Real-time monitoring of titer using ultraviolet signals

Abstract

The present invention relates to a method of monitoring titer in real time using an ultraviolet signal, in particular to a method of controlling, modulating, increasing or improving protein yield in a sample mixture comprising a target protein and an impurity, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during protein filtration in a harvest skid.

Description

[0001] This application is a divisional application of the invention application filed on March 26, 2019, with Chinese application number 201980022884.1 and entitled "Real-time monitoring of titer using ultraviolet signals". Technical Field

[0002] This disclosure relates to a method for monitoring the concentration of biomolecules, such as proteins, in a composition. Specifically, this disclosure relates to a method for monitoring, controlling, regulating, or increasing the protein yield in a composition using a real-time ultraviolet signal during protein filtration. Background Technology

[0003] Many therapeutic proteins (e.g., monoclonal antibodies (mAbs)) are currently under development, and many companies have multiple antibodies in their product pipelines. Basic unit operations (such as harvesting, protein A affinity chromatography, and additional purification steps) are used to purify the protein of interest.

[0004] The aim of upstream and recovery operations is to extract high-productivity therapeutic proteins during cell culture and recovery, and multiple online configurations are available for monitoring bioprocessing operations. See Whitford W., Julien C. Bioprocess Int. (5), S32–S45 (2007). Real-time monitoring and control of cell culture processes have recently been achieved. It has been shown that an increase in non-viable subpopulations in CHO cell cultures can predict the emergence of a stationary phase, suggesting the opportunity for fully automated cell culture processes and reliable and reproducible control of feed additions during culture proliferation. Sitton G., Srienc F.J. Biotechnol., 135 (2008), 174–180. Others have utilized multiple steps in the primary recovery process to remove biomass and clarify the feed stream for downstream column chromatography. Bink LR, Furey J. BioProcess Int. 8(3) 2010, 44–49, 57 (2010).

[0005] Some researchers have addressed the problem of increasing protein yield by addressing upstream steps to improve downstream yield. For example, others have attempted to reduce the mechanical stress on CHO cells by using peristaltic pumps and diaphragm pumps with magnetically levitated bearingless centrifugal pumps. Blaschczok K., et al., Chemie Ingenieur Technik, (85), 144-152 (2013). Others have evaluated proteomics approaches by studying the kinetics and fate of host cell proteins in the supernatant of cell lines that produce monoclonal antibodies during recovery and early downstream processing, including centrifugation, deep filtration, and protein A capture chromatography. Hogwood, CEM, et al., Biotechnol. Bioeng. 2013(110), 240–251. However, some processes require additional steps, such as fluorescent labeling, to identify protein concentrations and yields during purification. Ignatova and Gierasch, Proc Natl Acad Sci US A.; 101(2):523-8 (2004). Adding additional impurities may require additional purification steps that can affect the yield.

[0006] Therefore, real-time monitoring and control of the recycling process are still necessary to increase recycling yield and process robustness, quickly assess upstream performance, and facilitate immediate downstream processing during batch processing or in more critical continuous processes. Summary of the Invention

[0007] This paper discloses a novel real-time monitoring and control process and system designed and tested for filtration-based cell culture harvesting processes, such as deep filtration harvesting, for several therapeutic proteins. The methods described herein offer several advantages over existing technologies. First, the harvesting skid is designed to enable real-time monitoring and control of key process parameters and quality properties. Second, it uses a modeling approach to convert the online UV signal of the clarified bulk solution into real-time titers of the target product. Third, the harvesting process can be automated using this harvesting skid and real-time titers, improving process yield, robustness, and consistency. Finally, titer information is used to demonstrate cell culture performance and guide immediate downstream purification.

[0008] The core of this new technology is the application of real-time monitoring of UV signals during the harvesting process, converting online UV signals into real-time target protein concentrations. The model disclosed herein can be applied to several processes with different cellular characteristics and productivity levels. Using this system, the start and end of the collection of clarified bulk liquid can be quantitatively determined, which can significantly improve the robustness of the harvest and protein yield.

[0009] The method disclosed in this paper provides an in-depth understanding of the application of harvesting skids in the clarification process of cell cultures. The novel harvesting process disclosed herein improves protein yield while being scalable, automated, and applicable to multiple products with diverse properties. Real-time titer information can be used to demonstrate cell culture performance and guide immediate downstream processing.

[0010] This article discloses a method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture containing a target protein and impurities. The method includes monitoring a real-time ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process and automatically converting the UV signal into a target protein titer using an established model.

[0011] This article also discloses a method for controlling the collection of target proteins and improving protein yield in a sample mixture containing target proteins and impurities, the method comprising monitoring the real-time ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process.

[0012] In some implementations, the UV signal is continuously converted into the titer of the target protein based on an established model and automated control.

[0013] In some embodiments, the titer of the target protein is at least about 0.01 g / L, at least about 0.02 g / L, at least about 0.03 g / L, at least about 0.04 g / L, at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, and at least about 0.3 g / L. g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, to At least about 5.5 g / L, at least about 6 g / L, at least about 6.5 g / L, at least about 7 g / L, at least about 7.5 g / L, at least about 8 g / L, at least about 8.5 g / L, at least about 9 g / L, at least about 9.5 g / L, at least about 10 g / L, at least about 10.5 g / L, at least about 11 g / L, at least about 11.5 g / L, at least about 12 g / L, at least about 12.5 g / L, at least about 1 3 g / L, at least about 13.5 g / L, at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L, or at least about 20 g / L.

[0014] In some embodiments, the method disclosed herein further includes the following steps when the titer is at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, or at least about 6.5 g / L. The collection of the target protein begins at concentrations of at least 7 g / L, at least 7.5 g / L, at least 8 g / L, at least 8.5 g / L, at least 9 g / L, at least 9.5 g / L, at least 10 g / L, at least 10.5 g / L, at least 11 g / L, at least 11.5 g / L, at least 12 g / L, at least 12.5 g / L, at least 13 g / L, at least 13.5 g / L, at least 14 g / L, at least 14.5 g / L, at least 15 g / L, at least 15.5 g / L, at least 16 g / L, at least 16.5 g / L, at least 17 g / L, at least 17.5 g / L, at least 18 g / L, at least 18.5 g / L, at least 19 g / L, at least 19.5 g / L, or at least 20 g / L.

[0015] In some embodiments, the collected titers of the target protein are between about 0.05 g / L and about 20 g / L, between about 0.1 g / L and about 20 g / L, between about 0.2 g / L and about 20 g / L, between about 0.3 g / L and about 20 g / L, between about 0.4 g / L and about 20 g / L, between about 0.5 g / L and about 20 g / L, between about 0.6 g / L and about 20 g / L, and between about 0.7 g / L. Between approximately 0.8 g / L and approximately 20 g / L, between approximately 0.9 g / L and approximately 20 g / L, between approximately 1 g / L and approximately 20 g / L, between approximately 0.05 g / L and approximately 15 g / L, between approximately 0.1 g / L and approximately 15 g / L, between approximately 0.2 g / L and approximately 15 g / L, between approximately 0.3 g / L and approximately 15 g / L, between approximately 0.4 g / L and approximately 15 g / L... Between L, between about 0.5 g / L and about 15 g / L, between about 0.6 g / L and about 15 g / L, between about 0.7 g / L and about 15 g / L, between about 0.8 g / L and about 15 g / L, between about 0.9 g / L and about 15 g / L, or between about 1 g / L and about 15 g / L, between about 0.05 g / L and about 10 g / L, between about 0.1 g / L and about 10 g / L, between about 0.2 g / L... Between / L and about 10 g / L, between about 0.3 g / L and about 10 g / L, between about 0.4 g / L and about 10 g / L, between about 0.5 g / L and about 10 g / L, between about 0.6 g / L and about 10 g / L, between about 0.7 g / L and about 10 g / L, between about 0.8 g / L and about 10 g / L, between about 0.9 g / L and about 10 g / L, or between about 1 g / L and about 10 g / L.

[0016] In some embodiments, the method disclosed herein further includes stopping the collection of the target protein when the collection titer is below about 0.1 or 0.2 g / L.

[0017] In some embodiments, the target protein yield is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture.

[0018] In some embodiments, the target protein is derived from cells with a density of at least about 1 x 10⁻⁶. 6 cells / mL, at least approximately 5 x 10⁻⁶ 6cells / mL, at least approximately 1 x 10⁻⁶ 7 1.5 x 10⁻⁶ cells / mL, at least approximately 1.5 x 10� 7 1 cell / mL, at least approximately 2 x 10⁻⁶ 7 1 cell / mL, at least about 2.5 x 10⁻⁶ 7 1 cell / mL, at least approximately 3 x 10⁻⁶ cells / mL 7 1 cell / mL, at least approximately 3.5 x 10⁻⁶ cells / mL 7 cells / mL, at least approximately 4 x 10⁻⁶ 7 1 cell / mL, at least about 4.5 x 10⁻⁶ 7 1 cell / mL or at least about 5 x 10⁻⁶ cells / mL 7 Harvested from a culture medium of cells / mL.

[0019] In some embodiments, the protein filtration is depth filtration. In some embodiments, the depth filtration includes a primary depth filter and / or a secondary depth filter.

[0020] In some embodiments, the method disclosed herein further includes loading the sample mixture prior to the monitoring. In some embodiments, the method disclosed herein further includes rinsing the depth filter with water or a buffer prior to loading the cell culture and chasing the depth filter after loading the cell culture. In some embodiments, the method disclosed herein further includes chasing the sample mixture with phosphate-buffered saline (PBS) or other buffers. In some embodiments, the filtration-based cell culture harvesting process includes a harvesting sled. In some embodiments, the harvesting sled includes a control system wherein the control system automatically begins collecting the protein when a set titer is reached. In some embodiments, the harvesting sled includes a control system wherein the control system automatically begins collecting the protein when a set titer is reached. In some embodiments, the harvesting sled includes a control system wherein the control system automatically stops collecting the protein when a set titer is reached. In some embodiments, the control system regulates the flow rate of liquid through the harvesting sled. In some embodiments, the control system automatically drives a pump to increase the flow rate through the harvesting sled. In some embodiments, the control system automatically drives a pump to decrease the flow rate through the harvesting sled. In some embodiments, the methods disclosed herein do not include a gas venting step. In some embodiments, the target protein titer or the protein yield is not based on volume.

[0021] In some embodiments, this document discloses a method for increasing, controlling, or regulating the protein yield in a sample mixture containing a target protein and impurities, the method comprising (a) rinsing a harvesting sled with water; (b) loading the sample into the harvesting sled; (c) measuring the ultraviolet signal of the sample mixture during protein filtration in the harvesting sled as a real-time protein titer; (d) initiating protein collection based on the ultraviolet measurement and the real-time protein titer; (e) tracking the protein with PBS; and (f) stopping protein collection based on the ultraviolet measurement and the real-time protein titer; wherein the UV signal is correlated with the real-time protein titer during filtration.

[0022] In some implementations, the method further includes measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0023] In some embodiments, the method further includes measuring pressure using a pressure sensor. In some embodiments, the measured pressure range is -10 psi to 50 psi, -10 psi to 40 psi, -9 psi to 40 psi, -8 psi to 40 psi, -7 psi to 30 psi, -6 psi to -20 psi, -7 psi to 40 psi, -8 psi to 40 psi, -9 psi to 45 psi, -10 psi to -45 psi, or -7 psi to -45 psi.

[0024] In some embodiments, the method further includes measuring turbidity. In some embodiments, the measured turbidity ranges from 0 absorbance units (AU) to 2 AU.

[0025] In some embodiments, the method further includes measuring temperature. In some embodiments, the measured temperature range is 0°C to 70°C, 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 5°C to 70°C, 10°C to 70°C, 15°C to 70°C, 20°C to 70°C, 10°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, 20°C to 30°C, 35°C to 40°C, or 25°C to 45°C.

[0026] In some embodiments, the method further includes measuring flow rate. In some embodiments, the measured flow rate ranges are 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, 0 L / min to 110 L / min, 0 L / 0L / min to 120L / min, 0L / min to 130L / min, 0L / min to 140L / min, 0L / min to 150L / min, 0L / min to 160L / min, 0L / min to 170L / min, 0L / min to 180L / min, 0L / min to 190L / min, 0L / min to 200L / min, 0L / min to 250L / min, or 0L / min to 300L / min.

[0027] In some embodiments, the harvest sled includes one or more filters. In some embodiments, the filters include a primary depth filter and a secondary depth filter. In some embodiments, the sample mixture is selected from purified protein samples, clarified stock protein samples, cell culture samples, and any combination thereof.

[0028] In some embodiments, the protein is produced in a culture containing mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NSO), young hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0029] In some embodiments, the protein comprises an antibody or a fusion protein. In some embodiments, the protein is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, and an anti-IL8 antibody. In some embodiments, the protein is abatacept or berazepam.

[0030] In some embodiments, this document discloses a system for real-time monitoring and control of protein yield, wherein the system includes a sensor that measures a real-time UV signal of a sample mixture containing a target protein and impurities.

[0031] In some implementations, the system further includes sensors for measuring pressure, turbidity, temperature, flow rate, weight, or any combination thereof.

[0032] In some embodiments, a device includes a sensor configured to measure the UV signal of a sample mixture containing a target protein and impurities. In some embodiments, a processor is configured to control the collection of the target protein. In some embodiments, the processor is configured to use a target protein titer. In some embodiments, the processor is configured to use an established model to determine the cell culture harvesting process. In some embodiments, the cell culture harvesting process includes a filtration-based cell culture harvesting process. In some embodiments, a system includes a device containing a sensor configured to measure the UV signal of a sample mixture containing a target protein and impurities.

[0033] In some implementations, the disclosed system is provided for use in the methods described herein. Attached Figure Description

[0034] Figure 1 A shows an exemplary mechanical design and physical diagram of a harvesting sled. All values ​​are listed in inches.

[0035] Figure 2 A process flow diagram of the cell culture harvesting process using the new harvesting sled is shown. Various boxes indicate online measurement sensors, control modules, and physical instruments.

[0036] Figure 3 The experimental design described in this paper for modeling UV signals as product titers is illustrated.

[0037] Figure 4 A graphical comparison between the old and new harvesting methods is shown. Compared to the previous method, the new method eliminates the gas venting step. Simultaneously, the start and end of clarified liquid collection in the new method can be automatically controlled based on online UV readings and calculated titers. More specifically, the real-time target protein concentration during the harvesting process can be calculated using online UV sensor readings using the model generated and tested in this paper. Therefore, the cutoff point for liquid collection can be directly determined based on the calculated online target protein concentration. The calculation algorithm can be integrated into Delta V. TM In the control system, an automatic cutoff point is set to achieve the collection of clarified raw liquid.

[0038] Figure 5 Offline titer measurements against online UV signals are shown using serially diluted samples from GITR cell cultures.

[0039] Figure 6A and Figure 6B It shows the use of pure protein ( Figure 6A ) and clarified stock solution ( Figure 6BOffline titer measurements of online UV signals during small-scale harvesting processes. Online and offline UV titer values ​​were tested during the harvesting process.

[0040] Figure 7A , Figure 7B and Figure 7C The cell culture using anti-GITR antibody was shown. Figure 7A Abatacept cell culture ( Figure 7B ) and anti-CXCR4 antibody cell cultures ( Figure 7C Offline titer measurements of online UV signals during large-scale harvesting processes.

[0041] Figure 8A and Figure 8B The linear fit between offline titer measurements and online UV values ​​is shown. Figure 8A ); Linear fitting of predicted titers to actual titers based on UV ( Figure 8B ).

[0042] Figure 9A and Figure 9B The nonlinear fitting of offline titer measurements to online UV values ​​is shown. Figure 9A ); Linear fitting of predicted titers to actual titers based on UV ( Figure 9B ).

[0043] Figure 10 The average difference between the model-predicted titers and the actual titers of the seven molecules studied is shown (HPLC analysis). The seven molecules include Aba J, anti-CD73 antibody, anti-GITR antibody, anti-IL8 antibody, anti-CXCR4 antibody, anti-OX40 antibody, and anti-TIGIT antibody.

[0044] Figure 11 The diagram shows a comparison between online UV tracing, titer tracing obtained through UV signal modeling, and titers determined offline. The Y-axis displays the titer determined offline (g / L) or the titer based on UV modeling (g / L), and the X-axis displays time (min). Triangle lines represent online UV, square lines represent titers based on UV modeling (g / L), and diamond lines represent offline titers (g / L). Detailed Implementation

[0045] Various methods are provided for controlling, regulating, or increasing protein yield. These methods include using real-time measurements of ultraviolet (UV) signals of the sample mixture during purification steps, such as protein filtration in a harvesting sled, to control, regulate, or increase protein yield. The method utilizes the UV signal to provide the titer of the target protein according to the formula disclosed herein, which varies depending on whether collection occurs from the start of loading to the end of loading or after loading has ended.

[0046] This paper also discloses a variety of systems and devices related to the methods provided herein.

[0047] a. Terminology

[0048] It should be noted that the term "a" or "an" refers to one or more of the same entity; for example, "nucleotide sequence" should be understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "a"), "one or more," and "at least one" are used interchangeably herein.

[0049] Furthermore, the term “and / or” as used herein should be understood as a specific disclosure of each of two specified features or components being together or apart from the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0050] Similarly, unless the context explicitly states otherwise, the word "or" is intended to include "and". It should also be understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for descriptive purposes.

[0051] It should be understood that wherever the language “contains” is used to describe an aspect in this text, similar aspects described in terms of “consisting of” and / or “substantially consisting of” are also provided.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in connection with this disclosure. For example, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 2nd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0053] Units, prefixes, and symbols are represented in their form accepted by the Système International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the direction from amino to carboxyl. The headings provided herein are not intended to limit the various aspects of this disclosure, all of which can be obtained by referring to this specification in its entirety. Thus, the terms defined below are defined more completely by referring to the entire specification.

[0054] The term "about" is used herein to mean approximately, roughly, about, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the stated value. Thus, "about 10-20" means "about 10 to about 20". Typically, the term "about" can modify a numerical value by a variance of, for example, 10%, making it higher or lower than the stated value.

[0055] “Modeling” or “protein modeling” refers to a method of establishing a linear fit to determine the titer (e.g., in g / L) of a test protein. In one embodiment, modeling includes a method from the start of collection to the end of loading (e.g., up-tilt modeling). In another embodiment, modeling includes starting a chase to ending a collection (e.g., down-tilt modeling). In still other embodiments, modeling includes both up-tilt and down-tilt modeling.

[0056] "Protein yield" or "yield" refers to the total amount of protein recovered following the processes disclosed herein. Protein yield can be measured in grams or in a final concentration at a fixed volume (e.g., mg / ml). Yield percentage can also be measured as a percentage of the amount of starting protein (e.g., stock solution enzyme).

[0057] As used herein, the term "controlling protein yield" can refer to regulating, testing, or validating the final product (e.g., protein) collected during the processes disclosed herein. In some embodiments, controlling protein yield is achieved by changing the UV signal in real time to influence key process parameters and quality properties and to regulate protein yield. In some embodiments, controlling protein yield means maintaining a constant UV signal during the methods disclosed herein to obtain the desired protein yield.

[0058] As used herein, the term "regulating protein yield" refers to altering, changing, or modifying the final product (e.g., protein) collected during the process disclosed herein. Regulating protein yield alters the yield of the protein final product, which can be increased, decreased, or inhibited. In some embodiments, the process regulates protein yield, resulting in an increase in protein yield. In some embodiments, regulating protein yield is achieved by altering the UV signal in real time to influence key process parameters and quality properties and regulate protein yield.

[0059] The harvest sled described herein includes multiple sensors for real-time clarification and increased protein yield. The harvest sled, or “sled,” includes one or more pressure sensors, one or more flow sensors, one or more ultraviolet (UV) sensors, one or more weight sensors, one or more turbidity sensors, and / or one or more temperature sensors.

[0060] "Title" refers to the amount or concentration of a substance in a solution. As described in this article, upward-sloping and downward-sloping modeling are used to determine the titer.

[0061] As used in this article, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0062] The various aspects described herein are further described in detail in the following sections.

[0063] b. Methods and Applications

[0064] This disclosure is based on the ability to monitor and control key process parameters and quality properties in real time using UV. This method allows the online UV signal of the clarified stock solution to be converted into a real-time titer of the target product using modeling methods. This method can then be used to automate the harvesting process and improve process yield, robustness, and consistency. Titer information can also be used to demonstrate the performance of cell cultures and guide immediate processing for downstream purification. In some embodiments, this document discloses a method for controlling or regulating protein yield in a sample mixture containing a target protein and impurities, the method comprising real-time monitoring of the ultraviolet (UV) signal of the sample mixture during protein filtration in a harvesting skid.

[0065] In one embodiment, this disclosure includes a method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture containing a target protein and impurities. The method includes real-time monitoring of an ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process, and automatically converting the UV signal into a target protein titer using an established model. In another embodiment, the invention provides a method for controlling target protein collection and improving protein yield in a sample mixture containing a target protein and impurities, the method including real-time monitoring of an ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process.

[0066] This article also discloses a method for increasing or improving protein yield in a sample mixture containing target proteins and impurities, the method comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture harvesting process (e.g., protein filtration in a harvest sled).

[0067] Protein harvesting / purification involves multiple steps to isolate or purify a target protein from a mixture of proteins and impurities such as cells, cell culture medium, DNA, RNA, and other proteins. Clarifying the cell culture medium can be the first downstream unit operation in a detailed sequence of steps required to purify the target protein. A combination of centrifugation and / or filtration (e.g., deep filtration) is used for this operation. Therefore, the availability of large-scale filtration technologies (e.g., deep filtration) that allow for real-time monitoring of protein concentration can provide the ability to improve and streamline downstream processes.

[0068] Large-scale depth filtration systems are common in the biological treatment industry. In some implementations, depth filtration systems can utilize, for example... Figure 2 The harvest sled is shown. Before harvesting, the depth filter is rinsed with water or a suitable buffer to remove loose particles and extractables from the filter manufacturing process. The harvest sled may include one or more filters, such as a primary depth filter and a secondary depth filter. Cell culture medium containing the target protein can be obtained from the bioreactor and can be loaded (or pumped) onto one or more filters, such as a primary filter and a secondary filter. Real-time UV signals can then be measured after the loaded cell culture medium has passed through the filtration system (e.g., a primary or secondary filter). The filtered product can then be obtained in one or more tanks. After harvesting, the filter is rinsed again to recover valuable products retained in the shell. Subsequent rinsing can achieve harvest yields of 50% to 90% and ensure minimal product loss. Therefore, the method of the present invention aims to increase the yield of protein harvest by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25%.

[0069] In some implementations, the UV signal provides the titer of the target protein from the start of loading to the end of loading and / or after the end of loading until the end of filtration. In some implementations, the titer of the target protein from the start of loading to the end of loading can be calculated according to equation (I):

[0070] The predicted titer is calculated as a + b * (online UV signal). (I)

[0071] In some implementations, the titer of the target protein from the start of loading to the end of loading can be calculated according to Equation (I), which includes constants (a) and (b).

[0072] In some implementations, (a) is a value between 0 and -1.0. In some implementations, (a) is a value between -0.1 and -0.9. In some implementations, (a) is a value between -0.2 and -0.8. In some implementations, (a) is a value between -0.3 and -0.7. In some implementations, (a) is a value between -0.4 and -0.6.

[0073] In some implementations, (a) is a value between -0.2 and -0.5. In some implementations, (a) is a value between -0.25 and -0.45. In some implementations, (a) is a value between -0.30 and -0.40.

[0074] In some embodiments, (a) is a value between -0.5 and -0.9. In some embodiments, (a) is a value between -0.55 and -0.85. In some embodiments, (a) is a value between -0.60 and -0.80. In some embodiments, (a) is a value between -0.65 and -0.75.

[0075] In some embodiments, (a) is about -0.1. In some embodiments, (a) is about -0.15. In some embodiments, (a) is about -0.2. In some embodiments, (a) is about -0.25. In some embodiments, (a) is about -0.3. In some embodiments, (a) is about -0.35. In some embodiments, (a) is about -0.4. In some embodiments, (a) is about -0.45. In some embodiments, (a) is about -0.5. In some embodiments, (a) is about -0.55. In some embodiments, (a) is about -0.6. In some embodiments, (a) is about -0.65. In some embodiments, (a) is about -0.7. In some embodiments, (a) is about -0.75. In some embodiments, (a) is about -0.8. In some embodiments, (a) is about -0.85. In some embodiments, (a) is about -0.9. In some implementations, (a) is approximately -0.95. In some implementations, (a) is approximately -1.0.

[0076] In some embodiments, (a) is -0.35. In some embodiments, (a) is -0.69. In one embodiment, the cell type is DG44, and (a) is -0.35. In one embodiment, the cell type is CHOZN, and (a) is -0.69.

[0077] In some implementations, (b) is a value between 1.0 and 5.0. In some implementations, (b) is a value between 1.5 and 4.5. In some implementations, (b) is a value between 2.0 and 4.0. In some implementations, (b) is a value between 2.5 and 3.5.

[0078] In some embodiments, (b) is a value between 2.0 and 3.6. In some embodiments, (b) is a value between 2.1 and 3.5. In some embodiments, (b) is a value between 2.2 and 3.4. In some embodiments, (b) is a value between 2.3 and 3.3. In some embodiments, (b) is a value between 2.4 and 3.2. In some embodiments, (b) is a value between 2.5 and 3.1. In some embodiments, (b) is a value between 2.6 and 3.0. In some embodiments, (b) is a value between 2.7 and 2.9.

[0079] In some embodiments, (b) is a value between 3.3 and 4.8. In some embodiments, (b) is a value between 3.4 and 4.7. In some embodiments, (b) is a value between 3.5 and 4.6. In some embodiments, (b) is a value between 3.6 and 4.5. In some embodiments, (b) is a value between 3.7 and 4.4. In some embodiments, (b) is a value between 3.8 and 4.3. In some embodiments, (b) is a value between 3.9 and 4.2. In some embodiments, (b) is a value between 4.0 and 4.1.

[0080] In some embodiments, (b) is about 2.0. In some embodiments, (b) is about 2.1. In some embodiments, (b) is about 2.2. In some embodiments, (b) is about 2.3. In some embodiments, (b) is about 2.4. In some embodiments, (b) is about 2.5. In some embodiments, (b) is about 2.6. In some embodiments, (b) is about 2.7. In some embodiments, (b) is about 2.8. In some embodiments, (b) is about 2.9. In some embodiments, (b) is about 3.0. In some embodiments, (b) is about 3.1. In some embodiments, (b) is about 3.2. In some embodiments, (b) is about 3.3. In some embodiments, (b) is about 3.4. In some embodiments, (b) is about 3.5. In some embodiments, (b) is about 3.6. In some embodiments, (b) is about 3.7. In some embodiments, (b) is about 3.8. In some embodiments, (b) is about 3.9. In some embodiments, (b) is about 4.0. In some embodiments, (b) is about 4.1. In some embodiments, (b) is about 4.2. In some embodiments, (b) is about 4.3. In some embodiments, (b) is about 4.4. In some embodiments, (b) is about 4.5. In some embodiments, (b) is about 4.6. In some embodiments, (b) is about 4.7. In some embodiments, (b) is about 4.8. In some embodiments, (b) is about 4.9. In some embodiments, (b) is about 5.0.

[0081] In some embodiments, (b) is 2.88. In some embodiments, (b) is 4.06. In one embodiment, the cell type is DG44, and (b) is 2.88. In one embodiment, the cell type is CHOZN, and (b) is 4.06. In some embodiments, (a) is -0.35, and (b) is 2.88. In some embodiments, (a) is -0.69, and (b) is 4.06. In one embodiment, the cell type is DG44, and (a) is -0.35, and (b) is 2.88. In one embodiment, the cell type is CHOZN, and (a) is -0.69, and (b) is 4.06.

[0082] In other implementations, the titer of the target protein after loading and until filtration is completed can be calculated according to equation (II):

[0083] The model-predicted titer = A + B * exp(C * online UV signal). (II)

[0084] In some implementations, the titer of the target protein from the start of loading to the end of loading can be calculated according to Equation (II), which includes constants (A), (B) and (C).

[0085] In some implementations, (A) is a value between -2.5 and 1.0. In some implementations, (A) is a value between -2.0 and 0.5. In some implementations, (A) is a value between -1.5 and 0.0. In some implementations, (A) is a value between -1.0 and -0.5.

[0086] In some embodiments, (A) is a value between -1.5 and -0.4. In some embodiments, (A) is a value between -1.4 and -0.5. In some embodiments, (A) is a value between -1.3 and -0.6. In some embodiments, (A) is a value between -1.2 and -0.7. In some embodiments, (A) is a value between -1.1 and -0.8. In some embodiments, (A) is a value between -1.0 and -0.9.

[0087] In some embodiments, (A) is a value between -1.0 and 1.0. In some embodiments, (A) is a value between -0.9 and 0.9. In some embodiments, (A) is a value between -0.8 and 0.8. In some embodiments, (A) is a value between -0.7 and 0.7. In some embodiments, (A) is a value between -0.6 and 0.6. In some embodiments, (A) is a value between -0.5 and 0.5. In some embodiments, (A) is a value between -0.4 and 0.4. In some embodiments, (A) is a value between -0.3 and 0.3. In some embodiments, (A) is a value between -0.2 and 0.2. In some embodiments, (A) is a value between -0.1 and 0.1.

[0088] In some embodiments, (A) is about -2.0. In some embodiments, (A) is about -1.9. In some embodiments, (A) is about -1.8. In some embodiments, (A) is about -1.7. In some embodiments, (A) is about -1.6. In some embodiments, (A) is about -1.5. In some embodiments, (A) is about -1.4. In some embodiments, (A) is about -1.3. In some embodiments, (A) is about -1.2. In some embodiments, (A) is about -1.1. In some embodiments, (A) is about -1.0. In some embodiments, (A) is about -0.9. In some embodiments, (A) is about -0.8. In some embodiments, (A) is about -0.7. In some embodiments, (A) is about -0.6. In some embodiments, (A) is about -0.5. In some embodiments, (A) is about -0.4. In some embodiments, (A) is about -0.3. In some embodiments, (A) is about -0.2. In some embodiments, (A) is about -0.1. In some embodiments, (A) is about 0.1. In some embodiments, (A) is about 0.2. In some embodiments, (A) is about 0.3. In some embodiments, (A) is about 0.4. In some embodiments, (A) is about 0.5. In some embodiments, (A) is about 0.6. In some embodiments, (A) is about 0.7. In some embodiments, (A) is about 0.8. In some embodiments, (A) is about 0.9. In some embodiments, (A) is about 1.0.

[0089] In some embodiments, (A) is -0.95. In some embodiments, (A) is 0.02. In one embodiment, the cell type is DG44 and (A) is -0.95. In one embodiment, the cell type is CHOZN and (A) is 0.02.

[0090] In some implementations, (B) is a value between -1.5 and 2.5. In some implementations, (B) is a value between -1.0 and 2.0. In some implementations, (B) is a value between -0.5 and 1.5. In some implementations, (B) is a value between 0 and 1.0.

[0091] In some embodiments, (B) is a value between -0.5 and -0.4. In some embodiments, (B) is a value between -0.4 and -0.3. In some embodiments, (B) is a value between -0.3 and -0.2. In some embodiments, (B) is a value between -0.2 and -0.1. In some embodiments, (B) is a value between -0.1 and 0.0. In some embodiments, (B) is a value between 0.0 and 0.1. In some embodiments, (B) is a value between 0.1 and 0.2. In some embodiments, (B) is a value between 0.2 and 0.3. In some embodiments, (B) is a value between 0.3 and 0.4. In some embodiments, (B) is a value between 0.4 and 0.5. In some embodiments, (B) is a value between 0.5 and 0.6. In some embodiments, (B) is a value between 0.6 and 0.7. In some embodiments, (B) is a value between 0.7 and 0.8. In some embodiments, (B) is a value between 0.8 and 0.9. In some embodiments, (B) is a value between 0.9 and 1.0. In some embodiments, (B) is a value between 1.0 and 1.1. In some embodiments, (B) is a value between 1.1 and 1.2. In some embodiments, (B) is a value between 1.2 and 1.3. In some embodiments, (B) is a value between 1.3 and 1.4. In some embodiments, (B) is a value between 1.4 and 1.5.

[0092] In some embodiments, (B) is about -1.5. In some embodiments, (B) is about -1.4. In some embodiments, (B) is about -1.3. In some embodiments, (B) is about -1.2. In some embodiments, (B) is about -1.1. In some embodiments, (B) is about -1.0. In some embodiments, (B) is about -0.9. In some embodiments, (B) is about -0.8. In some embodiments, (B) is about -0.7. In some embodiments, (B) is about -0.6. In some embodiments, (B) is about -0.5. In some embodiments, (B) is about -0.4. In some embodiments, (B) is about -0.3. In some embodiments, (B) is about -0.2. In some embodiments, (B) is about -0.1. In some embodiments, (B) is about 0.1. In some embodiments, (B) is about 0.2. In some embodiments, (B) is about 0.3. In some embodiments, (B) is about 0.4. In some embodiments, (B) is about 0.5. In some embodiments, (B) is about 0.6. In some embodiments, (B) is about 0.7. In some embodiments, (B) is about 0.8. In some embodiments, (B) is about 0.9. In some embodiments, (B) is about 1.0. In some embodiments, (B) is about 1.1. In some embodiments, (B) is about 1.2. In some embodiments, (B) is about 1.3. In some embodiments, (B) is about 1.4. In some embodiments, (B) is about 1.5. In some embodiments, (B) is about 1.6. In some embodiments, (B) is about 1.7. In some embodiments, (B) is about 1.8. In some embodiments, (B) is about 1.9. In some embodiments, (B) is about 2.0.

[0093] In some embodiments, (B) is 0.86. In some embodiments, (B) is 0.13. In one embodiment, the cell type is DG44, and (B) is 0.86. In one embodiment, the cell type is CHOZN, and (B) is 0.13.

[0094] In some implementations, (C) is a value between 0 and 4.0. In some implementations, (C) is a value between 0.5 and 3.5. In some implementations, (C) is a value between 1.0 and 3.0. In some implementations, (C) is a value between 1.5 and 2.5.

[0095] In some embodiments, (C) is a value between 0.0 and 0.1. In some embodiments, (C) is a value between 0.1 and 0.2. In some embodiments, (C) is a value between 0.2 and 0.3. In some embodiments, (C) is a value between 0.3 and 0.4. In some embodiments, (C) is a value between 0.4 and 0.5. In some embodiments, (C) is a value between 0.5 and 0.6. In some embodiments, (C) is a value between 0.6 and 0.7. In some embodiments, (C) is a value between 0.7 and 0.8. In some embodiments, (C) is a value between 0.8 and 0.9. In some embodiments, (C) is a value between 0.9 and 1.0. In some embodiments, (C) is a value between 1.0 and 1.1. In some embodiments, (C) is a value between 1.1 and 1.2. In some embodiments, (C) is a value between 1.2 and 1.3. In some embodiments, (C) is a value between 1.3 and 1.4. In some embodiments, (C) is a value between 1.4 and 1.5. In some embodiments, (C) is a value between 1.5 and 1.6. In some embodiments, (C) is a value between 1.6 and 1.7. In some embodiments, (C) is a value between 1.7 and 1.8. In some embodiments, (C) is a value between 1.8 and 1.9. In some embodiments, (C) is a value between 1.9 and 2.0. In some embodiments, (C) is a value between 2.0 and 2.1. In some embodiments, (C) is a value between 2.1 and 2.2. In some embodiments, (C) is a value between 2.2 and 2.3. In some embodiments, (C) is a value between 2.3 and 2.4. In some embodiments, (C) is a value between 2.4 and 2.5. In some embodiments, (C) is a value between 2.5 and 2.6. In some embodiments, (C) is a value between 2.6 and 2.7. In some embodiments, (C) is a value between 2.7 and 2.8. In some embodiments, (C) is a value between 2.8 and 2.9. In some embodiments, (C) is a value between 2.9 and 3.0. In some embodiments, (C) is a value between 3.0 and 3.1. In some embodiments, (C) is a value between 3.1 and 3.2. In some embodiments, (C) is a value between 3.2 and 3.3. In some embodiments, (C) is a value between 3.3 and 3.4. In some embodiments, (C) is a value between 3.4 and 3.5. In some embodiments, (C) is a value between 3.5 and 3.6. In some embodiments, (C) is a value between 3.6 and 3.7. In some embodiments, (C) is a value between 3.7 and 3.8. In some embodiments, (C) is a value between 3.8 and 3.9. In some implementations, (C) is a value between 3.9 and 4.0.

[0096] In some embodiments, (C) is 1.21. In some embodiments, (C) is 2.41. In one embodiment, the cell type is DG44, and (C) is 1.21. In one embodiment, the cell type is CHOZN, and (C) is 2.41.

[0097] In some embodiments, A = -0.95, B = 0.86, and C = 1.21. In some embodiments, A = 0.02, B = 0.13, and C = 2.41. In one embodiment, the cell type is DG44, and (A) is -0.95, (B) is 0.86, and (C) is 1.21. In one embodiment, the cell type is CHOZN, and (A) is 0.02, (B) is 0.13, and (C) is 2.41.

[0098] In some embodiments, this document discloses a method for increasing, controlling, or regulating protein yield in a sample mixture containing a target protein and impurities, the method comprising (a) rinsing a harvesting sled with water; (b) loading a sample onto the harvesting sled; (c) measuring the ultraviolet signal of the sample mixture as a real-time determination of protein titer during protein filtration in the harvesting sled; (d) initiating protein collection based on the ultraviolet measurement and the real-time protein titer; (e) tracking the protein with PBS; and (f) stopping protein collection based on the ultraviolet measurement and the real-time protein titer; wherein the UV signal is correlated with the real-time protein titer during filtration.

[0099] In some embodiments, the method described herein includes water (e.g., RODI) rinsing. In some embodiments, the method includes loading a protein sample and initiating collection based on an online titer. In some embodiments, the method includes PBS pursuit and a final collection based on an online titer. The method disclosed herein does not include a gas venting step, unlike other methods.

[0100] In some implementations, the start and end of sample collection are automatically controlled based on online UV readings and calculated titers. In one particular implementation, the real-time target protein concentration during the harvest process is calculated using modeling via online UV sensor readings. In some implementations, the cutoff point for bulk collection is determined directly based on the calculated online target protein concentration. In some implementations, the calculation algorithm is integrated into Delta V. TM The control system is designed to achieve an automatic cutoff point for protein collection.

[0101] In some embodiments, the methods disclosed herein include a modeling step. In some embodiments, modeling includes offline titer measurements against an online UV signal using a series of diluted samples to establish a linear correlation between the UV signal and the titer. In some embodiments, the sample used for modeling is purified protein. In some embodiments, the sample used for modeling is stock protein containing contaminants. In some embodiments, the modeling is then used to control, regulate, increase, and / or improve protein yield.

[0102] In some embodiments, the methods disclosed herein include controlling, regulating, or increasing the yield of a target protein at a titer of at least about 0.01 g / L. In some embodiments, the titer is at least about 0.02 g / L. In some embodiments, the titer is at least about 0.03 g / L. In some embodiments, the titer is at least about 0.04 g / L. In some embodiments, the titer is at least about 0.05 g / L. In some embodiments, the titer is at least about 0.06 g / L. In some embodiments, the titer is at least about 0.07 g / L. In some embodiments, the titer is at least about 0.08 g / L. In some embodiments, the titer is at least about 0.09 g / L. In some embodiments, the titer is at least about 0.1 g / L. In some embodiments, the titer is at least about 0.2 g / L. In some embodiments, the titer is at least about 0.3 g / L. In some embodiments, the titer is at least about 0.4 g / L. In some embodiments, the titer is at least about 0.5 g / L. In some embodiments, the titer is at least about 0.6 g / L. In some embodiments, the titer is at least about 0.7 g / L. In some embodiments, the titer is at least about 0.8 g / L. In some embodiments, the titer is at least about 0.9 g / L. In some embodiments, the titer is at least about 1 g / L. In some embodiments, the titer is at least about 1.5 g / L. In some embodiments, the titer is at least about 2 g / L. In some embodiments, the titer is at least about 2.5 g / L. In some embodiments, the titer is at least about 3 g / L. In some embodiments, the titer is at least about 3.5 g / L. In some embodiments, the titer is at least about 4 g / L. In some embodiments, the titer is at least about 4.5 g / L. In some embodiments, the titer is at least about 5 g / L. In some embodiments, the titer is at least about 5.5 g / L. In some embodiments, the titer is at least about 6 g / L. In some embodiments, the titer is at least about 6.5 g / L. In some embodiments, the titer is at least about 7 g / L. In some embodiments, the titer is at least about 7.5 g / L. In some embodiments, the titer is at least about 8 g / L. In some embodiments, the titer is at least about 8.5 g / L. In some embodiments, the titer is at least about 9 g / L. In some embodiments, the titer is at least about 9.5 g / L. In some embodiments, the titer is at least about 10 g / L. In some embodiments, the titer is at least about 10.5 g / L. In some embodiments, the titer is at least about 11 g / L. In some embodiments, the titer is at least about 11.5 g / L. In some embodiments, the titer is at least about 12 g / L. In some embodiments, the titer is at least about 12.5 g / L. In some embodiments, the titer is at least about 13 g / L. In some embodiments, the titer is at least about 13.5 g / L.In some embodiments, the titer is at least about 14 g / L. In some embodiments, the titer is at least about 14.5 g / L. In some embodiments, the titer is at least about 15 g / L. In some embodiments, the titer is at least about 15.5 g / L. In some embodiments, the titer is at least about 16 g / L. In some embodiments, the titer is at least about 16.5 g / L. In some embodiments, the titer is at least about 17 g / L. In some embodiments, the titer is at least about 17.5 g / L. In some embodiments, the titer is at least about 18 g / L and at least about 18.5 g / L. In some embodiments, the titer is at least about 19 g / L. In some embodiments, the titer is at least about 19.5 g / L. In some embodiments, the titer is at least about 20 g / L.

[0103] In some embodiments, the methods disclosed herein include the collection of a target protein, depending on the titer of the target protein. In some embodiments, collection of the target protein begins when the titer is at least about 0.05 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.06 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.07 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.08 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.09 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.1 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.2 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.3 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 0.4 g / L. In some embodiments, target protein collection begins when the titer is at least about 0.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 0.6 g / L. In some embodiments, target protein collection begins when the titer is at least about 0.7 g / L. In some embodiments, target protein collection begins when the titer is at least about 0.8 g / L. In some embodiments, target protein collection begins when the titer is at least about 0.9 g / L. In some embodiments, target protein collection begins when the titer is at least about 1 g / L. In some embodiments, target protein collection begins when the titer is at least about 1.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 2 g / L. In some embodiments, target protein collection begins when the titer is at least about 2.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 3 g / L. In some embodiments, target protein collection begins when the titer is at least about 3.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 4 g / L. In some embodiments, target protein collection begins when the titer is at least about 4.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 5 g / L. In some embodiments, target protein collection begins when the titer is at least about 5.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 6 g / L. In some embodiments, target protein collection begins when the titer is at least about 6.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 7 g / L. In some embodiments, target protein collection begins when the titer is at least about 7.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 8 g / L.In some embodiments, target protein collection begins when the titer is at least about 8.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 9 g / L. In some embodiments, target protein collection begins when the titer is at least about 9.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 10 g / L. In some embodiments, target protein collection begins when the titer is at least about 10.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 11 g / L. In some embodiments, target protein collection begins when the titer is at least about 11.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 12 g / L. In some embodiments, target protein collection begins when the titer is at least about 12.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 13 g / L. In some embodiments, target protein collection begins when the titer is at least about 13.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 14 g / L. In some embodiments, target protein collection begins when the titer is at least about 14.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 15 g / L. In some embodiments, target protein collection begins when the titer is at least about 15.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 16 g / L. In some embodiments, target protein collection begins when the titer is at least about 16.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 17 g / L. In some embodiments, target protein collection begins when the titer is at least about 17.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 18 g / L. In some embodiments, target protein collection begins when the titer is at least about 18.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 19 g / L. In some embodiments, target protein collection begins when the titer is at least about 19.5 g / L. In some embodiments, target protein collection begins when the titer is at least about 20 g / L.

[0104] In some embodiments, the methods disclosed herein include the collection of a target protein at a titer within a specified range. In some embodiments, the collected target protein titer is between about 0.05 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.1 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.2 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.3 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.4 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.5 g / L and about 20 g / L. In some embodiments, the collected target protein titer is between about 0.6 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.7 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.8 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.9 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 1 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.05 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.1 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.2 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.3 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.4 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.5 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.6 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.7 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.8 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.9 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 1 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.05 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.1 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.2 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.3 g / L and about 10 g / L.In some embodiments, the collected target protein titer is between about 0.4 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 0.5 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 0.6 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 0.7 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 0.8 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 0.9 g / L and about 10 g / L. In some embodiments, the collected target protein titer is between about 1 g / L and about 10 g / L.

[0105] In some implementations, the method disclosed herein further includes stopping the collection of the target protein when the collection titer is below about 0.5 g / L.

[0106] In some embodiments, the method disclosed herein increases the yield of the target protein. In some embodiments, the target protein yield increases by at least about 1% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 2% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 3% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 4% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 5% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 6% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield increases by at least about 7% compared to the total protein yield. In some embodiments, the target protein yield increases by at least about 8% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 9% compared to the protein yield. In some embodiments, the target protein yield increases by at least about 10% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 11% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 12% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 13% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 14% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield increases by at least about 15% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield is increased by at least about 16% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield is increased by at least about 17% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield is increased by at least about 18% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time.In some embodiments, the target protein yield is increased by at least about 19% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield is increased by at least about 20% compared to the protein yield under conditions where the ultraviolet (UV) signal of the sample mixture is not monitored in real time.

[0107] In some embodiments, the ultraviolet (UV) signal of the sample mixture is measured to be between 0 and 2 AU. In other embodiments, the UV signal of the sample mixture is measured to be about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9 AU, about 1.0 AU, about 1.1 AU, about 1.2 AU, about 1.3 AU, about 1.4 AU, about 1.5 AU, about 1.6 AU, about 1.7 AU, about 1.8 AU, about 1.9 AU, or about 2.0 AU.

[0108] In some embodiments disclosed herein, the method includes protein filtration. In some embodiments, the method includes one or more filters. In some embodiments, protein filtration is depth filtration. In some embodiments, depth filtration includes a primary depth filter and a secondary depth filter. In some embodiments, depth filtration includes a primary depth filter.

[0109] In some implementations, the method includes loading the sample mixture before monitoring.

[0110] In some embodiments, the method includes rinsing a deep filter with buffer before loading cell cultures and tracking the deep filter after loading cell cultures. In some embodiments, the method includes tracking the sample mixture with phosphate-buffered saline (PBS). In some embodiments, the method includes a harvesting sled containing a control system that automatically initiates protein collection when the titer is above 0.5 g / L. In some embodiments, the method includes a harvesting sled containing a control system that automatically stops protein collection when the titer is below 0.5 g / L.

[0111] In some embodiments, the method includes a control system that regulates the flow rate of liquid through the harvesting skid. In some embodiments, the method includes a control system that automatically drives a pump to increase the flow rate through the harvesting skid. In some embodiments, the method includes a control system that automatically drives a pump to decrease the flow rate through the harvesting skid. In some embodiments, the method does not include a gas venting step.

[0112] In some implementations, the method includes a step of collecting protein yields that are not based on volume.

[0113] In some implementations, the methods disclosed herein include measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0114] In some embodiments, the method includes measuring pressure using a pressure sensor. In some embodiments, the measured pressure ranges from -10 psi to 50 psi, -10 psi to 40 psi, -9 psi to 40 psi, -8 psi to 40 psi, -7 psi to 30 psi, -6 psi to -20 psi, -7 psi to 40 psi, -8 psi to 40 psi, -9 psi to 45 psi, -10 psi to -45 psi, or -7 psi to -45 psi. In other embodiments, pressure may be measured at least once, twice, three times, four times, or five times, for example, before the primary filter, after the primary filter and before the secondary filter, after the secondary filter, after drainage, or any combination thereof.

[0115] In some embodiments, the method includes measuring turbidity. In some embodiments, the measured turbidity ranges from 0 absorbance units (AU) to 2 AU. In other embodiments, the measured turbidity is about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9 AU, about 1.0 AU, about 1.1 AU, about 1.2 AU, about 1.3 AU, about 1.4 AU, about 1.5 AU, about 1.6 AU, about 1.7 AU, about 1.8 AU, about 1.9 AU, or about 2.0 AU. In some embodiments, the turbidity is measured at least once, twice, three times, four times, or five times, for example, after a primary filter, after a secondary filter, or after both a primary and secondary filter. See also Figure 2 .

[0116] In some embodiments, the method includes measuring temperature. In some embodiments, the measured temperature ranges are 0°C to 70°C, 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 5°C to 70°C, 10°C to 70°C, 15°C to 70°C, 20°C to 70°C, 10°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, 20°C to 30°C, 35°C to 40°C, or 25°C to 45°C. In other embodiments, the temperature can be measured at any time during the filtration process, for example, at least once, twice, three times, four times, or five times, such as after the primary filter, after the secondary filter, or after both the primary and secondary filters. See also Figure 2 .

[0117] In some embodiments, the method includes measuring flow rate. In some embodiments, the measured flow rate ranges are 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, 0 L / min to 110 L / min, 0 L... 0 L / min to 120 L / min, 0 L / min to 130 L / min, 0 L / min to 140 L / min, 0 L / min to 150 L / min, 0 L / min to 160 L / min, 0 L / min to 170 L / min, 0 L / min to 180 L / min, 0 L / min to 190 L / min, 0 L / min to 200 L / min, 0 L / min to 250 L / min, or 0 L / min to 300 L / min. In other embodiments, the flow rate is measured at any time during the filtration process: before the primary filter, after the primary filter, before the secondary filter, after the secondary filter, or any combination thereof.

[0118] In some implementations, the liquid from the water source / bioreactor / PBS source is... The gravity pump drives the primary deep filter.

[0119] In some implementations, Delta V can be used, for example. TM The system calculates the cumulative flow volume using online flow sensor readings. In some implementations, the cumulative flow volume is used to determine the end of the water flushing. In some implementations, four pressure sensors are placed before the primary depth filter, secondary depth filter, pre-filter, and sterile filter. The pressure-flow control loop can operate based on the real-time pressure value before the primary depth filter. If the pressure value exceeds a certain threshold, Delta V... TM The automatic drive pump adjusts the flow rate. In some embodiments, two turbidity sensors are placed after the primary and secondary depth filters as indicators of filtrate quality. In some embodiments, a UV sensor is placed after the secondary depth filter; the value of this UV sensor is used to calculate the online target protein concentration and control the cutoff point for clarified stock solution collection. The weight of the upstream source and the downstream receiving container are monitored in real time and also displayed on DeltaV. TM Above. In some implementations, the monitor weighs between 0 and 550 kg and has a measurement accuracy of 0.01 kg.

[0120] In some embodiments, the protein is isolated from the source. In some embodiments, the sample mixture is selected from purified protein samples, clarified stock protein samples, cell culture samples, and any combination thereof. In some embodiments, the source is selected from cultured cells.

[0121] In some implementations, the cells are prokaryotes. In bacterial systems, many expression vectors can be advantageously selected depending on the intended use of the expressed protein molecule. For example, when producing large quantities of such a protein, a vector that directs the expression of high levels of an easily purified protein product may be needed to generate a pharmaceutical composition of the protein molecule.

[0122] In other embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NSO), young hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Madin-Darby bovine kidney cells (MDBK), and any combination thereof. In some embodiments, the cells are Chinese hamster ovary cells. In some embodiments, the cells are insect cells, such as fall armyworm (Spodoptera frugiperda) cells.

[0123] In other embodiments, the cells are mammalian cells. Such mammalian cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0124] In some embodiments, the mammalian cells are CHO cells. In some embodiments, the CHO cells are CHO-DG44, CHOZN, CHO / dhfr-, CHOK1SV GS-KO, or CHO-S. In some embodiments, the CHO cells are CHO-DG4. In some embodiments, the CHO cells are CHOZN.

[0125] Other suitable CHO cell lines disclosed herein include CHO-K (e.g., CHO K1), CHO pro3-, CHO P12, CHO-K1 / SF, DUXB11, CHO DUKX; PA-DUKX; CHO pro5; DUK-BII or derivatives thereof.

[0126] In some implementations, the cell density is at least about 1x10 6 The target protein is harvested from a culture medium at a cell density of at least about 5 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 5 x 10⁻⁶ cells / mL. 6 The target protein is harvested from a culture medium at a cell density of at least about 1 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 1 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 1.5 x 10⁻⁶ cells / mL. In some embodiments, the protein is harvested from a culture medium at a cell density of at least about 1.5 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 2 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 2 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 2.5 x 10⁻⁶ cells / mL. In some embodiments, the protein is harvested from a culture medium at a cell density of at least about 2.5 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 3 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 3 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 3.5 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 3.5 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least approximately 4 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least approximately 4 x 10⁻⁶ cells / mL. 7 The target protein is harvested from a culture medium containing at least 4.5 x 10^6 cells / mL. In some embodiments, the protein is harvested from a culture medium containing at least 4.5 x 10^6 cells / mL. 7 The target protein is harvested from a culture medium at a cell density of at least about 5 x 10⁻⁶ cells / mL. In some embodiments, the target protein is harvested from a culture medium at a cell density of at least about 5 x 10⁻⁶ cells / mL. 7 The target protein was harvested from a culture medium of cells per mL.

[0127] In some embodiments, the protein source is a stock protein. In some embodiments, the protein source is a composition comprising protein and non-protein components. The non-protein components may include DNA and other contaminants.

[0128] In some embodiments, the protein source is an animal. In some embodiments, the animal is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or a human). In some embodiments, the source is human tissue or cells. In some embodiments, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, cows, cats, or dogs). In some embodiments, such terms refer to pets or farm animals. In a particular embodiment, such terms refer to humans.

[0129] In some embodiments, the protein purified by the methods described herein is a fusion protein. A “fusion” or “fusion” protein contains a first amino acid sequence linked to a second amino acid sequence within a frame, the first amino acid sequence not naturally linked to the second amino acid sequence in nature. Amino acid sequences typically present in isolated proteins can be aggregated in a fusion polypeptide, or amino acid sequences typically present in the same protein can be placed in a new arrangement within the fusion polypeptide. Fusion proteins are produced, for example, by chemical synthesis or by generating and translating polynucleotides encoding peptide regions in a desired relationship. Fusion proteins may further contain a second amino acid sequence associated with the first amino acid sequence via covalent, non-peptide, or non-covalent bonds. After transcription / translation, a single protein is produced. Thus, multiple proteins or fragments thereof can be incorporated into a single polypeptide. “Operationally linked” is intended to indicate a functional connection between two or more elements. For example, an operational link between two polypeptides fuses the two polypeptides together within a frame to produce a single polypeptide fusion protein. In one particular aspect, the fusion protein further contains a third polypeptide that may contain a linker sequence, as discussed in further detail below.

[0130] In some embodiments, the protein purified by the methods described herein is an antibody. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramers comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody groups. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG). 2a or IgG 2b The antibody is an immunoglobulin molecule. In some embodiments, the antibody described herein is an IgG antibody or a class thereof (e.g., human IgG1 or IgG4) or a subclass thereof. In one specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In some embodiments, the antibody described herein is an IgG1 or IgG4 antibody.

[0131] In some embodiments, the protein described herein is referred to as an "antigen-binding domain," "antigen-binding region," "antigen-binding fragment," and similar terms, which refer to a portion of an antibody molecule containing amino acid residues (e.g., a complementarity-determining region (CDR)) that confer antigen-specificity to the antigen molecule. Antigen-binding regions can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans.

[0132] In some embodiments, the protein is an anti-LAG3 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IL8 antibody, or any combination thereof. In some embodiments, the protein is abatacept NGP. In other embodiments, the protein is berazepam NGP.

[0133] In some implementations, the protein is an antibody against GITR (a glucocorticoid-induced tumor necrosis factor receptor family-associated gene). In some embodiments, the anti-GITR antibody has a 6C8 CDR sequence, for example, a humanized antibody having a 6C8 CDR, such as that described in WO2006 / 105021; and antibodies containing the CDR of the anti-GITR antibody described in WO2011 / 028683; antibodies containing the CDR of the anti-GITR antibody described in JP2008278814; antibodies containing the CDR of the anti-GITR antibody described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, WO 2018 / 013818, or other anti-GITR antibodies described or mentioned herein, all of which are incorporated herein in their entirety.

[0134] In other embodiments, the protein is an anti-LAG3 antibody. Lymphocyte activation gene 3, also known as LAG-3, is a protein encoded by the LAG3 gene in humans. LAG3, discovered in 1990, is a cell surface molecule with multiple biological functions related to T cell function. It is an immune checkpoint receptor and is therefore a target of various drug development programs by pharmaceutical companies seeking to develop new therapies for cancer and autoimmune diseases. It is also developed alone as an anticancer drug in a soluble form. Examples of anti-LAG3 antibodies include, but are not limited to, those in WO 2017 / 087901 A2, WO 2016 / 028672 A1, WO2017 / 106129 A1, WO 2017 / 198741 A1, US 2017 / 0097333 A1, US 2017 / 0290914 A1, and US2017 / 0267759 A1, all of which are incorporated herein by reference in their entirety.

[0135] In some implementations, the protein is an anti-CXCR4 antibody. CXCR4 is a G1-coupled 7-transmembrane protein. CXCR4 is widely expressed on hematopoietic cells and is the major co-receptor for human immunodeficiency virus 1 (HIV-1) with CD4+. See Feng, Y., Broeder, CC, Kennedy, PE, and Berger, EA (1996) Science 272, 872-877. Examples of anti-CXCR4 antibodies include, but are not limited to, those in WO 2009 / 140124 A1, US 2014 / 0286936A1, WO 2010 / 125162 A1, WO 2012 / 047339 A2, WO 2013 / 013025 A2, WO 2015 / 069874 A1, WO2008 / 142303 A2, WO 2011 / 121040 A1, WO 2011 / 154580 A1, WO 2013 / 071068 A2, and WO2012 / 175576 ​​A1, all of which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the protein is an anti-CD73 (extracellular 5'-nucleotidase) antibody. In some embodiments, the anti-CD73 antibody inhibits the formation of adenosine. AMP degradation into adenosine leads to the creation of immunosuppressive and pro-angiogenic microenvironments in the tumor microenvironment, thereby promoting cancer onset and development. Examples of anti-CD73 antibodies include, but are not limited to, those in WO2017 / 100670 A1, WO 2018 / 013611 A1, WO 2017 / 152085 A1, and WO 2016 / 075176 A1, all of which are incorporated herein by reference in their entirety.

[0137] In some embodiments, the protein is an anti-TIGIT (T cell immune receptor with Ig and ITIM domains) antibody. TIGIT is a member of the immunoglobulin protein PVR (poliovirus receptor) family. TIGIT is expressed on several types of T cells, including follicular B helper T cells (TFH). The protein has been shown to bind to PVR with high affinity; this binding is thought to facilitate the interaction between TFH and dendritic cells to regulate T cell-dependent B cell responses. Examples of anti-TIGIT antibodies include, but are not limited to, antibodies in WO 2016 / 028656 A1, WO 2017 / 030823 A2, WO 2017 / 053748 A2, WO 2018 / 033798 A1, WO 2017 / 059095 A1, and WO 2016 / 011264 A1, all of which are incorporated herein by reference in their entirety.

[0138] In some implementations, the protein is an anti-OX40 (i.e., CD134) antibody. OX40 is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. OX40 plays a role in T cell antigen-presenting cell (APC) interactions and mediates the adhesion of activated T cells to endothelial cells. Examples of anti-OX40 antibodies include, but are not limited to, WO 2018 / 031490 A2, WO2015 / 153513 A1, WO 2017 / 021912 A1, WO 2017 / 050729 A1, WO 2017 / 096182 A1, WO 2017 / 134292 A1, WO 2013 / 038191 A2, WO 2017 / 096281 A1, WO 2013 / 028231 A1, WO 2016 / 057667 A1, WO 2014 / 148895 A1, WO 2016 / 200836 A1, WO 2016 / 100929 A1, WO 2015 / 153514 A1、WO 2016 / 002820 A1 and WO 2016 / 200835 A1, both of which are incorporated herein by reference in their entirety.

[0139] In some implementations, the protein is an anti-IL8 antibody. IL-8 is a chemokine that attracts neutrophils, basophils, and T cells, but not monocytes. It is also involved in neutrophil activation. In response to inflammatory stimuli, it is released from several cell types.

[0140] In some implementations, the protein is abatacept (as... (For Sale). Abatacept (also referred to as Aba in this document) is a drug used to treat autoimmune diseases such as rheumatoid arthritis by interfering with the immune activity of T cells. Abatacept is a fusion protein consisting of the Fc region of immunoglobulin IgG1 fused to the extracellular domain of CTLA-4. In order to activate T cells and generate an immune response, antigen-presenting cells must present two signals to T cells. One of these signals is the major histocompatibility complex (MHC) that binds to the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7-2).

[0141] In some implementations, the protein is berazip (trade name). Berazepam is a fusion protein composed of the Fc fragment of human IgG1 immunoglobulin linked to the extracellular domain of CTLA-4. Berazepam is a key molecule regulating T cell co-stimulation, selectively blocking T cell activation. It is designed to provide prolonged transplantation and transplant survival while limiting the toxicity of standard immunosuppressive regimens such as calcineurin inhibitors. It is compatible with abatacept. Only two amino acids are different.

[0142] c. System

[0143] In some embodiments, this document discloses a system for controlling, regulating, increasing, or improving protein yield in a sample mixture containing target proteins and impurities, the system comprising real-time monitoring of the ultraviolet (UV) signal of the sample mixture during protein filtration performed in a harvest sled.

[0144] The systems disclosed herein include one or more sensors. In some embodiments, the sensors include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

[0145] In some embodiments, the harvest sled is designed to integrate all sensors into a single cart, including pressure (4), UV (1), turbidity (2), temperature (2), and flow sensor (1). In some embodiments, the system includes three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controllers are built into the cart to accommodate a total of ten different sensors. In some embodiments, a gravity pump (e.g., A gravity pump is used to drive liquid to a depth filter and is mounted on a skid. In some implementations, the system is movable, lockable, and / or electronically stopable.

[0146] This document also provides systems (e.g., devices, such as harvesting sleds) that can be used in the above methods. In one embodiment, a system or device includes... Figure 1 A and / or Figure 1 Implementation scheme B. In one implementation scheme, a system or device includes... Figure 2 The implementation plan.

[0147] In some embodiments, this document discloses an apparatus for controlling, regulating, increasing, or improving the protein yield in a sample mixture containing a target protein and impurities. The apparatus may include one or more sensors. Sensors may include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

[0148] In some embodiments, the device is designed to integrate all sensors into the device, including a pressure (4), UV (1), turbidity (2), temperature (2), and flow sensor (1). In some embodiments, the device includes three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controller is built into the device to accommodate a total of ten different sensors. In some embodiments, a gravity pump (e.g., A gravity pump is used to drive liquid to a depth filter and is installed in the device. In some embodiments, the device is movable, lockable, and / or electronically stopable. The device may also include a processor configured to control the collection of the target protein. The processor may also be configured to change conditions of the device, such as temperature, pressure, turbidity, or flow rate. The processor may also be configured to control the collection of the target protein. In some embodiments, the processor may use an established model to determine the culture harvesting process. The cell culture harvesting process may include a filtration-based cell culture harvesting process. The processor may be configured to use the target protein titer. The device may be incorporated into a system for controlling, regulating, increasing, or improving the protein yield in a sample mixture containing the target protein and impurities.

[0149] d. Process

[0150] In one implementation, the system or device includes Figure 2 The implementation plan demonstrated the process of using this harvesting sled. Through... A gravity pump drives liquid from the water source / bioreactor / PBS source into the depth filter. The flow sensor is placed after the pump. Delta V TMThe cumulative flow volume is calculated using online flow sensor readings. This cumulative flow volume is used to determine the end of the water flushing process. Four pressure sensors are placed before the primary depth filter, secondary depth filter, pre-filter, and sterile filter. The pressure-flow control loop operates based on the real-time pressure value before the primary depth filter. Two turbidity sensors are placed after the primary and secondary depth filters as indicators of filtrate quality. A UV sensor is placed after the secondary depth filter to calculate the online target protein concentration and control the cutoff point for clarified stock solution collection. The weight of the upstream source and the downstream receiving container is monitored in real time and also displayed on the Delta V. TM superior.

[0151] The following examples are provided by way of example rather than limitation.

[0152] Example

[0153] Example 1: Harvest Sled Design

[0154] Harvesting sleds are used to control, regulate, increase, or improve protein yield in samples. Figure 1 A schematic diagram of the harvesting sled is shown. This sled is designed to integrate all sensors (including pressure (4), ultraviolet (1), turbidity (2), temperature (2), and flow sensor (1)) into a single trolley. See also Figure 2 Three PMAT controllers were built on the cart to house a total of ten different sensors used to drive the liquid to the depth filter. The gravity pump is also mounted on the sled. The harvest sled is designed to be movable, lockable, and capable of emergency stopping.

[0155] Table 1: Instruments used for designing skids

[0156]

[0157] Table 2: Instruments and materials used in the harvesting process

[0158]

[0159] The sensors used in the harvesting process have different functions. Pressure sensors monitor pressure during the process. A cascaded control system reduces the flow rate of the inlet pump when the pressure is too high. UV sensors monitor the UV signal after deep filtration during the process; this UV signal is converted into protein concentration to control the start and end of the concentrate collection. UV is measured at 280 nm.

[0160] Weight sensors monitor the weight of the upstream bioreactor and downstream receiver during the process, controlling loading and catching steps. Bioreactor load cell values ​​and receiver load cell values ​​are integrated into the harvest sled control system. Turbidity sensors measure turbidity at 880 nm, monitoring turbidity before and after deep filtration during the process. Turbidity breakthroughs can be observed if the deep filter becomes clogged. Temperature sensors monitor the temperature during the process. The harvesting process in this paper was conducted at ambient (room temperature).

[0161] The harvest sled process is used to purify proteins of interest from cell cultures. Through... A gravity pump drives liquid from the water source / bioreactor / PBS source to the primary depth filter. It has been demonstrated that, compared to the peristaltic pump P3P, [the following is unclear and likely incomplete:] Gravity pumps induced less cell death in CHO cell cultures. The flow sensor is placed after the pump. Delta V TM The accumulated flow volume is calculated using online flow sensor readings. This accumulated flow volume is used to determine the end of the water flushing process. Four pressure sensors are placed before the primary depth filter, secondary depth filter, pre-filter, and sterile filter P4P. The pressure-flow control loop operates based on the real-time pressure value before the primary depth filter. If the pressure value exceeds a certain threshold, Delta V... TM The automatic drive pump was adjusted to a lower speed. Two turbidity sensors were placed after the primary and secondary depth filters as indicators of filtrate quality. A UV sensor (whose values ​​were used to calculate the online target protein concentration and control the cutoff point for the collection of clarified stock solution) was placed after the secondary depth filter. The weight of the upstream source and the downstream receiving container were monitored in real time and also displayed on the Delta V. TM superior.

[0162] For each embodiment disclosed herein, the harvest sled uses each sensor to detect values ​​within the following range and accuracy.

[0163] Table 3.

[0164] sensor effect Measurement range Measurement accuracy pressure Monitor and control -7 to 30 psi Less than 0.9 psi flow Monitor and control 0 to 20 L / min Less than 0.18 L / min UV Monitor and control 0 to 2AU 0.02AU weight Monitor and control 0 to 550 kg 0.01kg Turbidity monitor 0 to 2AU 0.02AU temperature monitor 0 to 70℃ 0.2℃

[0165] Example 2: Converting UV signals into protein concentration

[0166] Compared to previous methods, the method disclosed herein eliminates the gas venting step. Simultaneously, the start and end of the collection of the clarified stock solution are automatically controlled based on online UV readings and calculated titers. See also Figure 3More specifically, the generated model is used to calculate the real-time target protein concentration during the harvesting process via online UV sensor readings. Therefore, the cutoff point for undiluted protein collection is directly determined based on the calculated online target protein concentration. The calculation algorithm has been integrated into Delta V. TM In the control system, an automatic cutoff point is obtained for the collection of clarified raw liquid.

[0167] The online UV sensor used in this harvest sled has an output absorbance of 0-2 AU. The path length of this UV sensor is adjusted to accommodate total target protein concentrations of 0-6 g / L within the 0-2 AU range. Other UV sensors or Flow VPE (C technology) can be used for determinations of higher concentrations.

[0168] To convert the online UV signal during the process into target protein concentration, a series of sequential steps were taken, such as... Figure 4 As shown.

[0169] In the first step, offline titer measurements against online UV signals were determined using serially diluted samples of D12 GITR cell cultures. Several components in the cell culture samples, including target proteins, HCPs (host cell proteins), and culture medium pigments, can affect the UV absorption signal. To simulate a real-world harvesting process (where the UV sensor measures the total absorbance of all these components), serial dilutions of the cell culture samples (D12 GITR cell cultures), rather than pure proteins, were used for UV sensor path length adjustment.

[0170] like Figure 5 As shown, the path length of the UV sensor was adjusted to cover a wide range of target protein concentrations observable during the harvest process. Due to the low accuracy of UV readings close to 2 AU (maximum output), the path length was reduced so that the UV reading at a titer of 5 g / L was approximately 1.6. Good linearity was observed, R0. 2 The value was 0.97. Therefore, serial dilutions of cell culture samples provide a strong correlation between UV readings and titers.

[0171] Example 3: Small-scale testing using pure protein and clarified stock solution

[0172] Small-scale testing was conducted using 2 L of pure protein (eTau) at a titer of 5.2 g / L. The depth filter was based on 60 L / m³. 2 The loading capacity (per primary filter) was scaled down. Offline samples after the secondary deep filter were harvested during the harvest process. Offline titer readings were plotted against online UV sensor values ​​to understand the relationship between purified protein concentration and online UV signal during the harvest process.

[0173] A second small-scale harvest experiment was conducted using 2 L of clarified stock solution (cell-free eTau cell culture) at a titer of 5 g / L. The depth filter was based on 60 L / m³. 2 The loading capacity (per primary filter) was scaled down. Offline samples were collected after the secondary deep filter during the harvest process. Offline titer readings were plotted against online UV sensor values ​​to understand the relationship between target protein concentrations (in a mixture of culture components) and online UV signals during the harvest process.

[0174] exist Figure 6A and Figure 6B The online UV and offline titer values ​​during the test harvest process are plotted. An "upward slope" data series was collected from the start of undiluted liquid collection to the end of loading; while a "downward slope" data series was collected from the start of pursuit to the end of undiluted liquid collection. (See figure...) Figure 6A and Figure 6B As shown, good linearity was observed for both the upward and downward sloping portions of the data. Therefore, when measuring other samples (e.g., Figure 6A Pure protein and Figure 6B When clarifying the original protein in the culture (in the form of a precipitate), serial dilutions of cell culture samples can provide a strong correlation between UV readings and titers.

[0175] However, the slopes are different for the upward and downward sloping sections, suggesting that different models may be needed for different stages of the harvesting process.

[0176] Example 4: Establishing a model using three large-scale cell culture processes

[0177] Three different cell lines were used for model establishment. These cell lines comprised different, large-scale cell culture processes (Aba NGP, GITR, and Next GenCXCR4) with varying characteristics (cell density, viability, titer, background noise, etc.). The cell lines were harvested using a harvesting sled to generate data for model establishment. A depth filter was applied based on 60-65 L / m³. 2 The loading capacity (per primary filter) varies proportionally. Offline samples are harvested after the secondary deep filter during the harvesting process. Offline titer readings and corresponding online UV sensor values ​​have been input into JMP software to generate a model. UV and titer values ​​are plotted on... Figure 7A , Figure 7B and Figure 7C In the middle section, good linearity is still observed for the upward-sloping portion of the data. However, curvature is observed for the downward-sloping portion.

[0178] During the loading of cell culture material, cells, target proteins, and background noise proteins all occupy the deep filter membrane space. When PBS pursuit begins, target proteins are washed out. As PBS pursuit continues, background noise proteins (such as HCPs) loosely bound to the deep filter membrane begin to be washed out along with the target proteins. Simultaneously, the release of HCPs from cell debris also leads to an increase in the background noise percentage (P5P). This may explain the difference in UV spectra between the clarified stock sample and the cell culture sample. In other words, for complex cell-containing materials, as PBS pursuit progresses, the contribution of target proteins to the total UV signal decreases, while background noise increases.

[0179] Based on these results, two separate models were developed to predict target protein concentrations using online UV signals: one was a linear model to fit data from the upward-sloping portion (from the start of collection to the end of loading), and the other was a non-linear model to fit data from the downward-sloping portion (from the start of pursuit to the end of collection).

[0180] A. Model fitting for the upwardly sloping portion.

[0181] For the upward-sloping portion of the data, the model includes a total of 22 samples. Offline titer values ​​are plotted against online UV values ​​(Figure 8). A linear fit is applied to the data. The R-squared value of the linear fit is... 2 The value is 0.98. Using this model, calculate the predicted titer value and compare it with the actual titer value. For example... Figure 8A and Figure 8B As shown, the fitted slope is very close to 1, and R0 2 It is 0.98.

[0182] To predict the target protein concentration from the start of collection to the end of loading, a linear model was generated: Model predicted titer = a + b * (online UV signal). The model constants a and b depend on the titer level. If the titer is approximately 3.5 g / L or less, then a = -0.35, b = 2.88; if the titer is approximately 3.5 g / L or higher, then a = -0.69, b = 4.06.

[0183] B. Model fitting for the downward-sloping portion

[0184] For the downward-sloping portion of the data, the model includes a total of 41 samples. A plot of offline titer values ​​relative to online UV values ​​is shown below. Figure 10 As shown. Nonlinear fitting was applied to the data. For the CHOZN and DG44 cell lines, the RMSE values ​​of the nonlinear fitting were 0.26 and 0.04, respectively. The predicted titer values ​​were calculated using the model and compared with the actual titer values. Figure 9A and Figure 9B The fitted slope is close to 1, and R0 2The values ​​are 0.97 and 0.99. Figure 9A and Figure 9B .

[0185] To predict the target protein concentration from the start of the hunt to the end of the collection, a nonlinear model was generated: Model predicted titer = A + B * exp(C * online UV signal). The model constants A, B, and C depend on the titer level. If the titer is approximately 3.5 g / L or less, then A = -0.95, B = 0.86, and C = 1.21; if the titer is approximately 3.5 g / L or higher, then A = 0.02, B = 0.13, and C = 2.41.

[0186] Example 5: Test models for four large-scale cell culture processes

[0187] Four large-scale (500L) cell culture processes (CD73, OX40, TIGIT, and IL8) were harvested. Deep filtration was scaled up based on preliminary data from a small scale. Offline samples following secondary deep filtration were collected during the harvest process for actual titer measurements. Online UV sensor values ​​were input into JMP software. Using this model, predicted titer values ​​were calculated based on the online UV sensor values ​​and compared with offline titer measurements.

[0188] Table 4: Cell culture process characteristics of the molecules tested in this report

[0189]

[0190]

[0191] Note: Viability is calculated as follows: Viability (%) = VCD on harvest day / Peak VCD * 100%.

[0192] Table 5. Model fit evaluation for the seven studied molecules

[0193]

[0194] The model was tested using four different large-scale (500L) cell culture harvesting processes, with initial titers of 0–0.1 g / L and final titers of 0.1–0.2 g / L. The model can test titers as low as 0.01 g / L based on a UV signal of 0.01 Au. The predicted titer values ​​were compared with the actual titer values ​​using JMP software. The RMSE values ​​for the model fit for each process are shown below. Figure 10 As shown.

[0195] The difference between the titer value predicted by the calculation model and the actual titer value is calculated. Figure 11The differences in the processes for each test are shown. The overall average difference ranges from 0.07 to 0.36 g / L, indicating that these models can be reliably applied to different processes with a variety of characteristics, as shown in Table 2.

[0196] A. Using online sensors to control the harvesting process and improve harvest yield.

[0197] Table 6: Yield Improvements of Seven Molecules in Studies Using the New Harvest Lever

[0198]

[0199] Calculate the yield during the harvest process using the following equation:

[0200] Yield = (Tit of clarified stock solution (g / L) * Volume of clarified stock solution (L)) * 100%

[0201] Titrate on the day of harvest in the bioreactor (g / L) * Bioreactor volume on the day of harvest (L)

[0202] As shown in Table 6, the yield using the new harvest sled is 2-5% higher than that using the old method.

[0203] In this study, a harvest sled for real-time monitoring and control was designed, and the deep-filtered harvest of several therapeutic proteins was examined.

[0204] Multiple online sensors were built into the harvest sled, and their real-time readings were integrated into Delta V. TM The system enables automated monitoring and control of key process parameters. A model was generated through a series of experimental steps to convert online UV signals during different stages of the harvest process into real-time target protein concentrations. These steps included adjusting the UV sensor path length, pure protein testing, and testing with complex cell culture samples. The model was then successfully tested on several large-scale harvest processes with a wide range of process characteristics, including background noise levels, product levels, total cell density, and viability.

[0205] This study utilizes a novel harvest sled and statistical model to quantitatively monitor and control the clarification process of cell cultures, significantly improving harvest robustness and protein yield. Online titer information is itself an important indicator of cell culture performance and can be used for real-time loading and determination of protein A chromatography in downstream processing.

[0206] Example 6: Real-time monitoring of new protein during protein harvest

[0207] Select a new target protein to clarify using the harvester's sled. First, as... Figure 2As shown, all sensors on the harvest sled are connected in series to monitor pressure, flow rate, UV, turbidity, and temperature during the harvest process. Secondly, the water source and... Gravity pump connection. Input total flow rate and velocity into Delta V. TM The deep filter flushing process is controlled. After reaching the total flow rate, the bioreactor source is connected to... A gravity pump is connected to begin loading cell cultures into the depth filter. Third, the UV prediction model constants for the upper tilt portion are input into Delta V. TM In the middle; and the collection start and end thresholds are input into Delta V. TM During loading, the online UV signal is converted into target protein concentration. Once the threshold is reached, the receiving container is connected to a sterile filter to collect the clarified stock solution. Fourth, after emptying the bioreactor, the PBS source is mixed with... A gravity pump is connected to initiate the pursuit step. Based on cell line type, the UV prediction model constants for the downward-sloping portion are input to Delta V. TM In the middle; and the collection end cutoff threshold is input into Delta V. TM During the pursuit, the online UV signal is converted into target protein concentration. Once a threshold is reached, the receiving container is disconnected from the process flow. Throughout the harvest process, pressure, turbidity, and temperature are monitored to indicate runaway problems.

[0208] Example 7: Confirming the online predicted titer from the online UV signal using offline titer analysis

[0209] The harvesting process begins with a water-for-injection (WFI) rinse of the depth filter. A UV sensor is connected to the outlet of the secondary depth filter. Once filtration stabilizes, a clear flow will be observed from the outlet; at this point, the UV sensor is reset to zero. After flushing the filter with the desired amount of WFI, the cell culture medium is connected to the filter inlet to begin loading. The online UV trace of the culture medium is monitored along the loading process. Along the loading process, a filtrate sample is removed and analyzed offline by titer determination. Figure 11 The titer trace was obtained by modeling based on the UV signal. The titer trace obtained by modeling matched the offline titer determination results well, and therefore could be used to start and end collections to improve process robustness and yield.

[0210] Unless otherwise stated, the practices described herein will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. Such techniques are well explained in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DN Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. US Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) APractical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods InEnzymology, Vols. 154and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986);); Crooks, Antisense drug Technology: Principles, strategies and applications, 2. nd Ed. CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0211] In some embodiments, this document discloses an apparatus for controlling, regulating, increasing, or improving the protein yield in a sample mixture containing a target protein and impurities. The apparatus may include one or more sensors. Sensors may include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

[0212] In some embodiments, the device is designed to integrate all sensors into the device, including a pressure (4), UV (1), turbidity (2), temperature (2), and flow sensor (1). In some embodiments, the device includes three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controller is built into the device to accommodate a total of ten different sensors. In some embodiments, a gravity pump (e.g., A gravity pump is used to drive liquid to a depth filter and is installed in the device. In some embodiments, the system is movable, lockable, and / or electronically stopable. The device may also include a processor configured to control the collection of the target protein. The processor may also be configured to change conditions of the device, such as temperature, pressure, turbidity, or flow rate. The processor may also be configured to control the collection of the target protein. In some embodiments, the processor may use an established model to determine the culture harvesting process. The cell culture harvesting process may include a filtration-based cell culture harvesting process. The processor may be configured to use the target protein titer. The device may be incorporated into a system for controlling, regulating, increasing, or improving the protein yield in a sample mixture containing the target protein and impurities.

[0213] All references cited above, as well as all references and amino acid or nucleotide sequences (e.g., GenBank numbers and / or Uniprot numbers) cited herein, are incorporated herein by reference in their entirety.

[0214] This disclosure relates to the following implementation plan.

[0215] 1. A method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture containing a target protein and impurities, comprising real-time monitoring of an ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process and automatically converting the UV signal into a target protein titer using an established model.

[0216] 2. A method for controlling the collection of target proteins and improving protein yield in a sample mixture containing target proteins and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture harvesting process.

[0217] 3. The method according to implementation scheme 1 or implementation scheme 2, wherein the UV signal is continuously converted into the titer of the target protein according to an established model and automatic control.

[0218] 4. The method according to embodiment 3, wherein the titer of the target protein is at least about 0.01 g / L, at least about 0.02 g / L, at least about 0.03 g / L, at least about 0.04 g / L, at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, or up to At least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L L, at least about 5.5 g / L, at least about 6 g / L, at least about 6.5 g / L, at least about 7 g / L, at least about 7.5 g / L, at least about 8 g / L, at least about 8.5 g / L, at least about 9 g / L, at least about 9.5 g / L, at least about 10 g / L, at least about 10.5 g / L, at least about 11 g / L, at least about 11.5 g / L, at least about 12 g / L, at least about 12.5 g / L, at least about 13 g / L, at least about 13.5 g / L, at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L, or at least about 20 g / L.

[0219] 5. The method according to embodiment 3 or embodiment 4, further comprising the following: when the titer is at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, at least about 6 g / L. 0.5g / L, at least about 7g / L, at least about 7.5g / L, at least about 8g / L, at least about 8.5g / L, at least about 9g / L, at least about 9.5g / L, at least about 10g / L, at least about 10.5g / L, at least about 11g / L, at least about 11.5g / L, at least about 12g / L, at least about 12.5g / L, at least about 13g / L, at least about 13.5g / L The target protein is collected at a concentration of at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L, or at least about 20 g / L.

[0220] 6. The method according to embodiment 5, wherein the titer of the collected target protein is between about 0.05 g / L and about 20 g / L, between about 0.1 g / L and about 20 g / L, between about 0.2 g / L and about 20 g / L, between about 0.3 g / L and about 20 g / L, between about 0.4 g / L and about 20 g / L, between about 0.5 g / L and about 20 g / L, and between about 0.6 g / L and about 20 g / L. Between approximately 0.7 g / L and approximately 20 g / L, between approximately 0.8 g / L and approximately 20 g / L, between approximately 0.9 g / L and approximately 20 g / L, between approximately 1 g / L and approximately 20 g / L, between approximately 0.05 g / L and approximately 15 g / L, between approximately 0.1 g / L and approximately 15 g / L, between approximately 0.2 g / L and approximately 15 g / L, between approximately 0.3 g / L and approximately 15 g / L, between approximately 0.4 g / L and approximately 1 g / L... Between 5 g / L, between about 0.5 g / L and about 15 g / L, between about 0.6 g / L and about 15 g / L, between about 0.7 g / L and about 15 g / L, between about 0.8 g / L and about 15 g / L, between about 0.9 g / L and about 15 g / L, or between about 1 g / L and about 15 g / L, between about 0.05 g / L and about 10 g / L, between about 0.1 g / L and about 10 g / L, and between about 0.2 g / L. Between g / L and about 10 g / L, between about 0.3 g / L and about 10 g / L, between about 0.4 g / L and about 10 g / L, between about 0.5 g / L and about 10 g / L, between about 0.6 g / L and about 10 g / L, between about 0.7 g / L and about 10 g / L, between about 0.8 g / L and about 10 g / L, between about 0.9 g / L and about 10 g / L, or between about 1 g / L and about 10 g / L.

[0221] 7. The method according to any one of embodiments 1 to 6, further comprising stopping the collection of the target protein when the collection titer is below about 0.1 or 0.2 g / L.

[0222] 8. The method according to any one of embodiments 1 to 7, wherein the target protein yield is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture.

[0223] 9. The method according to any one of embodiments 1 to 8, wherein the target protein is derived from cells with a density of at least about 1 x 10⁻⁶ cells / year. 6 cells / mL, at least approximately 5 x 10⁻⁶ 6 cells / mL, at least approximately 1 x 10⁻⁶ 7 Cells / mL, at least approximately 1.5 x 10⁻⁶ 7 1 cell / mL, at least approximately 2 x 10⁻⁶ 7 1 cell / mL, at least about 2.5 x 10⁻⁶ 7 1 cell / mL, at least approximately 3 x 10⁻⁶ cells / mL 7 1 cell / mL, at least approximately 3.5 x 10⁻⁶ cells / mL 7 cells / mL, at least approximately 4 x 10⁻⁶ 7 1 cell / mL, at least about 4.5 x 10⁻⁶ 7 1 cell / mL or at least about 5 x 10⁻⁶ cells / mL 7 Harvested from a culture medium of cells / mL.

[0224] 10. The method according to any one of embodiments 1 to 9, wherein the protein filtration is depth filtration.

[0225] 11. The method according to embodiment 10, wherein the depth filtration comprises a primary depth filter and / or a secondary depth filter.

[0226] 12. The method according to any one of embodiments 1 to 11, further comprising loading the sample mixture prior to the monitoring.

[0227] 13. The method according to any one of embodiments 1 to 12, further comprising rinsing the deep filter with water or a buffer solution before loading the cell culture, and chasing the deep filter after loading the cell culture.

[0228] 14. The method according to any one of embodiments 1 to 13, further comprising chasing the sample mixture with phosphate-buffered saline (PBS) or other buffer solutions.

[0229] 15. The method according to any one of embodiments 1 to 14, wherein the filter-based cell culture harvesting process includes a harvesting sled.

[0230] 16. The method according to embodiment 15, wherein the harvest sled includes a control system, wherein the control system automatically begins collecting the protein when a set titer is reached.

[0231] 17. The method according to embodiment 16, wherein the harvest sled includes a control system, wherein the control system automatically stops collecting the protein when a set titer is reached.

[0232] 18. The method according to embodiment 16 or 17, wherein the control system regulates the flow rate of liquid through the harvest sled.

[0233] 19. The method according to embodiment 18, wherein the control system automatically drives the pump to increase the flow rate through the harvest sled.

[0234] 20. The method according to embodiment 18, wherein the control system automatically drives the pump to reduce the flow rate through the harvest sled.

[0235] 21. The method according to any one of embodiments 1 to 20, wherein the method does not include the step of gas venting.

[0236] 22. The method according to any one of embodiments 1 to 21, wherein the target protein titer or the protein yield is not based on volume.

[0237] 23. A method for increasing, controlling, or regulating the protein yield in a sample mixture containing a target protein and impurities, comprising:

[0238] a) Rinse the harvest sled with water;

[0239] b) Load the sample into the harvest sled;

[0240] c) The ultraviolet (UV) signal of the sample mixture during protein filtration in the harvest sled is measured as the real-time protein titer;

[0241] d) Start collecting the protein based on UV measurements and real-time protein titers;

[0242] e) The protein was captured using PBS; and

[0243] f) Stop collecting the protein based on the UV measurement and the real-time protein titer;

[0244] During the filtration process, the UV signal is correlated with the real-time protein titer.

[0245] 24. The method according to any one of embodiments 1 to 23, further comprising measuring pressure, turbidity, temperature, flow rate or any combination thereof.

[0246] 25. The method according to embodiment 24, further comprising measuring pressure using a pressure sensor.

[0247] 26. The method according to embodiment 25, wherein the measured pressure ranges from -10 psi to 50 psi, -10 psi to 40 psi, -9 psi to 40 psi, -8 psi to 40 psi, -7 psi to 30 psi, -6 psi to -20 psi, -7 psi to 40 psi, -8 psi to 40 psi, -9 psi to 45 psi, -10 psi to -45 psi, or -7 psi to -45 psi.

[0248] 27. The method according to embodiment 24 further includes measuring turbidity.

[0249] 28. The method according to embodiment 27, wherein the turbidity measured ranges from 0 absorbance units (AU) to 2 AU.

[0250] 29. The method according to embodiment 24, further comprising measuring temperature.

[0251] 30. The method according to embodiment 29, wherein the measured temperature range is 0°C to 70°C, 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 5°C to 70°C, 10°C to 70°C, 15°C to 70°C, 20°C to 70°C, 10°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, 20°C to 30°C, 35°C to 40°C, or 25°C to 45°C.

[0252] 31. The method according to embodiment 25 further includes measuring flow rate.

[0253] 32. The method according to embodiment 31, wherein the measured flow rate ranges are 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, and 0 L / min to 110 L / min. n, 0 L / min to 120 L / min, 0 L / min to 130 L / min, 0 L / min to 140 L / min, 0 L / min to 150 L / min, 0 L / min to 160 L / min, 0 L / min to 170 L / min, 0 L / min to 180 L / min, 0 L / min to 190 L / min, 0 L / min to 200 L / min, 0 L / min to 250 L / min, or 0 L / min to 300 L / min.

[0254] 33. The method according to any one of embodiments 1 to 32, wherein the harvest sled includes one or more filters.

[0255] 34. The method according to embodiment 33, wherein the filter comprises a primary depth filter and a secondary depth filter.

[0256] 35. The method according to any one of embodiments 1 to 34, wherein the sample mixture is selected from pure protein samples, clarified stock protein samples, cell culture samples, and any combination thereof.

[0257] 36. The method according to any one of embodiments 1 to 35, wherein the protein is produced in a culture containing mammalian cells.

[0258] 37. The method according to embodiment 36, wherein the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), young hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0259] 38. The method according to embodiment 37, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

[0260] 39. The method according to embodiment 38, wherein the CHO cells are selected from CHO-DG44 cells, CHOZN cells, CHO / dhfr- cells, CHOK1SV GS-KO cells, and CHO-S cells.

[0261] 40. The method according to embodiment 38, wherein the mammalian cell is CHO-DG44, and wherein the target protein concentration is generated using a model-predicted titer, wherein the model-predicted titer includes constants (a) and (b).

[0262] 41. The method according to any one of embodiments 38 to 40, wherein (a) is -0.35 and (b) is 2.88.

[0263] 42. The method according to any one of embodiments 38 to 41, wherein the mammalian cell is CHO-DG44, and wherein the target protein concentration is generated using a model-predicted titer, wherein the model-predicted titer comprises constants (A), (B) and (C).

[0264] 43. The method according to implementation scheme 42, wherein (A) is -0.95, (B) is 0.86, and (C) is 1.21.

[0265] 44. The method according to embodiment 38, wherein the mammalian cell is CHOZN, and wherein the target protein concentration is generated using a model-predicted titer, wherein the model-predicted titer includes constants (a) and (b).

[0266] 45. The method according to implementation scheme 44, wherein (a) is -0.69 and (b) is 4.06.

[0267] 46. ​​The method according to any one of embodiments 38, 44 and 45, wherein the mammalian cell is CHOZN, and wherein the target protein concentration is generated using a model-predicted titer, wherein the model-predicted titer comprises constants (A), (B) and (C).

[0268] 47. The method according to implementation scheme 46, wherein (A) is 0.02, (B) is 0.13, and (C) is 2.41.

[0269] 48. The method according to any one of embodiments 1 to 47, wherein the protein comprises an antibody or a fusion protein.

[0270] 49. The method according to embodiment 48, wherein the protein is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, and an anti-IL8 antibody.

[0271] 50. The method according to embodiment 48, wherein the protein is abatacept or berazepam.

[0272] 51. A system for real-time monitoring and control of protein yield, wherein the system comprises a sensor for measuring a real-time UV signal of a sample mixture containing a target protein and impurities.

[0273] 52. The system according to embodiment 51, wherein the system further comprises a sensor for measuring pressure, turbidity, temperature, flow rate, weight, or any combination thereof.

[0274] 53. The system according to embodiment 51 or 52, which is used in the method according to any one of embodiments 1 to 50.

[0275] 54. An apparatus comprising a sensor configured to measure a UV signal of a sample mixture containing a target protein and impurities.

[0276] 55. The device according to embodiment 54, further comprising a processor configured to control the collection of the target protein.

[0277] 56. The device according to any one of embodiments 54 and 55, wherein the processor is configured to use target protein titers.

[0278] 57. The apparatus according to any one of embodiments 54 to 56, wherein the processor is configured to use an established model to determine the cell culture harvesting process.

[0279] 58. The apparatus according to any one of embodiments 54 to 57, wherein the cell culture harvesting process includes a filtration-based cell culture harvesting process.

[0280] 59. The system according to any one of embodiments 51 to 53, wherein the system includes the device according to any one of embodiments 54 to 58.

Claims

1. A method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture containing a target protein and impurities, comprising real-time monitoring of an ultraviolet (UV) signal of the sample mixture during a filter-based cell culture harvesting process and automatically converting the UV signal into a target protein titer using an established model.

2. A method for controlling the collection of target proteins and improving protein yield in a sample mixture containing target proteins and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture harvesting process.

3. The method according to claim 1 or claim 2, wherein the UV signal is continuously converted into the titer of the target protein according to an established model and automatic control.

4. The method of claim 3, wherein the titer of the target protein is at least about 0.01 g / L, at least about 0.02 g / L, at least about 0.03 g / L, at least about 0.04 g / L, at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, or to... At least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L L, at least about 5.5 g / L, at least about 6 g / L, at least about 6.5 g / L, at least about 7 g / L, at least about 7.5 g / L, at least about 8 g / L, at least about 8.5 g / L, at least about 9 g / L, at least about 9.5 g / L, at least about 10 g / L, at least about 10.5 g / L, at least about 11 g / L, at least about 11.5 g / L, at least about 12 g / L, at least about 12.5 g / L, at least about 13 g / L, at least about 13.5 g / L, at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L, or at least about 20 g / L.

5. The method according to claim 3 or claim 4, further comprising the following: when the titer is at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, at least about 6 g / L. 0.5g / L, at least about 7g / L, at least about 7.5g / L, at least about 8g / L, at least about 8.5g / L, at least about 9g / L, at least about 9.5g / L, at least about 10g / L, at least about 10.5g / L, at least about 11g / L, at least about 11.5g / L, at least about 12g / L, at least about 12.5g / L, at least about 13g / L, at least about 13.5g / L The target protein is collected at a concentration of at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L, or at least about 20 g / L.

6. The method of claim 5, wherein the titer of the collected target protein is between about 0.05 g / L and about 20 g / L, between about 0.1 g / L and about 20 g / L, between about 0.2 g / L and about 20 g / L, between about 0.3 g / L and about 20 g / L, between about 0.4 g / L and about 20 g / L, between about 0.5 g / L and about 20 g / L, or between about 0.6 g / L and about 20 g / L. Between approximately 0.7 g / L and approximately 20 g / L, between approximately 0.8 g / L and approximately 20 g / L, between approximately 0.9 g / L and approximately 20 g / L, between approximately 1 g / L and approximately 20 g / L, between approximately 0.05 g / L and approximately 15 g / L, between approximately 0.1 g / L and approximately 15 g / L, between approximately 0.2 g / L and approximately 15 g / L, between approximately 0.3 g / L and approximately 15 g / L, between approximately 0.4 g / L and approximately 1 g / L... Between 5 g / L, between about 0.5 g / L and about 15 g / L, between about 0.6 g / L and about 15 g / L, between about 0.7 g / L and about 15 g / L, between about 0.8 g / L and about 15 g / L, between about 0.9 g / L and about 15 g / L, or between about 1 g / L and about 15 g / L, between about 0.05 g / L and about 10 g / L, between about 0.1 g / L and about 10 g / L, and between about 0.2 g / L. Between g / L and about 10 g / L, between about 0.3 g / L and about 10 g / L, between about 0.4 g / L and about 10 g / L, between about 0.5 g / L and about 10 g / L, between about 0.6 g / L and about 10 g / L, between about 0.7 g / L and about 10 g / L, between about 0.8 g / L and about 10 g / L, between about 0.9 g / L and about 10 g / L, or between about 1 g / L and about 10 g / L.

7. The method according to any one of claims 1 to 6, further comprising stopping the collection of the target protein when the collection titer is below about 0.1 or 0.2 g / L.

8. The method according to any one of claims 1 to 7, wherein the target protein yield is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture.

9. The method according to any one of claims 1 to 8, wherein the target protein is derived from cells with a density of at least about 1 x 10⁻⁶ cells / year. 6 cells / mL, at least approximately 5 x 10⁻⁶ 6 cells / mL, at least approximately 1 x 10⁻⁶ 7 1.5 x 10^12 cells / mL, at least approximately 1.5 x 10^12 cells / mL 7 1 cell / mL, at least approximately 2 x 10⁻⁶ 7 1 cell / mL, at least about 2.5 x 10⁻⁶ 7 1 cell / mL, at least approximately 3 x 10⁻⁶ cells / mL 7 1 cell / mL, at least approximately 3.5 x 10⁻⁶ cells / mL 7 cells / mL, at least approximately 4 x 10⁻⁶ 7 1 cell / mL, at least about 4.5 x 10⁻⁶ 7 1 cell / mL or at least about 5 x 10⁻⁶ cells ... 7 Harvested from a culture medium of cells / mL.

10. The method according to any one of claims 1 to 9, wherein the protein filtration is depth filtration.

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