Real-time titer monitoring using ultraviolet signal

Abstract

To provide a method for controlling, adjusting, increasing, or improving a protein yield in a sample mixture containing a protein of interest and impurities, the method comprising monitoring an ultraviolet (UV) signal of the sample mixture in real time during protein filtration on a recovery skid.SOLUTION: A method for monitoring a concentration (titer) of a protein of interest in a sample mixture containing the protein of interest and impurities in real time, comprises: monitoring an ultraviolet (UV) signal of a sample mixture in real time and automatically converting the UV signal to a titer of the protein of interest during a filtration-based cell culture recovery process using an established model.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to methods for monitoring the concentration of a biomolecule (e.g., a protein) in a composition. In particular, the present disclosure relates to methods for monitoring, controlling, regulating, or increasing the yield of a protein from a composition using a real-time ultraviolet signal during filtration of the protein. [Background technology]

[0002] Hundreds of therapeutic proteins (e.g., monoclonal antibodies (mAbs)) are currently in development, and many companies have multiple antibodies in development. In addition to basic unit operations such as recovery, Protein A affinity chromatography and further purification steps are used to purify the protein of interest.

[0003] Upstream recovery operations aim for high productivity of therapeutic proteins in both cell culture and recovery processes and can utilize various online forms of monitoring bioprocessing operations. Whitford W., Julien C. Bioprocess Int. (5), S32-S45 (2007). Recently, real-time monitoring and control of cell culture processes has been performed. It has been shown that the onset of stationary phase can be predicted by the increase of nonviable subpopulations in CHO cultures, highlighting opportunities for fully automated cell culture processes as well as reliable and reproducible control of batch additions during culture expansion. Sitton G., Srienc FJ Biotechnol., 135 (2008), 174-180.Other primary recovery processes utilize multiple steps to remove biomass and purify the feed stream for downstream column chromatography. Bink LR, Furey J. BioProcess Int. 8(3) 2010, 44-49, 57 (2010).

[0004] There have been some efforts to improve protein yields in upstream steps to increase downstream yields. For example, attempts have been made to use peristaltic and diaphragm pumps to reduce the mechanical stress on CHO cells caused by magnetically levitated bearingless centrifugal pumps. Blaschczok K., et al. Chemie Ingenieur Technik, (85), 144-152 (2013). In addition, proteomic approaches were evaluated by investigating the dynamics and fate of host cell proteins in the supernatant of a monoclonal antibody producing cell line during harvest and early downstream processes such as centrifugation, depth 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 concentration and yield during the purification process. Ignatova and Gierasch, Proc Natl Acad Sci US A.; 101(2):523-8 (2004). The addition of additional impurities may necessitate additional purification steps that may affect yield. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, real-time monitoring and control of recovery processes is required to increase recovery rates and process reliability, rapidly assess upstream performance, and facilitate rapid downstream processing in batch or more critical continuous processes. [Means for solving the problem]

[0006] Described herein is a novel real-time monitoring and control process and system designed and tested for filtration-based cell culture harvest processes, e.g., depth filtration harvest, of several therapeutic proteins. The methods described herein provide several advantages over the prior art. First, the design of the harvest skid allows for real-time monitoring and control of key process parameters and quality attributes. Second, a modeling approach is used to convert the on-line UV signal of the clarified bulk into real-time titers of the target. Third, the harvest skid and real-time titers are used to automatically control the harvest process, improving process yield, reliability and consistency. Finally, the titer information is used to characterize cell culture performance and guide rapid processing of downstream purification.

[0007] The core of this new technology is the application of real-time monitoring of the UV signal during the harvest process and the conversion of the online UV signal to real-time concentration of the protein of interest. The model disclosed herein can be applied to processes with different cell characteristics and productivity levels. Using this system, the start and end of clarified bulk harvest can be quantitatively determined, thus significantly improving harvest reliability and protein yield.

[0008] The methods disclosed herein provide deep insight into the application of harvest skids in cell culture clarification processes. The novel harvest process disclosed herein improves protein yield while being scalable, automated, and applicable to multiple products with a wide range of properties. Real-time titer information can be used to characterize cell culture performance and rapidly guide downstream processing.

[0009] The present specification discloses a method for real-time monitoring of the concentration (titer) of a protein of interest in a sample mixture containing the protein of interest and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time and automatically converting the UV signal to a titer of the protein of interest during a filtration-based cell culture harvest process using an established model.

[0010] Further, the present specification discloses a method for controlling recovery of a protein of interest and improving protein yield in a sample mixture containing the protein of interest and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture recovery process.

[0011] In some embodiments, the UV signal is continuously converted to the titer of the protein of interest according to an established model and automated control.

[0012] In some embodiments, the titer of the protein of interest 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, 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. ...8 g / L, at least about 0.8 g / L, at least about 0.9 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 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 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 5.5g / L, at least about 6g / L, at least about 6.5g / 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, / 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.

[0013] In some embodiments, the methods disclosed herein provide a method for producing a medicament having a titer of 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 6.5 g / L, at least about 7 g / L, at least about 8 g / L, at least about 9 g / L, at least about 10 g / L, at least about 11 g / L, at least about 12 g / L, at least about 13 g / L, at least about 14 g / L, at least about 15 g / L, at least about 16 g / L, at least about 17 g / L, at least about 18 g / L, at least about 19 g / L, at least about 20 g / L, at least about 21 g / L, at least about 22 g / L, at least about 23 g / L, at least about 24 g / L, at least about 25 g / L, at least about 26 g / L, at least about 27 g / L, at least In some embodiments, the method further comprises initiating recovery of the protein of interest when the protein is 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.

[0014] In some embodiments, the titer at which the target protein is recovered is about 0.05 g / L to about 20 g / L, about 0.1 g / L to about 20 g / L, about 0.2 g / L to about 20 g / L, about 0.3 g / L to about 20 g / L, about 0.4 g / L to about 20 g / L, about 0.5 g / L to about 20 g / L, or about 0.6 g / L to about 20 g / L. L, about 0.7g / L to about 20g / L, about 0.8g / L to about 20g / L, about 0.9g / L to about 20g / L, about 1g / L to about 20g / L, about 0.05g / L ~ approx. 15g / L, approx. 0.1g / L ~ approx. 15g / L, approx. 0.2g / L ~ approx. 15g / L, approx. 0.3g / L ~ approx. 15g / L, approx. 0.4g / L ~ approx. 15g / L, about 0.5g / L to about 15g / L, about 0.6g / L to about 15g / L, about 0.7g / L to about 15g / L, about 0.8g / L to about 15g / L, about 0. 9g / L~about 15g / L, or about 1g / L~about 15g / L, about 0.05g / L~about 10g / L, about 0.1g / L~about 10g / L, about 0.2g / L~ Approx. 10g / L, Approx. 0.3g / L~Approx. 10g / L, Approx. 0.4g / L~Approx. 10g / L, Approx. 0.5g / L~Approx. 10g / L, Approx. 0.6g / L~Approx. 10g / L , about 0.7 g / L to about 10 g / L, about 0.8 g / L to about 10 g / L, about 0.9 g / L to about 10 g / L, or about 1 g / L to about 10 g / L.

[0015] In some embodiments, the methods disclosed herein further comprise stopping recovery of the protein of interest when the recovery titer is less than about 0.1 or 0.2 g / L.

[0016] In some embodiments, the yield of the protein of interest 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 not monitoring the ultraviolet (UV) signal of the sample mixture in real time.

[0017] In some embodiments, the protein of interest is at least about 1×10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 1 x 10 7 Cells / mL, at least approximately 1.5 x 10 7 Cells / mL, at least approximately 2 x 10 7 Cells / mL, at least approximately 2.5 x 10 7 Cells / mL, at least approximately 3 x 10 7 Cells / mL, at least approximately 3.5 x 10 7 Cells / mL, at least approximately 4 x 10 7 Cells / mL, at least approximately 4.5 x 10 7 cells / mL, or at least about 5 x 10 7 The medium is harvested with a cell density of 1000 cells / mL.

[0018] In some embodiments, the protein filtration is depth filtration. In some embodiments, the depth filtration comprises a primary depth filter and / or a secondary depth filter.

[0019] In some embodiments, the methods disclosed herein further comprise loading a sample mixture prior to monitoring. In some embodiments, the methods disclosed herein further comprise flushing the depth filter with water or a buffer before loading the cell culture and chasing the depth filter after loading the cell culture. In some embodiments, the methods disclosed herein further comprise chasing the sample mixture with phosphate buffered saline (PBS) or other buffer. In some embodiments, the filtration-based cell culture harvest process comprises a harvest skid. In some embodiments, the harvest skid comprises a control system that automatically initiates protein harvesting once a set titer is achieved. In some embodiments, the harvest skid comprises a control system that automatically stops protein harvesting once a set titer is achieved. In some embodiments, the control system regulates the flow rate of liquid passing through the harvest skid. In some embodiments, the control system automatically drives a pump to up-regulate the flow rate of the harvest skid. In some embodiments, the control system automatically drives a pump to down-regulate the flow rate of the harvest skid. In some embodiments, the methods disclosed herein do not include an air blowdown step. In some embodiments, the titer of the protein of interest or the yield of the protein is not volumetric.

[0020] In some embodiments, the method disclosed herein is a method for increasing, controlling, or regulating protein yield in a sample mixture containing a protein of interest and impurities, comprising: (a) flushing the collection skid with water; (b) loading the sample onto the collection skid; (c) measuring the ultraviolet (UV) signal of the sample mixture during protein filtration on the collection skid to obtain a real-time protein titer; (d) starting protein collection based on the UV measurement and real-time protein titer; (e) chasing the protein with PBS; and (f) stopping protein collection based on the UV measurement and real-time protein titer, wherein the UV signal correlates with the real-time protein titer during filtration.

[0021] In some embodiments, the method further comprises measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0022] In some embodiments, the method further includes measuring the pressure using a pressure sensor, hi some embodiments, the measured pressure is in the range of -10 pounds per square inch (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.

[0023] In some embodiments, the method further comprises measuring turbidity, hi some embodiments, the measured turbidity ranges from 0 absorbance units (AU) to 2 AU.

[0024] In some embodiments, the method further comprises measuring the temperature, in some embodiments, the measured temperature is in the range of 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.

[0025] In some embodiments, the method further comprises measuring a flow rate. In some embodiments, the measured flow rate is between 0 L / min and 20 L / min, between 0 L / min and 30 L / min, between 0 L / min and 40 L / min, between 0 L / min and 50 L / min, between 0 L / min and 60 L / min, between 0 L / min and 70 L / min, between 0 L / min and 80 L / min, between 0 L / min and 90 L / min, between 0 L / min and 100 L / min, between 0 L / min and 110 L / min, between 0 L / min and 120 L / min, between 0 L / min and 140 L / min, between 0 L / min and 160 L / min, between 0 L / min and 20 L / min, between 0 L / min and 220 L / min, between 0 L / min and 30 L / min, between 0 L / min and 40 L / min, between 0 L / min and 50 L / min, between 0 L / min and 60 L / min, between 0 L / min and 70 L / min, between 0 L / min and 80 L / min, between 0 L / min and 90 L / min, between 0 L / min and 100 L / min, between 0 L / min and 110 L / min, between 0 L / min and 160 L / min, between 0 L / min and 180 L / min, between 0 L / min and 240 L / min, between 0 L / min and 280 L / min, between 0 L / min and 320 L / min, between 0 L / min and 360 L / min, between 0 L / min and 40 L / min, between 0 L / min and 50 L / min, between 0 L / min and 60 L / min, between 0 L / min and 70 L / min ~120L / min, 0L / min-130L / min, 0L / min-140L / min, 0L / min-150L / min, 0L / min-160L / min, 0L / min-170L / min, 0L / min-180L / min, 0L / min-190L / min, 0L / min-200L / min, 0L / min-250L / min, or 0L / min-300L / min.

[0026] In some embodiments, the recovery skid comprises one or more filters. In some embodiments, the filters comprise a primary depth filter and a secondary depth filter. In some embodiments, the sample mixture is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof.

[0027] In some embodiments, the protein is produced in a culture comprising mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0028] 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 an abatacept or a belatacept.

[0029] In some embodiments, the present specification discloses a system for real-time monitoring and control of protein yield, comprising a sensor for measuring the real-time UV signal of a sample mixture containing a protein of interest and impurities.

[0030] In some embodiments, the system further comprises a sensor that measures pressure, turbidity, temperature, flow rate, weight, or any combination thereof.

[0031] In some embodiments, the device comprises a sensor configured to measure a UV signal of a sample mixture comprising the protein of interest and impurities. In some embodiments, the processor is configured to control recovery of the protein of interest. In some embodiments, the processor is configured to use a titer of the protein of interest. In some embodiments, the processor is configured to determine a cell culture harvest process using the established model. In some embodiments, the cell culture harvest process comprises a filtration-based cell culture harvest process. In some embodiments, the system comprises a device comprising a sensor configured to measure a UV signal of a sample mixture comprising the protein of interest and impurities.

[0032] In some embodiments, the disclosed systems are for use in the methods described herein. [Brief description of the drawings]

[0033] [Figure 1]FIG. 1A is a diagram of an exemplary recovery skid design. All values ​​are in inches. FIG. 1B is a photograph of the recovery skid.

[0034] [Diagram 2] Figure 2 shows a process flow diagram of the cell culture harvest process using the novel harvest skid. The various boxes represent the online measurement sensors, control modules, and physical instruments.

[0035] [Diagram 3] FIG. 3 shows the experimental design described herein for modeling UV signal versus product titer.

[0036] [Figure 4] Figure 4 shows a graphical comparison between the conventional and novel harvest methods. Compared with the conventional method, the air blow-down step is eliminated in the novel method. Meanwhile, the start and end of clarified bulk harvest in the novel method can be automatically controlled based on the online UV measurements and the calculated titer. More specifically, the real-time target protein concentration during the harvest process can be calculated by the online UV sensor readings using the model created and tested here. Therefore, the bulk harvest cut-off can be directly determined based on the calculated online target protein concentration. The calculation algorithm can be integrated into the Delta V™ control system to achieve the automated cut-off of clarified bulk harvest.

[0037] [Diagram 5] FIG. 5 shows offline titration versus online UV signal using serially diluted samples of GITR cell culture.

[0038] [Figure 6]Figures 6A and 6B show the online UV signal versus offline titer measurements for a small scale harvest process using pure protein (Figure 6A) and clarified bulk (Figure 6B). The online UV and offline titer values ​​during the test harvest process were plotted.

[0039] [Figure 7] Figure 7A, Figure 7B, and Figure 7C show offline titer measurements against online UV signal in a large-scale recovery process using anti-GITR antibody cell culture (Figure 7A), abatacept cell culture (Figure 7B), and anti-CXCR4 antibody cell culture (Figure 7C).

[0040] [Figure 8] 8A and 8B show a linear fit of offline titer measurements to online UV values ​​(FIG. 8A); a linear fit of UV-based predicted titers to actual titers (FIG. 8B).

[0041] [Figure 9] 9A and 9B show a non-linear fit of offline titer measurements to online UV values ​​(FIG. 9A); a linear fit of UV-based predicted titers to actual titers (FIG. 9B).

[0042] [Figure 10] FIG. 10 shows the average difference between model predicted and actual potencies (HPLC analysis) for seven molecules tested, including Aba J, anti-CD73 antibody, anti-GITR antibody, anti-IL8 antibody, anti-CXCR4 antibody, anti-OX40 antibody and anti-TIGIT antibody.

[0043] [Figure 11]FIG. 11 shows a comparison of the online UV trace, the titer trace achieved by modeling from the UV signal, and the titer determined offline. The Y-axis shows the titer measured offline (g / L) or the titer modeled based on UV (g / L), and the X-axis shows time (min). The triangular line shows the online UV, the square line shows the titer modeled based on UV (g / L), and the diamond line shows the offline titer (g / L). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description Various methods are provided that can be used to control, regulate, or increase protein yield. The methods include controlling, regulating, or increasing protein yield using real-time measurement of ultraviolet (UV) signal of a sample mixture during a purification step (e.g., protein filtration on a harvesting skid). The methods utilize the UV signal to determine the titer of the protein of interest according to a formula disclosed herein, which differs based on whether the harvest is from the start of loading to the end of loading or after the end of loading.

[0045] Also disclosed herein are various systems and devices related to the methods provided herein.

[0046] a.Terminology Terms with "a" or "an" refer to one or more. For example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0047] Furthermore, as used herein, "and / or" is intended to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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 (single); B (single); and C (single).

[0048] Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Furthermore, it should be understood that all base or amino acid size, and molecular weight or mass values ​​given for nucleic acids or polypeptides are approximate and are given for illustrative purposes.

[0049] Whenever an embodiment is described herein with the word "comprising," it is understood that similar embodiments that are otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure pertains.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide one of the common dictionary techniques for many of the terms used in this disclosure.

[0051] Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise noted, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not intended to be limiting of the various embodiments, but may be read with reference to the specification as a whole. Thus, the terms defined immediately below are fully defined with reference to the entire specification.

[0052] The term "about" is used herein to mean approximately, roughly, in the vicinity of, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. That is, "about 10 to 20" means "about 10 to about 20". In general, the term "about" can modify numerical values ​​above and below the stated value by a certain upper or lower (higher or lower) variation (e.g., 10 percent).

[0053] "Modeling" or "protein modeling" refers to a method of establishing a linear fit to determine the titer (e.g., g / L) of a test protein. In one embodiment, the modeling includes the method from the start of harvest to the end of loading (e.g., incline modeling). In another embodiment, the modeling includes the method from the start of chase to the end of harvest (e.g., decline modeling). In other embodiments, the modeling includes both incline and decline modeling.

[0054] "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 as a final concentration in a fixed volume (such as mg / ml). Additionally, percent yield can be measured as a percentage of the amount of starting protein (e.g., bulk enzyme).

[0055] As used herein, the term "protein yield control" refers to regulating, testing, or verifying the end product (e.g., protein) recovered during the processes disclosed herein. In some embodiments, protein yield control is achieved by altering the UV signal in real time to affect key process parameters and quality attributes to regulate protein yield. In some embodiments, protein yield control refers to maintaining a constant UV signal during the methods disclosed herein to achieve a desired protein yield.

[0056] The term "modulating protein yield" as used herein refers to altering the end product (e.g., protein) recovered during the processes disclosed herein. Modulating protein yield can change (increase, decrease, or prevent) the yield of the end protein product. In some embodiments, the process modulates protein yield, resulting in increased protein yield. Modulating protein yield is achieved by altering the UV signal in real time to affect key process parameters and quality attributes to adjust protein yield.

[0057] The harvest skid described herein comprises multiple sensors for real-time clarification and increased protein yield. The harvest skid, or "skid," comprises 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.

[0058] "Potency" refers to the amount or concentration of a substance in solution. Potency is determined using both incline and decline modeling, as described herein.

[0059] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0060] The various aspects described herein are described in further detail in the following subsections.

[0061] b. Method and Use The present disclosure is based on the ability of real-time UV monitoring and control of key process parameters and quality attributes. The method allows for the conversion of the online UV signal of the clarified bulk into real-time titers of the target using a modeling method. The method can then be used to automatically control the recovery process to improve process yield, reliability, and consistency. The titer information can also be used to characterize cell culture performance and guide downstream purification rapid processing. In some embodiments, the present disclosure discloses a method for controlling or regulating protein yield in a sample mixture containing a target protein and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during protein filtration in a recovery skid.

[0062] In one embodiment, the disclosure includes a method for monitoring the concentration (titer) of a protein of interest in a sample mixture containing the protein of interest and impurities in real time, comprising monitoring an ultraviolet (UV) signal of the sample mixture in real time and automatically converting the UV signal to a titer of the protein of interest using an established model during a filtration-based cell culture harvest process. In another embodiment, the disclosure provides a method for controlling the recovery of a protein of interest and improving the yield of the protein of interest in a sample mixture containing impurities, comprising monitoring an ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture harvest process.

[0063] The present specification also discloses a method for increasing or improving protein yield in a sample mixture containing a protein of interest and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during a filtration-based cell culture recovery process (e.g., protein filtration on a recovery skid).

[0064] Protein recovery / purification involves multiple steps to isolate or purify the protein of interest from a mixture of proteins and impurities (e.g., cells, cell culture medium, DNA, RNA, other proteins, etc.). Clarification of the cell culture fluid can be the first downstream operation in a series of elaborate steps required to purify the protein of interest. The operation uses a combination of centrifugation and / or filtration (e.g., depth filtration). The availability of large-scale filtration techniques, e.g., depth filtration, that allow real-time monitoring of protein concentration allows for improvements and simplification of downstream processes.

[0065] Large-scale depth filtration systems are common in the bioprocessing industry. In some embodiments, depth filtration systems can utilize a harvest skid, as shown in FIG. 2. Prior to harvest, the depth filter is flushed with water or an appropriate buffer to remove any particulates or extractables dislodged from the filter manufacturing process. The harvest skid can comprise one filter or multiple filters (e.g., a primary depth filter and a second depth filter). Cell culture media containing the protein of interest can be obtained from a bioreactor and loaded (or pumped) into one or more filters (e.g., a primary filter and a secondary filter). A real-time UV signal can then be measured after the loaded cell culture media passes through the filter system (e.g., the primary or secondary filter). The filtered product can then be obtained in one or more tanks. Once harvest is complete, the filter is flushed again to recover the valuable product retained in the housing. Recoveries of 50% to 90% can be achieved by flushing after use to minimize product loss. Thus, the methods of the invention are contemplated to improve protein recovery 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%.

[0066] In some embodiments, the UV signal provides the titer of the protein of interest from the start of loading to the end of loading and / or from the end of loading to the end of filtration. In some embodiments, the titer of the protein of interest from the start of loading to the end of loading can be calculated according to formula (I). Model predicted titer = a + b*(online UV signal) (I)

[0067] In some embodiments, the titer of a protein of interest from the start of loading to the end of loading can be calculated according to formula (I), which includes constants (a) and (b).

[0068] In some embodiments, (a) is a number between 0 and -1.0. In some embodiments, (a) is a number between -0.1 and -0.9. In some embodiments, (a) is a number between -0.2 and -0.8. In some embodiments, (a) is a number between -0.3 and -0.7. In some embodiments, (a) is a number between -0.4 and -0.6.

[0069] In some embodiments, (a) is a number between -0.2 and -0.5. In some embodiments, (a) is a number between -0.25 and -0.45. In some embodiments, (a) is a number between -0.30 and -0.40.

[0070] In some embodiments, (a) is a number between -0.5 and -0.9. In some embodiments, (a) is a number between -0.55 and -0.85. In some embodiments, (a) is a number between -0.60 and -0.80. In some embodiments, (a) is a number between -0.65 and -0.75.

[0071] 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 embodiments, (a) is about -0.95. In some embodiments, (a) is about -1.0.

[0072] 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.

[0073] In some embodiments, (b) is a number between 1.0 and 5.0. In some embodiments, (b) is a number between 1.5 and 4.5. In some embodiments, (b) is a number between 2.0 and 4.0. In some embodiments, (b) is a number between 2.5 and 3.5.

[0074] In some embodiments, (b) is a number between 2.0 and 3.6. In some embodiments, (b) is a number between 2.1 and 3.5. In some embodiments, (b) is a number between 2.2 and 3.4. In some embodiments, (b) is a number between 2.3 and 3.3. In some embodiments, (b) is a number between 2.4 and 3.2. In some embodiments, (b) is a number between 2.5 and 3.1. In some embodiments, (b) is a number between 2.6 and 3.0. In some embodiments, (b) is a number between 2.7 and 2.9.

[0075] In some embodiments, (b) is a number between 3.3 and 4.8. In some embodiments, (b) is a number between 3.4 and 4.7. In some embodiments, (b) is a number between 3.5 and 4.6. In some embodiments, (b) is a number between 3.6 and 4.5. In some embodiments, (b) is a number between 3.7 and 4.4. In some embodiments, (b) is a number between 3.8 and 4.3. In some embodiments, (b) is a number between 3.9 and 4.2. In some embodiments, (b) is a number between 4.0 and 4.1.

[0076] 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.

[0077] 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.

[0078] In another embodiment, the titer of the protein of interest from the end of loading to the end of filtration can be calculated according to formula (II). Model predicted titer = A + B*exp(C*online UV signal) (II)

[0079] In some embodiments, the titer of a protein of interest from the start of loading to the end of loading can be calculated according to formula (II), which includes constants (A), (B), and (C).

[0080] In some embodiments, (A) is a number between -2.5 and 1.0. In some embodiments, (A) is a number between -2.0 and 0.5. In some embodiments, (A) is a number between -1.5 and 0.0. In some embodiments, (A) is a number between -1.0 and -0.5.

[0081] In some embodiments, (A) is a number between -1.5 and -0.4. In some embodiments, (A) is a number between -1.4 and -0.5. In some embodiments, (A) is a number between -1.3 and -0.6. In some embodiments, (A) is a number between -1.2 and -0.7. In some embodiments, (A) is a number between -1.1 and -0.8. In some embodiments, (A) is a number between -1.0 and -0.9.

[0082] In some embodiments, (A) is a number between -1.0 and 1.0. In some embodiments, (A) is a number between -0.9 and 0.9. In some embodiments, (A) is a number between -0.8 and 0.8. In some embodiments, (A) is a number between -0.7 and 0.7. In some embodiments, (A) is a number between -0.6 and 0.6. In some embodiments, (A) is a number between -0.5 and 0.5. In some embodiments, (A) is a number between -0.4 and 0.4. In some embodiments, (A) is a number between -0.3 and 0.3. In some embodiments, (A) is a number between -0.2 and 0.2. In some embodiments, (A) is a number between -0.1 and 0.1.

[0083] 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.

[0084] In some embodiments, (A) is -0.95. In some embodiments, (A) is 0.02. In some embodiments, the cell type is DG44 and (A) is -0.95. In some embodiments, the cell type is CHOZN and (A) is 0.02.

[0085] In some embodiments, (B) is a number between -1.5 and 2.5. In some embodiments, (B) is a number between -1.0 and 2.0. In some embodiments, (B) is a number between -0.5 and 1.5. In some embodiments, (B) is a number between 0 and 1.0.

[0086] In some embodiments, (B) is a number between -0.5 and -0.4. In some embodiments, (B) is a number between -0.4 and -0.3. In some embodiments, (B) is a number between -0.3 and -0.2. In some embodiments, (B) is a number between -0.2 and -0.1. In some embodiments, (B) is a number between -0.1 and 0.0. In some embodiments, (B) is a number between 0.0 and 0.1. In some embodiments, (B) is a number between 0.1 and 0.2. In some embodiments, (B) is a number between 0.2 and 0.3. In some embodiments, (B) is a number between 0.3 and 0.4. In some embodiments, (B) is a number between 0.4 and 0.5. In some embodiments, (B) is a number between 0.5 and 0.6. In some embodiments, (B) is a number between 0.6 and 0.7. In some embodiments, (B) is a number between 0.7 and 0.8. In some embodiments, (B) is a number between 0.8 and 0.9. In some embodiments, (B) is a number between 0.9 and 1.0. In some embodiments, (B) is a number between 1.0 and 1.1. In some embodiments, (B) is a number between 1.1 and 1.2. In some embodiments, (B) is a number between 1.2 and 1.3. In some embodiments, (B) is a number between 1.3 and 1.4. In some embodiments, (B) is a number between 1.4 and 1.5.

[0087] 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.

[0088] In some embodiments, (B) is 0.86. In some embodiments, (B) is 0.13. In some embodiments, the cell type is DG44 and (B) is 0.86. In some embodiments, the cell type is CHOZN and (B) is 0.13.

[0089] In some embodiments, (C) is a number between 0 and 4.0. In some embodiments, (C) is a number between 0.5 and 3.5. In some embodiments, (C) is a number between 1.0 and 3.0. In some embodiments, (C) is a number between 1.5 and 2.5.

[0090] In some embodiments, (C) is a number between 0.0 and 0.1. In some embodiments, (C) is a number between 0.1 and 0.2. In some embodiments, (C) is a number between 0.2 and 0.3. In some embodiments, (C) is a number between 0.3 and 0.4. In some embodiments, (C) is a number between 0.4 and 0.5. In some embodiments, (C) is a number between 0.5 and 0.6. In some embodiments, (C) is a number between 0.6 and 0.7. In some embodiments, (C) is a number between 0.7 and 0.8. In some embodiments, (C) is a number between 0.8 and 0.9. In some embodiments, (C) is a number between 0.9 and 1.0. In some embodiments, (C) is a number between 1.0 and 1.1. In some embodiments, (C) is a number between 1.1 and 1.2. In some embodiments, (C) is a number between 1.2 and 1.3. In some embodiments, (C) is a number between 1.3 and 1.4. In some embodiments, (C) is a number between 1.4 and 1.5. In some embodiments, (C) is a number between 1.5 and 1.6. In some embodiments, (C) is a number between 1.6 and 1.7. In some embodiments, (C) is a number between 1.7 and 1.8. In some embodiments, (C) is a number between 1.8 and 1.9. In some embodiments, (C) is a number between 1.9 and 2.0. In some embodiments, (C) is a number between 2.0 and 2.1. In some embodiments, (C) is a number between 2.1 and 2.2. In some embodiments, (C) is a number between 2.2 and 2.3. In some embodiments, (C) is a number between 2.3 and 2.4. In some embodiments, (C) is a number between 2.4 and 2.5. In some embodiments, (C) is a number between 2.5 and 2.6. In some embodiments, (C) is a number between 2.6 and 2.7. In some embodiments, (C) is a number between 2.7 and 2.8. In some embodiments, (C) is a number between 2.8 and 2.9. In some embodiments, (C) is a number between 2.9 and 3.0. In some embodiments, (C) is a number between 3.0 and 3.1.In some embodiments, (C) is a number between 3.1 and 3.2. In some embodiments, (C) is a number between 3.2 and 3.3. In some embodiments, (C) is a number between 3.3 and 3.4. In some embodiments, (C) is a number between 3.4 and 3.5. In some embodiments, (C) is a number between 3.5 and 3.6. In some embodiments, (C) is a number between 3.6 and 3.7. In some embodiments, (C) is a number between 3.7 and 3.8. In some embodiments, (C) is a number between 3.8 and 3.9. In some embodiments, (C) is a number between 3.9 and 4.0.

[0091] In some embodiments, (C) is 1.21. In some embodiments, (C) is 2.41. In some embodiments, the cell type is DG44 and (C) is 1.21. In some embodiments, the cell type is CHOZN and (C) is 2.41.

[0092] 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, (a) is-0.95, (B) is 0.86, and (C) is 1.21. In one embodiment, the cell type is CHOZN, (a) is 0.02, (B) is 0.13, and (C) is 2.41.

[0093] In some embodiments, the present specification discloses a method for increasing, controlling, or regulating protein yield in a sample mixture containing a protein of interest and impurities, comprising: (a) flushing a collection skid with water; (b) loading a sample onto the collection skid; (c) measuring an ultraviolet (UV) signal of the sample mixture during protein filtration on the collection skid to obtain a real-time protein titer; (d) starting protein collection based on the UV reading and the real-time protein titer; (e) chasing the protein with PBS; and (f) stopping protein collection based on the UV reading and the real-time protein titer, wherein the UV signal during filtration correlates with the real-time protein titer.

[0094] In some embodiments, the methods described herein comprise a water (e.g., RODI) flush. In some embodiments, the methods comprise loading a protein sample and initiating collection based on online titer. In some embodiments, the methods comprise a PBS chase and terminating collection based on online titer. In comparison to other methods, the methods disclosed herein do not include an air blowdown step.

[0095] In some embodiments, the start and end of sample collection is automatically controlled based on online UV measurements and calculated titer. In certain embodiments, the real-time protein of interest concentration during the collection process is calculated by online UV sensor readings using modeling. In some embodiments, the bulk collection cutoff is determined based directly on the calculated online protein of interest concentration. In some embodiments, the calculation algorithm is integrated into the Delta V™ Control System to achieve the automatic protein collection cutoff.

[0096] In some embodiments, the methods disclosed herein include a step of modeling. In some embodiments, the modeling includes offline titration of the UV signal versus online titration using serially diluted samples to establish a linear correlation between UV signal and titration. In some embodiments, the sample used for modeling is a purified protein. In some embodiments, the sample used for modeling is a bulk protein including impurities. In some embodiments, modeling is then used to control, regulate, increase and / or improve the yield of the protein.

[0097] In some embodiments, the methods disclosed herein include controlling, regulating or improving the yield of a protein of interest having 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 potency is at least about 0.5 g / L. In some embodiments, the potency is at least about 0.6 g / L. In some embodiments, the potency is at least about 0.7 g / L. In some embodiments, the potency is at least about 0.8 g / L. In some embodiments, the potency is at least about 0.9 g / L. In some embodiments, the potency is at least about 1 g / L. In some embodiments, the potency is at least about 1.5 g / L. In some embodiments, the potency is at least about 2 g / L. In some embodiments, the potency is at least about 2.5 g / L. In some embodiments, the potency is at least about 3 g / L. In some embodiments, the potency is at least about 3.5 g / L. In some embodiments, the potency is at least about 4 g / L. In some embodiments, the potency is at least about 4.5 g / L. In some embodiments, the potency is at least about 5 g / L. In some embodiments, the potency is at least about 5.5 g / L. In some embodiments, the potency is at least about 6 g / L. In some embodiments, the potency is at least about 6.5 g / L. In some embodiments, the potency is at least about 7 g / L.In some embodiments, the potency is at least about 7.5 g / L. In some embodiments, the potency is at least about 8 g / L. In some embodiments, the potency is at least about 8.5 g / L. In some embodiments, the potency is at least about 9 g / L. In some embodiments, the potency is at least about 9.5 g / L. In some embodiments, the potency is at least about 10 g / L. In some embodiments, the potency is at least about 10.5 g / L. In some embodiments, the potency is at least about 11 g / L. In some embodiments, the potency is at least about 11.5 g / L. In some embodiments, the potency is at least about 12 g / L. In some embodiments, the potency is at least about 12.5 g / L. In some embodiments, the potency is at least about 13 g / L. In some embodiments, the potency is at least about 13.5 g / L. In some embodiments, the potency is at least about 14 g / L. In some embodiments, the potency is at least about 14.5 g / L. In some embodiments, the potency is at least about 15 g / L. In some embodiments, the potency is at least about 15.5 g / L. In some embodiments, the potency is at least about 16 g / L. In some embodiments, the potency is at least about 16.5 g / L. In some embodiments, the potency is at least about 17 g / L. In some embodiments, the potency is at least about 17.5 g / L. In some embodiments, the potency is at least about 18 g / L, at least about 18.5 g / L. In some embodiments, the potency is at least about 19 g / L. In some embodiments, the potency is at least about 19.5 g / L. In some embodiments, the potency is at least about 20 g / L.

[0098] In some embodiments, the methods disclosed herein include recovery of the protein of interest, which is dependent on the titer of the protein of interest. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.05 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.06 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.07 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.08 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.09 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.1 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.2 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 0.3 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.4 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.6 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.7 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.8 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 0.9 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 1 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 1.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 2 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 2.5 g / L.In some embodiments, collection of the protein of interest begins when the titer is at least about 3 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 3.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 4 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 4.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 5.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 6 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 6.5 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 7 g / L. In some embodiments, collection of the protein of interest begins when the titer is at least about 7.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 8 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 8.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 9 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 9.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 10 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 10.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 11 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 11.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 12 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 12.5 g / L.In some embodiments, recovery of the protein of interest begins when the titer is at least about 13 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 13.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 14 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 14.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 15 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 15.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 16 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 16.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 17 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 17.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 18 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 18.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 19 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 19.5 g / L. In some embodiments, recovery of the protein of interest begins when the titer is at least about 20 g / L.

[0099] In some embodiments, the methods disclosed herein include recovery of a protein of interest, the protein of interest having a range of titers. In some embodiments, the titer at which the protein of interest is recovered is between about 0.05 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.1 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.2 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.3 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.4 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.5 g / L and about 20 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.6 g / L and about 20 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.7 g / L and about 20 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.8 g / L and about 20 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.9 g / L and about 20 g / L. In some embodiments, the titer at which the target protein is recovered is between about 1 g / L and about 20 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.05 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.1 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.2 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.3 g / L and about 15 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.4 g / L and about 15 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.5 g / L and about 15 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.6 g / L and about 15 g / L. In some embodiments, the titer at which the protein of interest is recovered is between about 0.7 g / L and about 15 g / L.In some embodiments, the titer at which the target protein is recovered is between about 0.8 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.9 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 1 g / L and about 15 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.05 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.1 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.2 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.3 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.4 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.5 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.6 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.7 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.8 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 0.9 g / L and about 10 g / L. In some embodiments, the titer at which the target protein is recovered is between about 1 g / L and about 10 g / L.

[0100] In some embodiments, the methods disclosed herein further comprise stopping recovery of the protein of interest when the recovery titer is less than about 0.5 g / L.

[0101] In some embodiments, the yield of the protein of interest is increased by the methods disclosed herein. In some embodiments, the yield of the protein of interest is increased by at least about 1% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 2% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 3% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 4% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 5% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 6% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 7% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 8% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 9% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 10% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time.In some embodiments, the yield of the protein of interest is increased by at least about 11% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 12% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 13% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 14% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 15% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 16% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 17% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 18% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 19% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the protein of interest is increased by at least about 20% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time.

[0102] In some embodiments, the ultraviolet (UV) signal of the sample mixture is measured at 0 to 2 AU. In some embodiments, the ultraviolet (UV) signal of the sample mixture is measured at 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.

[0103] In some embodiments disclosed herein, the method comprises protein filtration. In some embodiments, the method comprises one or more filters. In some embodiments, the protein filtration is depth filtration. In some embodiments, the depth filtration comprises a primary depth filter and a secondary depth filter. In some embodiments, the depth filtration comprises a primary depth filter.

[0104] In some embodiments, the method includes loading the sample mixture prior to monitoring.

[0105] In some embodiments, the method includes flushing the depth filter with a buffer before loading the cell culture and chasing the depth filter after loading the cell culture. In some embodiments, the method includes chasing the sample mixture with phosphate buffered saline (PBS). In some embodiments, the method includes a harvest skid including a control system, the control system automatically starts harvesting the protein when the titer is greater than 0.5 g / L. In some embodiments, the method includes a control system, the control system automatically stops harvesting the protein when the titer is less than 0.5 g / L.

[0106] In some embodiments, the method includes a control system that adjusts the flow rate of liquid through the reclaim skid. In some embodiments, the method includes a control system that automatically drives a pump to up-regulate the flow rate through the reclaim skid. In some embodiments, the method includes a control system that automatically drives a pump to down-regulate the flow rate through the reclaim skid. In some embodiments, the method does not include an air blowdown step.

[0107] In some embodiments, the method includes recovering a non-volume yield of the protein.

[0108] In some embodiments, the methods disclosed herein include measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0109] In some embodiments, the method includes measuring the pressure using a pressure sensor. In some embodiments, the pressure is measured in the range of -10 pounds per square inch (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, the pressure may be measured at least 1, 2, 3, 4, or 5 times, for example, before the primary filter, after the primary filter and before the secondary filter, after the secondary filter, after draining, or any combination thereof.

[0110] In some embodiments, the method includes measuring turbidity. In some embodiments, turbidity is measured in the range of 0 absorbance units (AU) to 2 AU. In other embodiments, turbidity is measured at 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, turbidity can be measured at least once, twice, three times, four times, or five times during the filtration process, for example, after the primary filter, after the secondary filter, or after the primary filter and after the secondary filter. See FIG. 2.

[0111] In some embodiments, the method includes measuring the temperature. In some embodiments, the temperature is measured in the range of 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 may be measured at least once, twice, three times, four times, or five times during the filtration process, for example, after the primary filter, after the secondary filter, or after the primary filter and after the secondary filter. See FIG. 2.

[0112] In some embodiments, the method includes measuring a flow rate. In some embodiments, the flow rate is between 0 L / min and 20 L / min, 0 L / min and 30 L / min, 0 L / min and 40 L / min, 0 L / min and 50 L / min, 0 L / min and 60 L / min, 0 L / min and 70 L / min, 0 L / min and 80 L / min, 0 L / min and 90 L / min, 0 L / min and 100 L / min, 0 L / min and 110 L / min, 0 L / min and 12 L / min. The flow rate is measured in the range of 0 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.

[0113] In some embodiments, liquid from the water source / bioreactor / PBS source is pumped to the primary depth filter by a LEVITRONIX® gravity pump.

[0114] In some embodiments, a system such as Delta V™ can be used to calculate the volume of the flow indicator (flow totalizer) from the online flow sensor readings. In some embodiments, the volume of the flow totalizer is used to determine when the water flushing ends. In some embodiments, four pressure sensors are placed before the primary depth filter, the secondary depth filter, the prefilter, and the sterilizing filter. The pressure flow control loop works based on the real-time pressure value before the primary depth filter. When the pressure value exceeds a certain threshold, Delta V™ automatically drives the pump to downregulate the flow rate. In some embodiments, two turbidity sensors are placed after the primary and secondary depth filters as an indicator of filtration quality. In some embodiments, one UV sensor is placed after the secondary depth filter, the value of which is used to calculate the online target protein concentration and control the cutoff of the clarified bulk recovery. The weights of the upstream source and downstream receiving vessels in real time are monitored and displayed on the Delta V™. In some embodiments, the weight is monitored from 0 to 550 kg, with a measurement accuracy of 0.01 kg.

[0115] In some embodiments, the protein is isolated from a source. In some embodiments, the sample mixture is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof. In some embodiments, the source is selected from cultured cells.

[0116] In some embodiments, the cell is a prokaryote. In bacterial systems, some expression vectors can be advantageously selected depending on the use of the protein molecule to be expressed. For example, when producing large amounts of such proteins to generate pharmaceutical compositions of protein molecules, vectors that direct the expression of high levels of protein products that are easily purified are desirable.

[0117] In other embodiments, the cell is a eukaryotic organism. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), and any combination thereof. In one embodiment, the cell is a Chinese hamster ovary cell. In some embodiments, the cell is an insect cell (e.g., a Spodoptera frugiperda cell).

[0118] In other embodiments, the cell is a mammalian cell.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, NS0, 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.

[0119] In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the CHO cell is CHO-DG44, CHOZN, CHO / dhfr-, CHOK1SV GS-KO, or CHO-S. In some embodiments, the CHO cell is CHO-DG4. In some embodiments, the CHO cell is CHOZN.

[0120] Other suitable CHO cell lines disclosed herein include CHO-K, 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.

[0121] In some embodiments, the protein of interest is cultured at a cell density of about 1×106 In some embodiments, the protein of interest is recovered from a cell density of about 5×10 6 In some embodiments, the protein of interest is harvested from a cell density of about 1×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 1.5×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 2×10 7 In some embodiments, the protein of interest is recovered from a cell density of about 2.5×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 3×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 3.5×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 4×10 7 In some embodiments, the protein of interest is recovered from a cell density of about 4.5×10 cells / mL or more of culture medium. 7 In some embodiments, the protein of interest is recovered from a cell density of about 5×10 7 cells / mL or more are recovered from the medium.

[0122] In some embodiments, the protein source is bulk protein. In some embodiments, the protein source is a composition that includes protein components and non-protein components. The non-protein components may include DNA and other contaminants.

[0123] In some embodiments, the source of the protein is from an animal. In some embodiments, the animal is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). In some embodiments, the source is tissue or cells from a human. In certain 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 livestock. In certain embodiments, such terms refer to humans.

[0124] In some embodiments, the protein purified by the methods described herein is a fusion protein. A "fusion" or "fusion protein" comprises a first amino acid sequence linked in frame to a second amino acid sequence to which it is not naturally linked. Amino acid sequences that are normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide. Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship. Fusion proteins can further comprise a second amino acid sequence related to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond. A single protein is created by transcription / translation. In this way, multiple proteins or fragments thereof can be incorporated into a single polypeptide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operably linked link between two polypeptides is a fusion of both polypeptides together in frame to generate a single polypeptide fusion protein. In certain aspects, the fusion protein further comprises a third polypeptide, which can include a linker sequence, as described in more detail below.

[0125] In some embodiments, the protein purified by the methods described herein is an antibody. Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy 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, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab') 2 Antibodies include, but are not limited to, antibodies, fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, antibodies as described herein refer to polyclonal antibody populations. Antibodies include any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.) of immunoglobulin molecule, any class (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), ... 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 or IgA 2 etc.) or any subclass (e.g., IgG 2a or IgG 2b In certain embodiments, the antibody is a humanized monoclonal antibody. In another particular embodiment, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In certain embodiments, the antibody described herein is an IgG 1 or IgG 4 It is an antibody.

[0126] In some embodiments, the proteins described herein are referred to as "antigen-binding domains," "antigen-binding regions," "antigen-binding fragments," or similar terms, which refer to a portion of an antibody molecule that contains the amino acid residues that confer specificity to the antibody molecule for its antigen (e.g., complementarity determining regions (CDRs)). The antigen-binding region can be from any animal species, including rodents (e.g., mice, rats, or hamsters) or humans.

[0127] 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 belatacept NGP.

[0128] In some embodiments, the protein is an anti-GITR (glucocorticoid-induced tumor necrosis factor receptor family related gene) antibody. In some embodiments, the anti-GITR antibody is an antibody having the CDR sequences of 6C8, e.g., a humanized antibody having the CDRs of 6C8 (e.g., as described in WO2006 / 105021); an antibody comprising the CDRs of an anti-GITR antibody described, e.g., in WO2011 / 028683; an antibody comprising the CDRs of an anti-GITR antibody described in JP2008278814, an antibody comprising the CDRs of an anti-GITR antibody described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, or WO2018 / 013818, or any other anti-GITR antibody described or referenced herein, all of which are incorporated herein in their entirety.

[0129] In other embodiments, the protein is an anti-LAG3 antibody. Lymphocyte activation gene 3, also known as LAG-3, is a protein that in humans is encoded by the LAG3 gene. Discovered in 1990, LAG3 is a cell surface molecule that exerts diverse biological effects on T cell function. It is an immune checkpoint receptor and is the target of various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune diseases. In soluble form, it is also being developed as an anti-cancer drug in its own right. Examples of anti-LAG3 antibodies include, but are not limited to, the antibodies described in WO 2017 / 087901 A2, WO 2016 / 028672 A1, WO 2017 / 106129 A1, WO 2017 / 198741 A1, US 2017 / 0097333 A1, US 2017 / 0290914 A1, and US2017 / 0267759 A1, all of which are incorporated herein in their entirety.

[0130] In some embodiments, the protein is an anti-CXCR4 antibody. CXCR4 is a G1-associated seven-transmembrane protein. CXCR4 is widely expressed on cells of hematopoietic origin and is the major CD4+ coreceptor for human immunodeficiency virus 1 (HIV-1). 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, the antibodies of WO 2009 / 140124 A1, US 2014 / 0286936 A1, WO 2010 / 125162 A1, WO 2012 / 047339 A2, WO 2013 / 013025 A2, WO 2015 / 069874 A1, WO 2008 / 142303 A2, WO 2011 / 121040 A1, WO 2011 / 154580 A1, WO 2013 / 071068 A2, and WO 2012 / 175576 ​​A1, all of which are incorporated herein in their entirety.

[0131] In some embodiments, the protein is an anti-CD73 (ecto-5'-nucleotidase) antibody. Anti-CD73 antibodies inhibit the formation of adenosine. The breakdown of AMP to adenosine leads to the generation of an immunosuppressed pro-angiogenic niche within the tumor microenvironment that promotes the development and progression of cancer. Examples of anti-CD73 antibodies include, but are not limited to, those described in WO 2017 / 100670 A1, WO 2018 / 013611 A1, WO 2017 / 152085 A1, and WO 2016 / 075176 A1, all of which are incorporated herein in their entirety.

[0132] In some embodiments, the protein is an anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) antibody. TIGIT is a member of the PVR (poliovirus receptor) family of immunoglobulin proteins. TIGIT is expressed on several classes of T cells, including follicular B helper T cells (TFH). The protein has been shown to bind with high affinity to PVR, which is believed to aid in 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, the antibodies described 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 in their entirety.

[0133] In some embodiments, the protein is an anti-OX40 (i.e., CD134) antibody. OX40 is a cytokine of the tumor necrosis factor (TNF) ligand family. OX40 functions in T cell antigen-presenting cell (APC) interaction and mediates the adhesion of activated T cells to endothelial cells. Examples of anti-OX40 antibodies include those described in WO 2018 / 031490 A2, WO 2015 / 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, and WO 2015 / 153514 A1, WO 2016 / 002820 A1, and WO 2016 / 200835 A1, all of which are incorporated herein in their entireties.

[0134] In some embodiments, the protein is an anti-IL8 antibody. IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T cells, but not monocytes. It is also involved in neutrophil activation. IL-8 is released by several cell types in response to inflammatory stimuli.

[0135] In some embodiments, the protein is abatacept (sold as ORENCIA®). Abatacept (also abbreviated herein as Aba) 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 composed of the Fc region of immunoglobulin IgG1 fused to the extracellular domain of CTLA-4. For T cells to be activated and generate an immune response, antigen-presenting cells must present two signals to the T cells. One of these signals is the major histocompatibility complex (MHC) bound to the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7-2).

[0136] In some embodiments, the protein is belatacept (brand name NULOJIX®). Belatacept is a fusion protein composed of the Fc fragment of human IgG1 immunoglobulin bound to the extracellular domain of CTLA-4, a molecule important in regulating T cell costimulation, and selectively blocks the T cell activation process. It is intended to prolong graft survival while limiting the toxicity caused by standard immunosuppressive therapies such as calcineurin inhibitors. It differs from abatacept (ORENCIA®) ​​by only two amino acids.

[0137] c. System In some embodiments, the present specification discloses a system for controlling, regulating, increasing or improving protein yield in a sample mixture containing a protein of interest and impurities, comprising monitoring the ultraviolet (UV) signal of the sample mixture in real time during protein filtration on a recovery skid.

[0138] The systems disclosed herein comprise one or more sensors, hi some embodiments, the sensors comprise a pressure sensor, a UV sensor, a turbidity sensor, a temperature sensor, a flow sensor, and any combination thereof.

[0139] In some embodiments, the recovery skid is designed to integrate all sensors, including pressure sensor (4), UV sensor (1), turbidity sensor (2), temperature sensor (2), and flow sensor (1), into one cart. In some embodiments, the system comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controllers are built-on onto the cart, accommodating a total of 10 different sensors. In some embodiments, a gravity pump (such as a LEVITRONIX® gravity pump) is used to pump liquid to the depth filter and is attached to the skid cart. In some embodiments, the system is movable, lockable, and / or shuttable.

[0140] Also provided herein are systems (e.g., devices, e.g., recovery skids) that can be used in the above methods. In one embodiment, the system or device comprises the embodiment of Figure 1a and / or Figure 1b. In one embodiment, the system or device comprises the embodiment of Figure 2.

[0141] In some embodiments, the present specification discloses an apparatus for controlling, regulating, increasing or improving the yield of a protein in a sample mixture containing a protein of interest and impurities. The apparatus comprises one or more sensors. The sensor comprises a pressure sensor, a UV sensor, a turbidity sensor, a temperature sensor, a flow sensor, and any combination thereof.

[0142] In some embodiments, the device is designed to integrate all sensors into the device, including pressure sensor (4), UV sensor (1), turbidity sensor (2), temperature sensor (2), and flow sensor (1). In some embodiments, the device comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controller is built-in on the cart, accommodating a total of 10 different sensors. In some embodiments, a gravity pump (e.g., a LEVITRONIX® gravity pump, etc.) is used to pump liquid to the depth filter and is attached to the device. In some embodiments, the system is movable, lockable, and / or electrically shut off. The device may also comprise a processor configured to control the recovery of the protein of interest. Further, the processor may be configured to change the state of the device, e.g., temperature, pressure, turbidity, or flow rate. The processor may also be configured to control the recovery of the protein of interest. In some embodiments, the processor may use the established model to determine the harvest process of the culture. The cell culture harvest process may include a filtration-based cell culture harvest process. The processor can be configured to use the titer of the protein of interest. The device can be incorporated into a system for controlling, regulating, increasing or improving the yield of protein in a sample mixture containing the protein of interest and impurities.

[0143] d. Process In one embodiment, the system or device comprises the embodiment of FIG. 2 showing the process flow using a recovery skid. Liquid from the water source / bioreactor / PBS source was pumped to the depth filter by a LEVITRONIX® gravity pump. A LEVITRONIX® flow sensor was placed after the pump. Delta V™ calculated the flow totalizer volume from the online flow sensor readings. The flow totalizer volume was used to determine the end of water flushing. Four pressure sensors were placed before the primary depth filter, the secondary depth filter, the prefilter, and the sterilizing filter. The pressure flow control loop worked based on the real-time pressure value before the primary depth filter. Two turbidity sensors were placed after the primary and secondary depth filters as an indicator of filtrate quality. One UV sensor was placed after the secondary depth filter and was used to calculate the online protein of interest concentration and control the cutoff of the clarified bulk collection. Real-time upstream source and downstream receiving vessel weights were monitored and displayed on the Delta V™.

[0144] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0145] Example 1: Recovery skid design In some embodiments, a harvest skid was utilized to control, regulate, increase or improve the yield of protein in a sample. A schematic diagram of the harvest skid is shown in FIG. 1. The harvest skid was designed to integrate all sensors, including pressure sensor (4), UV sensor (1), turbidity sensor (2), temperature sensor (2), and flow sensor (1), on one cart. See FIG. 2. Three PMAT controllers were built on the cart to accommodate a total of 10 different sensors. A LEVITRONIX® gravity pump, used to pump liquid to the depth filter, was also mounted on the skid cart. The harvest skid was designed to be movable, lockable, and electronically shut off. [Table 1] [Table 2]

[0146] The sensors used for harvesting have separate functions: the pressure sensor monitors the pressure during the process and continuously controls the inflow to the pump, lowering the flow rate of the pump inflow if the pressure is too high; the UV sensor monitors the UV signal after depth filtration during the process and converts it into protein concentration to control the start and end of bulk harvesting. UV is measured at 280 nm.

[0147] Weight sensors monitor the weight of the upstream bioreactor and downstream receiver during the process and control the load and chase steps. The load cell values ​​of the bioreactor and receiver are integrated into the recovery skid control system. A turbidity sensor measuring turbidity at 880 nm monitors the turbidity before and after depth filtration during the process. If the depth filter is fouled, a spike in turbidity is observed. A temperature sensor monitors the temperature during the process. The recovery process here operates at ambient (room) temperature.

[0148] A recovery skid process was used to purify the protein of interest from cell culture. The liquid from the water source / bioreactor / PBS source was pumped to the primary depth filter by a LEVITRONIX® gravity pump. The LEVITRONIX® gravity pump proved to cause less cell death in CHO cell culture than the peristaltic pump P3P. A LEVITRONIX® flow sensor was placed after the pump. Delta V™ calculated the flow totalizer volume from the online flow sensor readings. The flow totalizer volume was used to determine the end time of water flushing. Four pressure sensors were placed before the primary depth filter, the secondary depth filter, the prefilter, and the sterilizing filter P4P. The pressure flow control loop worked based on the real-time pressure values ​​before the primary depth filter. When the pressure value exceeded a certain threshold, Delta V™ would automatically drive the pump to downregulate the pump speed. Two turbidity sensors were placed after the primary and secondary depth filters as an indicator of filtrate quality. One UV sensor was placed after the secondary depth filter and its value was used to calculate the on-line target protein concentration and control the clarified bulk recovery cut-off. Real-time upstream source and downstream receiving vessel weights were monitored and displayed on the Delta V™.

[0149] For each embodiment disclosed herein, the recovery skid uses each sensor to detect values ​​within the following ranges and accuracies: [Table 3]

[0150] Example 2. Conversion of UV signal to protein concentration Compared with the conventional method, the method disclosed herein eliminated the air blow-down step. Meanwhile, the start and end of clarified bulk harvest were automatically controlled based on the online UV measurements and the calculated titer. See Figure 3. More specifically, the real-time target protein concentration during the harvest process was calculated by the online UV sensor readings using the model developed. Thus, the bulk harvest cut-off was determined directly based on the calculated online target protein concentration. To realize the automated cut-off of clarified bulk harvest, the calculation algorithm was integrated into the Delta V™ control system.

[0151] The output absorbance of the online UV sensor used in this recovery skid was 0-2 AU. The path length of this UV sensor was adjusted to correspond to concentrations of 0-6 g / L of total target protein in the range of 0-2 AU. Other UV sensors or Flow VPE (C-technologies) can be used for higher concentration measurements.

[0152] To convert the online UV signal into the concentration of the target protein in the process, a series of steps were performed as shown in Figure 4 .

[0153] In the first step, we measured the offline titer against the online UV signal using serially diluted samples of D12 GITR cell culture. Several components in the cell culture sample, including the protein of interest, HCP (host cell proteins) and medium dyes, can contribute to the UV absorbance signal. To mimic the actual recovery process (where the UV sensor measures the total absorbance from all these components), we serially diluted the cell culture sample (D12 GITR cell culture) instead of pure protein and used it to adjust the path length of the UV sensor.

[0154] As shown in Figure 5, the path length of the UV sensor was adjusted to cover the wide range of target protein concentrations that may be observed during the recovery process. Because UV readings close to 2 AU (maximum output) have low accuracy, the path length was adjusted to achieve a UV reading of approximately 1.6 at a titer of 5 g / L. 2 Good linearity was observed, with a β-adjusted β-adjustment coefficient of 0.97. Thus, serial dilutions of cell culture samples provide a strong correlation between UV readings and titers.

[0155] Example 3. Small scale studies using pure protein and clarified bulk A small scale test was carried out using 2 L of pure protein (eTau) at a titer of 5.2 g / L. The depth filter was 60 L / m 2 The titer readings were scaled based on the loading amount (per primary filter). Offline samples after the secondary depth filter were collected during the harvest process. To understand the relationship between pure protein concentration and online UV signal during the harvest process, the offline titer readings were plotted with the online UV sensor values.

[0156] Further small scale recovery studies were carried out using 2 L of clarified bulk (depleted eTau cell culture) at a titer of 5 g / L. Depth filters were run at 60 L / m 2 The titer readings were scaled based on the loading amount (per primary filter). Offline samples after the secondary depth filter were collected during the harvest process. To understand the relationship between the concentration of the protein of interest (in the mixture of culture components) during the harvest process and the online UV signal, the offline titer readings were plotted with the online UV sensor values.

[0157] The online UV and offline titer values ​​during the tested harvest processes are plotted in Figure 6a and Figure 6b. The "incline" data series was collected from the start of bulk harvest to the end of loading, whereas the "decline" data series was collected from the start of the chase to the end of bulk harvest. As shown in Figure 6a and Figure 6b, good linearity was observed for both the incline and decline parts of the data in both processes. Thus, serial dilutions of cell culture samples result in a strong correlation between UV readings and titers when measuring other samples (e.g., pure protein in Figure 6a and clarified bulk protein in Figure 6b).

[0158] However, the slopes of the incline and decline parts are different, suggesting that separate models may be needed for different steps in the recovery process.

[0159] Example 4. Establishment of a model using three large-scale cell culture processes Three different cell lines were used to establish the model. These cell lines were constructed from different large-scale cell culture processes (Aba NGP, GITR, and Next Gen CXCR4) with different characteristics (cell density, viability, titer, background noise, etc.). The harvest skid was used to harvest the cell lines to generate data for model establishment. The depth filter scale was 60-65 L / m. 2 The titer loading was determined based on the amount of 10 ...

[0160] During loading of cell culture material, cells, target proteins, and background noise proteins all occupied space on the depth filter membrane. When the chase with PBS was started, the target proteins were washed away. As the PBS chase proceeded, background noise proteins such as HCPs that were loosely bound to the depth filter membrane began to be washed away together with the target proteins. At the same time, the release of HCPs from cell debris also contributed to the increase in the percentage of background noise P5P. This may be the reason for the difference in UV profiles between the clarified bulk samples and the cell culture samples. In other words, for complex cell-containing material, as the PBS chase proceeded, the target proteins had less and less impact on the total UV signal, along with the increase in background noise.

[0161] Based on these results, we established two separate models to predict the concentration of the target protein using the online UV signal: a linear model fitted to the data in the incline part (from the start of collection to the end of loading) and a nonlinear model fitted to the data in the decline part (from the start of chase to the end of collection).

[0162] A.Incline part model fit. For the incline portion of the data, a total of 22 samples were included in the model. In Figure 8, the offline titer values ​​are plotted against the online UV values. A linear fit was applied to the data. R 2 The R value was 0.98. Using this model, the predicted titers were calculated and compared to the actual titers. As shown in Figures 8a and 8b, the slope of the fit was very close to 1, with an R 2 was 0.98.

[0163] A linear model was created to predict the concentration of the target protein from the start of harvest to the end of the load. Model predicted titer = a + b * (online UV signal). The model constants a and b depend on the titer level. For titers below approximately 3.5 g / L, a = -0.35, b = 2.88. For titers above approximately 3.5 g / L, a = -0.69, b = 4.06.

[0164] B. Model fit of decline part For the decline portion of the data, a total of 41 samples were included in the model. In Figure 10, the offline titer values ​​are plotted against the online UV values. A nonlinear fit was applied to the data. The RMSE values ​​of the nonlinear fit were 0.26 and 0.04 for the CHOZN and DG44 cell lines, respectively. Using this model, the predicted titers were calculated and compared to the actual titers (Figures 9a and 9b). The slope of the fit was close to 1, with an R 2 were 0.97 and 0.99, respectively, Figures 9a and 9b.

[0165] A non-linear model was created to predict the concentration of the protein of interest from the start of the chase to the end of collection. Model predicted titer = A + B*exp(C*online UV signal). The model constants A, B, and C depend on the titer level. For titers below approximately 3.5 g / L, A=-0.95, B=0.86, C=1.21. For titers above approximately 3.5 g / L, A=0.02, B=0.13, C=2.41.

[0166] Example 5. Test model using four large-scale cell culture processes Four large-scale (500L) cell culture processes (CD73, OX40, TIGIT, IL8) were harvested. Depth filters were scaled up based on small-scale preliminary data. Offline samples after the secondary depth filter were collected during the harvest process for actual titer measurements. Online UV sensor values ​​were entered into JMP software. A model was used to calculate predicted titer values ​​based on the online UV sensor values ​​and compared to the offline titer measurements. [Table 4] [Table 5]

[0167] The model developed above was tested using four different large-scale (500 L) cell culture recovery processes (starting titers 0-0.1 g / L, ending titers 0.1-0.2 g / L). The model can be tested down to 0.01 g / L with a UV signal of 0.01 Au. The model predicted titers were compared with the actual titers using JMP software. The model fit RMSE values ​​for each process are shown in Figure 10.

[0168] The difference between the model predicted and actual titer values ​​was calculated and is shown in Figure 12 for each process tested. The overall average difference ranged from 0.07 to 0.36 g / L, suggesting that the model can be reliably applied to different processes with various characteristics as shown in Table 2. A. Use of on-line sensors to control the recovery process and improve recovery rates [Table 6]

[0169] The yield of the recovery process was calculated using the following formula:

number

[0170] As shown in Table 6, the yields using the new recovery skid were 2-5% higher than those using the conventional method.

[0171] In this study, real-time monitoring and control of a recovery skid was designed to investigate depth filtration recovery of several therapeutic proteins.

[0172] To achieve automated monitoring and control of key process parameters, online sensors were installed in the harvest skid and their real-time readings were integrated into the Delta V™ system. A model converting the online UV signal into real-time target protein concentration at different stages of the harvest process was developed in a series of experimental steps (adjusting the path length of the UV sensor, testing pure proteins, testing complex cell culture samples). The model was then successfully tested in several large-scale harvest processes with a wide range of process characteristics, including background noise levels, product levels, total cell density, and viability.

[0173] The novel harvest skid and statistical model developed in this study quantitatively monitored and controlled the cell culture clarification process, significantly improving harvest reliability and protein yield. The online titer information itself is an important indicator of cell culture performance and can be used to rapidly determine loading levels for Protein A chromatography in downstream processing.

[0174] Example 6. New protein real-time monitoring process during protein recovery A new target protein is selected to be clarified using this harvest skid. First, all sensors on the harvest skid are connected in-line as shown in Figure 2 to monitor pressure, flow rate, UV, turbidity and temperature during the harvest process. Second, the water source is connected to the LEVITRONIX® gravity pump. To control the flushing step of the depth filter, the total flow rate and flow rate are input into the Delta V™. After the total flow rate is reached, the bioreactor source is connected to the LEVITRONIX® gravity pump and the loading of the cell culture into the depth filter begins. Third, the UV prediction model constants for the incline portion are input into the Delta V™; the harvest start cutoff threshold is input into the Delta V™. The online UV signal is converted to the concentration of the target protein during loading. Once the threshold is reached, the receiving vessel is connected to the sterile filter and the clarified bulk is harvested. Fourth, after the bioreactor is emptied, the PBS source is connected to the LEVITRONIX® gravity pump to begin the chase step. UV prediction model constants for decline portion are entered into Delta V™ based on cell line type; harvest end cutoff threshold is entered into Delta V™. Online UV signal is converted to concentration of target protein during chase. When threshold is reached, receiving vessel is disconnected from process stream. Pressure, turbidity and temperature are monitored throughout the harvest process to indicate out of control problems.

[0175] Example 7. Confirmation of online predicted titers from online UV signal by offline titer analysis The recovery process was initiated by flushing the depth filter with water for injection (WFI). A UV sensor was connected to the outlet of the secondary depth filter. Once the filtration stabilized, a clear liquid flow was seen from the outlet; at that point the UV sensor reading was zero. After flushing the filter with the desired amount of WFI, cell culture media was connected to the filter inlet and loading was initiated. The online UV trace of the media was monitored along with loading. Filtrate samples were taken during loading and analyzed offline by titer assay. Figure 11 shows that the titer trace was achieved from the UV signal by modeling. The modeled titer trace was in good agreement with the offline titer assay results and can be used to initiate and terminate recovery for process reliability and yield improvement.

[0176] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art, and such techniques are fully explained in the literature. 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) A Practical 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 In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); ); Crooks, Antisense drug technology: Principles, strategies and applications, 2. nd See Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0177] In some embodiments, the present specification discloses an apparatus for controlling, regulating, increasing, or improving the yield of a protein in a sample mixture containing a protein of interest and impurities. The apparatus may comprise one or more sensors. The sensor may comprise a pressure sensor, a UV sensor, a turbidity sensor, a temperature sensor, a flow sensor, and any combination thereof.

[0178] In some embodiments, the device is designed to integrate all sensors into the device, including pressure sensor (4), UV sensor (1), turbidity sensor (2), temperature sensor (2), and flow sensor (1). In some embodiments, the device comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controller is built-in on the cart, accommodating a total of 10 different sensors. In some embodiments, a gravity pump (e.g., a LEVITRONIX® gravity pump, etc.) is used to pump liquid to the depth filter and is attached to the device. In some embodiments, the system is movable, lockable, and / or electrically shut off. The device may also comprise a processor configured to control the recovery of the protein of interest. Further, the processor may be configured to change the state of the device, e.g., temperature, pressure, turbidity, or flow rate. The processor may also be configured to control the recovery of the protein of interest. In some embodiments, the processor may use the established model to determine the harvest process of the culture. The cell culture harvest process may include a filtration-based cell culture harvest process. The processor can be configured to use the titer of the protein of interest. The device can be incorporated into a system for controlling, regulating, increasing or improving the yield of protein in a sample mixture containing the protein of interest and impurities.

[0179] All references cited above, as well as all references cited herein, and amino acid or nucleotide sequences (e.g., GenBank numbers and / or Uniprot numbers), are hereby incorporated by reference in their entirety.

Claims

1. A method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture containing the target protein and impurities, comprising monitoring an ultraviolet (UV) signal of the sample mixture in real time and automatically converting the UV signal to a titer of the target protein using an established model during a filtration-based cell culture recovery process including a recovery skid including a control system, wherein the UV signal is continuously converted to a titer of the target protein according to the established model and the control system, and the control system automatically starts recovery of the protein when a set titer for start recovery is achieved and automatically stops recovery of the protein when a set titer for stop recovery is achieved.

2. A method for controlling target protein recovery and improving protein yield in a sample mixture containing the target protein and impurities, comprising monitoring an ultraviolet (UV) signal of the sample mixture in real time and automatically converting the UV signal to a target protein titer using an established model during a filtration-based cell culture recovery process including a recovery skid including a control system, wherein the UV signal is continuously converted to a target protein titer according to the established model and control system, and the control system automatically starts protein recovery when a set start recovery titer is achieved and automatically stops protein recovery when a set stop recovery titer is achieved.

3. The method described in claim 1 or 2, wherein the titer of the recovered protein is 0.05 g / L to 20 g / L.

4. The method according to any one of claims 1 to 3, further comprising stopping the recovery of the target protein when the recovery titer is less than 0.1 or 0.2 g / L.

5. A method according to any one of claims 1 to 4, wherein the yield of the target protein is increased 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%, or at least 20% compared to the yield of the protein when the ultraviolet (UV) signal of the sample mixture is not monitored in real time.

6. The method of any one of claims 1 to 5, wherein the target protein is recovered from a culture medium having a cell density of at least 1x106 cells / mL, at least 5x106 cells / mL, at least 1x107 cells / mL, at least 1.5x107 cells / mL, at least 2x107 cells / mL, at least 2.5x107 cells / mL, at least 3x107 cells / mL, at least 3.5x107 cells / mL, at least 4x107 cells / mL, at least 4.5x107 cells / mL, or at least 5x107 cells / mL.

7. The method of claim 1, wherein the protein filtration is depth filtration.

8. The method of claim 7, wherein the depth filtration comprises a primary depth filter and / or a secondary depth filter.

9. The method of claim 1, further comprising loading the sample mixture prior to monitoring.

10. The method of claim 1, further comprising flushing the depth filter with water or a buffer before loading the cell culture and chasing the depth filter after loading the cell culture.

11. The method of claim 1, further comprising chasing the sample mixture with phosphate buffered saline (PBS) or other buffer.

12. The method of claim 1 or 2, wherein the control system adjusts the flow rate of liquid passing through the recovery skid.

13. The method of claim 12, wherein the control system automatically drives the pump to upwardly control the flow rate of the recovery skid.

14. The method of claim 12, wherein the control system automatically drives the pump to downwardly control the flow rate of the recovery skid.

15. The method of claim 1, wherein the method does not include an air blowdown step.

16. The method of claim 1, wherein the titer of the target protein or the protein yield is independent of volume.

17. A method for increasing, controlling, or regulating protein yield in a sample mixture containing a protein of interest and an impurity, comprising: (a) Flush the recovery skid with water; (b) loading the sample onto the recovery skid; (c) measuring the ultraviolet (UV) signal of the sample mixture during protein filtration on the recovery skid resulting in real-time protein titer; (d) triggering protein collection based on UV measurement and real-time protein titer; (e) chasing the protein with PBS; (f) Stopping protein collection based on UV measurement and real-time protein titer. wherein the UV signal is correlated with real-time protein titer during filtration and is continuously converted to a titer of the target protein according to an established model and automatic control, and the recovery skid includes a control system, which automatically starts protein recovery when a set start recovery titer is reached and automatically stops protein recovery when a set stop recovery titer is reached.

18. The method of any one of claims 1 to 17, further comprising measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

19. The method of any one of claims 1 to 18, wherein the recovery skid comprises one or more filters.

20. The method of claim 19, wherein the filter comprises a primary depth filter and a secondary depth filter.

21. The method of any one of claims 1 to 20, wherein the sample mixture is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof.

22. The method of any one of claims 1 to 21, wherein the protein is produced in a culture comprising mammalian cells.

23. The method of claim 22, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

24. The method of claim 23, wherein the CHO cells are selected from the group consisting of CHO-DG44 cells, CHOZN cells, CHO / dhfr- cells, CHOK1SV GS-KO cells, and CHO-S cells.

25. The method of claim 23, wherein the mammalian cell is CHO-DG44, the concentration of the target protein is obtained using a model predicted titer, the model predicted titer comprising constants (a) and (b), and the target protein is calculated according to formula (I): model predicted titer = a + b * (online UV signal).

26. The method of claim 25, wherein (a) is −0.35 and (b) is 2.

88.

27. The method of claim 23, wherein the mammalian cell is CHO-DG44, and the concentration of the target protein is obtained using a model predicted titer, the model predicted titer comprising constants (A), (B) and (C), and the target protein is calculated according to formula (II): model predicted titer = A + B * exp(C * online UV signal).

28. The method of claim 27, wherein (A) is -0.95, (B) is 0.86, and (C) is 1.

21.

29. The method of claim 23, wherein the mammalian cells are CHOZN, and the concentration of the target protein is obtained using a model predicted titer, the model predicted titer comprising constants (a) and (b), and the target protein is calculated according to formula (I): model predicted titer = a + b * (online UV signal).

30. The method of claim 29, wherein (a) is −0.69 and (b) is 4.

06.

31. The method of any one of claims 23, 29 and 30, wherein the mammalian cells are CHOZN, and the concentration of the target protein is obtained using a model predicted titer, the model predicted titer comprising constants (A), (B) and (C), and the target protein is calculated according to formula (I): model predicted titer = a + b * (online UV signal).

32. The method of claim 31, wherein (A) is 0.02, (B) is 0.13, and (C) is 2.

41.

33. A method according to any one of claims 1 to 32, wherein the protein comprises an antibody or a fusion protein.

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