology. [Figure 13] FIG. 13 shows a chart of residual glucose concentrations measured over time in cell line CHO6 according to one embodiment of the disclosed technology. [Figure 14] Figure 14 shows the predicted median values ​​calculated from the Janssen cell line database.

[0031]

number

[0032]

number

[0033] Certain implementations of the techniques of this disclosure are more fully described with reference to the accompanying drawings. However, the disclosed technology may be embodied in many different forms and may include, but is not limited to, any of the following: The present disclosure should not be construed as being limited to the embodiments described herein. The components described herein as comprising the It is intended that components that perform the same or similar functions as those described herein Many suitable components are intended to be included within the scope of the disclosed electronic devices and methods. Other components not described herein include, for example, These may include, but are not limited to, components developed after the development of the technique.

[0034] Also, a reference to one or more method steps may include any intervening steps expressly identified. It is also to be understood that the presence of additional or intervening method steps is not excluded.

[0035] Unless otherwise clear from the context, the term "a" or "an" entity refers to one of that entity. For example, "an amino acid" refers to one or more Thus, "a" (or "an"), "one or more" The terms "and "at least one" may be used interchangeably herein.

[0036] The term "nutrient" refers to the nutrients an organism needs to survive or grow, or otherwise provide biomass. Nutrients can refer to any compound, molecule, or substance used to add nutrients. a carbohydrate source (e.g., glucose, galactose, maltose, or fructose) or more complex sugars), amino acids, vitamins (e.g., B vitamins (e.g., Vitamins B12, A, E, riboflavin, thiamine, and biotin. In the present invention, one or more nutrients can be used as surrogate molecules to stimulate biological reactions. In some embodiments, the amount of total nutrient medium to be added to the vessel may be determined. The term may refer to monosaccharides, vitamins, and amino acids.

[0037] The term "amino acid" refers to the 20 standard amino acids: glycine, alanine, bacillin, thiamin ... Phosphorus, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan threonine, serine, asparagine, glutamine, tyrosine, cysteine, lysine , arginine, histidine, aspartic acid and glutamic acid, their single stereoisomers The term "amino acid" may refer to either the amino acid or its racemic mixture. Also known non-standard amino acids, e.g., 4-hydroxyproline, hydroxyproline , s-phosphocysteine, phosphotyrosine, ε-N,N,N-trimethyllysine, 3-methyl 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamate ε-N-acetyllysine, ω-N-methylarginine, N-acetylserine, N,N , N-trimethylalanine, N-formylmethionine, γ-aminobutyric acid, histamine, -pamine, thyroxine, citrulline, ornithine, β-cyanoalanine, homocysteine , azaserine, and S-adenosylmethionine. In some cases, the amino acid is glutamic acid, glutamine, lysine, tyrosine, or valine. In some embodiments, the amino acid is glutamate or glutamine.

[0038] The terms "nutrient medium," "feed medium," "supply," "total feed," and "total nutrient medium" are used interchangeably. The terms may be used interchangeably and refer to growing a cell line, propagating, and adding biomass to a cell line. Nutrient media may include "complete" media used to grow and culture cell lines. It can be distinguished from a substance or simple medium that is insufficient for the growth of bacteria and bacteria. Glucose or simple sugars are essential for growing cell lines in the absence of other necessary nutrients. It is not a nutrient medium in itself, as it is not sufficient.

[0039] In one aspect, the present invention provides a method for the production of glucose in a bioreactor, such as a CHO cell bioreactor. The present disclosure teaches a carbohydrate control algorithm that is used to balance the supply. The system utilizes data collected (automatically or non-automatically) to analyze the bioreactor Regulates glucose, thereby strengthening anti-glycation and preventing the biological effects of glucose deficiency Reduce reactor failure, dynamically measure cellular responses to glucose, and measure the response of any living organism (CH O) has advantages over the art in regulating glucose It is semi-automated or fully automated and organism-independent, so it can be used with other mammalian cells. (e.g., CAR-T, CHO, etc.). The system may, among other things, It is dynamic in that it periodically learns from glucose measurements. The system includes an algorithm that manages the glucose delivery system (as in a standard commercial kit). (These kits are suitable for use with this system.) Commercially available glucose feeding kits are available in many ways. (multiple feeding, various locations, continuous or discontinuous feeding possible). Use a continuous system such as a pre-programmed, non-feedback controlled continuous supply of nutrients. (The system simply divides the glucose bolus over time.) The glucose data can be automatically provided to the algorithm. Adjustments to feeding are based on this data and using a glucose analyzer. In embodiments, the disclosed system uses mammalian cells, yeast, or bacteria to produce glycoproteins. Used to manufacture commodity chemicals other than proteins.

[0040] In one embodiment, the system of the present disclosure involves feedback control during the cell culture process. This includes automated processes that

[0041] In one embodiment, both lactate and glucose are measured and a glucose target is achieved. Make stepwise changes (0.5g / L at a time).

[0042] In one embodiment, only glucose is measured. Simplify the process in the lab, allowing bioreactor operators to use algorithms to optimize glucose This makes it easier to provide adequate supplies.

[0043] In one embodiment, the algorithm uses only cell density measurements and is pre-programmed. The total glucose consumption value was calculated.

[0044] In one embodiment, the glucose target is calculated once a day. It may be updated to allow for multiple measurements, with the goal being for the next 24 hours.

[0045] The concept of this algorithm is to take the thought process used by a human operator and , using the measured glucose and lactate to increase or decrease glucose targets It comes from converting it into a flow diagram that makes incremental changes.

[0046] The glucose algorithm of the present disclosure is designed to mitigate the risk that a human operator may underestimate glucose ( glucose depletion event) or to ensure that glucose is not depleted. They tend to be more accurate than human operators because they tend to overestimate the target. The disclosed algorithms may potentially control glucose levels more efficiently than a human operator could. The algorithm is excellent at controlling the desired glucose concentration to be maintained. and may be set to a value of, for example, 1 to 2 g / L.

[0047] The methods of the present disclosure may be used for other monosaccharides, nutrients, etc. Non-limiting examples of saccharides include glutamic acid, galactose, lactate, and glutamine. It can be obtained.

[0048] According to the technology of the present disclosure, glucose can be minimized to avoid glycation.

[0049] In some embodiments, the methods disclosed herein involve a biological reaction that produces a biologic. In organ cell culture, to increase the amount of biologics produced or to improve the production of biologics The disclosed methods may include (a) reducing the amount of one or more nutrients in a bioreactor cell culture. (b) analyzing the concentration of one or more nutrients intermittently or continuously; and If the temperature is lower than 50°C, adding additional nutrient medium to the bioreactor cell culture may be included.

[0050] In some embodiments, a substantially stable distribution of amino acids throughout the bioreactor process is achieved. Additional nutrient medium may be added to the bioreactor cell culture in an amount sufficient to maintain the desired concentration. .

[0051] In some embodiments, the bioreactor cell culture is Chinese hamster ovary (C HO) cells, HEK-293 cells, or VERO cells. The biologic may be an antibody or antibody-like polypeptide.

[0052] In one embodiment, the method of the present invention may be carried out in the presence of any cell culture medium, for example The bioreactor process may be performed in serum-free media, protein-free media (including but not limited to, Protein-free media containing protein hydrolysates, or chemically defined media The reaction may be carried out in the presence of a solvent.

[0053] A variety of analytical devices may be used in the present invention. The analytical device may be a surrogate molecule or marker, e.g. For example, amino acids or other substances in the cell culture medium (e.g., vitamins, minerals, ions, sugars, etc.) The analytical equipment may include any device or process capable of detecting and / or quantifying substituents. The methods include gas chromatography, HPLC, cation exchange chromatography, and anion exchange chromatography, size exclusion chromatography, enzyme catalysis assays, and / or can be a device for performing a chemical reaction assay.

[0054] As shown in Figure 1, the production reactor 102 can contain a mammalian cell culture. Actor 102 may be a bioreactor, a cell culture reactor, or a sample bioreactor. The production reactor 102 can be a well plate, a shake flask, a benchtop vessel, or At least one commercial-scale (e.g., 15 kL) stainless steel reactor A reaction sample is taken from the production reactor 102 and fed to the nutrient supply control system 104. The nutritional delivery control system 110 may be sent to a glucose monitor that performs glucose measurements. The glucose measurement may include a glucose measurement system 104. The glucose measurement may be either offline or online. The nutritional delivery control system 110 may also be implemented by the glucose measurement system 104. Glucose goal prediction that receives glucose measurements from and performs a glucose goal prediction The nutritional supply control system 110 may include a glucose calculation system 106. 08, and then use the predicted glucose target to determine the amount of glucose to add. and may send instructions to the nutrition delivery system 120. The glucose measurement system 104, the glucose goal prediction system 106, and the glucose calculation system 10 The processes performed by 8 may be accomplished by one or more processors.

[0055] The nutrient supply system 120 supplies glucose from the glucose supply 112 to the production reactor 102. The blood glucose monitor may include a pump 111 that supplies a large amount of glucose.

[0056] FIG. 2 is a schematic diagram of an exemplary environment that may be used to implement one or more embodiments of the present disclosure. The nutrient supply control system 110 communicates with the production reactor via a network 180. 102 and the nutrition supply system 120. The nutrition supply control system 110 A feeding system 120 may be configured to provide one or more nutrients to the production reactor 102. can be instructed to.

[0057] Figure 3 shows a flow diagram of the glucose algorithm. At 302, the production reactor 102 may provide a sample. At 304, the glucose measurement system 104 may At 306, a glucose measurement can be performed. The target prediction system 106 can predict the amount of glucose to add to the production reactor 102. At 308, the glucose calculation system 108 calculates the correct amount of glucose to add. At 310, the correct amount of glucose is provided to the production reactor 102. This algorithm may be applicable to pre-culture. For example, This algorithm can be used to supply glucose to an enhanced seed train. The system may also be applicable to N-1 perfusion processes and production perfusion processes.

[0058] Figure 4 shows an example of an automated process. At 402, the production reactor 102 At 404, a glucose measurement can be performed, e.g., at 404 a for in-line glucose measurement (e.g., NovaFlex) or 404b for Raman The instrument can be used to perform offline pH and glucose and rhamnositometry. At 406, the nutrient delivery control system 110 may measure glucose delivery. At 408, the reactor control station may predict the predicted group feed target. At 410, the controller calculates the glucose delivery rate and may process the course delivery target. At 412, the controller may supply glucose to the production reactor 102. possible.

[0059] The methods disclosed herein may increase production in subsequent bioreactor cell cultures. In some embodiments, the method includes the step of producing an antibody (or other biologic) in a bioreactor. Increasing the amount of antibody (or other biologic) produced in cell culture or increasing the amount of antibody (or other biologics) production time. The method can be used with automated sampling equipment (e.g. (e.g., offline, online, in-line or at-line sample analysis) Analyze culture samples with or without extracting samples from the bioreactor. The method may include analyzing the culture sample (e.g., residual glucose) by an automated analyzer. The concentration of nutrients (or other surrogate markers) can be analyzed to generate data representing the amount of nutrients. The method may include using an algorithm or computer-based processing program. and the data generated (e.g., obtained from the assay of residual glucose from a sample) ), wherein the processed data is used to determine the amount of water added to the bioreactor. This method is used to determine the amount of additional nutrient medium to be added by an automated feeder. The method may include adding a determined amount of the determined nutrient medium to the bioreactor. This may include recording the time and amount of each nutrient medium addition.

[0060] Mammalian cells can include any mammalian cell that can be grown in culture. Examples of cells include CHO cells (CHO-K1, CHOK1SV (registered trademark), CH O DUKX-B11, CHO DG44, etc.), VERO, BHK, HeLa, CV1 (Cos, Cos-7, etc.), MDCK, 293, 3T3, C127, myeloma cell lines (e.g. mouse), PC12, HEK-293 cells (HEK-293T and HEK-293E, etc. Examples of such cells include PERC6, Sp2 / 0, NS0, and W138 cells. Mammalian cells derived from either the microbial community or the mammalian host may also be used. The cell culture may be Chinese hamster ovary (CHO) cells, HEK-293 cells, or It may contain VERO cells.

[0061] The steps of the methods of the present disclosure may be repeated and may occur at various intervals. In embodiments, the steps disclosed herein may include, throughout the bioreactor process: More than 10 times, or 10 to 1000 times, 20 to 500 times throughout the bioreactor process , or 30 to 100 times. In some embodiments, the steps Throughout the entire process, the time is approximately 4 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 6 hours, Every 8, 12, 16, 18, or 24 hours, or throughout the entire bioreactor process It may be repeated throughout the body about every 4 to 18 hours, or about every 10 minutes to about 6 hours. In certain embodiments, the method provides for the production of nutrients (e.g., gluten) in a period of about 1 day or after about 24 hours. Once daily, target concentrations of residual nutrients (e.g., residual glucose) are used. In certain embodiments, the method comprises measuring the amount of Using the target concentration of nutrients (glucose) produced after a period of time, the test is performed multiple times a day, e.g., once a day. This includes measuring the amount of residual nutrients (e.g., residual glucose) twice, three times, or four times a day. nothing.

[0062] The steps of the methods disclosed herein can be performed in a relatively short time, i.e., additional Sampling, analysis, and addition of nutrient medium can be done relatively quickly. In some embodiments, the steps of the disclosed methods are performed within about 1 minute to about 2 hours.

[0063] In some embodiments, the steps of the disclosed methods are performed by one or more automated devices. The terms "automatic," "automatically," or "automated" refer to the process of performing a task. necessary to initially prepare one or more devices for use or to automatically operate one or more devices. Human intervention, other than the human intervention or actions that may be required to maintain operation or represents one or more mechanical devices that perform one or more tasks without motion. A "mechanical device" that automatically performs a task may optionally include, for example, In controlling the timing, duration, frequency, type, and / or characteristics of the collected information that may be used therein for the purpose of making decisions that control and direct the execution of the device. It may include a computer and necessary instructions (code) for processing the data.

[0064] In various embodiments, "offline" analysis refers to data analysis relating to in-process conditions. Samples are taken from the production process so as not to convey real-time or near-real-time information. This refers to the permanent removal of a sample and analyzing it at a later time. In some embodiments, one or more analytical devices are used offline.

[0065] In one embodiment, the analytical device (or the sensor portion connected thereto) is The device or sensor part may be directly installed in a manufacturing unit or may be installed in a suitable It may be separated from the bioreactor or purification unit by a barrier or membrane.

[0066] In some embodiments, the analyzer may include a cellular analyzer to allow rapid determination of cell concentration. It may be a kit that can be placed in contact with a sample, such as a test strip. In some embodiments, the kits may include a surrogate marker or a specific concentration of a surrogate marker. a substrate that produces a chemical and / or enzyme-linked reaction that produces a detectable signal in the presence of The detectable signal may include, for example, a colorimetric change or other visual signal. In some embodiments, the analytical device is a disposable analytical device, e.g., a disposable test strip. Such kits can be easier to operate than other larger and more complex analytical devices. Such kits may also be useful for small-scale production, reducing the ease and cost of production. In cell culture growth, it can be useful to determine the optimal health and productivity of the culture. do.

[0067] A "conventional manufacturing process" is one that (a) produces a nutrient medium in the form of a bolus feed at a specified time; (b) glucose (or other single nutrient) is consumed This may include adding glucose (or other single nutrient) to the bioreactor in response to Conventional manufacturing processes result in low yields of bioproducts and / or low production efficiencies of bioproducts. In one embodiment, the system of the present disclosure utilizes a feedback control method. The concentration of one or more nutrients can be monitored and a The appropriate amount of total medium is added to the bioreactor. Monitoring is automatic and frequent. This allows for a significant increase in the yield of bioproducts.

[0068] In some embodiments, the amount of bioproduct produced is reduced compared to conventional manufacturing processes. In some embodiments, the amount of bioproduct produced can be significantly increased by using conventional methods. may be 10% to 100% greater than the amount of bioproduct produced by the manufacturing process In some embodiments, the amount of bioproduct produced by the methods of the present invention is greater than that of conventional bioproducts. 10%, 15%, 20%, or 25% of the amount of bioproduct produced by the manufacturing process , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80% , or in some cases 90% or 100% more.

[0069] In some embodiments, the degree of glycosylation of a biologic is determined by the degree of glycosylation of the biologic produced by a conventional manufacturing process. at least 10%, at least 15%, or less than the degree of glycation of the biologic At least 20%, at least 25%, at least 30%, at least 35%, at least 40% %, or at least 45% decrease.

[0070] In various embodiments, the glucose algorithms and methods described herein are based on a glucose level between 0 and 3 g. / L, 0.5-2g / L, 2-5g / L, less than 1g / L, or less than 2g / L one day after ingestion It may be effective in achieving a residual glucose concentration in the eye.

[0071] A variety of biological products are contemplated in the present invention. In some embodiments, biological products The agent may be an antibody, a recombinant protein, a glycoprotein, or a fusion protein. In some embodiments, the biologic may be a soluble protein. The biologic may be an antibody, antibody fragment, or modified antibody (e.g., multivalent antibody, domain deleted antibody, multivalent ... Monomeric antibodies, hinge-modified antibodies, stabilized antibodies, multispecific antibodies, linear antibodies, scFv, linked S cFv antibodies, multivalent linear antibodies, multivalent antibodies without Fc, Fab, multivalent Fab, etc. obtain.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of a method for manufacturing a semiconductor device according to the present invention; Reference will now be made in detail to exemplary embodiments. For convenience, like reference numerals will be used throughout the drawings to refer to like or similar parts. Used to refer to similar parts.

[0073] FIG. 5 is a block diagram of a nutrient supply control system 110 according to one aspect of the disclosed technology. The nutritional delivery control system 110 may include one or more processors 510. Glucose Measurement system 104, glucose goal prediction system 106 and glucose calculation system 1 The processes performed by 08 may be accomplished by one or more processors 510.

[0074] Referring to FIG. 5, a processor 510 executes stored instructions and reads stored data. Microprocessors, microcontrollers, digital signal processors, etc. that can operate based on The processor may include one or more of a processor, a co-processor, etc., or a combination thereof. The processor 510 is compatible with Intel's Pentium family or AMD's one or more existing microprocessors, such as the Turion™ family of microprocessors manufactured by The processor 510 may be a single core or may be a parallel processor. For example, the processor 510 may be a multi-core processor that simultaneously executes multiple processes. , a single-core processor configured with virtual processing technology. Processor 510 includes logical processors for executing and controlling multiple processes simultaneously. The processor 510 may run multiple software processes, applications, programs, and In order to realize the ability to execute, control, start, operate, store, etc. Those skilled in the art will appreciate that the present disclosure may be implemented using the same techniques as those disclosed herein. It will be appreciated that other types of processor configurations may be implemented that provide the above capabilities.

[0075] The non-transitory computer-readable medium 520, in some implementations, System 522, an application program (e.g., a web browser application) application, widget or gadget engine, and / or other applications as needed. one or more suitable types of memory (e.g., volatile or non-volatile memory, random access memory (RAM) ), read-only memory (ROM), programmable read-only memory (PROM), Erasable Programmable Read Only Memory (EPROM), Electrically Erasable Programmer EEPROM, magnetic disk, optical disk, floppy disk Disk, hard disk, removable cartridge, flash memory, independent disk redundancy In one embodiment, the processing techniques described herein may include , as a combination of executable instructions and data in a non-transitory computer-readable medium 520 The non-transitory computer-readable medium 520 may be implemented in accordance with one or more features of the embodiments of the present disclosure. It includes one or more memory devices that store data and instructions used to execute the features. The non-transitory computer readable medium 520 may also include a document management system, a Micros oft(TM) SQL database, SharePoint(TM) database, Or Acle™ database, Sybase™ database, or other relational database a memory controller for a relational or non-relational database, One or more pieces of data controlled by a device (e.g., a server) or software The non-transitory computer-readable medium 520 may include any combination of processors. When executed by the processor 510, it performs one or more processes consistent with embodiments of the present disclosure. In some embodiments, non-transitory computers may include software components that perform the The data-readable medium 520 may include one or more of the processes and functions associated with embodiments of the present disclosure. The non-transitory computer-readable medium 520 may include a database 524 for executing , including one or more programs 526 for performing one or more functions of the embodiments of the present disclosure. Additionally, the processor 510 may be configured to operate in conjunction with one or more processors located remotely from the system 110. For example, the system 110 may execute the program 526 when executed to implement the disclosed implementations. One or more remote programs 526 may be accessed to perform functions related to the embodiment. do.

[0076] The system 110 also receives signals or inputs from devices and provides signals or outputs to one or more Data is received and / or transmitted by the system 110 for provision to the device. One or more I / O devices 5 may be equipped with one or more interfaces that allow For example, the system 110 may include a data entry system 60. One or more keyboards, mouse devices, and touchscreens that allow the device to receive Smartphones, trackpads, trackballs, scroll wheels, digital cameras, An interface that may provide an interface to one or more input devices such as a microphone, a sensor, etc. The system 110 may include an interface component for processing images, video, data, or other information. The device 560 may include a graphical user interface 562.

[0077] In an exemplary embodiment of the disclosed technology, the system 110 may include a includes any number of hardware and / or software applications that run on the One or more I / O interfaces 560 receive data and and / or may be used to receive or collect user instructions. by one or more computer processors as desired in various embodiments of It may be processed and / or stored in one or more memory devices.

[0078] Network 180 is a network of interconnected computers, more commonly known as the Internet. Network 180 may include a network of computing devices. Any suitable network, including an individual connection over the Internet, such as a WiFi network In some embodiments, the network 180 may be of the radio frequency Identification (RFID), Near Field Communication (NFC), Bluetooth, Low Energy Bluetooth (trademark) (BLE), WiFi (trademark), ZigBee (trademark) , Ambient Backscatter Communication (ABC) protocol, direct connection such as USB, WAN, or LAN The information transmitted may be personal information. Security concerns may prevent these types of connections from being It may be provided that one or more of the sequences is encrypted or otherwise protected. However, in some embodiments, the information transmitted may not be personal. Therefore, network connectivity may be chosen for convenience over security. Network 180 is any type of computer network used to exchange data. For example, the network 180 may include an internetwork. Virtual private networks using private data networks and public networks Components in the network and / or system environment may affect the components of system 100. The network may be any other suitable connection that allows information to be sent and received between the components. 180 may also include a public switched telephone network (PSTN) and / or a wireless network. Network 180 may also be any network used to exchange data within a local area. Local networks, including computer networking configurations of the type, e.g., Wi Fi, Bluetooth, Ethernet, and system environment components may include other suitable network connections that allow the devices to interact with each other. [Example]

[0079] Example 1 Glucose feeding is a key parameter for optimizing the bioreactor process. Additionally, high cell density processes require significant amounts of glucose (in the range of 5-10 g / L per day). ) may be required, and the daily amount required varies depending on the cell density. , a variable glucose algorithm is used to predict daily glucose target requirements. The variable glucose algorithm can be deployed to measure the Viable Cell Density (VCD) and residual glucose. Your glucose readings are used to calculate your daily glucose goal. By implementing the algorithm, residual glucose concentrations measured one day after feeding in six cell lines were As a result, most residual glucose can be reduced to 0-3g / L. Concentrations of 0.5 to 2 g / L may be achieved.

[0080] The glucose supply target for the current day (d) is calculated by dividing the residual glucose target for the next day (d+1) by the glucose target for the next day (d+2). It can be calculated from the sum of the predicted glucose consumption from d to d+1 (Equation 1). Glucose goals d = predicted glucose consumption d+1 +Residual Glucose Target d+1 [1]

[0081] Figures 6A-6B show experimentally determined glucose algorithms by culture day. Two tables are shown, each showing residual glucose targets. Residual glucose targets are experimentally determined. The concentration may vary between 0.5 and 1.5 g / L depending on the day of culture.

[0082] Predicted glucose consumption is calculated for the next culture day (VCD) d+1 ) forecast VCD to the current day (d) It can be defined as multiplying the glucose consumption rate (Equation 2). Predicted glucose consumption d+1 = Specific glucose consumption rate d * Predictive VCD d+1 [2]

[0083] In this case, the specific glucose consumption rate is the glucose per cell per day from day d-1 to day d. The glucose consumption can be defined as the glucose concentration at d-1. It can be calculated as the difference between the target and the measured glucose concentration at d. The concentration of consumed glucose is the cumulative cell density change from d to d (ΔIVCD d ) can be normalized by multiplying it by the elapsed time from d-1 to d (Equation 3).

[0084]

number

[0085] The IVCD can be approximated in most cases using the logarithmic mean method. The simple mean method is can be used when does not change from d-1 to d.

[0086] Predicted VCD for d+1 (VCD d+1 ) is the change in IVCD from d-1 to d ( ΔIVCD d ) the median predicted fold change in ΔIVCD from d to d+1

[0087]

number

[0088]

number

[0089]

number

[0090] Experimental errors associated with cell counters can lead to overestimation and underestimation of individual VCD measurements. ΔIVCD d VCD as a measure of viable cell density d Use instead of Individual measurement errors reduce their impact on the IVCD and, therefore, glucose goal predictions.

[0091] Parameters to characterize the growth of cell lines in high titer media and feeds, namely , the median expected fold change in ΔIVCD and ΔIVCD / VCD on day d+1 was calculated as 1 It can be estimated from a database of 62 5L bioreactor runs. Six CHOK1SV® (Lonza) strains overexpressing the protein and with various phenotypes were identified. Sales AG) cell lines, which are abbreviated as CHO1, CHO2, CH These cells can be named CHO3, CHO4, CHO5, and CHO6. Figure 6C shows the predicted cell proliferation behavior. Figure 1 shows a table showing cell line and bioreactor run counts used to calculate activity. It should be noted that the target seeding density for these reactors is 500,000 viable cells / mL. This is a possible point.

[0092] The fold change in ΔIVCD from d to d+1 was, regardless of the cell line or source used, It can be calculated from all runs in the database as a function of culture day. Culture days (days) for 162 5L bioreactor runs from 6 cell lines using the same feedstocks d) The calculated mean and IQR of the fold change in ΔIVCD from day d to day d+1 are shown. Next, calculate the median and IQR of the data to obtain the expected median.

[0093]

number

[0094] The fold difference between ΔIVCD and VCD at d+1 may vary depending on the cell line or supply used. Regardless, it can be calculated from all runs in the database as a function of culture day. 162 5L bioreactor runs from 6 cell lines using titer media and feed The calculated mean and IQR of ΔIVCD / VCD on day d+1 up to the last culture day (d) are shown. Next, calculate the median and IQR of the data to obtain the expected median

[0095]

number

[0096] The error of the VCD prediction is the difference between the predicted VCD and the measured VCD on day d+1. This can be calculated using Equation 5.

[0097]

number

[0098] The percent error in VCD prediction was centered around 0 and varied by culture day, with an average of 0 to 9 9A-9B show that the percentage error of VCD prediction is less than 10% on average. This indicates that

[0099] The glucose algorithm performance was 5L as shown in Figures 10A-10C and 11A-11C. As shown in Figures 11B, 12, and 13, AMBR250 was used in five cell lines (CHO1, a bioreactor having CHO2, CHO3, CHO4, CHO5, and CHO6), and The results were evaluated with different high-potency feeds (JMF-1, JMF-5, and JaMS). Residual glucose can be measured approximately one day after feeding, and the data is used to calculate the demonstrated that the glucose concentration was controlled at 0.5-2 g / L in most cases.

[0100] In another embodiment, Equations 2 through 4 may be replaced with the following: Energy consumption d+1 can be defined according to Equation 2-1. The algorithm considers the time of day. It is fixed. Predicted glucose consumption d+1 = glucose consumption rate d * VCD d+1 Predicted fold change of * 1 Sun [2-1]

[0101] Glucose consumption rate d may be defined according to Equation 3-1.

[0102]

number

[0103] VCD d+1 The predicted fold change of 1 can be defined by Equation 4-1. It assumes the time of day.

[0104]

number

[0105] The values ​​in Equation 4-1 are calculated based on the High Titer medium and feed as shown in FIG. The database can be calculated from the Janssen cell line database using the Two 5L bioreactor runs were performed. Six cell lines with various phenotypes, i.e. These may include CHO1, CHO2, CHO3, CHO4, CHO5 and CHO6.

[0106] The predicted VCD of Eq. d+1 is the VCD of Equation 4-1 d+1 Predicted change in ΔIVCD d Multiply by It can be defined as

[0107] Residual glucose can be measured about a day after feeding, and the data is used to help the algorithm It has been demonstrated that in most cases the glucose concentration was controlled at 0.5-2 g / L.

[0108] Glucose can typically be controlled between 0.5 g / L and 2 g / L. 15 shows an exemplary flow process for calculating a course target. The process may receive the process signal, the VCD, and the sample time as inputs. The processor may determine whether the residual amount of glucose is greater than the target glucose. If not, in 1506, the amount of the cell culture process consumed between the previous day and the current day is Determine the amount of glucose consumed by determining the amount of glucose. If no, At 1508, a glucose monitor is configured to monitor the glucose level based on the difference between the predetermined glucose target and the residual amount of glucose. The amount of cell consumption is determined. In 1510, the integrated viable cell density IVCD is calculated. In step 12, the predetermined viable cell density for the next day is calculated based on the cumulative viable cell density. The specific glucose consumption rate is calculated by dividing the glucose consumption by ΔIVCD. In 1516, the specific glucose consumption rate is multiplied by the predetermined viable cell density for the next day. At 1518, the predicted glucose consumption is calculated. Calculate glucose goal by summing course consumption and predetermined glucose minimum do.

[0109] 2.0 Variable Glucose Delivery Sheet Instructions 2.1 AMBR250 Bioreactor Manual 2.1.1 First step The supply sheet can be found on the High Titer Implementation Resources SharePoint site. This site links to the SharePoint API-LM page. It has been done.

[0110] 2.1.2 Second step Before starting the run, as shown in Figure 16, SQL ID 1602 for each bioreactor and culture date To add a bioreactor SQL ID using a file, the macro uses the Follow the instructions as shown in 17. First, in the "Batch" tab, enter the generated Second, when you are done, press the "Update Batch ID" button 1702. Third, the bioreactor ID is entered into the "Input and Feed Targets" sheet.

[0111] 2.1.3 Third step As shown in Figure 18, for each AMBR250 bioreactor and culture day, the sampling Time 1802 (24 hour format hh:mm), measured VCD 1804 (10^6 Enter the number of cells / mL, and the glucose concentration in the bioreactor. was fed (g / L) using one of the macros.

[0112] The macros in the Excel file are for Vi-CELL, BioHT, MetaFLEX, and and AMBR250 instruments. To ensure accurate import of device file type values, please follow the sample naming requirements detailed below. For VCD measurements, Vi-CELL multifile or AMBR2 50 data tables can be imported. You can import MetaFLEX or BioHT files for configuration.

[0113] 2.1.3.1 Vi-CELL Multifile First, bioprocessing was performed on each bioreactor before sampling readings were taken. Second, the "Comments" section should be updated with the nomenclature to indicate the date of culture. D# must be used (e.g., culture day 1 = D1). Third, all readings The values ​​are saved in a single Excel multi-file. Multiple files are saved in the Vi-CELL software. It is created using software.

[0114] 2.1.3.2 BioHT text files The BioHT sample nomenclature is based on the Malvern API-LM format "Biological Reagents" Follow the "Device ID"_D#.

[0115] 2.1.3.3 MetaFLEX files First, enter "Bioreacto" in the "Patient ID" entry in MetaFLEX. Second, enter the culture date number in the "Patient Name" entry in MetaFLEX. (Just type #, not D#).

[0116] 2.1.3.4 AMBR250 Vi-CELL Value Vi-CELL values ​​from the AMBR250 are calculated daily via the AMBR250 software. The table is exported to the AMBR250 software. The column contains only the "Batch Name" and "Viable Cell Density" columns.

[0117] As shown in Figure 19, for the "Input and Supply Targets" sheet, select "Import Data File" After pressing the "Import" button 1902, the "Data to Import" window will appear as shown in Figure 20. Window 2000 will be displayed. The file for adding cell count and glucose values ​​is Press the appropriate "Browse" button 2002 and select the desired file, e.g., ViC for ViCell. ELL multi-file or AMBR250 ViCELL CSV file, MetaF MetaFLEX CSV file for lex and BioHT text for BioHT You can select a file by navigating to it. After selecting the file, Press the "Upload Data" button 2004. Enter and upload VCD and glucose values. and supply target” sheet.

[0118] For the AMBR250, MetaFLEX import allows you to import pH and sample data. The running time is entered.

[0119] Culture days are not exported from the AMB250. When you import the file, an additional dialog window will open, asking for the incubation date. Enter the culture day in the window (e.g. culture day 1 = 1).

[0120] 2.1.4 Fourth Step As shown in Figure 21, the feeding sheet lists the glucose target (g / L) for each AMBR250 container. ) and the operator enters SQ L, can be entered manually into the AMBR250 table or using a macro This can be semi-automatically entered into the AMBR250 table.

[0121] In the case of AMBR250, when you import a MetaFLEX file, the following appears: The pH 2202 and time 2204 are entered into the glucose feeding sheet. You can import the daily pH offset into the AMBR250 using This only works for MetaFLEX imports, not BioHT imports. pH is used to make some microscale bioreactors work easier. The online pH measured by the controller is included in the diagram in Figure 22. This can be verified with a fly pH measurement. If these differ, the online measurement is corrected. By including pH on the sheet, the user can select the Easily import your free-flow measurements. pH is used to calculate your glucose target. Not necessary.

[0122] 2.1.5 Fifth Step By using the "Export pH and Glucose" macro 2302, As shown in the figure, the glucose sheet displays the pH offline measurement file and the glucose Export two CSV files: a glucose measurement file and a glucose target file. Save the two tables to a flash drive.

[0123] If the MetaFLEX is not being used to measure glucose / pH, the pH The file will be blank.

[0124] 2.1.6 Sixth Step To enter your glucose target into the AMBR software, click on the left side as shown in Figure 24. Click on the "Tables" tab 2402 in the navigation bar.

[0125] 2.1.7 Seventh Step Next, on the right side of the screen, click on the "Data" tab 2502, as shown in FIG.

[0126] 2.1.8 Eighth Step Next, click the "Data Input..." button 2602 at the bottom right of the screen as shown in Figure 26. A dialog box will pop up. (Two ways - Export Master (using a keyboard or manually entering the data).

[0127] 2.1.9 Ninth Step In the dialog box, if you want to use the semi-automated macro method, select "Import data using a template" link 2702 (from step 2.1.5) (Available if glucose file can be exported from the If you want to use the method, click on "Enter data values."

[0128] 2.1.10 Tenth Step If using the manual approach, skip to step 2.1.12. If you use the Import Data Using Templates method, click the link 2702 Once selected, a new dialog box 2800 will appear, as shown in Figure 28. On the right side, select the "Load Data" button 2802. Once the table is loaded, all cells "Highlight" (click on the first cell and drag to the last cell) and select "Autodetect " button 2804.

[0129] 2.1.11 Eleventh Step The cells indicate the type of data found in each cell (bioreactor, data header, and data parameter). The color code is entered according to the parameter. All cells are coded correctly. If so, click "Save Data."

[0130] If using a MetaFLEX instrument, the Variable Glucose Algorithm Feed Sheet is You can export the measured pH in the saved file. Select "pH Offset" in the selection bar, select all reactors, and then select "pH Offset" Select "Import Data File" in the dialog box at the bottom left of the screen. Select "Exported pH File". Once the data is entered, Remove the deactivated bioreactor and click the "Save Data" button as shown in Figure 29. Select 2902.

[0131] 2.1.12 12th Step If the export button from 2.1.5 did not work, click "Enter data values" In the top right corner of the next window, click "Use current date and time" 3002 as shown in Figure 30. For each AMBR bioreactor, the measured glucose values ​​(g / L) were and glucose target value (g / L) 3006 into the table.

[0132] 2.1.13 13th step Finally, click "Save Data" 3008 at the bottom right of the screen as shown in Figure 30. do.

[0133] 2.2 5L Bioreactor Instructions 2.2.1 First step The supply sheet can be found on the High Titer Implementation Resources SharePoint site. This site links to the SharePoint API-LM page. It has been done.

[0134] 2.2.2 Second step Before starting a run, enter the SQL Bioreactor ID for each reactor and incubation date. The reactor ID 3102 is added using an embedded macro as shown in Figure 31. See Section 2 for information on using a macro to populate the bioreactor ID. Please refer to 1.2.

[0135] 2.2.3 Third step As shown in Figure 32, for each 5 L bioreactor and culture day, the sampling time was 320 2 (24-hour format hh:mm), measured VCD3204 (10^6 cells / m L) and the glucose concentration in the bioreactor (g / L), then enter the feed ( g / L).

[0136] As shown in Figure 33, the macro in the Excel file is used for Vi-CELL, BioHT, File import for external reading from MetaFLEX and AMBR250 instruments To accurately import values ​​for each device file type, Sample naming requirements detailed below must be followed. Multi-file or AMBR250 data tables can be imported. You can import MetaFLEX or BioHT files for course measurement. Cut.

[0137] 2.2.3.1 Vi-CELL Multifile Create a bioprocess for each bioreactor before taking sampling readings. The "Comments" section should be based on the nomenclature D# (e.g., culture day 1 = D1). All readings must be in a single Excel spreadsheet. Multiple files are created using the Vi-CELL software. will be done.

[0138] 2.2.3.2 BioHT text files The BioHT sample nomenclature is based on the Malvern API-LM format "Biological Reagents" Follow the "Device ID"_D#.

[0139] 2.2.3.3 MetaFLEX files The "Bioreactor ID" is entered into the "Patient ID" entry in MetaFLEX. Enter the culture date number in the "Patient Name" entry in taFLEX (enter only #, not D#)

[0140] Click the "Import Data File" button on the "Input and Supply Targets" sheet. As shown in Figure 4, the Import Data window 3400 is displayed. To select the file to add the trace values ​​to, press the appropriate "Browse" button 3402 and select the desired files (e.g., ViCELL Multifile for ViCell, ViCELL CSV , MetaFLEX CSV file for MetaFLEX and BioH for BioHT You can select a file by navigating to the file (a text file). After making your selection, press the "Upload Data" button. Enter the results into the "Force and Supply Targets" sheet.

[0141] 2.2.4 Fourth Step The feed sheet is created by the operator selecting "Glucose_Target" as shown in Figure 35. Calculate the glucose target (g / L) for each 5L container to enter into SQL under column 3502. do.

[0142] 2.2.5 Fifth Step In SQL (v2.28 or later), the measured glucose (g / L) is displayed as "Gluco Enter your glucose target in the "Glucose_Target" column 3600. " column 3602. The amount of glucose to be added to the bioreactor is entered in "Feed2_ The actual target for the bioreactor is calculated in the "Target" column 3604. The amount of glucose 3606 delivered is recorded.

[0143] FIG. 37 is an exemplary flowchart illustrating the process of controlling nutrient supply in a cell culture process. In 3702, a sample is received from a bioreactor containing a cell culture. In 3704, viable cell density and residual nutrient measurements are taken from the received sample. In 3706, the daily nutrient supply target can be determined based on the viable cell density and the residual nutrient. At 3708, the calculated daily nutritional goal may be calculated based on the measured values. Nutrients may be supplied to the bioreactor according to

[0144] In one embodiment, the process also comprises determining the daily residual nutrient concentration in the bioreactor. This may include maintaining the temperature within a range of

[0145] In one embodiment, the daily nutrient supply target is determined by the daily viable cell density and residual nutrient. It may be recalculated based on measurements.

[0146] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and and glutamine.

[0147] In one embodiment, the nutrient may include one or more simple sugars.

[0148] In one embodiment, measuring residual nutrients includes assaying nutrient concentrations within the bioreactor. obtain.

[0149] In one embodiment, the residual nutrient measurement is one of offline nutrient measurement and inline nutrient measurement. The method may include performing one or more of the following:

[0150] In one embodiment, residual nutrient measurements are performed using a NovaFlex instrument and a Raman probe. It may be performed by one or more.

[0151] In one embodiment, the bioreactor is a Chinese Hamster Ovary (CHO) cell bioreactor. For the production of biologics other than CHO, the equipment may be one or more of: Other mammalian cell types may be used, including recombinant cells, etc. Non-limiting examples include HEK, 293, and PerC6. Other non-mammalian cell types may also be used, such as yeast or bacteria.

[0152] In one embodiment, the cells in the bioreactor may be mammalian cells.

[0153] In one embodiment, the cells are CHO cells.

[0154] In one embodiment, the daily nutrient supply target is 100% bioreactions from at least six cell lines. The reactor is run at least according to a predetermined global average consumption value and growth profile. may also be calculated based in part on

[0155] FIG. 38 is another exemplary flow chart illustrating the process of controlling nutrient supply in a cell culture process. 38. In 3802, a sample is received from a container containing a cell culture. At 3804, viable cell density and residual nutrient measurements can be taken from the received sample. In 3806, the daily nutrient supply target can be determined based on the viable cell density and residual nutrient. In 3808, the calculated daily nutrient supply may be calculated based on the nutrient measurement. Nutrients may be supplied to the container according to the standard. In one embodiment, the container may be a flask.

[0156] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and and glutamine.

[0157] FIG. 39 shows the process of balancing glucose supply in the cell growth process. 3 is an exemplary flowchart. In 3902, a live cell is measured during a cell growth process. Cell density and glucose concentration may be determined periodically. The feed target may be adjusted periodically based on viable cell density and glucose concentration. In 6, glucose is periodically provided to the cell growth process according to the glucose supply target. can be provided.

[0158] FIG. 40 is an exemplary flowchart illustrating the process of controlling glucose supply in a cell culture process. 4 is a flow chart showing the flow of a sample from a production reactor containing a cell culture at 4002. At 4004, the glucose remaining amount can be determined from the received sample. In 4006, a sample is received from the production reactor. At 4008, the residual amount of glucose may be compared with a predetermined glucose target. In 4010, the amount of glucose consumed can be calculated. For example, the amount of glucose The consumption of glucose is increased when the residual glucose level is greater than the predetermined glucose target. This is determined by determining the amount of glucose consumed during the process between the previous day and the current day. In another example, the amount of glucose consumed may be determined by determining whether the remaining amount of glucose is greater than the predetermined glucose level. If below target, based on the difference between the predetermined glucose target and the residual amount of glucose possible.

[0159] In 4012, the integrated viable cell density can be calculated. In 4014, the integrated viable cell density In 4016, the glucose consumption rate can be calculated based on the predetermined viable cell density for the next day. and the specific glucose consumption rate can be calculated based on the integrated viable cell density. Predicted glucose consumption was calculated by multiplying the glucose consumption rate by the predetermined viable cell density for the next day. At 4020, a predetermined glucose consumption amount and a predetermined glucose minimum amount can be calculated. The glucose target may be calculated by summing the glucose amount. Glucose can be fed to the production reactor according to the target.

[0160] In one embodiment, feeding may occur daily.

[0161] FIG. 41 is an exemplary illustration showing a process for modulating the amount of glycosylation of a drug in a cell culture process. 4 is a flow chart showing a method for collecting sample from a production reactor containing a cell culture at 4102. In 4104, the residual amount of nutrients is determined from the received sample. At 4106, the amount of nutrients remaining since the last feeding may be measured. At 4108, a viable cell density may be determined from the received sample. At 110, the predicted consumption of nutrients until the next supply is calculated based on the consumption and production of nutrients. In 4112, the predicted nutrient consumption and the next Calculate the target amount of nutrients for the current feeding based on the predetermined residual nutrient targets from the previous feeding. At 4114, nutrients may be delivered to the bioreactor according to the calculated target amount of nutrients. can be supplied.

[0162] In one embodiment, the predicted viable cell density between the current feeding and the next feeding is determined by the determined viable cell density. The rate of nutrient consumption may be determined based at least in part on the density of the nutrient. The predicted consumption rate may be determined based, at least in part, on the predicted viable cell density and nutrient consumption rate. It can be calculated based on the following.

[0163] In one embodiment, feeding may occur daily.

[0164] In one embodiment, the nutrients are glucose, glutamate, galactose, lactate, and and glutamine.

[0165] In one embodiment, the nutrient may include one or more simple sugars.

[0166] FIG. 42 is an exemplary flowchart illustrating the process of controlling glucose supply in a cell culture process. At 4202, a glucose measurement may be determined. At 4204, At 4206, the current culture day can be determined. In 4208, the combination of the glucose measurement, lactate measurement and current incubation date A glucose target may be determined based on:

[0167] In one embodiment, the glucose measurement is less than 1 g / L and the lactate measurement is less than 1 g / L. If the glucose target is less than 5 g / L and the current culture day is 5, then the glucose target may be 5 g / L. .

[0168] In one embodiment, the glucose measurement is less than 1 g / L and the lactate measurement is less than 1 g / L. If the glucose level is >3 g / L and the current culture day is 5, the glucose target is 4.5 g / L. / L.

[0169] FIG. 43 is another exemplary diagram illustrating a process for controlling glucose supply in a cell culture process. 4302 is a flowchart of a process for extracting sample from a production reactor containing a cell culture. At 4304, the glucose concentration can be calculated from the received sample. The residual amount of glucose may be measured. At 4306, the residual amount of glucose may be compared with a predetermined glucose target. In 4308, the amount of glucose consumed can be calculated. For example, the amount of glucose The consumption of glucose is increased when the residual glucose level is greater than the predetermined glucose target. This is determined by determining the amount of glucose consumed during the process between the previous day and the current day. In another example, the amount of glucose consumed may be determined by determining whether the remaining amount of glucose is greater than the predetermined glucose level. If below target, based on the difference between the predetermined glucose target and the residual amount of glucose In 4310, the viable cell density for the current day can be determined. In 4312, the viable cell density for the previous day can be determined. In 4314, the viable cell density of the day and the viable cell density of the previous day can be determined. In 4316, the growth rate for the next day may be estimated based on the estimated growth rate. In 4318, the viable cell density for the next day can be predicted based on the integrated viable cell density. The cell density can be calculated in 4320 based on the glucose consumption rate and the integrated viable cell density. In 4322, the specific glucose consumption rate is calculated based on the next day's glucose consumption rate. By multiplying by a given viable cell density, predicted glucose consumption can be calculated. In 4, by summing the predetermined glucose consumption amount and the predetermined minimum glucose amount In 4326, the production reactor is adjusted according to the glucose target. The ethanol may be supplied with glucose.

[0170] Below is a list of abbreviations and definitions:

[0171] d may refer to the current cell culture day.

[0172] d+1 may refer to the next cell culture day.

[0173] d-1 may refer to the previous day of cell culture.

[0174] VCD may refer to viable cell density (cells / mL).

[0175] IVCD is the incorporated viable cell density (cells / mL * It can refer to the day.

[0176] ΔIVCD d is the change in IVCD (cell count) from day d-1 to day d * day / mL) Can point.

[0177] VCD d+1 may refer to the VCD on day d+1.

[0178] ΔIVCDd+1 is the change in IVCD (cell count) from day d to day d+1. * day / mL) possible.

[0179] IQR may refer to the interquartile range.

[0180] Glucose goals d may refer to the target concentration of glucose (g / L) to the feed reactor .

[0181] Glucose consumption rate d is the amount of glucose consumed per day from time d-1 to time d. It can refer to the amount of glucose (g / L / day).

[0182] Specific glucose consumption rate d is consumed per day by one cell from time d-1 to time d. This can refer to glucose (pg / (cell * day)).

[0183] Residual Glucose Target d+1 is the residual glutathione in the reactor at time d+1 (g / L) It may refer to the desired theoretical concentration of a course.

[0184] Predicted glucose consumption d+1 is the predicted glucose consumed at time d+1 (g / L) Can point.

[0185] VCD d+1 The predicted fold change of is derived from the database, from time d to time It can refer to the predicted fold change in VCD by d+1. death).

[0186] Predicted glucose consumption d+1 is consumed by the cell culture from time d to time d+1 It may refer to the concentration of glucose (g / L) that is predicted to be obtained.

[0187] Measured glucose d indicates the glucose concentration measured in the bioreactor on day d. possible.

[0188] ΔIVCD d+1 / VCD d+1 is the difference between ΔIVCD and VCD from d to d+1. It can refer to the magnification difference. If ΔIVCD=VCD, it is equal to 1.

[0189] ΔIVCD d+1 / ΔIVCD d can refer to the fold change in ΔIVCD from d to d+1. do.

[0190] Measured VCD d+1 may refer to the VCD measured at d+1.

[0191] The examples set forth herein disclose specific implementations of the disclosed technology, including the best mode. To illustrate, and to illustrate, the making and use of any device or system and any incorporated Enables those skilled in the art to practice certain embodiments of the disclosed technology, including performing the methods The patentable scope of particular embodiments of the technology of this disclosure is defined by the claims. The scope may include other examples that occur to those skilled in the art. If the invention has structural elements that are not different from the literal language of the claim, or if they are different from the language of the claim, If the scope of a claim includes equivalent structural elements that differ only slightly from the scope of the claim, It is intended that:

[0192] Certain embodiments of the techniques of this disclosure are described in terms of those currently believed to be most practical and in various forms. Although described in connection with various embodiments, the techniques of this disclosure are not limited to the embodiments of this disclosure. On the contrary, various modifications and equivalents included within the scope of the appended claims are intended to be included. It should be understood that the term "component" is intended to encompass configurations of Although terms are used herein, they are used in a generic and descriptive sense only and not for purposes of limitation. will be done.

[0193] Specific implementations of the techniques of this disclosure include systems and and the block and flow diagrams of the methods and / or computer program products. One or more blocks of the block diagrams and flow diagrams, and The combination of blocks in the block diagrams and flow diagrams may represent computer-executable program instructions. Similarly, it will be understood that some of the block and flow diagrams may be implemented by The blocks may not necessarily be performed in the order presented, according to some implementations of the techniques of this disclosure. It may not be necessary or even necessarily performed.

[0194] These computer program instructions may also be used by a computer or other programmable A computer readable medium capable of instructing a data processing device to function in a particular manner. The instructions stored in the computer readable memory may be stored in a Manufacture a product that includes instruction means for performing one or more of the functions specified in the flowchart blocks above. do.

[0195] Implementations of the techniques of this disclosure include computer readable program code or program instructions. To provide a computer program product including a computer usable medium embodied therein. the computer readable program code may be specified by one or more flow chart blocks. A computer program is adapted to be executed to perform one or more functions described in the RAM instructions may also be loaded into a computer or other programmable data processing device. The instructions executed on a computer or other programmable device may be one or more flow chart blocks. A sequence of actions that provides the elements or steps to implement the functionality specified in the block. The operation elements or steps are executed on a computer or other programmable device to A computer-implemented process may be generated.

[0196] Therefore, the blocks in the block diagrams and flow diagrams represent means for performing a particular function. combinations of elements or steps to perform a particular function, and Supports program instructions for executing functions. The blocks and combinations of blocks in the block diagrams and flow diagrams represent specific functions, elements, and or a special-purpose hardware-based computer system that performs the steps; or It is also understood that a program can be implemented using a combination of special-purpose hardware and computer instructions. Let's solve it.

Claims

1. 1. A method for controlling nutrient supply in a cell culture process, comprising: receiving a sample from a bioreactor containing a cell culture; determining viable cell density and residual nutrient measurements from the received sample; Calculating a daily nutrient supply target based on the viable cell density and the residual nutrient measurements. and Supplying nutrients to the bioreactor according to the calculated daily nutrient supply target. A method including:

2. and maintaining a daily residual nutrient concentration in the bioreactor within a predetermined range. The method of claim 1 .

3. determining the daily nutrient content based on the daily viable cell density and the residual nutrient measurement; The method of claim 1 or 2, further comprising recalculating the supply target.

4. The nutrients are glucose, glutamate, galactose, lactate, and glutamine. The method according to any one of claims 1 to 3, wherein the compound is selected from the group consisting of:

5. 4. The method of claim 1, wherein the nutrients comprise one or more monosaccharides.

6. wherein the residual nutrient measurement comprises assaying nutrient concentrations within the bioreactor. Item 6. The method according to any one of Items 1 to 5.

7. The residual nutrient measurement includes one or more of an offline nutrient measurement and an in-line nutrient measurement. The method of any one of claims 1 to 5, comprising performing

8. The residual nutrient measurements are performed by one or more of a NovaFlex instrument and a Raman probe. The method according to any one of claims 1 to 5, wherein the method is carried out by

9. The bioreactors are Chinese hamster ovary (CHO) cell bioreactors and 5 L bioreactors. The method according to any one of claims 1 to 8, wherein the reactor is one or more of:

10. The method according to any one of claims 1 to 9, wherein the cells in the bioreactor are mammalian cells. method.

11. 11. The method of claim 10, wherein the cells are CHO cells.

12. The daily nutrient supply target is a multiple of the bioreactor from at least six cell lines. At least partially based on global average consumption values ​​and growth profiles previously determined from several runs. The method according to any one of claims 1 to 11, wherein the calculated value is based on the following formula:

13. 1. A method for controlling nutrient supply in a cell culture process, comprising: receiving a sample from a container containing a cell culture; determining viable cell density and residual nutrient measurements from the received sample; Calculating a daily nutrient supply target based on the viable cell density and the residual nutrient measurements. and providing nutrients to said container in accordance with said calculated daily nutrient supply goal; A method comprising:

14. The method of claim 13, wherein the container is a flask.

15. The nutrients are glucose, glutamate, galactose, lactate, and glutamine.

15. The method of claim 13 or claim 14, wherein the

16. 1. A method for balancing glucose supply in a cell growth process, comprising: The viable cell density and glucose concentration measured during the cell growth process are determined periodically. and, periodically setting a glucose supply target for nutrients based on the viable cell density and the glucose concentration; and periodically supplying glucose to said cell growth process according to said glucose supply target. A method comprising:

17. 1. A method for controlling glucose supply in a cell culture process, comprising: receiving a sample from a bioreactor containing a cell culture; determining the residual amount of glucose from the received sample; determining a sample time at which a sample is received from the bioreactor; comparing said residual amount of glucose to a predetermined glucose target; Glucose consumption, If the residual amount of glucose is greater than the predetermined glucose target, By determining the amount of glucose consumed between the previous day and the current day during the culture process determining the glucose consumption; If the residual amount of glucose is less than or equal to the predetermined glucose target, The glucose consumption is determined based on the difference between the glucose target and the residual amount of glucose. determining, calculating by, Calculating the cumulative viable cell density; Calculating a predetermined viable cell density for the next day based on the integrated viable cell density; A specific glucose consumption rate is calculated based on the glucose consumption amount and the integrated viable cell density. To put out, The predicted glucose consumption rate is calculated by multiplying the specific glucose consumption rate by the predetermined viable cell density for the next day. Calculating the amount of glucose consumed, The predetermined glucose consumption amount and the predetermined minimum glucose amount are summed to determine the glucose target. Calculating, and feeding glucose to the bioreactor in accordance with the glucose target. Law.

18. 20. The method of claim 18, wherein said feeding occurs daily.

19. 1. A system for controlling nutrient supply in a cell culture process, comprising: The bioreactor includes a cell culture and a nutrient supply system that supplies nutrients to the bioreactor. a processor in communication with the device, determining viable cell density and residual nutrient measurements from samples withdrawn from the bioreactor; 、 Calculating a daily nutrient supply target based on the viable cell density and the residual nutrient measurements. death, The nutrients are supplied to the bioreactor according to the calculated daily nutrient supply target. a processor configured to direct the nutrition delivery system to deliver system.

20. The nutrient supply system provides a continuous or discontinuous supply of nutrients during the cell culture process.

20. The system of claim 19, wherein:

21. The nutrients are glucose, glutamate, galactose, lactate, and glutamine.

21. The system of claim 19 or claim 20, wherein the system is selected from:

22. 22. The method according to any one of claims 19 to 21, wherein the nutrients include one or more monosaccharides. Stem.

23. 1. A system for balancing glucose supply in a cell growth process, comprising: A processor in communication with a glucose supply system that supplies glucose during the cell growth process. a processor, Periodically determining the viable cell density and glucose concentration measured during the cell growth process. death, Periodically adjusting a glucose delivery target based on the viable cell density and the glucose concentration. Adjust, The glucose supply system is configured to supply the cell growth process in accordance with the glucose supply target. a processor configured to periodically instruct a process to deliver glucose. Yes, the system.

24. 1. A system for preventing glycation in a cell culture process, comprising: The bioreactor includes a cell culture and a nutrient supply system that supplies nutrients to the bioreactor. a processor in communication with the device, determining the residual amount of nutrients in a sample withdrawn from the bioreactor; determining the amount of the nutrient consumed since the last supply based on the remaining amount of the nutrient; determining the viable cell density within the sample; Based on the consumption of the nutrients and the viable cell density, the amount of the nutrients consumed until the next feeding is calculated. Calculate the predicted consumption of nutrients, Based on the predicted consumption of the nutrient and a predetermined remaining nutrient target before the next nutrient supply. calculating a target amount of said nutrient for the current nutritional supply; and supplying the nutrient to the bioreactor in accordance with the calculated target amount of the nutrient. A system having a processor configured to direct the nutrient delivery system.

25. 1. A method for modulating the amount of glycation of an agent in a cell culture process, comprising: receiving a sample from a bioreactor containing a cell culture; Determining nutrient residues from the received samples; determining the amount of the nutrient consumed since the last supply based on the remaining amount of the nutrient; 、 determining a viable cell density from the received sample; Based on the consumption of the nutrients and the viable cell density, the amount of the nutrients consumed until the next feeding is calculated. Calculating the expected consumption of nutrients; Based on the predicted consumption of the nutrient and a predetermined remaining nutrient target before the next nutrient supply. calculating a target amount of said nutrient for the current nutritional supply; supplying the nutrients to the bioreactor according to the calculated target amounts of the nutrients; A method comprising:

26. determining a time between the current feeding and the next feeding based at least in part on the determined viable cell density; determining a predicted viable cell density; determining a rate of nutrient consumption based at least in part on the amount of nutrient consumed; and calculating the predicted consumption rate based on the predicted viable cell density and the nutrient consumption rate; 26. The method of claim 25, further comprising:

27. 26. The method of claim 25, wherein said feeding occurs daily.

28. The nutrients are glucose, glutamate, galactose, lactate, and glutamine. The method according to any one of claims 25 to 27, wherein the compound is selected from the group consisting of:

29. The method according to any one of claims 25 to 27, wherein the nutrients include one or more monosaccharides. Law.

30. 1. A method for controlling glucose supply in a cell culture process, comprising: determining a glucose measurement; determining lactate measurements; Determining the current culture date, and A combination of the glucose measurement, the lactate measurement, and the current culture date. determining a glucose target based on the

31. The glucose measurement is less than 1 g / L and the lactate measurement is less than 1 g / L. and the glucose target is 5 g / L when the current culture day is day 5; 31. The method of claim 30.

32. The glucose measurement is less than 1 g / L and the lactate measurement is greater than 1 g / L / L and the current culture day is day 5, the glucose target is 4.5 31. The method of claim 30, wherein the HCl concentration is 1000 mg / L.

33. 1. A method for controlling glucose supply in a cell culture process, comprising: receiving a sample from a bioreactor containing a cell culture; determining the residual amount of glucose from the received sample; comparing said residual amount of glucose to a predetermined glucose target; Glucose consumption, If the residual amount of glucose is greater than the predetermined glucose target, By determining the amount of glucose consumed between the previous day and the current day during the culture process determining the glucose consumption; If the residual amount of glucose is less than or equal to the predetermined glucose target, The glucose consumption is determined based on the difference between the glucose target and the residual amount of glucose. determining, calculating by, Determining the viable cell density on the day; Determining the viable cell density the day before, estimating a proliferation rate based on the viable cell density on the day and the viable cell density on the previous day; predicting the cumulative viable cell density for the next day based on the estimated proliferation rate; Calculating a predetermined viable cell density for the next day based on the integrated viable cell density; A specific glucose consumption rate is calculated based on the glucose consumption amount and the integrated viable cell density. To put out, The predicted glucose consumption rate is calculated by multiplying the specific glucose consumption rate by the predetermined viable cell density for the next day. Calculating the amount of glucose consumed, The predetermined glucose consumption amount and the predetermined minimum glucose amount are summed to determine the glucose target. Calculating, and feeding glucose to the bioreactor in accordance with the glucose target. Law.

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