Real-time monitoring of titer using ultraviolet signals

Abstract

The present disclosure relates to methods for controlling, regulating, increasing, or improving protein yield in a sample mixture comprising a target protein and impurities, which include real-time monitoring of the ultraviolet (UV) signal of the sample mixture during protein filtration in a harvest skid.

Description

Field of the Invention

[0001] The present disclosure relates to a method for monitoring the concentration of biomolecules, such as proteins, in a composition. Specifically, the present disclosure relates to a method for monitoring, controlling, regulating, or increasing the protein yield in a composition using real-time ultraviolet signal monitoring during protein filtration. Background of the Invention

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

[0004] The purpose of upstream and recovery operations is high-productivity therapeutic proteins during cell culture and recovery processes, and multiple on-line configurations can be used to monitor bioprocess operations. See, Whitford W., Julien C. Bioprocess Int. (5), S32–S45 (2007). Real-time monitoring and control of cell culture processes have recently been achieved. It has been shown that an increase in the non-viable subpopulation in CHO cell cultures can predict the onset of the stationary phase, indicating the opportunity for a fully automated cell culture process and reliable and reproducible control of fed-batch addition during culture proliferation. Sitton G., Srienc F. J. Biotechnol., 135 (2008), 174-180. Others have utilized multiple steps during primary recovery to remove biomass and clarify the feed stream for downstream column chromatography. Bink L. R., Furey J. BioProcess Int. 8(3) 2010, 44–49, 57 (2010).

[0005] Some people solve the problem of increasing protein yield by solving upstream steps to increase downstream productivity. For example, others have tried to reduce the mechanical stress on CHO cells by the magnetically levitated bearingless centrifugal pump by using peristaltic pumps and diaphragm pumps. Blaschczok K., et al. Chemie Ingenieur Technik, (85), 144-152 (2013). Also, others have evaluated proteomic methods by studying the kinetics and fate of host cell proteins in the supernatant of cell lines producing monoclonal antibodies during recovery and early downstream processing, including centrifugation, depth filtration, and Protein A capture chromatography. Hogwood, C.E.M., 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 U S A.; 101(2):523-8 (2004). Adding additional impurities may require additional purification steps that can affect yield.

[0006] Accordingly, there remains a need to monitor and control the recovery process in real time to increase recovery yield and process robustness, rapidly evaluate upstream performance, and facilitate immediate downstream processing during batch processing or in more critical continuous processes. Summary of the Invention

[0008] Disclosed herein are new real-time monitoring and control processes and systems that are designed and verified for filtration-based cell culture harvest processes for several therapeutic proteins, such as depth filtration harvest. The methods described herein offer several advantages over the prior art. First, the harvest skid is designed to have the ability to monitor and control key process parameters and quality attributes in real time. Second, it uses a modeling method to convert the online UV signal of the clarified bulk solution into the real-time titer of the target product. Third, using this harvest skid and real-time titer can automatically control the harvest process and improve process yield, robustness, and consistency. Finally, titer information is used to demonstrate cell culture performance and guide the immediate processing of downstream purification.

[0009] The core of this new technology is to apply real-time monitoring of the UV signal during the harvest process and convert the online UV signal into the real-time target protein concentration. The models disclosed herein can be applied to several processes with different cell characteristics and productivity levels. Using this system, the start and end of the collection of the clarified bulk solution can be determined in a quantitative manner, which can significantly improve the robustness of the harvest and protein yield.

[0010] The methods disclosed herein provide insight into the use of harvest sleds in the process of cell culture clarification. The new harvest process disclosed herein improves protein yields while being scalable, automatically controllable, and applicable to multiple products with diverse properties. Real-time titer information can be used to demonstrate cell culture performance and guide immediate downstream processing.

[0011] Disclosed herein is a method for real-time monitoring of the concentration (titer) of a target protein in a sample mixture comprising the target protein and impurities, the method comprising monitoring a real-time ultraviolet (UV) signal of the sample mixture during a filtration-based cell culture harvest process and automatically converting the UV signal to a target protein titer using an established model.

[0012] Also disclosed herein is a method for controlling the collection of a target protein and improving the protein yield in a sample mixture comprising the target protein and impurities, the method comprising monitoring a real-time ultraviolet (UV) signal of the sample mixture during a filtration-based cell culture harvest process.

[0013] In some embodiments, the UV signal is continuously converted to the titer of the target protein according to an established model and automatic control.

[0014] In some embodiments, the titer of the target protein is at least about 0.01 g / L, at least about 0.02 g / L, at least about 0.03 g / L, at least about 0.04 g / L, at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, 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 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.

[0015] In some embodiments, the methods disclosed herein further comprise collecting the target protein when the titer is at least about 0.05 g / L, at least about 0.06 g / L, at least about 0.07 g / L, at least about 0.08 g / L, at least about 0.09 g / L, at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, at least about 6.5 g / L, at least about 7 g / L, at least about 7.5 g / L, at least about 8 g / L, at least about 8.5 g / L, at least about 9 g / L, at least about 9.5 g / L, at least about 10 g / L, at least about 10.5 g / L, at least about 11 g / L, at least about 11.5 g / L, at least about 12 g / L, at least about 12.5 g / L, at least about 13 g / L, at least about 13.5 g / L, at least about 14 g / L, at least about 14.5 g / L, at least about 15 g / L, at least about 15.5 g / L, at least about 16 g / L, at least about 16.5 g / L, at least about 17 g / L, at least about 17.5 g / L, at least about 18 g / L, at least about 18.5 g / L, at least about 19 g / L, at least about 19.5 g / L or at least about 20 g / L.

[0016] In some embodiments, the titer of the target protein collected is between about 0.05 g / L and about 20 g / L, between about 0.1 g / L and about 20 g / L, between about 0.2 g / L and about 20 g / L, between about 0.3 g / L and about 20 g / L, between about 0.4 g / L and about 20 g / L, between about 0.5 g / L and about 20 g / L, between about 0.6 g / L and about 20 g / L, between about 0.7 g / L and about 20 g / L, between about 0.8 g / L and about 20 g / L, between about 0.9 g / L and about 20 g / L, between about 1 g / L and about 20 g / L, between about 0.05 g / L and about 15 g / L, between about 0.1 g / L and about 15 g / L, between about 0.2 g / L and about 15 g / L, between about 0.3 g / L and about 15 g / L, between about 0.4 g / L and about 15 g / L, between about 0.5 g / L and about 15 g / L, between about 0.6 g / L and about 15 g / L, between about 0.7 g / L and about 15 g / L, between about 0.8 g / L and about 15 g / L, between about 0.9 g / L and about 15 g / L, or between about 1 g / L and about 15 g / L, between about 0.05 g / L and about 10 g / L, between about 0.1 g / L and about 10 g / L, between about 0.2 g / L and about 10 g / L, between about 0.3 g / L and about 10 g / L, between about 0.4 g / L and about 10 g / L, between about 0.5 g / L and about 10 g / L, between about 0.6 g / L and about 10 g / L, between about 0.7 g / L and about 10 g / L, between about 0.8 g / L and about 10 g / L, between about 0.9 g / L and about 10 g / L, or between about 1 g / L and about 10 g / L.

[0017] In some embodiments, the methods disclosed herein further comprise stopping the collection of the target protein when the collected titer is below about 0.1 or 0.2 g / L.

[0018] In some embodiments, the target protein yield is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% compared to the protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture.

[0019] In some embodiments, the target protein is from a cell density of at least about 1X10 6 cells / mL, at least about 5X10 6cells / mL, at least about 1X10 7 cells / mL, at least about 1.5X10 7 cells / mL, at least about 2X10 7 cells / mL, at least about 2.5X10 7 cells / mL, at least about 3X10 7 cells / mL, at least about 3.5X10 7 cells / mL, at least about 4X10 7 cells / mL, at least about 4.5X10 7 cells / mL, or at least about 5X10 7 cells / mL in the medium.

[0020] In some embodiments, the protein filtration is depth filtration. In some embodiments, the depth filtration includes a primary depth filter and / or a secondary depth filter.

[0021] In some embodiments, the methods disclosed herein further include loading the sample mixture prior to the monitoring. In some embodiments, the methods disclosed herein further include rinsing the depth filter with water or buffer prior to loading the cell culture and backflushing the depth filter after loading the cell culture. In some embodiments, the methods disclosed herein further include backflushing the sample mixture with phosphate buffered saline (PBS) or other buffer. In some embodiments, the filtration-based cell culture harvest process includes a harvest skid. In some embodiments, the harvest skid includes a control system, wherein when a set titer is reached, the control system automatically begins collecting the protein. In some embodiments, the harvest skid includes a control system, wherein when a set titer is reached, the control system automatically begins collecting the protein. In some embodiments, the harvest skid includes a control system, wherein when a set titer is reached, the control system automatically stops collecting the protein. In some embodiments, the control system regulates the flow rate of liquid through the harvest skid. In some embodiments, the control system automatically drives a pump to increase the flow rate through the harvest skid. In some embodiments, the control system automatically drives a pump to decrease the flow rate through the harvest skid. In some embodiments, the methods disclosed herein do not include a step of venting gas. In some embodiments, the target protein titer or the protein yield is not volume-based.

[0022] In some embodiments, provided herein is a method of increasing, controlling, or regulating protein yield in a sample mixture comprising a target protein and impurities, the method comprising (a) flushing a harvest skid with water; (b) loading the sample into the harvest skid; (c) measuring an ultraviolet (UV) signal of the sample mixture during protein filtration in the harvest skid as a real-time protein titer; (d) starting collection of the protein based on the UV metric and the real-time protein titer; (e) chasing the protein with PBS; and (f) stopping collection of the protein based on the UV metric and the real-time protein titer; wherein the UV signal is correlated with the real-time protein titer during filtration.

[0023] In some embodiments, the method further comprises measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0024] In some embodiments, the method further comprises measuring pressure using a pressure sensor. In some embodiments, the measured pressure ranges from -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.

[0025] In some embodiments, the method further comprises measuring turbidity. In some embodiments, the measured turbidity ranges from 0 absorbance units (AU) to 2 AU.

[0026] In some embodiments, the method further comprises measuring temperature. In some embodiments, the measured temperature ranges from 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.

[0027] In some embodiments, the method further includes measuring the flow rate. In some embodiments, the measured flow rate ranges from 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, 0 L / min to 110 L / min, 0 L / min to 120 L / min, 0 L / min to 130 L / min, 0 L / min to 140 L / min, 0 L / min to 150 L / min, 0 L / min to 160 L / min, 0 L / min to 170 L / min, 0 L / min to 180 L / min, 0 L / min to 190 L / min, 0 L / min to 200 L / min, 0 L / min to 250 L / min, or 0 L / min to 300 L / min.

[0028] In some embodiments, the harvest skid includes one or more filters. In some embodiments, the filter includes a primary depth filter and a secondary depth filter. In some embodiments, the sample mixture is selected from pure protein samples, clarified crude protein samples, cell culture samples, and any combination thereof.

[0029] In some embodiments, the protein is produced in a culture containing mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), simian kidney fibroblasts (COS-7), Madin-Darby bovine kidney cells (MDBK), or any combination thereof.

[0030] In some embodiments, the protein comprises an antibody or a fusion protein. In some embodiments, the protein is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, and an anti-IL8 antibody. In some embodiments, the protein is abatacept or belatacept.

[0031] In some embodiments, a system for real-time monitoring and control of protein yield is disclosed herein, wherein the system includes a sensor that measures the real-time UV signal of a sample mixture containing a target protein and impurities.

[0032] In some embodiments, the system further includes sensors that measure pressure, turbidity, temperature, flow rate, weight, or any combination thereof.

[0033] In some embodiments, a device includes a sensor configured to measure a UV signal of a sample mixture comprising a target protein and impurities. In some embodiments, the processor is configured to control the collection of the target protein. In some embodiments, the processor is configured to use the target protein titer. In some embodiments, the processor is configured to use an established model to determine a cell culture harvest process. In some embodiments, the cell culture harvest process includes a filtration-based cell culture harvest process. In some embodiments, a system includes a device comprising a sensor configured to measure a UV signal of a sample mixture comprising a target protein and impurities.

[0034] In some embodiments, the disclosed system is for use in the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A Shows the mechanical design of an exemplary harvest skid. All values are listed in inches. Figure 1B Shows a physical diagram of the harvest skid.

[0037] Figure 2 Shows a process flow diagram of a cell culture harvest process using the new harvest skid. The various boxes show on-line measurement sensors, control modules, and physical instruments.

[0038] Figure 3 Shows an experimental design for modeling a UV signal into a product titer as described herein.

[0039] Figure 4 Shows a graphical comparison between the old and new harvest methods. Compared to the previous method, the new method eliminates the gas venting step. Also, the start and end of clarified stock solution collection in the new method can be automatically controlled based on on-line UV readings and calculated titers. More specifically, a model generated and tested herein can be used to calculate the real-time target protein concentration during the harvest process from on-line UV sensor readings. Thus, the cut-off point for stock solution collection can be directly determined based on the calculated on-line target protein concentration. The calculation algorithm can be integrated into the Delta V TM control system to achieve an automatic cut-off point for clarified stock solution collection.

[0040] Figure 5 Shows an off-line titer measurement of a continuously diluted sample of a GITR cell culture against an on-line UV signal.

[0041] Figure 6A and Figure 6B Shows the use of pure protein ( Figure 6A ) and clarified stock solution ( Figure 6BOffline titer measurements of the on-line UV signal for small-scale harvest processes. On-line UV and offline titer values during the test harvest process are measured.

[0042] Figure 7A , Figure 7B and Figure 7C show the offline titer measurements of the on-line UV signal for large-scale harvest processes using anti-GITR antibody cell cultures ( Figure 7A ), abatacept cell cultures ( Figure 7B ), and anti-CXCR4 antibody cell cultures ( Figure 7C ).

[0043] Figure 8A and Figure 8B show the linear fits of the offline titer measurements against the on-line UV values ( Figure 8A ); the linear fits of the UV-based predicted titers against the actual titers ( Figure 8B ).

[0044] Figure 9A and Figure 9B show the non-linear fits of the offline titer measurements against the on-line UV values ( Figure 9A ); the linear fits of the UV-based predicted titers against the actual titers ( Figure 9B ).

[0045] Figure 10 shows the average difference (HPLC analysis) between the model-predicted titers and the actual titers of the seven molecules studied, which include Aba J, anti-CD73 antibody, anti-GITR antibody, anti-IL8 antibody, anti-CXCR4 antibody, anti-OX40 antibody, and anti-TIGIT antibody.

[0046] Figure 11 shows the comparison of the on-line UV tracer, the titer tracer obtained by UV signal modeling, and the offline-determined titer. The Y-axis shows the offline-determined titer (g / L) or the UV-modeled titer (g / L), and the X-axis shows time (min). The triangular line shows the on-line UV, the square line shows the UV-modeled titer (g / L), and the diamond line shows the offline titer (g / L). DETAILED DESCRIPTION OF THE INVENTION

[0048] A variety of methods are provided that can be used to control, regulate, or increase protein yield. The methods include using the ultraviolet (UV) signal of a sample mixture measured in real time during a purification step, such as during protein filtration in a harvest skid, to control, regulate, or increase protein yield. The method uses the UV signal to provide the titer of the target protein according to the formulas disclosed herein, which varies depending on whether collection occurs from the start to the end of loading or after the end of loading.

[0049] The present disclosure also discloses various systems and devices related to the methods provided herein.

[0050] a. Terms

[0051] It should be noted that the term "a" or "an" entity refers to one or more of such entities; for example, "nucleotide sequence" should be 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.

[0052] In addition, "and / or" as used herein should be understood to specifically disclose each of the two specified features or components, either together or not together with the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and". It should also be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate values and are provided for description purposes.

[0054] It should be understood that wherever aspects are described herein in terms of the language "comprising", similar aspects are also provided in terms of "consisting of" and / or "consisting essentially of".

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 2nd Edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many of the terms used in this disclosure to those skilled in the art.

[0056] Units, prefixes, and symbols are expressed in their accepted form of the Système International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxyl direction. The headings provided herein are not limitations of the various aspects of the disclosure, and the various aspects of the disclosure can be obtained by referring to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0057] The term "about" is used herein to mean approximate, roughly, around, or in the vicinity thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical values. Thus, "about 10 - 20" means "about 10 to about 20". Generally, the term "about" can modify a numerical value by a variance of higher or lower (greater or less) than, for example, 10% such that the value is higher or lower than the stated value.

[0058] "Modeling" or "protein modeling" refers to a method of establishing a linear fit to determine the titer of a test protein (e.g., in g / L). In one embodiment, modeling includes the method from start collection to end loading (e.g., up - slope modeling). In another embodiment, modeling includes starting the chase to end collection (e.g., down - slope modeling). In other embodiments, modeling includes both up - slope modeling and down - slope modeling.

[0059] "Protein yield" or "yield" refers to the total amount of protein recovered after the processes disclosed herein. Protein yield can be measured in grams or as the final concentration in a fixed volume (e.g., mg / ml). The percentage yield can also be measured as a percentage of the amount of starting protein (e.g., stock enzyme).

[0060] The term "control protein yield" as used herein can refer to regulating, testing, or validating the final product (e.g., protein) collected during the processes disclosed herein. In some embodiments, control of protein yield is achieved by changing the UV signal in real - time to affect key process parameters and quality attributes and regulating the protein yield. In some embodiments, control of protein yield refers to maintaining a constant UV signal during the methods disclosed herein to obtain the desired protein yield.

[0061] As used herein, the term "regulating protein yield" refers to altering, changing, or modifying the end product (e.g., protein) collected during the processes disclosed herein. Regulating protein yield changes the yield of the protein end product, which can be increased, decreased, or inhibited. In some embodiments, the process regulates protein yield, which results in an increase in protein yield. In some embodiments, regulating protein yield is achieved by changing the UV signal in real time to affect key process parameters and quality attributes and regulate protein yield.

[0062] The harvest skid as described herein includes a plurality of sensors for real-time clarification and protein yield increase. The harvest skid or "skid" includes one or more pressure sensors, one or more flow sensors, one or more ultraviolet (UV) sensors, one or more weight sensors, one or more turbidity sensors, and / or one or more temperature sensors.

[0063] "Titer" refers to the amount or concentration of a substance in a solution. As described herein, up-tilt modeling and down-tilt modeling are used to determine the titer.

[0064] As used herein, the terms "ug" and "uM" may be used interchangeably with "μg" and "μM", respectively.

[0065] The various aspects described herein are further described in detail in the following subsections.

[0066] b. Methods and Uses

[0067] This disclosure is based on the ability to monitor and control key process parameters and quality attributes in real time using UV. This method allows the online UV signal of the clarified feedstock to be converted into the real-time titer of the target product using a modeling method. Then, this method can be used to automatically control the harvest process and improve process yield, robustness, and consistency. Titer information can also be used to demonstrate the performance of the cell culture and guide the immediate processing of downstream purification. In some embodiments, a method for controlling or regulating protein yield in a sample mixture containing a target protein and impurities is disclosed herein, the method comprising monitoring in real time the ultraviolet (UV) signal of the sample mixture during protein filtration in a harvest skid.

[0068] In one embodiment, this disclosure includes a method for real-time monitoring of the target protein concentration (titer) in a sample mixture containing a target protein and impurities, the method comprising, during a filtration-based cell culture harvest process, monitoring in real time the ultraviolet (UV) signal of the sample mixture and automatically converting the UV signal into the target protein titer using an established model. In another embodiment, the present invention provides a method for controlling the collection of a target protein and improving the protein yield in a sample mixture containing a target protein and impurities, the method comprising monitoring in real time the ultraviolet (UV) signal of the sample mixture during a filtration-based cell culture harvest process.

[0069] The present disclosure also provides a method for increasing or improving the protein yield in a sample mixture containing a target protein and impurities, the method comprising monitoring in real time the ultraviolet (UV) signal of the sample mixture during a filtration-based cell culture harvest process (such as protein filtration in a harvest skid).

[0070] Protein harvest / purification involves multiple steps of separating or purifying a target protein from a mixture of the protein and impurities such as cells, cell culture medium, DNA, RNA, other proteins, etc. Clarifying the cell culture broth can be the first downstream unit operation in the detailed sequence of steps required for purifying the target protein. A combination of centrifugation and / or filtration (such as depth filtration) is used for this operation. Thus, the availability of large-scale filtration techniques (such as depth filtration) that can monitor the real-time protein concentration can provide the ability to improve and simplify the downstream process.

[0071] Large-scale depth filtration systems are common in the bioprocessing industry. In some embodiments, the depth filtration system can utilize a harvest skid as Figure 2 shown. Prior to harvest, the depth filter is rinsed with water or an appropriate buffer to remove loose particles and extractables from the filter manufacturing process. The harvest skid can include one filter or multiple filters, such as a primary depth filter and a secondary depth filter. The cell culture medium containing the target protein can be obtained from a bioreactor and loaded onto (or pumped through) one or more filters, such as the primary filter and the secondary filter. Then, the real-time UV signal can be measured after the loaded cell culture medium has passed through the filtration system (such as the primary filter or the secondary filter). Then, the filtered product can be obtained in one or more tanks. After harvest is complete, the filter is rinsed again to recover valuable product retained in the housing. Subsequent rinses for reuse can achieve a harvest yield of 50% to 90% and ensure minimal product loss. Thus, the method of the present invention aims to increase the yield of protein harvest by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24% or at least 25%.

[0072] In some embodiments, the UV signal provides the titer of the target protein from the start of loading to the end of loading and / or after the end of loading until the end of filtration. In some embodiments, the titer of the target protein from the start of loading to the end of loading can be calculated according to Equation (I):

[0073] Titer predicted by the model = a + b*(online UV signal). (I)

[0074] In some embodiments, the titer of the target protein from the start of loading to the end of loading can be calculated according to formula (I), which includes constants (a) and (b).

[0075] In some embodiments, (a) is a value between 0 and -1.0. In some embodiments, (a) is a value between -0.1 and -0.9. In some embodiments, (a) is a value between -0.2 and -0.8. In some embodiments, (a) is a value between -0.3 and -0.7. In some embodiments, (a) is a value between -0.4 and -0.6.

[0076] In some embodiments, (a) is a value between -0.2 and -0.5. In some embodiments, (a) is a value between -0.25 and -0.45. In some embodiments, (a) is a value between -0.30 and -0.40.

[0077] In some embodiments, (a) is a value between -0.5 and -0.9. In some embodiments, (a) is a value between -0.55 and -0.85. In some embodiments, (a) is a value between -0.60 and -0.80. In some embodiments, (a) is a value between -0.65 and -0.75.

[0078] In some embodiments, (a) is approximately -0.1. In some embodiments, (a) is approximately -0.15. In some embodiments, (a) is approximately -0.2. In some embodiments, (a) is approximately -0.25. In some embodiments, (a) is approximately -0.3. In some embodiments, (a) is approximately -0.35. In some embodiments, (a) is approximately -0.4. In some embodiments, (a) is approximately -0.45. In some embodiments, (a) is approximately -0.5. In some embodiments, (a) is approximately -0.55. In some embodiments, (a) is approximately -0.6. In some embodiments, (a) is approximately -0.65. In some embodiments, (a) is approximately -0.7. In some embodiments, (a) is approximately -0.75. In some embodiments, (a) is approximately -0.8. In some embodiments, (a) is approximately -0.85. In some embodiments, (a) is approximately -0.9. In some embodiments, (a) is approximately -0.95. In some embodiments, (a) is approximately -1.0.

[0079] 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.

[0080] In some embodiments, (b) is a value between 1.0 and 5.0. In some embodiments, (b) is a value between 1.5 and 4.5. In some embodiments, (b) is a value between 2.0 and 4.0. In some embodiments, (b) is a value between 2.5 and 3.5.

[0081] In some embodiments, (b) is a value between 2.0 and 3.6. In some embodiments, (b) is a value between 2.1 and 3.5. In some embodiments, (b) is a value between 2.2 and 3.4. In some embodiments, (b) is a value between 2.3 and 3.3. In some embodiments, (b) is a value between 2.4 and 3.2. In some embodiments, (b) is a value between 2.5 and 3.1. In some embodiments, (b) is a value between 2.6 and 3.0. In some embodiments, (b) is a value between 2.7 and 2.9.

[0082] In some embodiments, (b) is a value between 3.3 and 4.8. In some embodiments, (b) is a value between 3.4 and 4.7. In some embodiments, (b) is a value between 3.5 and 4.6. In some embodiments, (b) is a value between 3.6 and 4.5. In some embodiments, (b) is a value between 3.7 and 4.4. In some embodiments, (b) is a value between 3.8 and 4.3. In some embodiments, (b) is a value between 3.9 and 4.2. In some embodiments, (b) is a value between 4.0 and 4.1.

[0083] 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.

[0084] 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, (a) is -0.35, and (b) is 2.88. In one embodiment, the cell type is CHOZN, (a) is -0.69, and (b) is 4.06.

[0085] In other embodiments, the titer of the target protein from the end of loading until the end of filtration can be calculated according to Equation (II):

[0086] The titer predicted by the model = A + B * exp(C * online UV signal). (II)

[0087] In some embodiments, the titer of the target protein from the start to the end of loading can be calculated according to formula (II), which includes constants (A), (B), and (C).

[0088] In some embodiments, (A) is a value between -2.5 and 1.0. In some embodiments, (A) is a value between -2.0 and 0.5. In some embodiments, (A) is a value between -1.5 and 0.0. In some embodiments, (A) is a value between -1.0 and -0.5.

[0089] In some embodiments, (A) is a value between -1.5 and -0.4. In some embodiments, (A) is a value between -1.4 and -0.5. In some embodiments, (A) is a value between -1.3 and -0.6. In some embodiments, (A) is a value between -1.2 and -0.7. In some embodiments, (A) is a value between -1.1 and -0.8. In some embodiments, (A) is a value between -1.0 and -0.9.

[0090] In some embodiments, (A) is a value between -1.0 and 1.0. In some embodiments, (A) is a value between -0.9 and 0.9. In some embodiments, (A) is a value between -0.8 and 0.8. In some embodiments, (A) is a value between -0.7 and 0.7. In some embodiments, (A) is a value between -0.6 and 0.6. In some embodiments, (A) is a value between -0.5 and 0.5. In some embodiments, (A) is a value between -0.4 and 0.4. In some embodiments, (A) is a value between -0.3 and 0.3. In some embodiments, (A) is a value between -0.2 and 0.2. In some embodiments, (A) is a value between -0.1 and 0.1.

[0091] 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.

[0092] In some embodiments, (A) is -0.95. In some embodiments, (A) is 0.02. In one embodiment, the cell type is DG44 and (A) is -0.95. In one embodiment, the cell type is CHOZN and (A) is 0.02.

[0093] In some embodiments, (B) is a value between -1.5 and 2.5. In some embodiments, (B) is a value between -1.0 and 2.0. In some embodiments, (B) is a value between -0.5 and 1.5. In some embodiments, (B) is a value between 0 and 1.0.

[0094] In some embodiments, (B) is a value between -0.5 and -0.4. In some embodiments, (B) is a value between -0.4 and -0.3. In some embodiments, (B) is a value between -0.3 and -0.2. In some embodiments, (B) is a value between -0.2 and -0.1. In some embodiments, (B) is a value between -0.1 and 0.0. In some embodiments, (B) is a value between 0.0 and 0.1. In some embodiments, (B) is a value between 0.1 and 0.2. In some embodiments, (B) is a value between 0.2 and 0.3. In some embodiments, (B) is a value between 0.3 and 0.4. In some embodiments, (B) is a value between 0.4 and 0.5. In some embodiments, (B) is a value between 0.5 and 0.6. In some embodiments, (B) is a value between 0.6 and 0.7. In some embodiments, (B) is a value between 0.7 and 0.8. In some embodiments, (B) is a value between 0.8 and 0.9. In some embodiments, (B) is a value between 0.9 and 1.0. In some embodiments, (B) is a value between 1.0 and 1.1. In some embodiments, (B) is a value between 1.1 and 1.2. In some embodiments, (B) is a value between 1.2 and 1.3. In some embodiments, (B) is a value between 1.3 and 1.4. In some embodiments, (B) is a value between 1.4 and 1.5.

[0095] 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.

[0096] In some embodiments, (B) is 0.86. In some embodiments, (B) is 0.13. In one embodiment, the cell type is DG44 and (B) is 0.86. In one embodiment, the cell type is CHOZN and (B) is 0.13.

[0097] In some embodiments, (C) is a value between 0 and 4.0. In some embodiments, (C) is a value between 0.5 and 3.5. In some embodiments, (C) is a value between 1.0 and 3.0. In some embodiments, (C) is a value between 1.5 and 2.5.

[0098] In some embodiments, (C) is a value between 0.0 and 0.1. In some embodiments, (C) is a value between 0.1 and 0.2. In some embodiments, (C) is a value between 0.2 and 0.3. In some embodiments, (C) is a value between 0.3 and 0.4. In some embodiments, (C) is a value between 0.4 and 0.5. In some embodiments, (C) is a value between 0.5 and 0.6. In some embodiments, (C) is a value between 0.6 and 0.7. In some embodiments, (C) is a value between 0.7 and 0.8. In some embodiments, (C) is a value between 0.8 and 0.9. In some embodiments, (C) is a value between 0.9 and 1.0. In some embodiments, (C) is a value between 1.0 and 1.1. In some embodiments, (C) is a value between 1.1 and 1.2. In some embodiments, (C) is a value between 1.2 and 1.3. In some embodiments, (C) is a value between 1.3 and 1.4. In some embodiments, (C) is a value between 1.4 and 1.5. In some embodiments, (C) is a value between 1.5 and 1.6. In some embodiments, (C) is a value between 1.6 and 1.7. In some embodiments, (C) is a value between 1.7 and 1.8. In some embodiments, (C) is a value between 1.8 and 1.9. In some embodiments, (C) is a value between 1.9 and 2.0. In some embodiments, (C) is a value between 2.0 and 2.1. In some embodiments, (C) is a value between 2.1 and 2.2. In some embodiments, (C) is a value between 2.2 and 2.3. In some embodiments, (C) is a value between 2.3 and 2.4. In some embodiments, (C) is a value between 2.4 and 2.5. In some embodiments, (C) is a value between 2.5 and 2.6. In some embodiments, (C) is a value between 2.6 and 2.7. In some embodiments, (C) is a value between 2.7 and 2.8. In some embodiments, (C) is a value between 2.8 and 2.9. In some embodiments, (C) is a value between 2.9 and 3.0. In some embodiments, (C) is a value between 3.0 and 3.1. In some embodiments, (C) is a value between 3.1 and 3.2. In some embodiments, (C) is a value between 3.2 and 3.3. In some embodiments, (C) is a value between 3.3 and 3.4. In some embodiments, (C) is a value between 3.4 and 3.5. In some embodiments, (C) is a value between 3.5 and 3.6. In some embodiments, (C) is a value between 3.6 and 3.7. In some embodiments, (C) is a value between 3.7 and 3.8. In some embodiments, (C) is a value between 3.8 and 3.9. In some embodiments, (C) is a value between 3.9 and 4.0.

[0099] In some embodiments, (C) is 1.21. In some embodiments, (C) is 2.41. In one embodiment, the cell type is DG44 and (C) is 1.21. In one embodiment, the cell type is CHOZN and (C) is 2.41.

[0100] In some embodiments, A = -0.95, B = 0.86, and C = 1.21. In some embodiments, A = 0.02, B = 0.13, and C = 2.41. In one embodiment, the cell type is DG44 and (A) is -0.95, (B) is 0.86, and (C) is 1.21. In one embodiment, the cell type is CHOZN and (A) is 0.02, (B) is 0.13, and (C) is 2.41.

[0101] In some embodiments, provided herein is a method of increasing, controlling, or regulating protein yield in a sample mixture comprising a target protein and an impurity, the method comprising (a) flushing a harvest skid with water; (b) loading a sample onto the harvest skid; (c) measuring an ultraviolet signal of the sample mixture during protein filtration in the harvest skid as a real-time determination of protein titer; (d) initiating collection of the protein based on the ultraviolet metric and the real-time protein titer; (e) chasing the protein with PBS; and (f) stopping collection of the protein based on the ultraviolet metric and the real-time protein titer; wherein the UV signal is correlated with the real-time protein titer during filtration.

[0102] In some embodiments, the methods described herein include flushing with water (e.g., RODI). In some embodiments, the method includes loading a protein sample and initiating collection based on an on-line titer. In some embodiments, the method includes PBS chasing and a final collection based on an on-line titer. Compared to other methods, the methods disclosed herein do not include a gas venting step.

[0103] In some embodiments, the start and end of sample collection are automatically controlled based on on-line UV readings and calculated titers. In a particular embodiment, the real-time target protein concentration during the harvest process is calculated by using modeling through on-line UV sensor readings. In some embodiments, the cut-off point for bulk collection is determined directly based on the calculated on-line target protein concentration. In some embodiments, a computational algorithm is integrated into the Delta V TM control system to achieve an automatic cut-off point for protein collection.

[0104] In some embodiments, the methods disclosed herein include a modeling step. In some embodiments, modeling includes offline titer measurements against the online UV signal using serially diluted samples to establish a linear correlation between the UV signal and the titer. In some embodiments, the sample used for modeling is a purified protein. In some embodiments, the sample used for modeling is a crude protein containing contaminants. In some embodiments, the modeling is then used to control, regulate, increase, and / or improve protein yield.

[0105] In some embodiments, the methods disclosed herein include controlling, modulating, or increasing the production of a target protein, the titer of which is at least about 0.01 g / L. In some embodiments, the titer is at least about 0.02 g / L. In some embodiments, the titer is at least about 0.03 g / L. In some embodiments, the titer is at least about 0.04 g / L. In some embodiments, the titer is at least about 0.05 g / L. In some embodiments, the titer is at least about 0.06 g / L. In some embodiments, the titer is at least about 0.07 g / L. In some embodiments, the titer is at least about 0.08 g / L. In some embodiments, the titer is at least about 0.09 g / L. In some embodiments, the titer is at least about 0.1 g / L. In some embodiments, the titer is at least about 0.2 g / L. In some embodiments, the titer is at least about 0.3 g / L. In some embodiments, the titer is at least about 0.4 g / L. In some embodiments, the titer is at least about 0.5 g / L. In some embodiments, the titer is at least about 0.6 g / L. In some embodiments, the titer is at least about 0.7 g / L. In some embodiments, the titer is at least about 0.8 g / L. In some embodiments, the titer is at least about 0.9 g / L. In some embodiments, the titer is at least about 1 g / L. In some embodiments, the titer is at least about 1.5 g / L. In some embodiments, the titer is at least about 2 g / L. In some embodiments, the titer is at least about 2.5 g / L. In some embodiments, the titer is at least about 3 g / L. In some embodiments, the titer is at least about 3.5 g / L. In some embodiments, the titer is at least about 4 g / L. In some embodiments, the titer is at least about 4.5 g / L. In some embodiments, the titer is at least about 5 g / L. In some embodiments, the titer is at least about 5.5 g / L. In some embodiments, the titer is at least about 6 g / L. In some embodiments, the titer is at least about 6.5 g / L. In some embodiments, the titer is at least about 7 g / L. In some embodiments, the titer is at least about 7.5 g / L. In some embodiments, the titer is at least about 8 g / L. In some embodiments, the titer is at least about 8.5 g / L. In some embodiments, the titer is at least about 9 g / L. In some embodiments, the titer is at least about 9.5 g / L. In some embodiments, the titer is at least about 10 g / L. In some embodiments, the titer is at least about 10.5 g / L. In some embodiments, the titer is at least about 11 g / L. In some embodiments, the titer is at least about 11.5 g / L. In some embodiments, the titer is at least about 12 g / L. In some embodiments, the titer is at least about 12.5 g / L. In some embodiments, the titer is at least about 13 g / L. In some embodiments, the titer is at least about 13.5 g / L.In some embodiments, the titer is at least about 14 g / L. In some embodiments, the titer is at least about 14.5 g / L. In some embodiments, the titer is at least about 15 g / L. In some embodiments, the titer is at least about 15.5 g / L. In some embodiments, the titer is at least about 16 g / L. In some embodiments, the titer is at least about 16.5 g / L. In some embodiments, the titer is at least about 17 g / L. In some embodiments, the titer is at least about 17.5 g / L. In some embodiments, the titer is at least about 18 g / L, at least about 18.5 g / L. In some embodiments, the titer is at least about 19 g / L. In some embodiments, the titer is at least about 19.5 g / L. In some embodiments, the titer is at least about 20 g / L.

[0106] In some embodiments, the methods disclosed herein include collecting the target protein, which depends on the titer of the target protein. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.05 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.06 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.07 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.08 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.09 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.1 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.2 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.3 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.4 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.6 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.7 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.8 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 0.9 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 1 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 1.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 2 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 2.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 3 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 3.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 4 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 4.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 5.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 6 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 6.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 7 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 7.5 g / L. In some embodiments, collection of the target protein is initiated when the titer is at least about 8 g / L.In some embodiments, collection of the target protein begins when the titer is at least about 8.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 9 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 9.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 10 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 10.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 11 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 11.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 12 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 12.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 13 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 13.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 14 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 14.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 15 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 15.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 16 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 16.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 17 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 17.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 18 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 18.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 19 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 19.5 g / L. In some embodiments, collection of the target protein begins when the titer is at least about 20 g / L.

[0107] In some embodiments, the methods disclosed herein include the collection of a target protein, wherein the titer of the target protein is within a certain range. In some embodiments, the titer of the collected target protein is between about 0.05 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.1 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.2 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.3 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.4 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.5 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.6 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.7 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.8 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.9 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 1 g / L and about 20 g / L. In some embodiments, the titer of the collected target protein is between about 0.05 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.1 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.2 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.3 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.4 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.5 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.6 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.7 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.8 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.9 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 1 g / L and about 15 g / L. In some embodiments, the titer of the collected target protein is between about 0.05 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.1 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.2 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.3 g / L and about 10 g / L.In some embodiments, the titer of the collected target protein is between about 0.4 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.5 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.6 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.7 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.8 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 0.9 g / L and about 10 g / L. In some embodiments, the titer of the collected target protein is between about 1 g / L and about 10 g / L.

[0108] In some embodiments, the methods disclosed herein further comprise stopping the collection of the target protein when the collected titer is below about 0.5 g / L.

[0109] In some embodiments, the yield of the target protein is increased by the methods disclosed herein. In some embodiments, the yield of the target protein is increased by at least about 1% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 2% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 3% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 4% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 5% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 6% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 7% compared to the protein yield. In some embodiments, the yield of the target protein is increased by at least about 8% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 9% compared to the protein yield. In some embodiments, the yield of the target protein is increased by at least about 10% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 11% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 12% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 13% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 14% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 15% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 16% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 17% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the yield of the target protein is increased by at least about 18% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time.In some embodiments, the target protein yield is increased by at least about 19% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time. In some embodiments, the target protein yield is increased by or at least about 20% compared to the protein yield in the case where the ultraviolet (UV) signal of the sample mixture is not monitored in real time.

[0110] In some embodiments, the ultraviolet (UV) signal of the sample mixture is measured, which is from 0 to 2 AU. In other embodiments, the UV signal of the sample mixture is measured, which is about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9 AU, about 1.0 AU, about 1.1 AU, about 1.2 AU, about 1.3 AU, about 1.4 AU, about 1.5 AU, about 1.6 AU, about 1.7 AU, about 1.8 AU, about 1.9 AU or about 2.0 AU.

[0111] In some embodiments disclosed herein, the method includes protein filtration. In some embodiments, the method includes one or more filters. In some embodiments, the protein filtration is depth filtration. In some embodiments, the depth filtration includes a primary depth filter and a secondary depth filter. In some embodiments, the depth filtration includes a primary depth filter.

[0112] In some embodiments, the method includes loading the sample mixture before monitoring.

[0113] 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 comprising a control system, wherein when the titer is higher than 0.5 g / L, the control system automatically starts collecting the protein. In some embodiments, the method includes a harvest skid comprising a control system, wherein when the titer is lower than 0.5 g / L, the control system automatically stops collecting the protein.

[0114] In some embodiments, the method includes a control system that regulates the flow rate of the liquid through the harvest skid. In some embodiments, the method includes a control system that automatically drives a pump to increase the flow rate through the harvest skid. In some embodiments, the method includes a control system that automatically drives a pump to decrease the flow rate through the harvest skid. In some embodiments, the method does not include the step of gas venting.

[0115] In some embodiments, the method includes the step of collecting the protein yield not based on volume.

[0116] In some embodiments, the methods disclosed herein include measuring pressure, turbidity, temperature, flow rate, or any combination thereof.

[0117] In some embodiments, the method includes measuring pressure using a pressure sensor. In some embodiments, the measured pressure ranges from -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 can be measured at least once, twice, three times, four times, or five times, such as before a primary filter, after the primary filter and before a secondary filter, after the secondary filter, after drainage, or any combination thereof.

[0118] In some embodiments, the method includes measuring turbidity. In some embodiments, the measured turbidity ranges from 0 absorbance units (AU) to 2 AU. In other embodiments, the measured turbidity is about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9 AU, about 1.0 AU, about 1.1 AU, about 1.2 AU, about 1.3 AU, about 1.4 AU, about 1.5 AU, about 1.6 AU, about 1.7 AU, about 1.8 AU, about 1.9 AU, or about 2.0 AU. In some embodiments, turbidity is measured at least once, twice, three times, four times, or five times, such as after a primary filter, after a secondary filter, or after a primary filter and after a secondary filter. See Figure 2 。

[0119] In some embodiments, the method includes measuring temperature. In some embodiments, the measured temperature ranges from 0 °C to 70 °C, 0 °C to 60 °C, 0 °C to 50 °C, 0 °C to 40 °C, 5 °C to 70 °C, 10 °C to 70 °C, 15 °C to 70 °C, 20 °C to 70 °C, 10 °C to 60 °C, 20 °C to 50 °C, 20 °C to 40 °C, 20 °C to 45 °C, 30 °C to 40 °C, 35 °C to 40 °C, 20 °C to 30 °C, 35 °C to 40 °C, or 25 °C to 45 °C. In other embodiments, the temperature can be measured at any time during the filtration process, such as at least once, twice, three times, four times, or five times, such as after a primary filter, after a secondary filter, or after a primary filter and after a secondary filter. See Figure 2 。

[0120] In some embodiments, the method includes measuring a flow rate. In some embodiments, the measured flow rate ranges from 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, 0 L / min to 110 L / min, 0 L / min to 120 L / min, 0 L / min to 130 L / min, 0 L / min to 140 L / min, 0 L / min to 150 L / min, 0 L / min to 160 L / min, 0 L / min to 170 L / min, 0 L / min to 180 L / min, 0 L / min to 190 L / min, 0 L / min to 200 L / min, 0 L / min to 250 L / min, or 0 L / min to 300 L / min. In other embodiments, the flow rate is measured at any time during the filtration process: before the primary filter, after the primary filter, before the secondary filter, after the secondary filter, or any combination thereof.

[0121] In some embodiments, the liquid from the water source / bioreactor / PBS source is driven by a gravity pump to the primary depth filter.

[0122] In some embodiments, a system such as Delta V TM can be employed to calculate the cumulative volume of the flow rate through the online flow sensor readings. In some embodiments, the cumulative volume of the flow rate is used to determine the end of the water rinse. In some embodiments, four pressure sensors are placed before the primary depth filter, the secondary depth filter, the pre-filter, and the sterile filter. The pressure-flow control loop can operate based on the real-time pressure value before the primary depth filter. If the pressure value exceeds a certain threshold, Delta V TM automatically drives the pump to decrease the flow rate. In some embodiments, two turbidity sensors are placed after the primary and secondary depth filters as indicators of the filtrate quality. In some embodiments, a UV sensor is placed after the secondary depth filter, and the value of this UV sensor is used to calculate the online target protein concentration and control the cut-off point for collecting the clarified stock solution. The weights of the real-time upstream source and the downstream receiving container are monitored and also displayed on DeltaV TM . In some embodiments, the weight is a monitor from 0 to 550 kg with a measurement accuracy of 0.01 kg.

[0123] In some embodiments, proteins are separated from a source. In some embodiments, the sample mixture is selected from pure protein samples, clarified crude protein samples, cell culture samples, and any combination thereof. In some embodiments, the source is selected from cultured cells.

[0124] In some embodiments, the cells are prokaryotes. In a bacterial system, depending on the intended use of the expressed protein molecule, many expression vectors can be advantageously selected. For example, when large amounts of such a protein are to be produced, for generating a pharmaceutical composition of the protein molecule, a vector that directs the expression of a high level of a protein product that is easy to purify may be required.

[0125] In other embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), simian kidney fibroblasts (COS-7), Madin-Darby bovine kidney cells (MDBK), and any combination thereof. In some embodiments, the cells are Chinese hamster ovary cells. In some embodiments, the cells are insect cells, such as Spodoptera frugiperda cells.

[0126] In other embodiments, the cells are mammalian cells. Such mammalian cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0, CRL7O3O, COS (such as COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0127] In some embodiments, the mammalian cells are CHO cells. In some embodiments, the CHO cells are CHO-DG44, CHOZN, CHO / dhfr-, CHOK1SV GS-KO, or CHO-S. In some embodiments, the CHO cells are CHO-DG4. In some embodiments, the CHO cells are CHOZN.

[0128] Other suitable CHO cell lines disclosed herein include CHO-K (such as CHO K1), CHO pro3-, CHO P12, CHO-K1 / SF, DUXB11, CHO DUKX; PA-DUKX; CHO pro5; DUK-BII, or derivatives thereof.

[0129] In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 1x10 6 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 5x10 6 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 1x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 1.5x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 2x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 2.5x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 3x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 3.5x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 4x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 4.5x10 7 cells / mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 5x10 7 cells / mL.

[0130] In some embodiments, the source of the protein is a crude protein. In some embodiments, the source of the protein is a composition comprising protein and non-protein components. The non-protein components can include DNA and other contaminants.

[0131] 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 from human tissue or cells. 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 farm animals. In certain embodiments, such terms refer to humans.

[0132] In some embodiments, the protein purified by the methods described herein is a fusion protein. A "fusion" or "fused" protein comprises a first amino acid sequence joined in-frame to a second amino acid sequence, where the first amino acid sequence is not naturally joined to the second amino acid sequence in nature. 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 fusion polypeptide in a new arrangement. Fusion proteins are produced, for example, by chemical synthesis or by generating and translating a polynucleotide encoding peptide regions in the desired relationship. A fusion protein can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent bond, a non-peptide bond, or a non-covalent bond. After transcription / translation, a single protein is made. 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 operable linkage between two polypeptides fuses the two polypeptides in-frame to produce a single polypeptide fusion protein. In a particular aspect, the fusion protein further comprises a third polypeptide that can comprise a linker sequence, as discussed in further detail below.

[0133] In some embodiments, the protein purified by the methods described herein is an antibody. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetravalent antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain - antibody heavy chain pairs, intracellular antibodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the foregoing. In certain embodiments, the antibodies described herein refer to a polyclonal antibody population. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies or a class thereof (e.g., human IgG1 or IgG4) or a subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In certain embodiments, the antibodies described herein are IgG1 or IgG4 antibodies.

[0134] In some embodiments, the proteins described herein are "antigen-binding domains", "antigen-binding regions", "antigen-binding fragments", and like terms, which refer to a portion of an antibody molecule that contains the amino acid residues (e.g., complementarity determining regions (CDRs)) that confer specificity for an antigen molecule against the antigen. The antigen-binding region can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans.

[0135] 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.

[0136] 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 has the CDR sequence of 6C8, e.g., a humanized antibody having the CDRs of 6C8, e.g., as described in WO2006 / 105021; and an antibody comprising the CDRs of the anti-GITR antibody described in WO2011 / 028683; an antibody comprising the CDRs of the anti-GITR antibody described in JP2008278814, an antibody comprising the CDRs of the anti-GITR antibody described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, WO 2018 / 013818, or other anti-GITR antibodies described or mentioned herein, all of which are incorporated herein by reference in their entirety.

[0137] In other embodiments, the protein is an anti-LAG3 antibody. Lymphocyte activation gene 3, also known as LAG-3, is a protein encoded by the LAG3 gene in humans. LAG3 was discovered in 1990 and is a cell surface molecule with multiple biological roles in T cell function. It is an immune checkpoint receptor and is thus the target of multiple drug development programs by pharmaceutical companies seeking to develop new therapies for cancer and autoimmune disorders. It has also been developed as an anti-cancer drug in soluble form alone. Examples of anti-LAG3 antibodies include, but are not limited to, the antibodies in WO 2017 / 087901 A2, WO 2016 / 028672 A1, WO2017 / 106129 A1, WO 2017 / 198741 A1, US 2017 / 0097333 A1, US 2017 / 0290914 A1, and US2017 / 0267759 A1, all of which are incorporated herein by reference in their entirety.

[0138] In some embodiments, the protein is an anti-CXCR4 antibody. CXCR4 is a 7-transmembrane protein coupled to G1. CXCR4 is widely expressed on cells of hematopoietic origin and is a major co-receptor for human immunodeficiency virus type 1 (HIV-1) with CD4+. See Feng, Y., Broeder, C.C., Kennedy, P.E., and Berger, E.A. (1996) Science 272, 872-877. Examples of anti-CXCR4 antibodies include, but are not limited to, the antibodies in WO 2009 / 140124 A1, US 2014 / 0286936A1, WO 2010 / 125162 A1, WO 2012 / 047339 A2, WO 2013 / 013025 A2, WO 2015 / 069874 A1, WO2008 / 142303 A2, WO 2011 / 121040 A1, WO 2011 / 154580 A1, WO 2013 / 071068 A2, and WO2012 / 175576 A1, all of which are incorporated herein by reference in their entirety.

[0139] In some embodiments, the protein is an anti-CD73 (ecto-5'-nucleotidase) antibody. In some embodiments, the anti-CD73 antibody inhibits the formation of adenosine. Degradation of AMP to adenosine leads to the generation of an immunosuppressive and pro-angiogenic niche in the tumor microenvironment, thus promoting the onset and development of cancer. Examples of anti-CD73 antibodies include, but are not limited to, the antibodies in WO2017 / 100670 A1, WO 2018 / 013611 A1, WO 2017 / 152085 A1, and WO 2016 / 075176 A1, all of which are incorporated herein by reference in their entirety.

[0140] In some embodiments, the protein is an anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) antibody. TIGIT is a member of the immunoglobulin protein PVR (poliovirus receptor) family. TIGIT is expressed on several types of T cells, including follicular helper T cells (TFH). The protein has been shown to bind to PVR with high affinity; this binding is thought to contribute to 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 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.

[0141] 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 plays a role in T cell antigen-presenting cell (APC) interactions and mediates the adhesion of activated T cells to endothelial cells. Examples of anti-OX40 antibodies include, but are not limited to, WO 2018 / 031490 A2, 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, WO 2015 / 153514 A1, WO 2016 / 002820 A1, and WO 2016 / 200835 A1, all of which are incorporated herein in their entirety.

[0142] In some embodiments, the protein is an anti-IL8 antibody. IL-8 is a chemokine that can attract neutrophils, basophils, and T cells, but not monocytes. It is also involved in neutrophil activation. In response to inflammatory stimuli, it is released from several cell types.

[0143] In some embodiments, the protein is abatacept (under For sale). Abatacept (also simply referred to as Aba herein) 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. To activate T cells and generate an immune response, antigen-presenting cells must present two signals to T cells. One of these signals is the major histocompatibility complex (MHC) that binds to the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7-2).

[0144] In some embodiments, the protein is belatacept (trade name ). Belatacept is a fusion protein composed of the Fc fragment of human IgG1 immunoglobulin linked to the extracellular domain of CTLA-4. Belatacept is an important molecule that regulates T cell co-stimulation and can selectively block the T cell activation process. It is designed to provide extended graft and transplant survival while limiting the toxicity produced by standard immunosuppressive regimens (such as calcineurin inhibitors). It differs from abatacept by only 2 amino acids.

[0145] c. System

[0146] In some embodiments, a system for controlling, regulating, increasing, or improving the protein yield in a sample mixture containing a target protein and impurities is disclosed herein. The system includes real-time monitoring of the ultraviolet (UV) signal of the sample mixture during protein filtration performed in a harvest skid.

[0147] The system disclosed herein includes one or more sensors. In some embodiments, the sensors include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

[0148] In some embodiments, the harvest skid is designed to integrate all sensors into a trolley, and the sensors include pressure (4), UV (1), turbidity (2), temperature (2), and flow sensors (1). In some embodiments, the system includes three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controllers are built on the trolley to accommodate a total of ten different sensors. In some embodiments, a gravity pump (e.g., gravity pump) is used to drive the liquid to a depth filter and is installed on the skid. In some embodiments, the system is movable, lockable, and / or electronically stoppable.

[0149] The present disclosure also provides a system (e.g., a device, e.g., a harvest sled) that can be used in the above methods. In one embodiment, a system or device includes the embodiments in FIGS. 1a and / or 1b. In one embodiment, a system or device includes Figure 2 embodiments of

[0150] In some embodiments, the present disclosure provides a device for controlling, regulating, increasing, or improving protein yield in a sample mixture containing a target protein and impurities. The device can include one or more sensors. The sensors can include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

[0151] In some embodiments, the device is designed to integrate all sensors into the device, the sensors including pressure (4), UV (1), turbidity (2), temperature (2), and flow sensors (1). In some embodiments, the device includes three PMAT (Pressure Monitor Alarm Transmitter) controllers. In some embodiments, the PMAT controllers are built into the device to accommodate a total of ten different sensors. In some embodiments, a gravity pump (e.g., gravity pump) is used to drive liquid to a depth filter and is installed in the device. In some embodiments, the device is movable, lockable, and / or electronically stoppable. The device can also include a processor configured to control the collection of the target protein. The processor can also be configured to change the conditions of the device, such as temperature, pressure, turbidity, or flow. The processor can also be configured to control the collection of the target protein. In some embodiments, the processor can use an established model to determine the cell culture harvest process. The cell culture harvest process can include a filtration-based cell culture harvest process. The processor can be configured to use the target protein titer. The device can be incorporated into a system for controlling, regulating, increasing, or improving protein yield in a sample mixture containing a target protein and impurities.

[0152] d. Process

[0153] In one embodiment, the system or device includes Figure 2 embodiments of which demonstrate the process flow using such a harvest sled. By a gravity pump drives liquid from a water source / bioreactor / PBS source into a depth filter. A TMCalculate the flow cumulative volume through the online flow sensor readings. The flow cumulative volume is used to determine the end of the water rinse. Four pressure sensors are placed in front of the primary depth filter, secondary depth filter, pre-filter, and sterile filter. The pressure-flow control loop operates based on the real-time pressure value before the primary depth filter. Two turbidity sensors are placed after the primary and secondary depth filters as indicators of the filtrate quality. A UV sensor is placed after the secondary depth filter to calculate the online target protein concentration and control the cut-off point for collecting the clarified stock solution. The upstream source weight and the weight of the downstream receiving container are monitored in real-time and also displayed on Delta V TM on it.

[0154] The following embodiments are provided by way of example and not limitation. Embodiment

[0155] Embodiment 1: Harvest Skid Design

[0156] To control, regulate, increase, or improve the protein yield in a sample, a harvest skid is utilized. Figure 1 shows a schematic diagram of the harvest skid. The harvest skid is designed to integrate all sensors (including pressure (4), ultraviolet (1), turbidity (2), temperature (2), and flow sensors (1)) into a trolley. See Figure 2 . Three PMAT controllers are built into the trolley to accommodate a total of ten different sensors. The gravity pump used to drive the liquid to the depth filter is also installed on the skid. The harvest skid is designed to be movable, lockable, and capable of emergency stop.

[0157] Table 1: Instruments Used for Designing the Skid

[0158]

[0159] Table 2: Instruments and Materials Used During Harvest

[0160]

[0161] The sensors used for harvest have different functions. The pressure sensors monitor the pressure during the process. The cascade controls the inlet pump to reduce the flow rate of the inlet pump when the pressure is too high. The UV sensor monitors the UV signal after depth filtration during the process; this UV signal is converted to protein concentration to control the start and end of collecting the stock solution. The UV is the measurement value at 280 nm.

[0162] The weight of the upstream bioreactor and the weight of the downstream receiver are monitored during the weight sensor monitoring process; the loading and hunting steps are controlled. The bioreactor load cell value and the receiver load cell value have been integrated into the harvest skid control system. The turbidity sensor measures the turbidity at 880 nm and monitors the turbidity before and after depth filtration during the process. If the depth filter becomes clogged, a turbidity breakthrough can be observed. The temperature sensor monitors the temperature during the process. The harvest process herein is carried out at ambient (room) temperature.

[0163] The harvest skid process is used to purify the protein of interest from the cell culture. Through The gravity pump drives the liquid from the water source / bioreactor / PBS source to the primary depth filter. It has been confirmed that, compared with the peristaltic pump P3P, the gravity pump causes less cell death in CHO cell cultures. The flow sensor is placed behind the pump. Delta V TM Calculates the flow cumulative volume through the online flow sensor readings. The flow cumulative volume is used to determine the end of the water rinse. Four pressure sensors are placed in front of the primary depth filter, secondary depth filter, pre-filter, and sterile filter P4P. The pressure-flow control loop works based on the real-time pressure value before the primary depth filter. If the pressure value exceeds a certain threshold, Delta V TM will automatically drive the pump to lower the pump speed. Two turbidity sensors are placed after the primary and secondary depth filters as an indicator of the filtrate quality. A UV sensor (whose value is used to calculate the online target protein concentration and control the cut-off point for collecting the clarified stock solution) is placed after the secondary depth filter. The weight of the real-time upstream source and the weight of the downstream receiving container are monitored and also displayed on Delta V TM screen.

[0164] For each embodiment disclosed herein, the harvest skid uses each sensor to detect values within the following ranges and accuracies.

[0165] Table 3.

[0166] Sensor Function Measurement range Measurement accuracy Pressure Monitor and control -7 to 30 psi Less than 0.9 psi Flow rate Monitor and control 0 to 20 L / min Less than 0.18 L / min UV Monitor and control 0 to 2 AU 0.02 AU Weight Monitor and control 0 to 550 kg 0.01 kg Turbidity Monitor 0 to 2 AU 0.02 AU Temperature Monitor 0 to 70 °C 0.2℃

[0167] Example 2: Converting the UV signal to protein concentration

[0168] Compared with the previous method, the method disclosed herein eliminates the gas venting step. At the same time, the start and end of collecting the clarified stock solution are automatically controlled according to the online UV readings and the calculated titer. See Figure 3. More specifically, the generated model is used to calculate the real-time target protein concentration during the harvesting process based on the online UV sensor readings. Therefore, the cut-off point for the collection of the stock solution is directly determined according to the calculated online target protein concentration. The calculation algorithm has been integrated into the Delta V TM control system to obtain an automatic cut-off point for the collection of clarified stock solution.

[0169] The online UV sensor used in this harvesting skid has an output absorbance of 0 - 2 AU. The path length of this UV sensor is adjusted to accommodate a total target protein concentration of 0 - 6 g / L within the range of 0 - 2 AU. Other UV sensors or Flow VPE (C technology) can be used for the determination of higher concentrations.

[0170] To convert the online UV signal in the process into the target protein concentration, a series of consecutive steps are taken, as Figure 4 shown.

[0171] In the first step, off-line titer measurements were determined for a series of diluted samples of the D12GITR cell culture against the online UV signal. Several components in the cell culture sample, including the target protein, HCP (host cell protein), and media pigments, can affect the UV absorption signal. To simulate the real-life harvesting process (where the UV sensor measures the total absorbance of all these components), the cell culture sample (D12GITR cell culture) rather than pure protein was serially diluted and used for the adjustment of the UV sensor path length.

[0172] As Figure 5 shown, the path length of the UV sensor was adjusted to cover a wide range of target protein concentrations that can be observed during the harvesting process. Since the UV reading accuracy is low near 2 AU (the maximum output), the path length was lowered so that the UV reading was approximately 1.6 at a titer of 5 g / L. Good linearity was observed, with R 2 being 0.97. Therefore, the serial dilution of the cell culture sample provides a strong correlation between the UV reading and the titer.

[0173] Example 3: Small-scale testing using pure protein and clarified stock solution

[0174] Small-scale testing was carried out using 2 L of pure protein (eTau) with a titer of 5.2 g / L. The depth filter was scaled down based on a loading capacity of 60 L / m 2 (per primary filter). Off-line samples were harvested after the secondary depth filter during the harvesting process. The off-line titer readings were plotted against the online UV sensor values to understand the relationship between the pure protein concentration and the online UV signal during the harvesting process.

[0175] A second small-scale harvest trial was conducted using 2 L of clarified stock solution (cell-free eTau cell culture) with a titer of 5 g / L. The depth filters were scaled down based on a loading capacity of 60 L / m 2 (per primary filter). Offline samples were collected after the secondary depth filter during the harvest process. The offline titer readings were plotted against the online UV sensor values to understand the relationship between the target protein concentration (in the mixed species of the culture components) and the online UV signal during the harvest process.

[0176] The online UV and offline titer values during the test harvest process are plotted in Figures 6a and 6b. The "up-slope" data series was collected from the start of stock solution collection to the end of loading; while the "down-slope" data series was collected from the start of chase to the end of stock solution collection. As shown in Figures 6a and 6b, good linearity was observed for both processes for the up-slope and down-slope portions of the data. Thus, serial dilution of the cell culture samples can provide a strong correlation between the UV readings and the titer when measuring other samples (e.g., pure protein in Figure 6a and clarified stock solution protein in Figure 6b).

[0177] However, the slopes are different for the up-slope and down-slope portions, indicating that different models may be required for different stages of the harvest process.

[0178] Example 4: Model building using three large-scale cell culture processes

[0179] Three different cell lines were used for model building. These cell lines included different, large-scale cell culture processes (Aba NGP, GITR, and Next GenCXCR4) with different characteristics (cell density, viability, titer, background noise, etc.). The cell lines were harvested using the harvest skid to generate data for model building. The depth filters were scaled proportionally based on a loading capacity of 60 - 65 L / m 2 (per primary filter). Offline samples were collected after the secondary depth filter during the harvest process. The offline titer readings and the corresponding online UV sensor values were input into JMP software to generate a model. The UV and titer values are plotted in Figures 7a, 7b, and 7c. Good linearity was still observed for the up-slope portion of the data. However, for the down-slope portion, curvature was observed.

[0180] During the loading of cell culture materials, cells, target proteins, and background noise proteins all occupy the depth filter membrane space. When the PBS chase begins, the target proteins are washed out. As the PBS chase progresses, background noise proteins (such as HCP) loosely bound to the depth filter membrane start to be washed out together with the target proteins. At the same time, the release of HCP from cell debris also leads to an increase in the background noise percentage P5P. This may be the reason for the difference in the UV spectra between the clarified bulk sample and the cell culture sample. In other words, for complex cell-containing substances, as the PBS chase progresses, the contribution of the target protein to the total UV signal becomes smaller and smaller, while the background noise becomes higher and higher.

[0181] Based on these results, two separate models were established to predict the target protein concentration using the online UV signal: one is a linear model to fit the data from the upward-sloping part (from the start of collection to the end of loading), and the other is a non-linear model to fit the data from the downward-sloping part (from the start of the chase to the end of collection).

[0182] A. Model fitting for the upward-sloping part.

[0183] For the upward-sloping part of the data, a total of 22 samples were included in the model. The offline titer values were plotted against the online UV values (Figure 8). A linear fit was applied to the data. The R 2 value was 0.98. Using this model, the predicted titer values were calculated and compared with the actual titer values. As shown in Figures 8a and 8b, the fitting slope was very close to 1, and the R 2 was 0.98.

[0184] To predict the target protein concentration from the start of collection to the end of loading, a linear model was generated: the predicted titer of the model = a + b * (online UV signal). The model constants a and b depend on the titer level. If the titer is about 3.5 g / L or less, then a = -0.35 and b = 2.88; if the titer is about 3.5 g / L or higher, then a = -0.69 and b = 4.06.

[0185] B. Model fitting for the downward-sloping part

[0186] For the downward-sloping part of the data, a total of 41 samples were included in the model. The offline titer values were plotted against the online UV values, as Figure 10 shown. A non-linear fit was applied to the data. For the CHOZN and DG44 cell lines, the RMSE values of the non-linear fit were 0.26 and 0.04, respectively. Using this model, the predicted titer values were calculated and compared with the actual titer values (Figures 9a and 9b). The fitting slope was close to 1, and the R 2 was 0.97 and 0.99. Figures 9a and 9b.

[0187] To predict the target protein concentration collected from the start of the chase to the end, a non-linear model was generated: Predicted titer by the model = A + B * exp(C * online UV signal). The model constants A, B, and C depend on the titer level. If the titer is about 3.5 g / L or less, then A = -0.95, B = 0.86, C = 1.21; if the titer is about 3.5 g / L or higher, then A = 0.02, B = 0.13, C = 2.41.

[0188] Example 5: Test models for four large-scale cell culture processes

[0189] Four large-scale (500 L) cell culture processes (CD73, OX40, TIGIT, and IL8) were harvested. The depth filters were scaled up based on preliminary small-scale data. Offline samples were collected after the secondary depth filters during the harvest process for actual titer measurements. The online UV sensor values were input into JMP software. Using this model, the predicted titer values were calculated based on the online UV sensor values and compared with the offline titer measurement values.

[0190] Table 4: Cell culture process characteristics of the molecules tested in this report

[0191]

[0192] Note: The viability calculation here is as follows: Viability (%) = VCD on the harvest day / peak VCD * 100%.

[0193] Table 5. Model fit evaluation for seven studied molecules

[0194]

[0195] The above-generated model was tested using four different large-scale (500 L) cell culture harvest processes, where the starting titer was 0 - 0.1 g / L and the ending titer was 0.1 - 0.2 g / L. The model can be tested down to 0.01 g / L based on a UV signal of 0.01 Au. The predicted titer values by the model were compared with the actual titer values using JMP software. The model fit RMSE values for each process are as Figure 10 shown.

[0196] The difference between the predicted titer values by the model and the actual titer values was calculated. Figure 12 shows the differences for each tested process. The range of the overall average difference was 0.07 - 0.36 g / L, indicating that these models can be reliably applied to different processes with various characteristics, as shown in Table 2.

[0197] A. Using an online sensor to control the harvest process and improve the harvest yield

[0198] Table 6: Yield Improvement of Seven Molecules in Studies Using the New Harvest Skid

[0199]

[0200] The yield of the harvest process was calculated using the following equation:

[0201]

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

[0203] In this study, a harvest skid with real-time monitoring and control was designed, and the depth filtration harvest of several therapeutic proteins was examined.

[0204] Multiple online sensors were built into the harvest skid, and their real-time readings were integrated into the Delta V TM system to obtain automatic monitoring and control of key process parameters. A model for converting the online UV signal during different stages of the harvest process into real-time target protein concentration was generated in a series of experimental steps, which included: adjusting the UV sensor path length, pure protein testing, and complex cell culture sample testing. Then, the model was successfully tested using several large-scale harvest processes with a large number of process characteristics, including background noise level, product level, total cell density, and viability.

[0205] Using this new type of harvest skid and statistical model generated in this study, the clarification process of cell cultures was monitored and controlled in a quantitative manner, which significantly improved harvest robustness and protein yield. Online titer information itself is an important indicator of cell culture performance and can be used for immediate loading determination of protein A chromatography in downstream processing.

[0206] Example 6: Process of Real-Time Monitoring of New Proteins during Protein Harvest

[0207] A new target protein was selected to be clarified using the harvest skid. First, as Figure 2 shown, all the sensors on the harvest skid were connected in series to monitor pressure, flow rate, UV, turbidity, and temperature during the harvest process. Second, the water source was connected to a gravity pump. The total flow rate and flow velocity were input into the Delta V TM to detect the depth filter flushing step. After reaching the total flow rate, the bioreactor source was connected to a gravity pump to start loading the cell culture into the depth filter. Third, the UV prediction model constant for the upper inclined part was input into the Delta V TM ; and the collection start cut-off threshold was input into the Delta V TMDuring loading, the on-line UV signal is converted to the target protein concentration. After reaching the threshold, the receiving vessel is connected to a sterile filter to collect the clarified bulk solution. Fourth, after emptying the bioreactor, the PBS source is connected to a gravity pump to initiate the chase step. Based on the cell line type, the UV prediction model constant for the lower slope portion is input into Delta V TM ; and the collection end cut-off threshold is input into Delta V TM During the chase, the on-line UV signal is converted to the target protein concentration. Once the threshold is reached, the receiving vessel is disconnected from the process stream. During the entire harvest process, pressure, turbidity, and temperature are monitored to indicate out-of-control issues.

[0208] Example 7: Confirming the On-line Predicted Titer from the On-line UV Signal by Off-line Titer Analysis

[0209] The harvest process begins with a water for injection (WFI) rinse of the depth filter. The UV sensor is connected to the outlet of the secondary depth filter. Once filtration is stable, clear flow is seen at the outlet; at this point, the UV sensor is zeroed. After rinsing the desired amount of WFI through the filter, the cell culture medium is connected to the filter inlet to initiate loading. The on-line UV trace of the culture medium is monitored along the loading process. Along the loading process, filtrate samples are taken and analyzed off-line by titer determination. Figure 11 Shows the titer trace obtained by modeling based on the UV signal. The titer trace obtained by modeling matches well with the off-line titer determination results and can thus be used to start and end collection to improve process robustness and yield.

[0210] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. 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., N.Y.); 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 MouseEmbryo,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,(1986););Crooks,Antisense drug Technology:Principles,strategies and applications,2. nd Ed.CRC Press(2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0211] In some embodiments, an apparatus for controlling, regulating, increasing, or improving protein yield in a sample mixture comprising a target protein and impurities is disclosed herein. The apparatus may include one or more sensors. The sensors may include pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.

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

[0213] All references cited above, as well as all references and amino acid or nucleotide sequences cited herein (e.g., GenBank accession numbers and / or Uniprot accession 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, the method comprising, during a filtration-based cell culture harvest process, real-time monitoring of the ultraviolet (UV) signal of the sample mixture and automatically converting the UV signal into the target protein titer using an established model, the filtration-based cell culture harvest process comprising a control system, wherein according to the established model and the control system, the UV signal is continuously converted into the titer of the target protein, wherein the target protein is produced in a culture containing a mammal, and wherein the target protein concentration is generated using the titer predicted by the model, the titer predicted by the model comprising: (1) Constants (a) and (b), wherein the target protein concentration is calculated according to Equation (I): Model predicted titer = a + b*(online UV signal); and (i) The mammalian cell is CHO-DG44, and (a) is -0.35 and (b) is 2.88; or (ii) The mammalian cell is CHOZN, and (a) is -0.69 and (b) is 4.06; or (2) Constants (A), (B) and (C), wherein the target protein is calculated according to Equation (II): Model predicted titer = A + B*exp(C*online UV signal); and (i) The mammalian cell is CHO-DG44, and (A) is -0.95, (B) is 0.86, and (C) is 1.21; or (ii) The mammalian cell is CHOZN, and (A) is 0.02, (B) is 0.13, and (C) is 2.41, wherein the target protein comprises an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody or an anti-IL8 antibody; or wherein the target protein comprises abatacept or belatacept.

2. A method for controlling the collection of a target protein and improving the protein yield in a sample mixture containing the target protein and impurities, the method comprising, during a filtration-based cell culture harvest process, real-time monitoring of the ultraviolet (UV) signal of the sample mixture, the filtration-based cell culture harvest process comprising a control system, wherein according to the established model and the control system, the UV signal is continuously converted into the titer of the target protein, wherein the target protein is produced in a culture containing a mammal, and wherein the target protein concentration is generated using the titer predicted by the model, the titer predicted by the model comprising: (1) Constants (a) and (b), wherein the target protein concentration is calculated according to Equation (I): Model predicted titer = a + b*(online UV signal); and (i) The mammalian cell is CHO-DG44, and (a) is -0.35 and (b) is 2.88; or (ii) The mammalian cell is CHOZN, and (a) is -0.69 and (b) is 4.06; or (2) Constants (A), (B), and (C), where the target protein is calculated according to formula (II): Model-predicted titer = A + B * exp(C * on-line UV signal); and (i) The mammalian cell is CHO-DG44, and (A) is -0.95, (B) is 0.86, and (C) is 1.21; or (ii) The mammalian cell is CHOZN, and (A) is 0.02, (B) is 0.13, and (C) is 2.41, where the target protein comprises an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, or an anti-IL8 antibody; or where the target protein comprises abatacept or belatacept.

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

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

5. The method according to claim 4, wherein the titer of the target protein collected is between 0.05 g / L and 20 g / L, between 0.1 g / L and 20 g / L, between 0.2 g / L and 20 g / L, between 0.3 g / L and 20 g / L, between 0.4 g / L and 20 g / L, between 0.5 g / L and 20 g / L, between 0.6 g / L and 20 g / L, between 0.7 g / L and 20 g / L, between 0.8 g / L and 20 g / L, between 0.9 g / L and 20 g / L, between 1 g / L and 20 g / L, between 0.05 g / L and 15 g / L, between 0.1 g / L and 15 g / L, between 0.2 g / L and 15 g / L, between 0.3 g / L and 15 g / L, between 0.4 g / L and 15 g / L, between 0.5 g / L and 15 g / L, between 0.6 g / L and 15 g / L, between 0.7 g / L and 15 g / L, between 0.8 g / L and 15 g / L, between 0.9 g / L and 15 g / L, or between 1 g / L and 15 g / L, between 0.05 g / L and 10 g / L, between 0.1 g / L and 10 g / L, between 0.2 g / L and 10 g / L, between 0.3 g / L and 10 g / L, between 0.4 g / L and 10 g / L, between 0.5 g / L and 10 g / L, between 0.6 g / L and 10 g / L, between 0.7 g / L and 10 g / L, between 0.8 g / L and 10 g / L, between 0.9 g / L and 10 g / L, or between 1 g / L and 10 g / L.

6. The method according to claim 1 or 2, further comprising stopping the collection of the target protein when the collected titer is below 0.1 or 0.2 g / L.

7. The method according to claim 1 or 2, 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 protein yield without real-time monitoring of the ultraviolet (UV) signal of the sample mixture.

8. The method according to claim 1 or 2, wherein the target protein is harvested from a culture medium having a cell density of at least 1 × 10 6 cells / mL, at least 5 × 10 6 cells / mL, at least 1 × 10 7 cells / mL, at least 1.5 × 10 7 cells / mL, at least 2 × 10 7 cells / mL, at least 2.5 × 10 7 cells / mL, at least 3 × 10 7 cells / mL, at least 3.5 × 10 7 cells / mL, at least 4 × 10 7 cells / mL, at least 4.5 × 10 7 cells / mL or at least 5 × 10 7 cells / mL.

9. The method according to claim 1 or 2, wherein the protein filtration is depth filtration.

10. The method according to claim 9, wherein the depth filtration comprises a primary depth filter and / or a secondary depth filter.

11. The method according to claim 1 or 2, further comprising loading the sample mixture before the monitoring.

12. The method according to claim 1 or 2, further comprising rinsing the depth filter with water or buffer before loading the cell culture, and backflushing the depth filter after loading the cell culture.

13. The method according to claim 1 or 2, further comprising backflushing the sample mixture with phosphate buffered saline (PBS) or other buffer.

14. The method according to claim 1 or 2, wherein the control system regulates the flow rate of the liquid through the harvest skid.

15. The method according to claim 14, wherein the control system automatically drives the pump to increase the flow rate through the harvest skid.

16. The method according to claim 14, wherein the control system automatically drives the pump to decrease the flow rate through the harvest skid.

17. The method according to claim 1 or 2, wherein the method does not include the step of gas venting.

18. The method according to claim 1 or 2, wherein the target protein titer or the protein yield is not volume-based.

19. A method for increasing, controlling, or regulating the protein yield in a sample mixture comprising a target protein and impurities, comprising a) rinsing a harvest skid including a control system with water; b) loading the sample into the harvest skid; c) measuring the ultraviolet (UV) signal of the sample mixture during protein filtration in the harvest skid as a real-time protein titer; d) starting to collect the protein based on the UV measurement value and the real-time protein titer; e) backflushing the protein with PBS; and f) stopping collecting the protein based on the UV measurement value and the real-time protein titer; wherein during the filtration, the UV signal is related to the real-time protein titer, wherein the target protein is produced in a culture comprising a mammal, and wherein the target protein concentration is generated using a model-predicted titer, the model-predicted titer comprising: (1) constants (a) and (b), wherein the target protein concentration is calculated according to Equation (I): Model-predicted titer = a + b*(online UV signal); and (i) the mammalian cell is CHO-DG44, and (a) is -0.35 and (b) is 2.88; or (ii) the mammalian cell is CHOZN, and (a) is -0.69 and (b) is 4.06; or (2) constants (A), (B), and (C), wherein the target protein is calculated according to Equation (II): Model-predicted titer = A + B*exp(C*online UV signal); and (i) the mammalian cell is CHO-DG44, and (A) is -0.95, (B) is 0.86, and (C) is 1.21; or (ii) the mammalian cell is CHOZN, and (A) is 0.02, (B) is 0.13, and (C) is 2.41, wherein the target protein comprises an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody or an anti-IL8 antibody; or wherein the target protein comprises abatacept or belatacept.

20. The method according to claim 1, 2 or 19, further comprising measuring pressure, turbidity, temperature, flow rate or any combination thereof during the method.

21. The method according to claim 20, further comprising measuring pressure using a pressure sensor during the method.

22. The method according to claim 21, wherein the measured pressure ranges from -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.

23. The method according to claim 20, further comprising measuring turbidity during the method.

24. The method according to claim 23, wherein the measured turbidity ranges from 0 absorbance units (AU) to 2 AU.

25. The method according to claim 20, further comprising measuring temperature during the method.

26. The method according to claim 25, wherein the measured temperature ranges from 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.

27. The method according to claim 21, further comprising measuring flow rate during the method.

28. The method according to claim 27, wherein the measured flow rate ranges from 0 L / min to 20 L / min, 0 L / min to 30 L / min, 0 L / min to 40 L / min, 0 L / min to 50 L / min, 0 L / min to 60 L / min, 0 L / min to 70 L / min, 0 L / min to 80 L / min, 0 L / min to 90 L / min, 0 L / min to 100 L / min, 0 L / min to 110 L / min, 0 L / min to 120 L / min, 0 L / min to 130 L / min, 0 L / min to 140 L / min, 0 L / min to 150 L / min, 0 L / min to 160 L / min, 0 L / min to 170 L / min, 0 L / min to 180 L / min, 0 L / min to 190 L / min, 0 L / min to 200 L / min, 0 L / min to 250 L / min, or 0 L / min to 300 L / min.

29. The method according to claim 14, wherein the harvesting sled comprises one or more filters.

30. The method according to claim 19, wherein the harvesting sled comprises one or more filters.

31. The method according to claim 29 or 30, wherein the filter comprises a primary depth filter and a secondary depth filter.

32. The method according to claim 1, 2 or 19, wherein the sample mixture comprises a pure protein sample, a clarified crude protein sample, a cell culture sample, or any combination thereof.

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