Product quality attribute measurement

Abstract

A system for measuring a product quality attribute of an analyte of a biological sample includes: a first flow control device; a sample purification device; a second flow control device in fluid communication with the first and second sample analyzers, wherein the first sample analyzer comprises a first chromatographic column; and a control unit configured such that during operation of the system, the control unit adjusts a configuration of the second flow control device to direct a portion of the biological sample to one of the first and second sample analyzers, and determining a product quality attribute of an analyte of the biological sample based on the analysis of the portion of the biological sample by the one of the first and second sample analyzers.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080080043.9 (application date: 2020 / 9 / 22, invention name: product quality attribute measurement).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 904,682, filed on September 23, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to systems and methods for product quality attribute measurement of samples, including samples harvested from a continuous biomanufacturing system. Background Art

[0005] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce therapeutically or commercially important proteins. Integrated continuous biomanufacturing is an important aspect of reducing the costs associated with such protein-based therapies. Monitoring systems are used in biomanufacturing to evaluate various biological products and process conditions. Summary of the invention

[0006] Integrated continuous biomanufacturing of therapeutic protein substances and other biomolecules offers great promise for the future production of life-saving drugs and for facilitating widespread adoption of therapies that rely on the availability of such biomolecules. Dual-column and multi-column chromatography systems with various configurations are available for industrial-scale biomanufacturing. In such systems, analysis of the eluate from the chromatography system can be used to determine a variety of product quality attributes, to monitor and regulate a variety of bioprocessing conditions.

[0007] The present disclosure features methods and systems for determining one or more product quality attributes of analytes in biological samples, including samples harvested from a bioreactor and offline samples introduced into the system in series or in parallel. A variety of product quality attributes can be measured, including but not limited to analyte concentration, analyte charge variation or heterogeneity, analyte aggregation, and analyte integrity or purity. The system can include a sample analyzer with different types of chromatographic columns that are dedicated to the measurement of specific product quality attributes. The measured product quality attributes can be used to provide feedback and control of biomanufacturing process related parameters and operations.

[0008] In one aspect, the disclosure features a system for measuring a product quality attribute of an analyte of a biological sample, the system featuring: a first flow control device; a sample purification device in fluid communication with the first flow control device; a second flow control device in fluid communication with the first flow control device, the sample purification device, and first and second sample analyzers, wherein the first sample analyzer includes a first chromatographic column; and a control unit coupled to the first and second flow control devices and configured such that during operation of the system, the control unit: (a) adjusts a configuration of the first flow control device to direct a portion of the biological sample from the first flow control device into the sample purification device or into the second flow control device such that the portion of the biological sample is received by the second flow control device; (b) adjusts a configuration of the second flow control device to direct the portion of the biological sample to one of the first and second sample analyzers; and (c) determines a product quality attribute of the analyte of the biological sample based on an analysis of the portion of the biological sample by the one of the first and second sample analyzers.

[0009] Embodiments of the system may include any one or more of the following features.

[0010] The first chromatographic column may be a cation exchange chromatographic column, a size exclusion chromatographic column, or a reverse phase chromatographic column. The sample purification device may include an affinity chromatographic column.

[0011] The second sample analyzer may include a quantitative detector configured to generate an electrical signal representing the amount of the analyte in the biological sample. The first chromatographic column may be in fluid communication with the quantitative detector, and the quantitative detector may be configured to generate an electrical signal representing the amount of the analyte in the elution stream from the first chromatographic column.

[0012] The first sample analyzer may include a quantitative detector in fluid communication with the first chromatographic column and configured to generate an electrical signal representative of the amount of the analyte in the elution stream from the first chromatographic column. The second sample analyzer may include a second chromatographic column, and the second chromatographic column may be different from the first chromatographic column and may be one of a cation exchange chromatographic column, a size exclusion chromatographic column, a reverse phase chromatographic column, and a hydrophilic interaction chromatographic column.

[0013] The second flow control device can be fluidically connected to a third sample analyzer including a third chromatography column, and the third chromatography column can be different from the first and second chromatography columns and can be one of a cation exchange chromatography column, a size exclusion chromatography column, a reverse phase chromatography column, and a hydrophilic interaction chromatography column.

[0014] The second flow control device may be in fluid communication with four additional sample analyzers, each of which is characterized in that it includes a chromatographic column that is different from the first chromatographic column and from the chromatographic columns of other sample analyzers of the four additional sample analyzers.

[0015] The product quality attribute of the analyte may be the concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variation or heterogeneity of the analyte in the biological sample, or a measure of purity or integrity of the analyte in the biological sample.

[0016] The affinity chromatography column may be one of a protein A chromatography column, a protein G chromatography column, and a receptor binding column. The analyte may include a protein (eg, an antibody) in the biological sample.

[0017] The system may include a column manager in fluid communication with the first and second sample analyzers and the second flow control device and coupled to the control unit, wherein the control unit is configured to adjust a configuration of the column manager to direct the portion of the biological sample into one of the first and second sample analyzers.

[0018] The system may include a column manager in fluid communication with the first, second, and third sample analyzers and the second flow control device, and coupled to the control unit, wherein the control unit may be configured to adjust a configuration of the column manager to direct the portion of the biological sample into one of the first, second, third, and fourth sample analyzers.

[0019] The portion of the biological sample may be a first portion, and the product quality attribute may be a first product quality attribute, and the control unit may be configured such that during operation of the system, the control unit: (d) adjusts the configuration of the first flow control device to direct a second portion of the biological sample from the first flow control device to the sample purification device or the second flow control device so that the second portion of the biological sample is received by the second flow control device; (e) adjusts the configuration of the second flow control device to direct the second portion of the biological sample to one of the first and second sample analyzers that did not receive the first portion of the biological sample; and (f) determines a second product quality attribute of the analyte of the biological sample based on analysis of the second portion of the biological sample by the one of the first and second sample analyzers that received the second portion of the biological sample. The first and second product quality attributes may be different, and the first and second product quality attributes may each be selected from the group consisting of a concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variation or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

[0020] The portion of the biological sample may be a first portion and the product quality attribute may be a first product quality attribute, and the control device may be configured to repeat steps (a)-(c) for another portion of the biological sample to determine two different product quality attributes of the analyte of the biological sample.

[0021] The portion of the biological sample may be a first portion and the product quality attribute may be a first product quality attribute, and the control device may be configured to repeat steps (a)-(c) for two other portions of the biological sample to determine three different product quality attributes of the analyte of the biological sample.

[0022] The portion of the biological sample may be a first portion and the product quality attribute may be a first product quality attribute, and the control device may be configured to repeat steps (a)-(c) for three other portions of the biological sample to determine four different product quality attributes of the analyte of the biological sample.

[0023] The product quality attributes can each be selected from the group consisting of a concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variation or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

[0024] The system may include a sampling device coupled to the control unit and configured to receive the biological sample and deliver the portion of the biological sample to the first fluid control device. The sampling device may include a container interface configured to receive the biological sample in a container. The sampling device may include a fluid channel configured to receive the biological sample, and the control unit may be configured such that during operation of the system, the control unit sends a signal to the sampling device to cause the sampling device to discharge the portion of the biological sample from the fluid channel into the first flow control device.

[0025] The system may include a pump that is fluidly connected to the second flow control device, the first and second sample analyzers, and first and second buffer reservoirs associated with the first and second sample analyzers, respectively, wherein the control unit and the pump are configured so that during operation of the system, when the portion of the biological sample is directed into one of the first and second sample analyzers, the pump delivers a buffer solution from the corresponding associated buffer reservoir to one of the first and second sample analyzers.

[0026] The system may include a pump that is fluidly connected to the second flow control device, the first, second and third sample analyzers, and first, second and third buffer reservoirs associated with the first, second and third sample analyzers, respectively, wherein the control unit and the pump are configured so that during operation of the system, when the portion of the biological sample is directed into one of the first, second and third sample analyzers, the pump delivers a buffer solution from the corresponding associated buffer reservoir to the one of the first, second and third sample analyzers.

[0027] The system may include a pump in fluid communication with the second flow control device, the first sample analyzer, and a buffer reservoir associated with the first sample analyzer, wherein the first chromatographic column is a cation exchange column, and wherein the control unit and the pump are configured such that during operation of the system, the pump delivers an acetate buffer to the first chromatographic column to propagate the portion of the biological sample along the first chromatographic column. The acetate buffer may have a pH of 4.0 or less.

[0028] The biological sample can be a harvested culture medium extracted from a bioreactor. The biological sample can be an intermediate solution or a product solution from a biomanufacturing system. The biological sample can be a portion of a cell culture.

[0029] The quantitative detector may include a diode array detector, a spectral detector configured to measure absorbance information of the portion of the biological sample, a fluorescence detector, and / or a mass spectrometry detector.

[0030] Embodiments of the system may also include any other features described herein, including any combination of features disclosed individually in different embodiments, unless explicitly stated otherwise.

[0031] In another aspect, the disclosure features a system for measuring multiple product quality attributes of an analyte of a biological sample, the system featuring a first flow control device; a sample purification device comprising a purification chromatography column in fluid communication with the first flow control device; a second flow control device in fluid communication with the first flow control device and the sample purification device; a first sample analyzer comprising a first chromatography column in fluid communication with the second flow control device; a second sample analyzer comprising a second chromatography column in fluid communication with the second flow control device; a third sample analyzer comprising a third chromatography column in fluid communication with the second flow control device; a fourth sample analyzer comprising a quantitative detector; and a control unit coupled to the first and second flow control devices and configured such that during operation of the system, the control unit: (a) adjusts the configuration of the first flow control device to (a) directing a first portion of the biological sample from the first flow control device into the sample purification device or into the second flow control device so that the portion of the biological sample is received by the second flow control device; (b) adjusting the configuration of the second flow control device to direct the first portion of the biological sample to one of the first, second, third, and fourth sample analyzers; (c) determining a first product quality attribute of an analyte of the biological sample based on analysis of the portion of the biological sample by the one of the first, second, third, and fourth sample analyzers; and (d) repeating steps (a)-(c) for three additional portions of the biological sample, adjusting the configuration of the second flow control device so that each portion of the biological sample is directed to a different one of the sample analyzers to determine a total of four product quality attributes of the analyte of the biological sample.

[0032] Embodiments of the system may include any one or more of the following features.

[0033] The four product quality attributes may be different. The first, second and third chromatographic columns may be different types of columns. The first chromatographic column may be a cation exchange column, the second chromatographic column may be a size exclusion column, and the third chromatographic column may be a reverse phase column or a hydrophilic interaction column.

[0034] The first sample analyzer may determine information regarding a measure of charge variation or heterogeneity of the analyte in the biological sample, the second sample analyzer may determine information regarding a measure of aggregation of the analyte in the biological sample, the third sample analyzer may determine information regarding a measure of purity or integrity of the analyte in the biological sample, and the fourth sample analyzer may determine information regarding the concentration of the analyte in the biological sample.

[0035] The four product quality attributes may include a measure of charge variation or heterogeneity of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of purity or integrity of the analyte in the biological sample, and a concentration of the analyte in the biological sample. The first chromatographic column may be a cation exchange chromatographic column, the second chromatographic column may be a size exclusion chromatographic column, and the third chromatographic column may be a reverse phase chromatographic column.

[0036] The sample purification device may include an affinity chromatography column. The analyte may include a protein in the biological sample. The protein may include an antibody in the biological sample.

[0037] The system may include a column manager in fluid communication with the first, second, and third sample analyzers and the second flow control device and coupled to the control unit, wherein the control unit is configured to adjust a configuration of the column manager to direct the portion of the biological sample into one of the first, second, and third sample analyzers. The system may include a sampling device coupled to the control unit and configured to receive the biological sample and deliver the portion of the biological sample to the first fluid control device.

[0038] The quantitative detector may include one of a diode array detector, a spectral detector configured to measure absorbance information of the portion of the biological sample, a fluorescence detector, and a mass spectrometry detector.

[0039] Embodiments of the system may also include any other features described herein, including any combination of features disclosed individually in different embodiments, unless explicitly stated otherwise.

[0040] In another aspect, the disclosure features a method for measuring a product quality attribute of an analyte of a biological sample, the method comprising obtaining a biological sample by extracting the biological sample from an operating bioreactor or from a purification device in fluid communication with the operating bioreactor, directing a first portion of the biological sample to a first sample analyzer, and obtaining information about a first product quality attribute of the analyte of the biological sample by analyzing the first portion of the biological sample in the first sample analyzer, directing a second portion of the biological sample to a second sample analyzer, and obtaining information about a second product quality attribute of the analyte of the biological sample by analyzing the second portion of the biological sample in the second sample analyzer, wherein the first and second product quality attributes are different, and wherein at least one of the first and second product quality attributes includes a measure of charge variation or heterogeneity of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of purity or integrity of the analyte in the biological sample, and a concentration of the analyte in the biological sample.

[0041] Embodiments of the method may also include any other features described herein, including any combination of features disclosed individually in different embodiments, unless explicitly stated otherwise.

[0042] definition

[0043] The term "unit operation" is a term of art that refers to a functional step that can be performed in a process for manufacturing a therapeutic protein drug from a liquid culture medium. For example, an operation unit can be filtration (e.g., removing contaminating bacteria, yeast, viruses or mycobacteria and / or particulate matter from a fluid containing a recombinant therapeutic protein), capture, removal of epitope tags, purification, holding or storage, polishing, virus inactivation, adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein, and removing unwanted salts.

[0044] The term "cycle of chromatography" or "chromatographic cycle" is a term of art and means all steps performed in a round of chromatography using a single chromatography column. For example, a chromatography cycle can include the steps of equilibrating the chromatography column with a buffer, passing a sample comprising a recombinant protein through the chromatography column, eluting the recombinant protein from the chromatography column, and washing the chromatography column by passing a denaturing buffer through the chromatography column. Additional examples of steps performed in a chromatography cycle are described herein. Further examples of steps performed in a chromatography cycle are also well known in the art.

[0045] The term "capture" means steps performed to partially purify or separate (e.g., at least or about 5% pure by weight (e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least or about 95%) pure), concentrate, and stabilize a recombinant therapeutic protein from one or more other components present in a liquid culture medium or a diluted liquid culture medium (e.g., culture medium proteins or one or more other components present in or secreted from mammalian cells (e.g., DNA, RNA, or other proteins)). Typically, capture is performed using a resin that binds the recombinant therapeutic protein (e.g., by using affinity chromatography). Non-limiting methods for capturing a recombinant therapeutic protein from a liquid culture medium or a diluted liquid culture medium are described herein, and other methods are known in the art. The recombinant therapeutic protein can be captured from a liquid culture medium using at least one chromatographic column and / or chromatographic membrane (e.g., any chromatographic column and / or chromatographic membrane described herein).

[0046] The term "purification" means a step performed to separate a recombinant therapeutic protein from one or more other impurities (e.g., major impurities) or components present in a fluid containing the recombinant therapeutic protein (e.g., liquid culture medium proteins or one or more other components present in or secreted from mammalian cells (e.g., DNA, RNA or other proteins, endotoxins, viruses, etc.). For example, purification can be performed during or after an initial capture step. Purification can be performed using a resin, membrane, or any other solid support that binds the recombinant therapeutic protein or contaminants (e.g., by using affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). The recombinant therapeutic protein can be purified from a fluid containing the recombinant therapeutic protein using at least one chromatographic column and / or chromatographic membrane (e.g., any chromatographic column or chromatographic membrane described herein).

[0047] The term "refining" is a term of art and means a step performed to remove residual trace or small amounts of contaminants or impurities from a fluid containing a recombinant therapeutic protein close to the final desired purity. For example, refining can be performed by passing the fluid containing the recombinant therapeutic protein through one or more chromatographic columns or one or more membrane absorbers, which selectively bind the target recombinant therapeutic protein or small amounts of contaminants or impurities present in the fluid containing the recombinant therapeutic protein. In this example, the eluate / filtrate of the one or more chromatographic columns or one or more membrane absorbers contains the recombinant therapeutic protein.

[0048] The term "filtration" means removing at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of undesirable biological contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses, or mycobacteria) and / or particulate matter (e.g., precipitated proteins) from a liquid (e.g., a liquid culture medium or fluid in any system or process described herein).

[0049] The term "eluate / filtrate" is a term of art and means the fluid released from a chromatography column or chromatography membrane, which fluid contains a detectable amount of the recombinant therapeutic protein.

[0050] The term "isolate" or "isolated" in certain contexts means at least partially purified or purified (e.g., at least or about 5%, such as at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95% pure by weight) from one or more other components present in a filtrate (e.g., a filtrate produced using the presently described methods), such as one or more components of DNA, RNA and / or other proteins present in the filtrate. Non-limiting methods for separating proteins from filtrates are described herein, and other methods are known in the art.

[0051] The term "integrated process" means a process that uses structural elements that act synergistically to achieve a specific result (eg, production of a therapeutic protein drug from a liquid culture medium).

[0052] The term "continuous process" means a process in which a fluid is continuously fed through at least a portion of a system. For example, in any of the exemplary continuous biomanufacturing systems described herein, a liquid culture medium containing a recombinant therapeutic protein is continuously fed into the system, and a therapeutic protein drug substance is fed out of the system while the system is operating. In another example, a continuous process is a process in which a liquid culture medium containing a recombinant therapeutic protein is continuously fed from a bioreactor through a first MCCS. Another example of a continuous process is a process in which a liquid culture medium containing a recombinant therapeutic protein is continuously fed from a bioreactor through a first and a second MCCS. Additional examples include a process in which a liquid culture medium containing a recombinant therapeutic protein is continuously fed through a first MCCS, a process in which a liquid culture medium containing a recombinant therapeutic protein is continuously fed through a first and a second MCCS, or a process in which a fluid containing a recombinant therapeutic protein is continuously fed through a second MCCS.

[0053] The term "biological manufacturing system" or "bio-manufacturing system" refers to a system used to produce biological drugs.

[0054] The term "biopharmaceutical" means any therapeutic substance made or obtained from a living organism or its products for the prevention, diagnosis or treatment of a pathology. Thus, a biopharmaceutical or biopharmaceutical is a medical drug produced using biotechnology, for example, a protein (e.g., a recombinant therapeutic protein) or a nucleic acid (DNA, RNA or antisense oligonucleotide) for therapeutic or in vivo diagnostic purposes.

[0055] The term "multi-column chromatography system" or "MCCS" means a system having a total of two or more interconnected or switched chromatographic columns and / or chromatographic membranes. A non-limiting example of a multi-column chromatography system is a periodic countercurrent chromatography system (PCC), which comprises a total of two or more interconnected or switched chromatographic columns and / or chromatographic membranes. Other examples of multi-column chromatography systems are described herein and are known in the art.

[0056] The term "mammalian cell" means any cell from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow or rabbit). In some embodiments, the mammalian cell can be, for example, an immortalized cell, a differentiated cell or an undifferentiated cell.

[0057] The term "cell culture" means a plurality of mammalian cells (eg, any of the mammalian cells described herein) suspended in a liquid culture medium (eg, any of the liquid culture medium described herein). The cell culture can have a cell density of greater than about 0.1 x 106 cells / mL (e.g., greater than about 1.0 x 106 cells / mL, greater than about 5.0 x 106 cells / mL, greater than about 10 x 106 cells / mL, greater than about 15 x 106 cells / mL, greater than about 20 x 106 cells / mL, greater than about 25 x 106 cells / mL, greater than about 30 x 106 cells / mL, greater than about 35 x 106 cells / mL, greater than about 40 x 106 cells / mL, greater than about 45 x 106 cells / mL, greater than about 50 x 106 cells / mL, greater than about 55 x 106 cells / mL, greater than about 60 x 106 cells / mL, greater than about 65 x 106 cells / mL, greater than about 70 x 106 cells / mL, greater than about 75 x 106 cells / mL, greater than about 76 x 106 cells / mL, greater than about 77 x 106 cells / mL, greater than about 78 x 106 cells / mL, greater than about 79 x 106 cells / mL, greater than about 80 x 106 cells / mL, greater than about 81 x 106 cells / mL, greater than about 82 x 106 cells / mL, greater than about 83 x 106 cells / mL, greater than about 84 x 106 cells / mL, greater than about 86 x 106 cells / mL, greater than about 88 x 106 cells / mL, greater than about 89 x 106 cells / mL, greater than about 90 x 106 cells / mL, greater than about 91 x 106 cells / mL, greater than about 92 x 106 cells / mL, greater than about 93 x 106 cells / mL 106 cells / mL, greater than about 80x106 cells / mL, greater than about 85x 106 cells / mL, greater than about 90x 106 cells / mL, greater than about 95x 106 cells / mL, greater than about 100x 106 cells / mL).

[0058] The terms "culturing" or "cell culture" mean the maintenance or growth of mammalian cells in a liquid medium under a controlled set of physical conditions.

[0059] The term "liquid culture medium" means a fluid containing enough nutrients to grow mammalian cells in an in vitro culture medium. For example, the liquid culture medium may include one or more of the following: amino acids (e.g., 20 kinds of amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvic acid, lipoic acid, magnesium, glucose, sodium, potassium, ions, copper, zinc, selenium, and other necessary trace metals and sodium bicarbonate. The liquid culture medium may contain serum from mammals. In some cases, the liquid culture medium does not contain serum or other extracts (liquid culture medium of determined components) from mammals. The liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Non-limiting examples of liquid culture mediums are described herein, and other examples are known in the art and are commercially available.

[0060] The term "immunoglobulin" means a polypeptide comprising an immunoglobulin having an amino acid sequence (e.g., a variable domain sequence, a framework sequence, or a constant domain sequence) of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids). An immunoglobulin may, for example, include a light chain immunoglobulin of at least 15 amino acids, such as a heavy chain immunoglobulin of at least 15 amino acids. An immunoglobulin may be an isolated antibody (e.g., IgG, IgE, IgD, IgA, or IgM). An immunoglobulin may be a subclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). An immunoglobulin may be an antibody fragment, such as a Fab fragment, a F(ab')2 fragment, or a scFv fragment. An immunoglobulin may also be a bispecific antibody or a trispecific antibody, or a dimer, trimer, or multimer antibody, or a diabody, or An immunoglobulin may also be an engineered protein (eg, a fusion protein) comprising at least one immunoglobulin domain.Non-limiting examples of immunoglobulins are described herein, and other examples of immunoglobulins are known in the art.

[0061] The term "recombinant therapeutic protein" or "recombinant protein" refers to any therapeutic protein obtained by recombinant DNA technology. As used herein, "recombinant therapeutic protein" includes, for example, antibodies or antibody fragments, enzymes, engineered proteins, or immunogenic proteins or protein fragments.

[0062] The term "protein fragment" or "polypeptide fragment" means a portion of a polypeptide sequence that is at least or about 4 amino acids, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids in length, or more than 20 amino acids in length. Recombinant protein fragments can be produced using any of the processes described herein.

[0063] The term "engineered protein" means a polypeptide that is not naturally encoded by an endogenous nucleic acid present in an organism (e.g., a mammal). Examples of engineered proteins include enzymes (e.g., having one or more amino acid substitutions, deletions, insertions, or additions that increase the stability and / or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins comprising at least one recombinant scaffold sequence.

[0064] The term "secreted protein" or "secreted recombinant protein" means a protein (e.g., a recombinant protein) that, when translated in a mammalian cell, initially comprises at least one secretion signal sequence and is at least partially secreted into the extracellular space (e.g., liquid culture medium) in the mammalian cell at least in part by enzymatic cleavage of the secretion signal sequence. A "secreted" protein need not be completely dissociated from the cell to be considered a secreted protein.

[0065] The term "perfusion bioreactor" means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein the culture of the cells present in the bioreactor includes periodic or continuous removal of the first liquid culture medium and simultaneous or shortly thereafter addition of substantially the same volume of a second liquid culture medium to the bioreactor. In some examples, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium removed and added over an incremental time period (e.g., a time period of about 24 hours, a time period between about 1 minute and about 24 hours, or a time period greater than 24 hours) during the culture period (e.g., a daily refeed rate of the culture medium). The fraction of culture medium removed and replaced each day can vary depending on the specific cells cultured, the initial inoculation density, and the cell density at a particular time. "RV" or "reactor volume" means the volume of culture medium present at the beginning of the culture process (e.g., the total volume of culture medium present after inoculation).

[0066] The term "fed-batch bioreactor" is a term of art and means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein the cultivation of the cells present in the bioreactor includes the periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without substantially or significantly removing the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch cultivation, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch cultivation, the second liquid culture medium is added as a dry powder.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. Suitable methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of this paper's subject matter. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0068] The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the detailed description, drawing description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is a schematic diagram of an example of a system for determining a product quality attribute of an analyte of a biological sample.

[0070] Figure 2 is a schematic diagram of an example of a sample manager.

[0071] Figure 3 is a flow chart illustrating an exemplary set of steps for determining a product quality attribute of an analyte of a biological sample.

[0072] Figures 4A-4C is a schematic diagram showing an exemplary configuration of a flow control device.

[0073] Figure 5A is a graph showing potency chromatograms of a plurality of samples.

[0074] Figure 5B is a table showing the calculated mass loadings and concentrations for a number of samples.

[0075] Fig. 6A is a graph showing potency chromatograms of a plurality of samples.

[0076] Figure 6B is a set of tables with measured peak information for different samples.

[0077] Figure 7 is a graph showing the measured chromatograms of a set of samples.

[0078] Figure 8 is a schematic diagram illustrating an example of a biomanufacturing system.

[0079] Fig. 9 is a schematic diagram showing an example of a three-column switching technique.

[0080] Fig.10 Comparison of three titer methods for therapeutic monoclonal antibodies in 100L bioreactors (MIMICS-mPQA-blue; CEDEX TM Figure 2. Graphs of the MIMICs-mPQA (bioanalyzer - red; Octet - green). All data points plotted on MIMICs-mPQA are frozen harvests. Data gaps represent samples that were not analyzed.

[0081] Fig.11 Comparison of three titer methods (MIMICS-mPQA-blue; CEDEX- TM Figure 2. Graphs of the MIMICs-mPQA (bioanalyzer - red; Octet - green). All data points plotted on MIMICs-mPQA are frozen harvests. Data gaps represent samples that were not analyzed.

[0082] Fig.12 Comparison of three titer titration methods (MIMICS-mPQA-blue; CEDEX- TM Figure 2. Graphs of the MIMICs-mPQA (bioanalyzer - red; Octet - green). All data points plotted on MIMICs-mPQA are frozen harvests. Data gaps represent samples that were not analyzed.

[0083] Fig.13 Figure 1 is a graph comparing two aggregation methods (MIMICS-mPQA and offline method) for a 100L bioreactor of a therapeutic monoclonal antibody. The graph compares two assay results: monomer percentage and high molecular weight (HMW) percentage. All data points plotted on MIMICS-mPQA are from frozen harvest. Data gaps represent samples that were not analyzed.

[0084] Fig.14Figure 1 is a graph comparing two aggregation methods (MIMICS-mPQA and offline method) for a 3L bioreactor (V09) for a therapeutic monoclonal antibody. The graph compares two assay results: monomer percentage and high molecular weight (HMW) percentage. All data points plotted on MIMICS-mPQA are from frozen harvests. Data gaps represent samples that were not analyzed.

[0085] Fig.15 Figure 1 is a graph comparing two aggregation methods (MIMICS-mPQA and offline method) for a 3L bioreactor (V12) of a therapeutic monoclonal antibody. The graph compares two assay results: monomer percentage and high molecular weight (HMW) percentage. All data points plotted on MIMICS-mPQA are from frozen harvests. Data gaps represent samples that were not analyzed.

[0086] Fig.16 Figure 2 is a graph comparing the total % purity of the two purity methods (MIMICS-mPQA and offline method) from all bioreactors (100L, 3L V09, 3L V12) of a therapeutic monoclonal antibody. All data points plotted on MIMICS-mPQA are from frozen harvests. Data gaps represent samples that were not analyzed.

[0087] The same symbols in the drawings represent the same elements. DETAILED DESCRIPTION

[0088] introduction

[0089] Industrial-scale biomanufacturing can be performed in a variety of configurations in dual-column and multi-column chromatography systems. In these complex systems, product yield, quality, and scrap rate are functions of a large number of process-related parameters and steps. During the manufacture of therapeutic proteins and other commercially valuable biomolecules, product results can be strongly affected by these parameters and steps. Therefore, proper control of such parameters and steps is an important aspect of large-scale manufacturing. For example, features and aspects of biomanufacturing systems are disclosed in PCT Patent Application Publication No. WO2014 / 137903, the entire contents of which are incorporated herein by reference.

[0090] Monitoring of bioreactor harvest, intermediate solution streams and / or product facilitates appropriate control of biomanufacturing parameters, including automated control. Conventional monitoring techniques include, for example, UV absorbance measurements.

[0091] Unfortunately, these methods may drift over measurement periods of several days due to factors such as temperature, humidity, ambient light intensity, and local sample inhomogeneities. Furthermore, such methods may not allow for multiple quantities to be calculated or otherwise determined. In complex biomanufacturing environments, multiple quantities are often evaluated in order to provide appropriate feedback information for the adjustment of process parameters.

[0092] The present disclosure features systems and methods that can be used to determine the values ​​of multiple product quality attributes. The system can be implemented as a two-dimensional chromatography system. Along a first dimension of the system, a portion of a biological sample can be optionally purified using a sample purification device, which can include a chromatographic column. Along a second dimension of the system, the portion of the sample can then be directed to one of a plurality of different sample analyzers to determine the product quality attributes of the sample. Other portions of the sample can be directed to different sample analyzers to determine different product quality attributes of the sample.

[0093] Product quality attribute analysis system

[0094] Figure 1 1 is a schematic diagram showing an example of a measurement system 100 for measuring multiple product quality attributes. The system 100 includes a sample manager 102, a first pump 104 (e.g., a binary pump), a first flow control device 106, a sample purification device 108, a second flow control device 110, a column manager 112, a plurality of sample analyzers 114a-114d, a detector 116, a second pump 118 (e.g., a quaternary pump), solvent / buffer reservoirs 120a-120d, and a control unit 122. The sample manager 102, the pump 104, the first flow control device 106, the second flow control device 110, the column manager 112, and the detector 116 can be connected to the control unit 122 via communication lines 124a-124g.

[0095] During operation, sample manager 102 receives biological samples for analysis. Sample manager 102 can be implemented in various ways. For example, in some embodiments, sample manager includes a container receiver, which is configured to receive the sample in a container. Suitable containers include, for example, vials, test tubes and other sealed or unsealed containers. In certain embodiments, the sample can be carried by a single hole or multi-hole plate, and the container receiver is configured to receive such a plate. Sample manager 102 can optionally include a transfer mechanism for transferring part of the biological sample to the first flow control device 106. Suitable transfer mechanisms include, but are not limited to, a sample injection device based on a syringe and a single channel or multi-channel fluid transfer device. Examples of suitable sample managers include Waters H Class Sample Manager and Waters Process Sample Manager (both available from Waters Corp., Milford, MA) with a flow through needle.

[0096] In certain embodiments, sample manager 102 receives a biological sample from a sampling device that is in fluid communication with a bioreactor, a fluid conduit, or another component of a biomanufacturing system. For example, the biological sample can be harvested directly from a bioreactor (thus corresponding to a harvested sample of a growth medium), or can be a solution or culture medium extracted from another location in the biomanufacturing system.

[0097] Figure 2 1 is a schematic diagram of an example of a sample manager 102 configured to receive a biological sample directly from a sampling device. The sample manager 102 includes an inlet 202, a holding conduit 204, and a gate valve 206 coupled to the control unit 122 via a control line 124c. The biological sample is introduced into the sample manager 102 through the inlet 202, and the sample is maintained before being delivered to the first flow control device 106 within the holding conduit 204. To deliver a portion of the sample to the first flow control device 106, the control unit 122 sends a signal to the gate valve 206. The gate valve 206 opens, discharging a portion of the sample from the holding conduit 204 into the outlet conduit 208. The discharged portion of the sample is then pumped (e.g., by the first pump 104) to the first flow control device 106.

[0098] Typically, sample manager 102 can receive a variety of different biological samples. In some embodiments, as described above, the biological sample corresponds to a harvested portion of a growth medium from a bioreactor. In certain embodiments, the biological sample corresponds to a process fluid or medium extracted from another location in a biomanufacturing system, such as a product or intermediate-containing solution sampled before or after a purification stage in a biomanufacturing system.

[0099] In certain embodiments, system 100 can be used to determine product quality attributes for cell line development, and the biological sample corresponds to a portion of a cell culture, a cell culture medium, a fluid suspension of cells, or another type of sample in which cells, cell breakdown products, cell metabolites, and / or cell culture impurities are present.

[0100] The system 100 determines product quality attributes of one or more analytes in a biological sample received by the sample manager 102. In general, attributes can be determined for a variety of different types of analytes. For example, in some embodiments, the system 100 determines product quality attributes of protein analytes, including but not limited to antibodies (including monospecific, bispecific, and trispecific antibodies), non-antibody proteins, fusion proteins, and / or Fab fragments.

[0101] In some embodiments, the analyte is a recombinant therapeutic protein. Non-limiting examples of recombinant therapeutic proteins that can be analyzed using the systems and methods disclosed herein include immunoglobulins (including light and heavy chain immunoglobulins, antibodies or antibody fragments (e.g., any antibody fragment described herein)), enzymes (e.g., galactosidase (e.g., α-galactosidase), Myozyme or Cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF) or interferon α or β) or immunogenic or antigenic proteins or protein fragments (e.g., proteins for vaccines). Recombinant therapeutic proteins can be engineered antigen-binding polypeptides comprising at least one multifunctional recombinant protein scaffold (see, e.g., Gebauer et al., Current Opin. Chem. Biol. 13: 245-255, 2009; and recombinant antigen-binding proteins described in U.S. Patent Application No. 2012 / 0164066 (incorporated herein in their entirety by reference)).

[0102] Non-limiting examples of recombinant therapeutic proteins that are antibodies include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, acizumab、alemtuzumab、alirocumab、altumomab、amatuximab、amatuximab、anatumomab、anrukinzumab、apolizumab、arcitumomab、atinumab、tocilizumab、basilimab asilizimab, bectumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, biciromab, canakinumab, certolizumab, cetuximab, cixutumumab, daclizumab , denosumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomabtiuxetan), igovomab, imgatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab.

[0103] Other non-limiting examples of recombinant therapeutic proteins that can be analyzed include alglucosidase alfa, laronidase, abatacept, galsulase, luteinizing hormone alfa, anti-hemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, insulin or insulin analogs, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human serum albumin, erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin-11, laronidase, idursuphase, galsulphase, alpha-1 proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alfa (e.g., Other examples of recombinant proteins that can be produced by the method of the present invention include acid α-glucosidase, alglucosidase alpha (e.g. and ), α-L-iduronidase (such as ), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucosidase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (such as and ), galactosylceramidase, α-galactosidase-A (such as ), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

[0104] As described above, in some embodiments, the analyte is a component of a cell, and the methods and systems described herein can be used for cell line process development. Examples of such cells include, but are not limited to, bacteria (e.g., gram-negative bacteria), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, or Arxula adeninivorans), or mammalian cells. Mammalian cells can be cells grown in suspension or adherent cells. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells or CHO-K1 cells), Sp2.0, myeloma cells (e.g., NS / 0), B cells, hybridoma cells, T cells, human embryonic kidney (HEK) cells (e.g., HEK 293E and HEK 293F), African green monkey kidney epithelial cells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK).

[0105] Mammalian cells can contain recombinant nucleic acids encoding recombinant therapeutic proteins (e.g., nucleic acids stably integrated into the genome of mammalian cells). Non-limiting examples of recombinant nucleic acids encoding exemplary recombinant therapeutic proteins are described below, and the recombinant therapeutic proteins are recombinant therapeutic proteins that can be produced using the methods described herein. In some cases, the mammalian cells cultured in a bioreactor (e.g., any bioreactor described herein) are from a larger culture.

[0106] Nucleic acids encoding recombinant therapeutic proteins can be introduced into mammalian cells using a variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentivirus, adenovirus or retrovirus infection) and electroporation. In some cases, nucleic acids encoding recombinant therapeutic proteins cannot be stably integrated into the chromosomes of mammalian cells (transient transfection), while in other cases, nucleic acids are integrated. Alternatively or additionally, nucleic acids encoding recombinant therapeutic proteins can be present in plasmids and / or mammalian artificial chromosomes (e.g., human artificial chromosomes). Alternatively or additionally, viral vectors (e.g., lentivirus, retrovirus or adenovirus vectors) can be used to introduce nucleic acids into cells. Nucleic acids can be operably connected to promoter sequences (e.g., strong promoters, such as β-actin promoters and CMV promoters, or inducible promoters). If desired, the vector comprising nucleic acids can also include a selective marker (e.g., a gene that imparts resistance to mammalian cells hygromycin, puromycin or neomycin).

[0107] In some embodiments, the recombinant therapeutic protein is a secreted protein that is released into the extracellular medium by mammalian cells. For example, the nucleic acid sequence encoding a soluble recombinant therapeutic protein may include a sequence encoding an N-terminal or C-terminal secretory signal peptide of the recombinant therapeutic protein, which is cleaved by an enzyme present in mammalian cells and subsequently released into the extracellular medium.

[0108] After the biological sample is received by the sample manager 102, the sample is delivered by the first pump 104 through the conduit 126 to the first flow control device 106. The first flow control device 106 is connected to the control unit 122 via the control line 124b. Generally, the biological sample can be delivered to a plurality of different outputs via the first flow control device 106. In a first configuration, the first flow control device 106 delivers the biological sample directly to the second flow control device 110 via the conduit 128. In another configuration, the first flow control device 106 delivers the biological sample to the sample purification device 108 via the conduit 130. The control unit 122 is configured to adjust the configuration of the first flow control device 106 to direct the biological sample to any destination depending on the desired analysis mode of the biological sample.

[0109] The first flow control device 106 can be implemented in various ways. In some embodiments, for example, the first flow control device 106 can be implemented as a multi-way valve. Suitable valves include, for example, the 2-position, 6-port UltraLife switching valve of IDEX MX Series II (available from IDEX Corp., Lake Forest, IL). In certain embodiments, the first flow control device 106 can be implemented as a multi-channel fluidic device having an input and / or output manifold and an electronically controllable flow regulator.

[0110] When the biological sample is directed to the sample purification device 108, the biological sample is at least partially purified before the sample is analyzed in the system 100. Purification can be performed in a variety of ways, but generally includes removing one or more non-analyte components from the sample. Alternatively, or in addition, purification of the biological sample can also include concentration of the analyte in the sample, and separation of one analyte from one or more other analytes in the sample.

[0111] The sample purification device 108 can be implemented in various ways. In some embodiments, for example, the sample purification device 108 is implemented as a chromatographic device and is characterized by one or more chromatographic columns. Suitable chromatographic columns for the sample purification device 108 include, for example, affinity chromatography columns. The term "affinity chromatography" refers to a type of chromatography that captures and separates analyte molecules (e.g., recombinant protein analytes) based on affinity. Affinity chromatography refers to the use of affinity chromatography resins (e.g., affinity chromatography resins including protein ligands (e.g., protein A or protein G)). In some embodiments, affinity chromatography includes pseudo-affinity chromatography resins. In some embodiments, affinity chromatography resins include cofactor ligands, substrate ligands, metal ligands, product ligands, or aptamer ligands. Typically, affinity chromatography resins can include any receptor or ligand with affinity for any biological analyte, including DNA, oligonucleotides. In some embodiments, affinity chromatography resins can include single domain antibody fragments from Camelidae for purification of gene therapy vectors. As another example, the affinity chromatography column can be an adeno-associated virus (AAV) affinity chromatography column.

[0112] Non-limiting examples of affinity chromatography resins can include a protein or peptide ligand (e.g., between about 5 amino acids to about 100 amino acids, between about 5 amino acids to about 90 amino acids, between about 5 amino acids to about 80 amino acids, between about 5 amino acids to about 70 amino acids, between about 5 amino acids to about 60 amino acids, between about 5 amino acids to about 50 amino acids, between about 5 amino acids to about 40 amino acids, between about 5 amino acids to about 30 amino acids, or between about 5 amino acids to about 20 amino acids), a small molecule substrate or cofactor for an enzyme, an aptamer, an inhibitor (e.g., a competitive protein inhibitor), or a metal.

[0113] Non-limiting examples of protein A affinity chromatography resins include: GE MabSelect SuRe TM(a highly cross-linked agarose resin with a particle size of 85 μm, with epoxy functional groups attaching protein A to the agarose), JJSR LifeSciences Amsphere ProA JWT203 (a porous polymethacrylate resin with a particle size of approximately 50 μm, with epoxy functional groups attaching protein A to the polymethacrylate), and Kaneka KanCap A (a highly cross-linked cellulose with a particle size of 65-85 μm, with protein A attached to the cellulose by reductive amination).

[0114] For an affinity chromatography column, such as a Protein A column, the steps in an affinity chromatography cycle may include the steps of loading the affinity column, such as a Protein A chromatography column, with a fluid containing the analyte, washing the column to remove unwanted biological material (e.g., contaminating proteins and / or small molecules), eluting the target recombinant protein bound to the column, and re-equilibrating the column.

[0115] Any individual step in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any individual steps in a chromatography cycle can include a buffer gradient. Any combination of various well-known aspects of a single chromatography cycle can be used in these methods in any combination, for example, one or more different chromatography resins, one or more flow rates, one or more buffers, one or more void volumes of a column, one or more bed volumes of a column, one or more volumes of buffer used in each step, one or more volumes of fluid containing the target protein, and the amount and type of one or more buffers used in each step.

[0116] In some embodiments, the Protein A column can be loaded with 1x phosphate buffered saline (PBS) at a pH of about 7 (e.g., about pH 7.0 to about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, or about pH 7.9). In some embodiments, the Protein A column can be loaded with 1x PBS at a pH of about 7.2.

[0117] In some embodiments, the Protein A column is eluted with a buffer comprising about 50 mM to about 200 mM citrate phosphate (e.g., about 50 mM to about 190 mM, about 50 mM to about 180 mM, about 50 mM to about 170 mM, about 50 mM to about 160 mM, about 50 mM to about 150 mM, about 50 mM to about 140 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 75 mM to about 200 mM, about 75 mM to about 190 mM, about 50 mM to about 180 mM, about 50 mM to about 170 mM, about 50 mM to about 160 mM, about 50 mM to about 150 mM, about 50 mM to about 140 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 0mM, about 75mM to about 180mM, about 75mM to about 170mM, about 75mM to about 160mM, about 75mM to about 150mM, about 75mM to about 140mM, about 75mM to about 130mM, about 75mM to about 120mM, about 75mM to about 110mM, about 75mM to about 100mM, about 75mM to about 90mM, about 75mM to about 80mM, about 100mM to about 200mM, about 100mM to about 190mM, about 100mM to about 180mM, about 100mM to about 170mM, about 100mM to about 160mM, about 100mM to about 150mM, about 100mM to about 140mM, about 100mM to about about 130mM, about 100mM to about 120mM, about 100mM to about 110mM, about 125mM to about 200mM, about 125mM to about 190mM, about 125mM to about 180mM, about 125mM to about 170mM, about 125mM to about 160mM, about 125mM to about 150mM, about 125mM to about 140mM, about 125mM to about 130mM, about 150mM to about 200mM, about 150mM to about 190mM, about 150mM to about 180mM, about 150mM to about 170mM, about 150mM to about 160mM, about 175mM to about 200mM, about 175mM to about 190mM, about 175mM to about 180mM M to about 180mM, or about 180mM to about 200mM), about 50mM to about 200mM NaCL (e.g., about 50mM to about 190mM, about 50mM to about 180mM, about 50mM to about 170mM, about 50mM to about 160mM, about 50mM to about 150mM, about 50mM to about 140mM, about 50mM to about 130mM, about 50mM to about 120mM, about 50mM to about 110mM, about 50mM to about 100mM, about 50mM to about 90mM, about 50mM to about 80mM, about 50mM to about 70mM, about 50mM to about 60mM, about 75mM to about 200mM, about 75mM to about 190mM,about 75mM to about 180mM, about 75mM to about 170mM, about 75mM to about 160mM, about 75mM to about 150mM, about 75mM to about 140mM, about 75mM to about 130mM, about 75mM to about 120mM, about 75mM to about 110mM, about 75mM to about 100mM, about 75mM to about 90mM, about 75mM to about 80mM, about 100mM to about 200mM, about 100mM to about 190mM, about 100 mM to about 180mM, about 100mM to about 170mM, about 100mM to about 160mM, about 100mM to about 150mM, about 100mM to about 140mM, about 100mM to about 130mM, about 100mM to about 120mM, about 100mM to about 110mM, about 125mM to about 200mM, about 125mM to about 190mM, about 125mM to about 180mM, about 125mM to about 170mM, about 125mM to about 160mM 0mM, about 125mM to about 150mM, about 125mM to about 140mM, about 125mM to about 130mM, about 130mM to about 200mM, about 130mM to about 190mM, about 130mM to about 180mM, about 130mM to about 170mM, about 130mM to about 160mM, about 130mM to about 150mM, about 140mM to about 200mM, about 140mM to about 190mM, about 140mM to about 180mM, about 1 40mM to about 170mM, about 140mM to about 160mM, about 140mM to about 150mM, about 150mM to about 200mM, about 150mM to about 190mM, about 150mM to about 180mM, about 150mM to about 170mM, about 150mM to about 160mM, about 175mM to about 200mM, about 175mM to about 190mM, about 175mM to about 180mM, or about 180mM to about 200mM), pH about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.4, about pH 2 to about pH 3.2, about pH 2 to about pH 3.0, about pH 2 to about pH 2.8, about pH 2 to about pH 2.6, about pH 2 to about pH 2.4, about pH 2 to about pH 2.2, about pH 3 to about pH 4, about pH 3 to about pH 3.8, about pH 3 to about pH 3.6, about pH 3 to about pH 3.4, or about pH 3 to about pH 3.2).

[0118] In some embodiments, about 10 mM to about 100 mM (e.g., 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 15 mM to about 100 mM, about 15 mM to about 90 mM, about 15 mM to about 80 mM, about 15 mM to about 70 mM, about 15 ... mM to about 60mM, about 15mM to about 50mM, about 15mM to about 40mM, about 15mM to about 30mM, about 15mM to about 20mM, about 20mM to about 100mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 20mM to about 25mM, about 30mM to about 100mM, about 30mM to about 90mM, about 30mM to about 80mM, about 30mM to about 70mM, about 30mM to about 60mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM to about 100mM, about 50mM to about 90mM mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, or about 90 mM to about 100 mM) of sodium acetate at a pH of about The protein A column is eluted from a pH range of about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about pH 3.75, about pH 2 to about pH 3.6, about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.4, about pH 2 to about pH 3.2, about pH 2 to about pH 3.0, about pH 2 to about pH 2.8, about pH 2 to about pH 2.6, about pH 2 to about pH 2.4, about pH 2 to about pH 2.2, about pH 3 to about pH 4, about pH 3 to about pH 3.8, about pH 3 to about pH 3.75, about pH 3 to about pH 3.6, about pH 3 to about pH 3.4, or about pH 3 to about pH 3.2).

[0119] Chromatographic analysis using this type of chromatographic column can include, for example, sequential chromatographic steps that typically perform loading, washing, elution, and regeneration of the chromatographic column. Any of the exemplary flow rates, buffer volumes, and / or time lengths assigned to each sequential chromatographic step described herein can be used for any of these various sequential chromatographic steps.

[0120] In some embodiments, a single chromatography column or a single chromatography membrane comprising a resin capable of capturing an analyte is loaded, for example, between about 5 minutes to about 90 minutes (e.g., between about 10 minutes to about 90 minutes, between about 15 minutes to 80 minutes, between about 20 minutes to 80 minutes, between about 30 minutes to about 80 minutes, between about 40 minutes to about 80 minutes, and between about 50 minutes to 80 minutes).

[0121] After the analyte is loaded onto the post, the post is washed with at least one wash buffer. At least one (e.g., two, three or four) wash buffers are used to elute all compounds that are not the analyte from the post without interfering with the interaction of the analyte with the resin.

[0122] The wash buffer can be passed through the column at a flow rate of between about 0.1 mL / min to about 25 mL / min (e.g., between about 0.2 mL / min to about 20 mL / min, between about 0.5 mL / min to about 20 mL / min, between about 0.2 mL / min to about 15 mL / min, between about 0.5 mL / min to about 15 mL / min, between about 0.5 mL / min to about 10 mL / min, between about 0.5 mL / min to about 14 mL / min, between about 1.0 mL / min to about 25.0 mL / min, between about 1.0 mL / min to about 15.0 mL / min).

[0123] The volume of wash buffer used (e.g., the combined total volume of wash buffers used when more than one wash buffer is used) can be, for example, between about 1X column volume (CV) to about 15X CV (e.g., between about 1X CV to about 14X CV, between about 1X CV to about 13X CV, between about 1X CV to about 12X CV, between about 1X CV to about 11X CV, between about 2X CV to about 11X CV, between about 3X CV to about 11X CV, between about 4X CV to about 11X CV, between about 5X CV to about 11X CV, or between about 5X CV to about 10X CV). The total time for washing can be, for example, between about 2 minutes to about 3 hours (e.g., between about 2 minutes to about 2.5 hours, between about 2 minutes to about 2.0 hours, between about 5 minutes to about 1.5 hours, between about 10 minutes to about 1.5 hours, between about 10 minutes to about 1.25 hours, between about 20 minutes to about 1.25 hours, or between about 30 minutes to about 1 hour).

[0124] After washing the column, the analyte is eluted from the column by passing the elution buffer through the column. The elution buffer can be passed through the column at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.1 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, about 0.5 mL / min to about 6.0 mL / min, about 1.0 mL / min to about 5.0 mL / min, about 0.5 mL / min to about 14 mL / min, about 1.0 mL / min to about 25.0 mL / min, about 1.0 mL / min to about 15.0 mL / min). The volume of elution buffer used to elute the analyte from the column can be, for example, from about 1X column volume (CV) to about 15X CV (e.g., between about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV). The total time for elution can be, for example, between about 0.1 minutes and about 3 hours (e.g., between about 2 minutes and about 2.5 hours, between about 2 minutes and about 2.0 hours, between about 2 minutes and about 1.5 hours, between about 2 minutes and about 1.5 hours, between about 2 minutes and about 1.25 hours, between about 2 minutes and about 1.25 hours, between about 2 minutes and about 1 hour, between about 2 minutes and about 40 minutes, between about 10 minutes and about 40 minutes, between about 20 minutes and about 40 minutes, between about 0.1 minutes and about 10 minutes).

[0125] Non-limiting examples of elution buffers that can be used depend on the capture mechanism and / or the analyte. For example, the elution buffer can contain different concentrations of salt (e.g., increased salt concentration), different pH (e.g., increased or decreased salt concentration), or molecules that will compete with the analyte for binding to the resin. Examples of such elution buffers are described above.

[0126] After analyte is eluted from the chromatographic column, regeneration buffer can be used to balance the chromatographic column. Regeneration buffer can be passed through the post with a flow rate of, for example, about 0.1 mL / minute to about 25 mL / minute (for example, about 0.2 mL / minute to about 20 mL / minute, about 0.5 mL / minute to about 20 mL / minute, about 0.2 mL / minute to about 15 mL / minute, about 0.5 mL / minute to about 15 mL / minute, about 0.5 mL / minute to about 10 mL / minute, about 0.5 mL / minute to about 6.0 mL / minute, about 1.0 mL / minute to about 5.0 mL / minute, about 0.5 mL / minute to about 14 mL / minute, about 1.0 mL / minute to about 25.0 mL / minute, about 5.0 mL / minute to about 15.0 mL / minute, or about 1.0 mL / minute to about 15.0 mL / minute).

[0127] The volume of regeneration buffer used to equilibrate the column can be, for example, from about 1X column volume (CV) to about 15X CV (e.g., between about 1X CV to about 14X CV, about 1X CV to about 13X CV, about 1X CV to about 12X CV, about 1X CV to about 11X CV, about 2X CV to about 11X CV, about 3X CV to about 11X CV, about 2X CV to about 5X CV, about 4X CV to about 11X CV, about 5X CV to about 11X CV, or about 5X CV to about 10X CV).

[0128] In some embodiments, the sample purification device 108 includes a single affinity chromatography column. In certain embodiments, the sample purification device 108 includes multiple affinity chromatography columns. When multiple columns are used, the columns can be different (e.g., including different chromatographic resins), or can be the same. In addition, multiple columns can be loaded and / or eluted with the same solvent and buffer, or with different solvents and / or buffers. Each column in the multi-column sample purification device can include any one or more chromatographic resins described herein, and can be loaded and / or eluted with any one or more different solvents and buffers described herein.

[0129] As described above, the biological sample is delivered to the second flow control device 110 directly from the first flow control device 106 or through the sample purification device 108. The second flow control device 110 receives the biological sample and directs the biological sample along one of a plurality of flow paths based on a control signal from the control unit 122 transmitted on the control line 124d. The second flow control device 110 can generally be implemented in the same manner as the first flow control device 106 described above.

[0130] Typically, the second flow control device 110 directs the biological sample to one of a plurality of sample analyzers, such as Figure 1 As shown. Generally, the system 100 may include 2 or more sample analyzers (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more, 15 or more, or even more). Each sample analyzer is associated with the measurement of a product quality attribute of an analyte of a biological sample. Figure 1 , four different sample analyzers 114a-114d are shown by way of example. However, it should be understood that the system 100 may include any number of sample analyzers, depending on the number of product quality attributes to be measured.

[0131] In some embodiments, a particular sample analyzer does not include a chromatographic column. Figure 1 In the embodiment, the sample analyzer 114a does not include a chromatographic column. The biological sample can be delivered directly from the second flow control device 110 to the sample analyzer 114a. More specifically, when a product quality attribute associated with the sample analyzer 114a is to be measured, the control unit 122 sends a control signal to the second flow control device 110, adjusts the configuration of the second flow control device 110, and causes the biological sample to be delivered to the sample analyzer 114a. For the analyte in the biological sample, the product quality attribute associated with the sample analyzer 114a is measured.

[0132] In certain embodiments, a particular sample analyzer includes a chromatographic column. Figure 1 In the example, each of the sample analyzers 114b-114d includes a chromatographic column. As described above, in order to deliver the biological sample to such a sample analyzer, the sample may be delivered directly from the second flow control device 110 under the control of the control unit 122.

[0133] Optionally, in some embodiments, the system 100 optionally includes a column manager 112 that receives the biological sample from the second flow control device 110 and directs it to a sample analyzer. The column manager is particularly useful in a system that includes multiple sample analyzers having chromatographic columns. Figure 1 As shown, the column manager 112 can be coupled to the control unit 122 via a control line 124f. To introduce a biological sample into a sample analyzer associated with a particular product quality attribute to be measured, the control unit 122 can send a control signal to the column manager 112 to adjust the configuration of the column manager 112 to introduce the biological sample into the flow path of the sample analyzer.

[0134] The column manager 112 may optionally communicate with one or more reservoirs (for illustration purposes, in FIG. Figure 1The second pump 118 is connected to the fluid of the four containers 120a-120d shown in the figure, and the second pump can also be optionally connected to the control unit 122 through the control line 124e. When the biological sample is introduced into a specific sample analyzer, the control unit 122 also sends a control signal to the second pump 118 to cause the second pump 118 to introduce a flow of a suitable loading buffer, elution buffer or other suitable solvent or solution into the specific sample analyzer. Figure 1 Four reservoirs are shown by way of example, but it should be generally understood that the second pump 118 can be in fluid communication with 2 or more reservoirs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, 20 or more, or even more).

[0135] The second pump can generally be implemented in a variety of ways. For example, in some embodiments, a suitable second pump 118 is a Waters H-Class Bio Quaternary Pump (Waters Corp., Milford, MA) with an additional solvent selection valve modification.

[0136] After the biological sample is loaded onto the column of the selected chromatographic sample analyzer, the column is developed and eluted, and the eluate is analyzed to provide information about product quality attributes associated with the sample analyzer of the analytes of the biological sample.

[0137] The analysis of the eluate can be performed in a variety of ways. In some embodiments, for example, the system 100 includes a detector 116 in fluid communication with the outlet of the chromatographic column of the sample analyzer. The detector 116 detects the analyte in the eluate and provides measurement information to the control unit 122 via control line 124g. The control unit 122 uses the measurement information (which typically corresponds to a chromatogram and / or chromatographic information, such as detected peak heights, areas, and times) to determine the value of a specific product quality attribute of the analyte of the biological sample.

[0138] A variety of detectors can generally be used with the system 100. In some embodiments, the detector 116 corresponds to a photodiode array detector. Suitable diode array detectors for use in the system 100 include, but are not limited to, the Waters Photodiode Array Detector (Waters Corp., Milford, MA).

[0139] Other types of detectors may also be used. For example, in some embodiments, detector 116 corresponds to an absorption spectrometer that measures the absorbance of a biological sample (e.g., in at least one of the ultraviolet, visible, and infrared regions of the electromagnetic spectrum). In certain embodiments, detector 116 may be implemented as a fluorescence detector and include a light source for directing illumination light onto the biological sample, and a detection element for measuring fluorescence emission from the sample. In some embodiments, detector 116 may be implemented as a mass spectrometer, in which the biological sample is ionized, and the distribution of ions is resolved by mass to determine the abundance information of the biological sample. In certain embodiments, detector 116 may be implemented as a multi-angle light scattering detector or a refraction angle detector.

[0140] In some embodiments, each column-based sample analyzer can be in fluid communication with a different dedicated detector. Figure 1 As shown, two or more column-based sample analyzers can be in fluid communication with a common detector. That is, detector 116 can be shared between two or more sample analyzers because system 100 analyzes only a portion of a biological sample at a time.

[0141] In some embodiments, detector 116 is effectively used as a sample analyzer. For example, in Figure 1 In the example embodiment, the sample analyzer 114a may include a fluid conduit extending between the second flow control device 110 and the detector 116. When a sample is delivered to the sample analyzer 114a, the sample simply propagates through the fluid conduit and is then analyzed directly by the detector 116.

[0142] In general, in system 100, each sample analyzer is dedicated to measuring a specific product quality attribute of an analyte in a biological sample. The specific product quality attribute to be determined is selected by control unit 122, which adjusts the configuration of second flow control device 110 to direct a portion of the biological sample to one of the sample analyzers.

[0143] The system 100 can generally be configured to measure any number of product quality attributes, depending on the number of sample analyzers present in the system. For example, in certain embodiments, the system 100 can measure 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more, 12 or more, 15 or more, 20 or more, or even more) product quality attributes of analytes in a biological sample.

[0144] Product quality attributes

[0145] As described above, the system 100 can be used to measure a variety of product quality attributes of analytes in a biological sample. Depending on the nature of the sample analyzer present in the system 100, a variety of product quality attributes can be measured.

[0146] (a) Concentration or potency

[0147] In some embodiments, system 100 includes a sample analyzer for measuring the concentration or titer of an analyte in a biological sample. The concentration or titer of the analyte can be measured directly in the biological sample using any of the different types of detectors described above. Thus, for example, to measure Figure 1 To measure the concentration or titer of a biological sample, a portion of the biological sample can be delivered directly from the second flow control device 110 to the detector 116, that is, the sample analyzer can effectively be a fluid conduit extending between the second flow control device 110 and the detector 116.

[0148] (b) Charge variation or heterogeneity

[0149] In some embodiments, system 100 includes a sample analyzer that measures charge variation or heterogeneity of analytes in a biological sample. Charge variation / heterogeneity can be determined, for example, by a sample analyzer that includes a cation exchange column for performing cation exchange chromatography on the analytes in the sample.

[0150] The term "cation exchange chromatography" refers to a type of ion exchange chromatography that uses negatively charged ion exchange resins to separate molecules based on charge differences. In some embodiments, the cation exchange chromatography column is a strong cation exchange chromatography column, such as a HiTrap SP HP cation exchange chromatography column, a Mono S cation exchange chromatography column, or a Thermo MAbPac strong cation exchange chromatography column.

[0151] A chromatography cycle using a cation exchange chromatography column (e.g., a strong cation exchange chromatography column) wherein the analyte is bound to the chromatography resin in a loading step may include the steps of loading the column with a fluid comprising the analyte, washing the column to remove unwanted biological material, eluting the analyte bound to the column, and re-equilibrating the column. In certain embodiments, a chromatography cycle using a cation exchange chromatography column wherein unwanted biological material is bound to the chromatography resin in a loading step and the analyte is not bound may include the steps of loading the column with a fluid comprising a target protein, collecting the target recombinant protein in the flow-through, and re-equilibrating the column.

[0152] Any individual step in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any individual steps in a chromatography cycle can include a buffer gradient. Any combination of various well-known aspects of a single chromatography cycle can be used in these methods in any combination, for example, one or more different chromatography resins, one or more flow rates, one or more buffers, one or more void volumes of a column, one or more bed volumes of a column, one or more volumes of buffer used in each step, one or more volumes of fluid containing the target protein, and the amount and type of one or more buffers used in each step.

[0153] In some embodiments, the cation exchange column is loaded with about 50 mM to about 120 mM citrate phosphate (e.g., about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, or about 120 mM), about 100 mM to about 150 mM NaCl (e.g., about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mM), and the pH is between about pH 3 and about pH 4 (e.g., about pH 3.2, about pH 3.4, about pH 3.6, about pH 3.8, or about pH 4).

[0154] In some embodiments, about 10 mM to about 100 mM (e.g., 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 15 mM to about 100 mM, about 15 mM to about 90 mM, about 15 mM to about 80 mM, about 15 mM to about 70 mM, about 15 ... mM to about 60mM, about 15mM to about 50mM, about 15mM to about 40mM, about 15mM to about 30mM, about 15mM to about 20mM, about 20mM to about 100mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 20mM to about 25mM, about 30mM to about 100mM, about 30mM to about 90mM, about 30mM to about 80mM, about 30mM to about 70mM, about 30mM to about 60mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM to about 100mM, about 50mM to about 90mM mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, or about 90 mM to about 100 mM) of sodium acetate at a pH of about The protein A column is eluted from a pH range of about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about pH 3.75, about pH 2 to about pH 3.6, about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.4, about pH 2 to about pH 3.2, about pH 2 to about pH 3.0, about pH 2 to about pH 2.8, about pH 2 to about pH 2.6, about pH 2 to about pH 2.4, about pH 2 to about pH 2.2, about pH 3 to about pH 4, about pH 3 to about pH 3.8, about pH 3 to about pH 3.75, about pH 3 to about pH 3.6, about pH 3 to about pH 3.4, or about pH 3 to about pH 3.2).

[0155] In some embodiments, the cation exchange column is prepared with about 10 mM to about 100 mM triacetate (e.g., about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 15 mM to about 100 mM, about 15 mM to about 90 mM, about 15 mM to about 80 mM, about 15 mM to about 70 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 10 mM to about 40 mM, about 15 mM to about 30 mM, about 10 mM to about 20 mM, about 15 mM to about 10 0mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 30mM to about 100mM, about 30mM to about 90mM, about 30mM to about 80mM, about 30mM to about 70mM, about 30mM to about 60mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM M to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, or about 90 mM to about 100 mM), and about 10 mM to about 100 mM (e.g., about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 1 0mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 20mM, about 15mM to about 100mM, about 15mM to about 90mM, about 15mM to about 80mM, about 15mM to about 70mM, about 15mM to about 60mM, about 15mM to about 50, about 15mM to about 40mM, about 15mM to about 30mM, about 15mM to about 20mM, about 20mM to about 100mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM,about 20mM to about 25mM, about 25mM to about 100mM, about 25mM to about 90mM, about 25mM to about 80mM, about 25mM to about 70mM, about 25mM to about 60mM, about 25mM to about 50mM, about 25mM to about 40mM, about 25mM to about 30mM, about 30mM to about 100mM, about 30mM to about 90mM, about 30mM to about 80mM, about 30mM to about 70mM, about 30mM to about 60mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM to about 100mM, about 50mM to about 90mM, about 50mM to about 80mM, about 50mM to about 70mM, about 50mM to about 60mM, about 60mM to about 100mM, about 60mM to about 90mM, about 60mM to about 80mM, about 60mM to about 70mM, about 70mM to about 100mM, about 70mM to about 90mM, about 70mM to about 80mM, about 80mM to about 100mM, about 80mM to about 90mM, or about 90mM to about 100mM) of NaCl at a pH of about 7 to about pH 10 (e.g., about pH 7.2 to about pH 9.8, about pH 7.2 to about pH 9.6, about pH 7.2 to about pH 9.4, about pH 7.2 to about pH 9.2, about pH 7.2 to about pH 9, about pH 7.2 to about pH 8.8, about pH 7.2 to about pH 8.6, about pH 7.2 to about pH 8.4, about pH 7.2 to about pH 8.2, about pH 7.2 to about pH 8, about pH 7.2 to about pH 7.8, about pH 7.2 to about pH 7.6, about pH 7.2 to about pH 7.4, about pH 8 to about pH 10, about pH 8 to about pH 9.8, about pH 8 to about pH 9.6, about pH 8 to about pH 9.4, about pH 8 to about pH 9.2, about pH 8 to about pH 9, about pH 8 to about pH 9 8.8, about pH 8 to about pH 8.6, about pH 8 to about pH 8.4, about pH 8 to about pH 8.2, about pH 9 to about pH 10, about pH 9 to about pH 9.2, about pH 9 to about pH 9.4, about pH 9 to about pH 9.6, about pH 9 to about pH 9.8, about pH 9.5 to about pH 10, or about pH 9.5 to about pH 9.8).

[0156] In some embodiments, the cation exchange column is prepared with about 10 mM to about 50 mM (e.g., 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM), and about 20 mM to about 50 mM NaCl (e.g., 20 mM, about 25 mM, about 30 mM, about 40 mM, or about 50 mM), at about pH 8 to about pH 10 (e.g., about pH 8 to about 9.8, about 8 to about 9.6, about pH 8 to about pH 9.4, about pH 8 to about pH 9.2, about pH 8 to about pH 9, about pH 8 to about pH 8.8, about pH 8 to about pH 8.6, about pH 8 to about pH 8.4, about pH 8 to about pH 8.2, about pH 9 to about pH 10, about pH 9 to about pH 9.8, about pH 9 to about pH 9.6, about pH 9 to about pH 9.4, about pH 9 to about pH 9.2, or about pH 9.4 to about pH 10).

[0157] (c) Aggregation

[0158] In some embodiments, system 100 includes a sample analyzer that measures aggregation of analytes in a biological sample. The extent of aggregation can be determined, for example, by a sample analyzer that includes a size exclusion column for performing size exclusion chromatography on the analytes in the sample.

[0159] The term "size exclusion chromatography column" or "molecular sieve chromatography" refers to a chromatographic column in which analyte and other components are separated by size and / or molecular weight. In some embodiments, a size exclusion chromatography column is used to separate protein aggregates, such as protein polymers (e.g., dimers and trimers). Non-limiting examples of size exclusion chromatography columns include: Sephadex G-10, Sephadex G-25, Sephadex G-50, Sephadex G-75, Sephadex G-100, Sephadex G-150, Sephadex G-200, Sepharose 2B, Sepharose 4B, Sepharose 6B, Bio-gel P-300, and Waters BEH SEC 200A.

[0160] In some embodiments, the size exclusion column is loaded with 1x phosphate buffered saline (PBS) at a pH of about 7 (e.g., about pH 7.0 to about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, or about pH 7.9). In some embodiments, the size exclusion column is loaded with 1x PBS at a pH of about 7.2.

[0161] (d) Integrity or purity

[0162] In some embodiments, system 100 includes a sample analyzer that measures the integrity or purity of an analyte in a biological sample. The integrity or purity can be determined, for example, by a sample analyzer that includes a reverse phase column for performing reverse phase chromatography on the analyte in the sample.

[0163] The term "reverse phase chromatography" or "hydrophobic chromatography" refers to a type of chromatography that includes a hydrophobic stationary phase. Non-limiting examples of reverse phase chromatography columns are known in the art and include, for example, Sepax Opalshell-C18. Non-limiting examples of hydrophobic ligands include aliphatics, such as C2, C4, C8, C10, C12, C16, and C18, and polyphenyls.

[0164] A chromatography cycle using a reverse phase chromatography column may include the steps of loading the column with a fluid containing the analyte, washing the column to remove unwanted biological material, eluting the analyte bound to the column, and re-equilibrating the column.

[0165] Any individual step in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any individual steps in a chromatography cycle can include a buffer gradient. Any combination of various well-known aspects of a single chromatography cycle can be used in these methods in any combination, for example, one or more different chromatography resins, one or more flow rates, one or more buffers, one or more void volumes of a column, one or more bed volumes of a column, one or more volumes of buffer used in each step, one or more volumes of fluid containing the target protein, and the amount and type of one or more buffers used in each step.

[0166] In some embodiments, the reverse phase column contains about 0.01% to about 1% (e.g., about 0.01% to about 0.8%, about 0.01% to about 0.6%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.02% to about 1%, about 0.02% to about 0.8%, about 0.02% to about 0.6%, about 0.02% to about 0.5%, about 0.02% to about 0.4%, about 0.02% to about 0. 2%, about 0.02% to about 0.1%, about 0.05% to about 1%, about 0.05% to about 0.8%, about 0.05% to about 0.6%, about 0.05% to about 0.5%, about 0.05% to about 0.4%, about 0.05% to about 0.2%, about 0.05% to about 0.1%, about 0.06% to about 1%, about 0.06% to about 0.8%, about 0.06% to about 0.6%, about 0.06% to about 0.5%, about 0.06% to about 0.4%, about 0.06% to about 0. 2%, about 0.06% to about 0.1%, about 0.08% to about 1%, about 0.08% to about 0.8%, about 0.08% to about 0.6%, about 0.08% to about 0.5%, about 0.08% to about 0.4%, about 0.08% to about 0.2%, about 0.08% to about 1%, about 0.1% to about 1%, about 0.1% to about 0.8%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, about 0.1% to about 0.2%, about 0.2% to about 1%, about 0.2% to about 0.8%, about 0.2% to about 0.6%, about 0.2% to about 0.5%, about 0.2% to about 0.4%, about 0.4% to about 1%, about 0.4% to about 0.8%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 0.8%, about 0.5% to about 0.6%, about 0.6% to about 1%, about 0.6% to about 0.8%, or about 0.8% to about 1%) of trifluoroacetic acid (TFA).

[0167] In some embodiments, the reverse phase column is used in about 0.01% to about 1% (e.g., about 0.01% to about 0.8%, about 0.01% to about 0.6%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.02% to about 1%, about 0.02% to about 1% (e.g., about 0.01% to about 0.8%, about 0.01% to about 0.6%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.02% to about 1%) in about 1:10 to about 10:120 isopropanol (IPA):acetonitrile (ACN) (e.g., about 1:50, 1:90, 1:100, 1:120, 10:50, 10:90, or 10:120). % to about 0.8%, about 0.02% to about 0.6%, about 0.02% to about 0.5%, about 0.02% to about 0.4%, about 0.02% to about 0.2%, about 0.02% to about 0.1%, about 0.05% to about 1%, about 0.05% to about 0.8%, about 0.05% to about 0.6%, about 0.05% to about 0.5%, about 0.05% to about 0.4%, about 0.05% to about 0.2%, about 0.05% to about 0.1%, about 0.06% to about 1%, about 0.06% to about 0.8%, about 0.06% to about 0.6 %, about 0.06% to about 0.5%, about 0.06% to about 0.4%, about 0.06% to about 0.2%, about 0.06% to about 0.1%, about 0.08% to about 1%, about 0.08% to about 0.8%, about 0.08% to about 0.6%, about 0.08% to about 0.5%, about 0.08% to about 0.4%, about 0.08% to about 0.2%, about 0.08% to about 1%, about 0.1% to about 1%, about 0.1% to about 0.8%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4 %, about 0.1% to about 0.2%, about 0.2% to about 1%, about 0.2% to about 0.8%, about 0.2% to about 0.6%, about 0.2% to about 0.5%, about 0.2% to about 0.4%, about 0.4% to about 1%, about 0.4% to about 0.8%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 0.8%, about 0.5% to about 0.6%, about 0.6% to about 1%, about 0.6% to about 0.8%, or about 0.8% to about 1%) of trifluoroacetic acid (TFA).

[0168] In some embodiments, the reverse phase column is eluted with about 0.01% to about 0.2% (e.g., about 0.01%, about 0.02%, about 0.04%, about 0.05%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.15%, about 0.16%, about 0.18%, or about 0.2%) trifluoroacetic acid (TFA) in about 10:90 to about 1:80 isopropanol (IPA):acetonitrile (ACN).

[0169] In some embodiments, about 10 mM to about 100 mM (e.g., 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 15 mM to about 100 mM, about 15 mM to about 90 mM, about 15 mM to about 80 mM, about 15 mM to about 70 mM, about 15 ... mM to about 60mM, about 15mM to about 50mM, about 15mM to about 40mM, about 15mM to about 30mM, about 15mM to about 20mM, about 20mM to about 100mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 60mM, about 20mM to about 50mM, about 20mM to about 40mM, about 20mM to about 30mM, about 20mM to about 25mM, about 30mM to about 100mM, about 30mM to about 90mM, about 30mM to about 80mM, about 30mM to about 70mM, about 30mM to about 60mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM to about 100mM, about 50mM to about 90mM mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, or about 90 mM to about 100 mM) of sodium acetate at a pH of about The reverse phase column is eluted from a pH of about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about pH 3.75, about pH 2 to about pH 3.6, about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.4, about pH 2 to about pH 3.2, about pH 2 to about pH 3.0, about pH 2 to about pH 2.8, about pH 2 to about pH 2.6, about pH 2 to about pH 2.4, about pH 2 to about pH 2.2, about pH 3 to about pH 4, about pH 3 to about pH 3.8, about pH 3 to about pH 3.75, about pH 3 to about pH 3.6, about pH 3 to about pH 3.4, or about pH 3 to about pH 3.2).

[0170] (e) Antibody reduction

[0171] In some embodiments, system 100 includes a sample analyzer that measures the reduction of an antibody analyte in a biological sample. In a biomanufacturing production platform, reductive degradation of an antibody product is a problem, and the system described herein can be used to measure the reduction of an antibody to feedback-regulate a manufacturing process parameter. Antibody reduction can be determined, for example, by a sample analyzer that includes a reversed-phase column for performing reversed-phase chromatography on an analyte in a sample. Any reversed-phase column resin, buffer, pH value, and other operating conditions discussed herein can be used in conjunction with the reversed-phase chromatography in a sample analyzer.

[0172] In some embodiments, a hydrophilic interaction chromatography column can be used to measure product quality attributes of sample analytes. In certain embodiments, a hydrophobic interaction chromatography column can be used to measure product quality attributes, such as oxidation analysis. In some embodiments, a lectin column can be used to obtain product quality attributes related to glycosylation information.

[0173] In certain embodiments, enzyme chromatography columns can be used for peptide mapping to measure product quality attributes. Reversed phase chromatography separations can be used to separate peptide analytes for analysis.

[0174] Analysis of product quality attributes using system 100 can generally be accomplished in near real time to provide timely control feedback for adjustment of various biomanufacturing process conditions and parameters. In some embodiments, for example, the concentration or potency attribute of an analyte of a biological sample can be determined in 10 minutes or less (e.g., 9 minutes or less, 8 minutes or less, 7 minutes or less, 6 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less) from initial introduction of a portion of the sample on a suitable sample analyzer to determination of the attribute value.

[0175] In some embodiments, a charge change or heterogeneity attribute of an analyte of a biological sample can be determined in 70 minutes or less (e.g., 65 minutes or less, 60 minutes or less, 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less) from the initial introduction of a portion of the sample on a suitable sample analyzer to the determination of the attribute value.

[0176] In some embodiments, the aggregation property of an analyte of a biological sample can be determined in 30 minutes or less (e.g., 28 minutes or less, 26 minutes or less, 24 minutes or less, 22 minutes or less, 20 minutes or less, 18 minutes or less, 16 minutes or less, 14 minutes or less, 12 minutes or less, 10 minutes or less) from the initial introduction of a portion of the sample on a suitable sample analyzer to the determination of the property value.

[0177] In certain embodiments, the integrity or purity attribute of an analyte of a biological sample can be determined in 30 minutes or less (e.g., 28 minutes or less, 26 minutes or less, 24 minutes or less, 22 minutes or less, 20 minutes or less, 18 minutes or less, 16 minutes or less, 14 minutes or less, 12 minutes or less, 10 minutes or less) from the initial introduction of a portion of the sample on a suitable sample analyzer to the determination of the attribute value.

[0178] For an analysis cycle in which the value of each of the above four product quality attributes of the analyte is determined, the time interval from the initial introduction of the first portion of the biological sample into the first sample analyzer to the determination of the value of the fourth attribute can be 150 minutes or less (e.g., 130 minutes or less, 110 minutes or less, 105 minutes or less, 100 minutes or less, 95 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less).

[0179] Figure 3 300, which includes a series of exemplary steps for determining a product quality attribute of an analyte of a biological sample. In a first step 302, a sample is received in a sample manager. As described above, the sample can be received offline in a vial, well plate, or other container, from which it is extracted and injected into the first flow control device 106. The sample can also be received from an online sampling device and held in a fluid circuit or channel, from which a portion of the biological sample is injected into the first flow control device 106.

[0180] Next, in step 304, a portion of the biological sample is injected into the first flow control device 106. The injected sample portion may optionally be purified in step 306. Whether purified or not, the portion of the biological sample is delivered to the second flow control device 110 and then delivered from the second flow control device to a sample analyzer associated with a product quality attribute of interest in step 308.

[0181] In step 310, the portion of the biological sample is analyzed in the sample analyzer to determine the relevant product quality attribute value of the analyte in the sample. After the product quality attribute value is determined, if all product quality attribute values ​​for the sample analysis cycle have not been determined (see step 312), control returns to step 304 and another portion of the biological sample is injected into the first flow control device 106.

[0182] Alternatively, if all product quality attribute values ​​have been determined in step 312 , the attribute values ​​may optionally be transmitted by control unit 122 to the master controller for use in adjusting one or more biomanufacturing process parameters. The analysis cycle then ends at step 316 .

[0183] The method of delivering the biological sample from the first flow control device 106 directly to the second flow control device 110 or to the second flow control device 110 via the sample purification device 108 depends on, among other things, the nature of the flow control device. Figures 4A-4C An example of delivering a biological sample between first and second flow control devices when the first and second flow control devices are implemented as multi-way valves is shown.

[0184] Figure 4A is a schematic diagram of the first flow control device 106, which is configured to be in a first position, where ports 1 and 2 are connected, ports 3 and 4 are connected, and ports 5 and 6 are connected. The biological sample is injected into port 2 from the sample manager 102. Port 2 is connected to port 1, which in turn is connected to the second flow control device 110 and the column manager 112. As a result, the sample is delivered directly to the second flow control device 110 without passing through the sample purification device 108.

[0185] Figure 4B and Figure 4C is a schematic diagram showing first and second flow control devices 106 and 110 configured to deliver a biological sample to a sample purification device 108. Figure 4B In the embodiment, the first flow control device is configured such that the device is in the second position, ports 2 and 3 are connected, ports 4 and 5 are connected, and ports 1 and 6 are connected. The second flow control device is configured in the first position, ports 1 and 2 are connected, ports 3 and 4 are connected, and ports 5 and 6 are connected. In addition, the sample purification device 108 is connected between ports 1 and 4, port 3 is connected to a waste container, and port 2 is connected to port 3 of the first flow control device. Figure 4B The configuration shown is used to load a biological sample onto a column of a sample purification device 108, with a loading buffer introduced through port 1 of the first flow control device.

[0186] Once the biological sample is loaded onto the sample purification device column, e.g. Figure 4CThe configuration of the first and second flow control devices is adjusted as shown to elute the biological sample from the sample purification device column. In this configuration, the second flow control device is adjusted to a different configuration, where ports 2 and 3 are connected, ports 4 and 5 are connected, and ports 1 and 6 are connected. In addition, port 6 of the second flow control device 110 is connected to port 1 of the first flow control device 106. In order to elute the analyte from the sample purification device column, the elution buffer is delivered to port 5 of the second flow control device, and the eluted analyte is delivered to the second flow control device and then enters one of the sample analyzers in fluid communication with the second flow control device.

[0187] Analyzing biological samples to determine the value of product quality attributes according to the systems and methods described herein is advantageous in various aspects. For example, the described system can serve as a single collection platform for many different product quality attributes throughout process development (e.g., from cell line development to drug product development), maintaining data continuity and consistency in the collection method.

[0188] In addition, measurements of product quality attributes can be used for spectral model validation and ongoing model verification. For example, using periodically measured product quality attributes, the spectral model can be maintained by verifying the predictive accuracy of chemometric methods using online FTIR and / or Raman measurements.

[0189] The automated nature of the system described herein can be used to eliminate tedious, repetitive analytical steps that would otherwise be performed manually, thereby saving time and reducing the likelihood that operator error will affect the value of the measured attribute. Similarly, integrated sample purification can be performed online, greatly reducing the amount of time required to obtain a purer biological sample.

[0190] The systems and methods described herein can be used to analyze harvested culture media directly at a location close to the bioreactor in a manufacturing system. In some embodiments, feedback of information about measured product quality attributes (e.g., by control unit 122) can provide more direct and timely control of manufacturing process parameters, which can be adjusted to increase product yield, reduce scrap rates, and otherwise improve the efficiency of the biomanufacturing process.

[0191] In addition to applications in direct assessment of product quality attributes in bioreactor management, the attribute values ​​can also be used in cell line process development. For example, the methods described herein can be used to determine product quality attributes of monoclonal antibody therapeutics to analyze and compare different clones.

[0192] Integration and Regulation of Biomanufacturing Systems

[0193] The systems disclosed herein can be integrated with biomanufacturing systems to provide feedback control of various components and steps in the synthesis and purification processes of various bioproducts.

[0194] An integrated and fully continuous process for manufacturing therapeutic protein drugs and other substances may include, for example, providing a liquid culture medium containing a substantially cell-free recombinant therapeutic protein, which is then fed into a first multi-column chromatography system (MCCS1). The next step involves capturing the recombinant therapeutic protein in the liquid culture medium using MCCS1, and then continuously feeding the MCCS1 eluate containing the recombinant therapeutic protein into a second multi-column chromatography system (MCCS2), and using MCCS2 to purify and finely process the protein. The resulting eluate from MCCS2 is considered to be a therapeutic protein drug. The process is integrated and can be run continuously from the liquid culture medium to the eluate from MCCS2 (i.e., a therapeutic protein drug).

[0195] Biomanufacturing systems are typically used to perform the above-described processes. For example, such systems may include MCCS1 (which includes an inlet) and MCCS2 (which includes an outlet). In these systems, the first and second MCCSs are in fluid communication with each other. These systems are also configured so that a fluid can be conveyed into the inlet, through the first MCCS and the second MCCS, and out of the manufacturing system through the outlet.

[0196] Such a system can provide continuous, time-efficient production of therapeutic drugs from liquid culture medium. For example, the elapsed time between feeding a fluid (e.g., liquid culture medium) containing a therapeutic protein into the first MCCS and eluting the therapeutic protein drug (containing the therapeutic protein) from the outlet of the second MCCS can be, for example, between about 4 hours and about 48 hours.

[0197] Figure 8 is a schematic diagram showing an example of a biomanufacturing system. System 1 includes a first MCCS, a four-column periodic countercurrent chromatography system (PCCS) 2, wherein three of the four columns of the four-column PCCS 2 (columns 3, 4, and 5) perform a unit operation of capturing a recombinant therapeutic protein from a fluid containing the recombinant therapeutic protein (e.g., a liquid culture medium substantially free of mammalian cells), and one of the columns of PCCS 2 (column 6) performs a unit operation of inactivating viruses present in an eluate from columns 3, 4, and 5 of PCCS 2 containing the recombinant therapeutic protein. Columns 3, 4, and 5 may contain resins that utilize a protein A binding capture mechanism. Column 6 is capable of maintaining a fluid at a pH of about 3.75 for about 1 hour. PCCS 1 also has an inlet 7. Inlet 7 may be, for example, an orifice for receiving fluid into PCCS 1.

[0198] The system 1 further includes a second MCCS, which is a PCCS 8, which includes three chromatography columns 9, 10, and 11 and a chromatography membrane 12. The columns 9, 10, and 11 in the PCCS 8 may contain cation exchange resins. The chromatography membrane 12 in the PCCS 8 may contain cation exchange resins. The PCCS 8 further includes a fluid conduit 13 disposed between the columns 9, 10, and 11 in the PCCS 8 and the chromatography membrane 12 in the PCCS 8. The PCCS 8 further includes an online buffer adjustment reservoir 14 in fluid communication with the fluid conduit 13 and configured such that a buffer contained in the online buffer adjustment reservoir 14 is introduced into a fluid present in the fluid conduit 13. The PCCS 8 further includes an outlet 15. The outlet 15 may be, for example, an orifice that allows fluid to be discharged from the PCCS 8.

[0199] The system 1 may further include a fluid conduit 16 disposed between the PCCS 2 and the PCCS 8. The system 1 may also include an online buffer regulation reservoir 17 in fluid communication with the fluid conduit 16, which is configured so that a buffer contained in the online buffer regulation reservoir 17 can be introduced into the fluid present in the fluid conduit 16. The system 1 may also include a filter 18 disposed in the fluid conduit 16 to filter the fluid present in the fluid conduit 16. The system 1 may also include a cut-off groove 19 disposed in the fluid conduit 16 and configured to contain any fluid in the fluid conduit 16 that cannot be easily fed into the PCCS 8.

[0200] The system 1 may further include a pump system 20 in fluid communication with the inlet 7. The pump system 20 may include a pump 21 for pushing a fluid into the inlet 7. The system 1 may also include a fluid conduit 22 disposed between the pump 21 and the inlet 7. The system 1 may also include a filter 23 disposed in the fluid conduit 22 to filter a fluid (e.g., a liquid culture medium) present in the fluid conduit 22. The system 1 may also include a cut-off groove 24 disposed in the fluid conduit 22, which is configured such that the cut-off groove 24 is in fluid communication with the fluid conduit 22 and is capable of storing any fluid present in the fluid conduit 22 that cannot enter the inlet 7.

[0201] The system 1 may further include a bioreactor 25 and a fluid conduit 26 disposed between the bioreactor 25 and the pump 21. A filtration system 27 may be disposed in the fluid conduit 26 to filter the liquid culture medium present in the fluid conduit 26 (eg, to remove cells therefrom).

[0202] The first MCCS (PCCS2) includes an inlet through which a fluid (e.g., a substantially cell-free liquid culture medium) can be delivered into the first MCCS. The inlet can be any structure known in the art for such a purpose. It can include, for example, threads, ribs, or seals that allow insertion of a fluid conduit, such that after the fluid conduit is inserted into the inlet, the fluid will enter the first MCCS through the inlet without a significant amount of fluid leaking out of the inlet.

[0203] The first MCCS includes at least two chromatographic columns, at least two chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane, and an inlet. For example, the first MCCS may include a total of four chromatographic columns, or three chromatographic columns and one chromatographic membrane, or any other exemplary MCCS described herein, or having one or more of any exemplary features (in any combination) of the MCCS described herein.

[0204] One or more chromatographic columns and / or one or more chromatographic membranes present in the first MCCS may contain one or more of various resins. For example, the resin contained in one or more of the one or more chromatographic columns and / or one or more chromatographic membranes present in the first MCCS may be a resin that utilizes a capture mechanism (e.g., a protein A binding capture mechanism, a protein G binding capture mechanism, an antibody or antibody fragment binding capture mechanism, a substrate binding capture mechanism, a cofactor binding capture mechanism, an aptamer binding capture mechanism, and / or a tag binding capture mechanism). The resin contained in one or more of the one or more chromatographic columns and / or one or more chromatographic membranes of the first MCCS may be a cation exchange resin, an anion exchange resin, a molecular sieve resin, or a hydrophobic interaction resin, or any combination thereof. Other examples of resins that can be used to purify recombinant therapeutic proteins are known in the art and may be contained in one or more of the one or more chromatographic columns and / or one or more chromatographic membranes present in the first MCCS. One or more chromatographic columns and / or chromatographic membranes present in the first MCCS may contain the same and / or different resins (e.g., any resin described herein or known in the art for purification of recombinant proteins).

[0205] The two or more chromatography columns and / or chromatography resins present in the first MCCS can perform one or more unit operations (e.g., capturing recombinant therapeutic proteins, purifying recombinant therapeutic proteins, polishing recombinant therapeutic proteins, inactivating viruses, adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein, or filtering a fluid containing a recombinant therapeutic protein). In a non-limiting example, the first MCCS can perform unit operations of capturing recombinant therapeutic proteins from a fluid (e.g., a liquid culture medium) and inactivating viruses present in a fluid containing a recombinant therapeutic protein. The first MCCS can perform any combination of two or more unit operations described herein or known in the art.

[0206] One or more chromatographic columns and / or one or more chromatographic membranes present in the first MCCS can be connected or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). The first MCCS may also include one or more (e.g., two, three, four, or five) pumps (e.g., automated, such as automatic peristaltic pumps). A column switching event can be triggered by detecting the level of a recombinant therapeutic protein in a fluid (e.g., input and / or eluate into and / or from one or more of the one or more chromatographic columns and / or chromatographic membranes in the first MCCS), a specific liquid (e.g., buffer) volume, or a specific elapsed time through the fluid passing through the first MCCS. Column switching generally refers to a mechanism that allows at least two different chromatographic columns and / or chromatographic membranes in a MCCS (e.g., two or more different chromatographic columns and / or chromatographic membranes present in a MCCS (e.g., a first or second MCCS)) to pass through different steps (e.g., equilibration, loading, elution, or washing) substantially simultaneously during at least a portion of the process.

[0207] The PCCS2 as the first MCCS may include four chromatographic columns, wherein the first three columns perform a unit operation of capturing a recombinant therapeutic protein from a fluid (e.g., a liquid culture medium), and the fourth column of the PCCS performs a unit operation of inactivating viruses in a fluid containing the recombinant therapeutic protein. The PCCS as the first MCCS may use a column switching mechanism. The PCC system may employ an improved Systems (GE Healthcare, Piscataway, NJ) that are capable of running up to, for example, four, five, six, seven, or eight columns or more.

[0208] Column switching events can be triggered by detecting the concentration of a particular protein or other substance in a fluid eluting from a column of PCCS 2 or PCCS 8, flowing through a filter in an MCCS, contained in a cut-off channel in an MCCS, or flowing through a conduit in an MCCS (e.g., between MCCS 1 and MCCS 2). The measurement system disclosed herein can be used to measure the concentration of such proteins and communicate this concentration information to a controller in system 1, which initiates events such as column switching, filtration, and fluid transfer in system 1.

[0209] The first MCCS may be equipped with: one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) measurement systems configured to obtain infrared spectral information of the process fluid (e.g., system 100), one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) valves, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) pH meters, and / or one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) conductivity meters. The first MCCS may also be equipped with a controller that executes an operating system that utilizes software (e.g., Unicorn-based software (GE Healthcare, Piscataway, NJ) or other software that implements similar functionality) to determine when column switching should occur (e.g., based on concentration information from infrared spectroscopy measurements, liquid volume, or elapsed time) and to affect (trigger) column switching events. The measurement system may optionally be placed at the inlet of one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the one or more chromatographic columns and / or the one or more chromatographic membranes in the first MCCS, and / or at the outlet of one or more chromatographic columns and / or the one or more chromatographic membranes in the first MCCS.

[0210] The first MCCS may further include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) online buffer conditioning reservoirs and / or buffer reservoirs. In other examples, the first MCCS may include one or more (e.g., two, three, four, five, or six) cut-off slots that can accommodate fluids that cannot easily enter one or more chromatographic columns and / or chromatographic membranes in the first MCCS. The systems described herein may contain one or more cut-off slots (e.g., the cut-off slots described herein) in the first and / or second MCCS. Other examples of the systems described herein do not include a cut-off slot in the first MCCS or the second MCCS, or do not include a cut-off slot in the entire system. Other examples of the systems include a maximum of one, two, three, four, or five cut-off slots in the entire system.

[0211] In some embodiments, the first MCCS may include a virus inactivation device. For example, referring to Figure 8 In some embodiments, the first MCCS includes a virus inactivation device 6 (i.e., in place of the column 6 described above). The virus inactivation device 6 is configured to inactivate viruses and viral vectors used in the biomanufacturing process. In some embodiments, for example, the virus inactivation device 6 includes a mixing vessel. Alternatively, in some embodiments, for example, the device 6 includes a piston flow inactivation system. Each example of these virus inactivation devices helps to eliminate active viruses and viral vectors from the process fluid in the first MCCS.

[0212] The second MCCS includes at least two chromatographic columns, at least two chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane, and an outlet. For example, the second MCCS may include a total of four chromatographic columns, three chromatographic columns and one chromatographic membrane, or any other exemplary MCCS described herein, or may have one or more of any exemplary features (in any combination) of the MCCS described herein. The one or more chromatographic columns and / or one or more chromatographic membranes present in the second MCCS may have one or more of the following: any shape, size, volume (bed volume) and / or unit operation described herein. The resin contained in one or more of the one or more chromatographic columns and / or one or more chromatographic membranes present in the second MCCS may be a resin that utilizes a capture mechanism (e.g., protein A binding capture mechanism, protein G binding capture mechanism, antibody or antibody fragment binding capture mechanism, substrate binding capture mechanism, cofactor binding capture mechanism, label binding capture mechanism, and / or aptamer binding capture mechanism). Useful resins include, for example, cation exchange resins, anion exchange resins, molecular sieve resins, and hydrophobic interaction resins. The one or more chromatography columns and / or chromatography membranes present in the second MCCS may contain the same and / or different resins (eg, any resin described herein or known in the art for recombinant protein purification).

[0213] One or more chromatographic columns and / or one or more chromatographic membranes present in the second MCCS may perform one or more unit operations (e.g., any unit operation described herein or any combination of the unit operations described herein). In a non-limiting example, the second MCCS may perform a unit operation of purifying a recombinant therapeutic protein from a fluid and finely treating a recombinant therapeutic protein present in a fluid containing the recombinant therapeutic protein. In other non-limiting examples, the second MCCS may perform unit operations of purifying a recombinant therapeutic protein present in a fluid, finely treating a recombinant therapeutic protein present in a fluid, and filtering a fluid containing the recombinant therapeutic protein. In another example, the second MCCS may perform unit operations of purifying a recombinant therapeutic protein present in a fluid, finely treating a recombinant therapeutic protein present in a fluid, filtering a fluid containing the recombinant therapeutic protein, and adjusting the ion concentration and / or pH of a fluid containing the recombinant therapeutic protein. The second MCCS may perform any combination of two or more unit operations described herein or known in the art.

[0214] The second MCCS may also include one or more (eg, two, three, four, or five) pumps (eg, automated, such as automated peristaltic pumps).

[0215] One or more chromatographic columns and / or one or more chromatographic membranes present in the second MCCS may be connected or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). A column switching event may be triggered by detecting the level of a recombinant therapeutic protein or other substance (measured by infrared spectroscopy and analyzed using a chemometric model as discussed above to determine the level of a recombinant therapeutic protein in a fluid passing through the second MCCS (e.g., input and / or eluate into and / or from one or more of the one or more chromatographic columns and / or chromatographic membranes in the second MCCS), a specific volume of liquid (e.g., a buffer), or a specific elapsed time.

[0216] The PCCS 8 forming the second MCCS may contain three columns (performing a unit operation of purifying a recombinant therapeutic protein from a fluid) and a chromatographic membrane (performing a unit operation of polishing a recombinant therapeutic protein present in a fluid). For example, the three columns that perform the unit operation of purifying a recombinant therapeutic protein from a fluid may contain, for example, cation exchange resins, and the chromatographic membrane that performs the unit operation of polishing may contain a cation exchange resin. The PCCS as the second MCCS may use a column switching mechanism. For example, the PCCS may utilize a modified HPLC system that is capable of operating up to, for example, four, five, six, seven, or eight columns, or more. system (GE Healthcare, Piscataway, NJ).

[0217] Similar to the first MCCS, the second MCCS may also be equipped with: one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) infrared spectroscopy measurement systems, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) valves, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) pH meters, and / or one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) conductivity meters. The one or more measurement systems transmit the measured concentration information of the protein or other substance in the fluid to the controller, which uses the concentration information to determine whether to trigger a column switching event. The second MCCS may be equipped with an operating system executed by the controller that receives the concentration information, and the operating system uses software (e.g., Unicorn-based software, GE Healthcare, Piscataway, NJ) to determine when a column switching event should occur (e.g., based on infrared spectroscopy measurements, liquid volume, or elapsed time) and initiate a column switching event. In examples where the second MCCS includes one or more infrared spectroscopic measurement systems, the measurement systems may optionally be placed at the inlets of one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the one or more chromatographic columns and / or one or more chromatographic membranes in the second MCCS, and / or at the outlets of one or more chromatographic columns and / or one or more chromatographic membranes in the second MCCS.

[0218] The second MCCS may further include one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) online buffer conditioning reservoirs and / or buffer reservoirs. In other examples, the second MCCS may include one or more (e.g., two, three, four, five, or six) cut-off slots (e.g., any cut-off slots described herein) that can accommodate fluids that cannot easily enter one or more chromatography columns and / or chromatography membranes in the second MCCS.

[0219] The second MCCS includes an outlet through which the therapeutic protein drug can leave the system. The outlet may include, for example, threads, ribs, or seals that allow insertion of a fluid conduit, or a vial designed to hold or store the therapeutic protein drug. The outlet may contain a surface that can be used to seal a sterile vial or other such storage container on the outlet to allow the recombinant protein drug to flow directly into the sterile vial or storage container.

[0220] Any fluid conduit described herein can be, for example, a tube made of, for example, polyethylene, polycarbonate, or plastic. The fluid conduit disposed between the first MCCS and the second MCCS can further include one or more of any combination of the following: one or more online buffer regulation reservoirs, which are in fluid communication with the fluid conduit and are positioned so that the buffer stored in the one or more online buffer regulation reservoirs is added to the fluid present in the fluid conduit; a cut-off groove (e.g., any one or more cut-off grooves described herein), which is in fluid communication with the fluid conduit and is positioned so that it can accommodate any excess fluid present in the fluid conduit that cannot be easily fed to the second MCCS; and one or more filters, which are disposed in the fluid conduit so that they can filter the fluid present in the fluid conduit (e.g., remove bacteria). Any online buffer regulation reservoir may contain, for example, a volume of buffer between about 0.5L and 50L (e.g., at a temperature of or below 50°C, 37°C, 25°C, 15°C, or 10°C).

[0221] The systems described herein may optionally include a fluid conduit disposed between the final chromatography column or chromatography membrane and the outlet in the second MCCS. The systems described herein may further include one or more filters in fluid connection with the fluid conduit disposed between the final chromatography column or chromatography membrane and the outlet in the second MCCS, such that the filters can remove, for example, precipitated material, particulate matter, or bacteria from the fluid present in the fluid conduit disposed between the final chromatography column or chromatography membrane and the outlet in the second MCCS.

[0222] Some examples of the systems provided herein also include a bioreactor in fluid connection with the inlet of the first MCCS.Any exemplary bioreactor described herein or known in the art can be used in the systems of the present invention.

[0223] Some examples of the systems provided herein also include a pump system. The pump system may include one or more of the following: one or more (e.g., two, three, four, five, six, seven, eight, nine or ten) pumps (e.g., any pump described herein or known in the art), one or more (e.g., two, three, four or five) filters (e.g., any filter described herein or known in the art), one or more (e.g., two, three, four, five, six, seven, eight, nine or ten) UV detectors, and one or more (e.g., two, three, four or five) break tanks (e.g., any break tank described herein). Some examples of the systems provided herein further include a fluid conduit (e.g., any exemplary fluid conduit described herein or known in the art) disposed between the pump and the inlet of the first MCCS. In some examples, this particular fluid conduit may include one or more (e.g., two, three or four) pumps (e.g., any pump described herein or known in the art) and / or one or more (e.g., two, three or four) break tanks (e.g., any exemplary break tank described herein), wherein these pumps and / or break tanks are in fluid connection with the fluid present in the fluid conduit.

[0224] Some examples of the systems described herein further include another fluid conduit connected to the fluid conduit between the pump and the inlet, wherein one end of the other fluid conduit is fluidically connected to the bioreactor and the other end is fluidically connected to the fluid conduit between the pump and the inlet. The additional fluid conduit may include a filter that is capable of removing cells from the liquid culture medium taken from the bioreactor (e.g., ATF cell retention system).

[0225] The aforementioned biomanufacturing system allows the continuous production of therapeutic protein drugs. For example, the system provided herein allows the yield percentage of recombinant therapeutic protein (from starting material, such as starting liquid culture medium) to be greater than about 70%, greater than about 80%, greater than about 82%, greater than about 84%, greater than about 86%, greater than about 88%, greater than about 90%, greater than about 92%, greater than about 94%, greater than about 96% or greater than about 98%. The system described herein can also cause the yield percentage of recombinant therapeutic protein (from starting material, such as starting liquid culture medium) to be between about 80% to about 90%, between about 82% to about 90%, between about 84% to about 90%, between about 84% to about 88%, between about 84% to about 94%, between about 82% to about 92%, or between about 85% to about 95%.

[0226] The systems described herein can also result in the production of therapeutic protein drug products containing a recombinant therapeutic protein at a concentration greater than about 1.0 mg / mL, for example, greater than about 15 mg / mL, greater than about 20 mg / mL, greater than about 25 mg / mL, greater than about 30 mg / mL, greater than about 35 mg / mL, greater than about 40 mg / mL, greater than about 45 mg / mL, greater than about 50 mg / mL, greater than about 55 mg / mL, greater than about 60 mg / mL, greater than about 65 mg / mL, greater than about 70 mg / mL, greater than about 75 mg / mL, greater than about 80 mg / mL, greater than about 85 mg / mL, greater than about 90 mg / mL, greater than about 100 mg / mL, greater than about 125 mg / mL, or greater than about 150 mg / mL.

[0227] As described above, in some embodiments, the first and / or second MCCS can be a periodic countercurrent chromatography system (PCCS). PCCS can, for example, include two or more chromatographic columns (e.g., three columns or four columns), which are switched to allow continuous elution of recombinant therapeutic proteins from two or more chromatographic columns. PCCS can include two or more chromatographic columns, two or more chromatographic membranes, or at least one chromatographic column and at least one chromatographic membrane. Column operation is usually composed of loading, washing, elution and regeneration steps. In PCCS, multiple columns are used to discretely and continuously run the same steps in a cyclic manner. Since the columns are operated in series, the flow through one column and the washing liquid from the column are captured by another column. This unique feature of PCCS allows the loading of resin to be close to its static binding capacity rather than dynamic binding capacity, which is typical during batch mode chromatography.

[0228] An example of a three-column switching technique for a PCCS containing three columns is shown in Fig. 9 One cycle is defined as three complete column operations that produce an elution pool from each of the three columns used in the column switching technique. Once all steps in the cycle are completed, the cycle restarts. Due to the continuous cycle and elution, the fluid entering the PCCS is processed continuously, while the elution of the recombinant therapeutic protein from each column is discrete and periodic.

[0229] To advance from one step in the PCCS cycle to another, as Fig. 9 The exemplary cycle shown in FIG. 1 employs a column switching strategy. Fig. 9 The three columns of the exemplary PCCS system shown employ two automatic switching operations per column, the first of which is associated with initial product breakthrough and the second coincides with column saturation. Determining when a column switching operation should occur is based on information about the concentration of the recombinant therapeutic protein in the eluate from each chromatographic column of the PCCS.

[0230] As described above, a suitable sample analyzer can be used to determine the concentration of the recombinant therapeutic protein in the eluate from the PCCS column. The concentration information, which acts as a feedback control for the biomanufacturing system, is transmitted by the control unit 122 to the MCCS controller, which initiates the column switch after determining that the switch is authorized.

[0231] For example, during column loading, the PCC control system can determine the baseline concentration of the therapeutic protein material eluting from the column (which is typically a zero concentration) using the infrared spectroscopy measurement system discussed above. During active elution, as the protein material breaks through, the measured protein concentration increases (e.g., above the baseline concentration). The system continues to monitor the increasing protein concentration, and when the concentration reaches a predetermined threshold, flow from column 1 is directed to column 2 rather than to waste. Nominally, this occurs at time t 1 .

[0232] As the feed into column 1 continues, column 1 is eventually almost saturated with protein product. At this point, the protein concentration in the measured eluate has reached another predetermined value, which occurs at time t 2 At this point, the MCCS controller switches the inlet feed to column 2.

[0233] The above-mentioned column switching strategy allows for uniform loading of the column regardless of the feed product concentration and capacity. Similar switching of columns can be implemented based on the detected recombinant protein levels in the eluate from each column. Column switching can also be based on the elapsed time or amount of fluid (e.g., buffer) passing through one or more chromatographic columns and / or chromatographic membranes in the first or second MCCS.

[0234] In addition to providing feedback information to control column switching events, the measurement systems disclosed herein can also provide feedback information for adjusting various other biomanufacturing steps and operating parameters. An example of such an adjustment is the controlled adjustment of buffer concentrations at various stages of a biomanufacturing process.

[0235] Typically, during the use of two or more MCCS in any process described herein, one or more (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) different types of buffers may be employed. As is known in the art, the one or more types of buffers used in two or more MCCS in the process described herein will depend on: the resins present in the one or more chromatographic columns and / or one or more chromatographic membranes of the two or more MCCS (e.g., the first MCCS and the second MCCS), the recombinant therapeutic protein, and the unit operation (e.g., any exemplary unit operation described herein) - which is performed by the specific one or more chromatographic columns and / or one or more chromatographic membranes of the two or more MCCS. During the use of two or more MCCS in any process described herein, the volume and type of buffer employed can also be determined by one skilled in the art (e.g., as discussed in more detail below). For example, the volume and one or more types of buffers employed during the use of two or more MCCS in any process described herein can be selected to optimize one or more of the following in the recombinant protein drug product: the total yield of the recombinant therapeutic protein, the activity of the recombinant therapeutic protein, the purity level of the recombinant therapeutic protein, and the removal of biological contaminants (e.g., the absence of active viruses, mycobacteria, yeast, bacteria, or mammalian cells) from a fluid containing the recombinant therapeutic protein.

[0236] The unit operation of adjusting the ionic concentration and / or pH of a fluid containing a recombinant therapeutic protein can be performed using a MCCS (e.g., a first and / or second MCCS) that includes and utilizes a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) to add new or additional buffer solution to the fluid containing the recombinant therapeutic protein (e.g., between columns in a single MCCS, or after the last column in the second-to-last MCCS (e.g., a first MCCS) and before feeding the fluid containing the recombinant therapeutic protein to the first column of the next MCCS (e.g., a second MCCS). The in-line buffer adjustment reservoir can be of any size (e.g., greater than 100 mL) and can contain any buffer solution (e.g., a buffer solution having one or more of the following: an increased or decreased pH compared to the fluid containing the recombinant therapeutic protein; an increased or decreased ionic (e.g., salt) concentration compared to the fluid containing the recombinant therapeutic protein; and / or an increased or decreased concentration of an agent that competes with the recombinant therapeutic protein for binding to a resin present in at least one chromatographic column or at least one chromatographic membrane of the MCCS (e.g., a first or second MCCS)).

[0237] In some embodiments, the MCCS controller determines the amount of the buffer solution added to the process fluid based on the concentration or titer information of the analyte in the biological sample. For example, the solute used for such measurement can be a buffer solution component or a process fluid component, and its concentration is related to the fluid buffer composition, the pH of the process fluid, and / or the ionic strength of the process fluid. The measurement of the concentration information of the component is provided to the MCCS controller as feedback information, which uses the feedback information to determine when and how much one or more buffer solutions are discharged into the process fluid. The infrared spectroscopy measurement system can usually be positioned at any position in the biomanufacturing system for measuring the process fluid to provide the MCCS controller with feedback information related to the buffer.

[0238] In certain embodiments, the antibody concentration information of the process fluid can be used to control the rate at which the cell culture is introduced into the bioreactor. Specifically, by determining the antibody concentration value in the process fluid harvested from the bioreactor, the MCCS controller can adjust the rate at which the cell culture flows out into the bioreactor. Regulation in this manner allows control of the volumetric productivity of the cell density and specific productivity derived from the bioreactor. For a fixed perfusion rate, this type of regulation allows control of the antibody concentration in the process fluid so that the MCCS1 will receive an approximately constant amount of product per unit time. In other words, regulating this property can be used to ensure that the product generation rate in the bioreactor remains approximately constant within a specific time period.

[0239] In some embodiments, determination of certain quality attributes associated with a process fluid may be used by the MCCS controller to determine whether the biomanufacturing system is operating within acceptable parameters, or whether during operation, the system is outside of one or more acceptable parameter ranges.

[0240] For each of the one or more quality attributes, a range of acceptable values ​​may be established through a calibration procedure. These ranges effectively establish operating conditions for the system under which the biological product is produced at an acceptable rate and purity level, while the production of byproducts and other undesirable substances is at an acceptably low level. When the system is operated outside of one or more ranges, product yield and / or purity may decrease, the rate / amount of undesirable substance production may increase, the rate of reagent consumption may increase, and / or other undesirable effects or conditions may occur.

[0241] The quality attributes determined for the process fluid at one or more locations within the system can be used to ensure that the system operates within an acceptable range of these operating parameters. If the determined values ​​of one or more quality attributes fall outside of the established acceptable range, the MCCS controller identifies that a potential fault condition exists.

[0242] To address a fault condition, the MCCS controller (or another system controller connected to the MCCS controller) can adjust any operating parameter of the biomanufacturing system to modify its operation, thereby also adjusting the quality attribute values ​​so that they fall within an acceptable range. Corrective actions of this nature ensure that the system can actively remain within an established set or range of operating conditions based on the feedback provided by the determined quality attribute values.

[0243] In certain embodiments, if the MCCS controller (or another system controller connected to the MCCS controller) determines that the system is so far from its acceptable range of operating conditions that returning the system to the acceptable range of conditions would be difficult or even impossible, or would result in other undesirable consequences, the controller may transmit a control signal to the bioreactor to suspend production and discharge its contents to waste. In such cases, the production process has deviated so far from the acceptable range of operating conditions for the system that effective corrective action is impractical or impossible. By simply draining the contents of the bioreactor, the system can save considerable time by restarting the production process, rather than attempting to adjust an ongoing production process that may have irreparably deviated from the acceptable range of conditions.

[0244] In addition, feedback can be provided to the MCCS controller (or another system controller) based on the measured values ​​of one or more bioreactor medium components (e.g., glucose concentration, glutamine concentration, lactate concentration, and ammonium ion concentration), which can then be used to adjust reactor conditions to ensure that cell viability, product yield, and other performance indicators are maintained within target ranges. The controller can adjust any one or more process parameters based on the values ​​of bioreactor medium components in a manner similar to the adjustments made based on the values ​​of product quality attributes and other measured quantities.

[0245] Hardware and software implementation

[0246] The control unit 122 may be configured to perform any of the control functions described herein and may be implemented in hardware, software, or a combination of hardware and software. The control unit 122 typically includes at least one electronic processor connected to a memory unit, a storage device, an output device (e.g., a display), and a human-machine interface device (e.g., a keyboard, a mouse, a touch pad, a touch-sensitive display). The control unit 122 receives information as electrical signals from system components along some or all of the control lines shown herein and transmits electrical control signals to system components along the control lines.

[0247] The method steps and control functions described herein may be implemented in a computer program using a standard or proprietary programming language. Such a program is designed to be executed by the control unit 122 (e.g., an electronic processor of the control unit) and causes the control unit to perform the steps and functions described. Each program may be stored on a computer readable storage medium (e.g., an optical, magnetic, solid-state, rewritable or other permanent storage medium). An example of a proprietary programming language that may be used to provide instructions to the control unit 122 is Software Suite (Waters Corp., Milford, MA).

[0248] Example

[0249] The following examples are provided to further illustrate various aspects of the foregoing disclosure, but are not intended to otherwise limit any feature of the claims, or to limit any aspect of the embodiments, unless expressly stated otherwise.

[0250] Example 1.

[0251] System 100 was used to analyze anti-TGFβ from a perfusion bioreactor operating in an intensified perfusion process. The system included a Process Sample Manager (PSM), a Column Heater (CH), a Column Manager (CM) containing two 1-position 9-port valves, a Binary Pump (BSM), a Quaternary Pump (QSM) with an additional solvent selection valve on the "D" line, a PDA detector (PDA), a 2-position 10-port column selection valve equipped with a 5 mL stainless steel sample holding loop (all obtained from Waters Corp., Milford, MA), and two IDEX auxiliary 2-position 6-port switching valves.

[0252] The harvest from the bioreactor was automatically sampled using MAST (Modular Automated Sampling Technology) (obtained from Lonza, Basel, Switzerland) connected to a liquid handler (obtained from Gilson, Middleton, WI). The MAST system draws approximately 40 mL of harvest via a positive displacement pump to empty the line before placing 10 mL of harvest into a 12 mL clear glass bottle on the liquid handler platform. The liquid handler then transfers 7.5 mL of harvest to the system 100 by reinjecting the material into a PEEK tubing transfer line connecting the liquid handler to the aspiration device. The sample is transferred directly into a 5 mL sample holding loop where it is held until sample analysis is performed. Once the sample transfer is complete, the host computer sends a signal to the control unit 122 to initialize the 3 Method set specified in the software.

[0253] The method setup run for this example includes loop loading functionality, two injections on the Protein A (Pro A) column of the sample purification device at two mass loads, two injections on the size exclusion column at 2 mass loads, an intermediate water wash method between the separation techniques (Pro A and size exclusion chromatography (SEC)) of the common system components, and a final wash cycle with 20% methanol and complete segmentation to ensure that the cycle is in the right position to accept more sample. It is important to note that whenever a secondary column (such as SEC) is mentioned, it should be assumed that the method includes online purification of Pro A unless otherwise stated.

[0254] For each injection, 1 mL of harvest was withdrawn from the holding loop and transferred to the 50 μL sample injection loop for full loop injection on the system. To test different mass loadings, full loop injections as well as online 1:2 dilutions using 1X phosphate buffered saline (PBS) pH 7.2 buffer were performed.

[0255] Sample purification for titer analysis was performed on a Thermo POROS A 2.1 x 30 mm, 20 μm column (ie a Protein A affinity column). Tables 1 and 2 show the gradient details for the first and second pumps.

[0256] Table 1: First (binary) pump method parameters for potency analysis

[0257]

[0258] Table 2: Second (quaternary) pump method parameters for potency analysis

[0259]

[0260]

[0261] For the method shown in Table 1, buffer A was 1X Dulbecco's phosphate buffered saline, pH 7.2, and buffer B was 20 mM sodium phosphate, 1 M sodium chloride, and 7.5% isopropanol, pH 8.0. For the method shown in Table 2, buffer A was 0.1 M phosphate-citrate, 0.14 M sodium chloride, pH 3.2.

[0262] The system is configured to direct the purified sample directly to the detector 116 (PDA detector) without passing through any column-based sample analyzer. The separation is monitored at an absorbance of 280 nm. The protein A column is maintained in ambient environment at room temperature. After detection in the PDA detector, the measured chromatogram is integrated to calculate the area under the curve corresponding to the peak of the eluted protein at about 2.5 minutes. The area of ​​the monoclonal antibody is then quantified by using a calibration curve, which is plotted against the area by mass loading of 50 μg to 2 μg on the column.

[0263] Figure 5A is a graph showing the potency chromatograms of a pure sample and a 1:2 diluted sample, Figure 5B is a table showing the calculated mass loading and concentrations for 6 different runs at each dilution ratio. The data show excellent reproducibility between runs, both visually and in the calculated results.

[0264] Size exclusion separation was performed on a Waters UPLC BEH SEC 4.6 x 300 mm, 1.7 μm, and Aggregation analysis was performed on the column. Tables 3 and 4 show the gradient details for the first and second pumps.

[0265] Table 3: First (binary) pump method parameters for aggregation analysis

[0266]

[0267]

[0268] Table 4: Second (quaternary) pump method parameters for aggregation analysis

[0269]

[0270] For the method shown in Table 3, buffer A was 1X Dulbecco's phosphate buffered saline, pH 7.2, and buffer B was 20 mM sodium phosphate, 1 M sodium chloride, and 7.5% isopropanol, pH 8.0. For the method shown in Table 4, buffer A was 0.1 M phosphate-citrate and 0.14 M sodium chloride, pH 3.2, and buffer B was 1X Dulbecco's phosphate buffered saline, pH 7.2.

[0271] The column manager is configured to introduce the sample into the sample analyzer using a size exclusion chromatography column. The separation of the analyte and other components is monitored at an absorbance of 280 nanometers. The size exclusion column is maintained at 25°C in a 30 cm column heater while the protein A column is in the environment. When detected in the PDA detector 116, the recorded chromatogram is integrated to calculate the area under the curve corresponding to the peaks of high molecular weight species (before the main peak), main species and low molecular weight species (after the main peak).

[0272] Fig. 6A is a graph showing the potency chromatograms of the pure sample and the 1:2 diluted sample, and Figure 6B A table with measured peak information for 6 different samples in each of two dilutions is shown. The data shows excellent reproducibility between runs, both visually and in the calculated results. There are some differences in peak percentages between mass loadings, which may be caused by the column itself.

[0273] Figure 7 is a graph showing measured chromatograms from experiments where samples were obtained every 1.5 hours for a total of 12 samples. For these experiments, only 1 mass load was delivered to the Protein A and Size Exclusion Chromatography columns: a 1:2 online dilution was performed on the sample manager with 1X PBS, pH 7.2. The potency and aggregation product quality attributes showed excellent reproducibility across the 12 samples.

[0274] For reverse phase analysis, 0.1% trifluoroacetic acid in water (mobile phase A), 0.1% trifluoroacetic acid in 10% isopropanol, 90% acetonitrile (mobile phase B), and 20 mM sodium acetate pH 3.75 (Protein A column eluate) were used on a Waters BioResolve Polyphenyl 2.1x100mm, 2.7μm, Separation was performed on a column. For strong cation exchange analysis, separations were performed on a Thermo MAbPac SCX-10RS, 2.1x150mm, 5μm column. The mobile phases were 20mM sodium acetate pH 3.75 (mobile phase A and protein A eluate) and 20mM Tris acetate, 25mM sodium chloride pH 9.8 (mobile phase B). Currently, this method uses gradient curvature to create an "S" shaped gradient for optimal peak resolution.

[0275] The above method is used to analyze samples online. As described above, the system 100 can also analyze offline samples using, for example, a sample manager flow-through needle module (available from Waters Corp., Milford, MA). Using a flow-through needle, the sample is injected directly from a vial or well plate. The injection volume can be varied between 1 μL and 100 μL as needed. This flexibility allows the mass load on the column to be changed without online dilution. In addition, there is no sample holding loop in the system version because all sample volumes are contained in the vial or well plate, and multiple injections are allowed per vial.

[0276] Example 2. Application of MIMICS-mPQA in process development and online process monitoring of pilot-scale bioreactors

[0277] Experiment Overview

[0278] The MIMICS-mPQA platform was applied to a 100L scale run of an anti-TGFβ monoclonal therapeutic antibody. Harvest samples were run on the MIMICS-mPQA platform to collect online product quality information for 3 attributes: potency, aggregation, and purity / integrity.

[0279] The samples analyzed were harvest material (post-ATF) from the 100L bioreactor and 2 satellite 3L bioreactors. For the analysis, a Protein A affinity column was used for potency determinations, a Protein A column in tandem with a size exclusion (SEC) column was used for aggregation analysis, and a Protein A column in tandem with a reversed phase (RP) column was used for purity / integrity information. Testing was performed over a 4-week period during which samples from all reactors in each cohort were run over a period of approximately 4 harvest days. During the analysis period, the system mobile phase was replaced as needed once consumed.

[0280] For each harvest day of each bioreactor test, 0.75 mL of material was transferred to an autosampler vial and placed on the MIMICS-mPQA system. Each sample was injected on the system for 1 analytical cycle to quantify 3 target attributes. The injection volume was maintained at 20 μL, but the mass loading on each column was different, targeting the linear region of each method by applying different online dilution factors ranging from 1:2 to 1:10 based on previous development experience. In brief, the load target on the Pro A column was 10-25 μg, the load target on the SEC column was 20-50 μg, and the load target on the RP column was 3-7 μg.

[0281] For the Pro A titer method, one standard curve was run at the beginning of the full campaign and again at a second time point after the Pro A buffer exchange, for a total of 2 standard curves throughout the campaign. The standard curve was constructed by in-line dilution of 2.5 mg / mL stock anti-TGFβ to generate a curve ranging from 2-50 μg on the column.

[0282] Stocks used for the standard curve were previously diluted in previous runs and stored in subfluidic at -80 °C for the MIMICS-mPQA activity.

[0283] result

[0284] The data captured from Pro A quantification were also compared to two offline quantitative titer measurements: titer measured on Octet using ProA biosensor tips and titer measured by CEDEX. TM The titer was measured by bioanalyzer. Figure 10-12 Comparable performance of MIMICS-mPQA titers compared to offline methods in 100-L and 3-L bioreactors was demonstrated.

[0285] In general, the trends of the three methods are consistent. TM When the bioanalyzer methods were compared, the % difference for all runs was less than 10%, which is an acceptable CV for an analytical method. CEDEX TM Bioanalyzers are commonly used for daily upstream process monitoring for potency measurements. The comparability of MIMICS-mPQA to this technology provides an orthogonal tool for mammalian culture-based potency determinations. Another advantage of MIMICS-mPQA is the reduction of manual handling of samples and potential bias from sample storage when the test is performed offline.

[0286] The aggregate data captured in this experiment were compared to an offline SEC method run for normal process monitoring. Data for each bioreactor were presented in Figure 2. Figure 13-15 shown.

[0287] Overall, the trends between the offline analysis method and MIMICS-mPQA are comparable. Both methods show that aggregation levels were below 2.5% throughout the campaign. The MIMICS-mPQA system allowed for more data points to be acquired for this campaign than would normally be tested in offline analysis. This increased sampling allowed for a deeper look at the fluctuations in aggregation throughout the campaign.

[0288] Purity data obtained from MIMICS-mPQA analysis were also compared with offline purity methods used for process monitoring, such as Fig.16The offline method is based on a CE-SDS approach that is orthogonal to the purity measurement performed by MIMICS-mPQA.

[0289] like Fig.16 As shown, the MIMICS-mPQA system provides an overall lower absolute percentage purity compared to the offline method. Despite the absolute difference in percentage, the overall trend of the two methods is consistent.

[0290] Overall, the MIMICS-mPQA system was successfully applied in an online monitoring mode for a 100 L pilot-scale bioreactor project against TGFβ. The system allowed for increased sampling compared to offline analysis, which further allowed for a better understanding of the process during an active process. In addition, the titer and aggregation methods were highly comparable compared to offline analysis, providing confidence in the on-site analysis compared to the results obtained in the analytical laboratory. The MIMICS-mPQA system also allowed for reduced sample consumption and faster throughput, as there was no need for pre-purification of Protein A prior to analysis, as this step was part of the overall MIMICS-mPQA workflow. Finally, the system was able to operate for 4 weeks with minimal incidents, further enhancing the understanding of instrument robustness and column stability.

[0291] In summary, the present invention includes but is not limited to the following items:

[0292] 1. A system for measuring a product quality attribute of an analyte of a biological sample, the system comprising:

[0293] a first flow control device;

[0294] a sample purification device in fluid communication with the first flow control device;

[0295] a second flow control device in fluid communication with the first flow control device, the sample purification device, and first and second sample analyzers, wherein the first sample analyzer includes a first chromatographic column; and

[0296] a control unit coupled to the first and second flow control devices and configured to cause

[0297] During operation of the system, the control unit:

[0298] (a) adjusting the configuration of the first flow control device to direct a portion of the biological sample from the first flow control device to the sample purification device or the second flow control device, so that the portion of the biological sample is purified by the second flow control device;

[0299] take over;

[0300] (b) adjusting the configuration of the second flow control device to direct the portion of the biological sample to one of the first and second sample analyzers; and

[0301] (c) determining a product quality attribute of an analyte of the biological sample based on the analysis of the portion of the biological sample by said one of the first and second sample analyzers.

[0302] 2. A system according to claim 1, wherein the first chromatography column is a cation exchange chromatography column.

[0303] 3. A system according to item 1, wherein the first chromatographic column is a size exclusion chromatography column.

[0304] 4. A system according to item 1, wherein the first chromatographic column is a reverse phase chromatographic column.

[0305] 5. A system according to item 1, wherein the sample purification device comprises an affinity chromatography column.

[0306] 6. A system according to item 1, wherein the second sample analyzer includes a quantitative detector configured to generate an electrical signal representative of the amount of analyte in the biological sample.

[0307] 7. A system according to claim 6, wherein the first chromatographic column is fluidically connected to the quantitative detector, and wherein the quantitative detector is configured to generate an electrical signal representing the amount of analyte in the elution flow from the first chromatographic column.

[0308] 8. A system according to claim 1, wherein the first sample analyzer includes a quantitative detector, which is connected to the first chromatographic column fluid and is configured to generate an electrical signal representing the amount of analyte in the elution flow from the first chromatographic column.

[0309] 9. A system according to claim 1, wherein the second sample analyzer comprises a second chromatographic column, and

[0310] wherein the second chromatographic column is different from the first chromatographic column and is a cation exchange chromatographic column

[0311] One of a column, a size exclusion chromatography column, a reversed phase chromatography column, and a hydrophilic interaction chromatography column.

[0312] 10. A system according to claim 9, wherein the second flow control device is fluidically connected to a third sample analyzer comprising a third chromatographic column, and wherein the third chromatographic column is different from the first and second chromatographic columns and is one of a cation exchange chromatographic column, a size exclusion chromatographic column, a reverse phase chromatographic column, and a hydrophilic interaction chromatographic column.

[0313] 11. A system according to claim 1, wherein the second flow control device is fluidically connected to four additional sample analyzers, each of the four additional sample analyzers comprising a chromatographic column different from the first chromatographic column and different from the chromatographic columns of other sample analyzers in the four additional sample analyzers.

[0314] 12. A system according to item 1, wherein the product quality attribute of the analyte is the concentration of the analyte in the biological sample.

[0315] 13. A system according to item 1, wherein the product quality attribute of the analyte is a measure of the aggregation of the analyte in the biological sample.

[0316] 14. A system according to claim 1, wherein the product quality attribute of the analyte is a measure of charge variation or heterogeneity of the analyte in the biological sample.

[0317] 15. A system according to claim 1, wherein the product quality attribute of the analyte is a measure of the purity or integrity of the analyte in the biological sample.

[0318] 16. The system of claim 5, wherein the affinity chromatography column is one of a protein A chromatography column, a protein G chromatography column, and a receptor binding column.

[0319] 17. A system according to item 1, wherein the analyte comprises a protein in the biological sample.

[0320] 18. A system according to item 1, wherein the protein comprises an antibody in the biological sample.

[0321] 19. The system according to claim 9 further comprises a column manager which is fluidly connected to the first and second sample analyzers and the second flow control device and is connected to the control unit, wherein the control unit is configured to adjust the configuration of the column manager to direct the portion of the biological sample into one of the first and second sample analyzers.

[0322] 20. A system according to claim 10, further comprising a column manager which is fluidically connected to the first, second, and third sample analyzers and the second flow control device, and is connected to the control unit, wherein the control unit is configured to adjust the configuration of the column manager to direct the portion of the biological sample into one of the first, second, third, and fourth sample analyzers.

[0323] 21. A system according to claim 1, wherein the portion of the biological sample is a first portion, and

[0324] The product quality attribute is a first product quality attribute, and wherein the control unit is configured

[0325] Such that during operation of the system, the control unit:

[0326] (d) adjusting the configuration of the first flow control device to direct a second portion of the biological sample from the first flow control device to the sample purification device or the second flow control device, so that the second portion of the biological sample is purified by the second flow control device.

[0327] Control device receiving;

[0328] (e) adjusting the configuration of the second flow control device to direct the second portion of the biological sample to one of the first and second sample analyzers that did not receive the first portion of the biological sample; and

[0329] (f) determining a second product quality attribute of the analyte of the biological sample based on an analysis of the second portion of the biological sample by the one of the first and second sample analyzers that received the second portion of the biological sample.

[0330] 22. A system according to claim 21, wherein the first and second product quality attributes are different, and wherein the first and second product quality attributes are each selected from the concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variation or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

[0331] 23. A system according to claim 9, wherein the portion of the biological sample is a first portion, and

[0332] The product quality attribute is a first product quality attribute, and the control device is configured to repeat steps (a)-(c) on another portion of the biological sample to determine two different product quality attributes of the analyte of the biological sample.

[0333] 24. A system according to claim 9, wherein the portion of the biological sample is a first portion, and

[0334] The product quality attribute is a first product quality attribute, and the control device is configured to repeat steps (a)-(c) for two other portions of the biological sample to determine three different product quality attributes of the analyte of the biological sample.

[0335] 25. A system according to claim 10, wherein the portion of the biological sample is a first portion, and

[0336] The product quality attribute is a first product quality attribute, and the control device is configured to repeat steps (a)-(c) on another portion of the biological sample to determine two different product quality attributes of the analyte of the biological sample.

[0337] 26. A system according to claim 10, wherein the portion of the biological sample is a first portion, and

[0338] The product quality attribute is a first product quality attribute, and the control device is configured to repeat steps (a)-(c) for three other portions of the biological sample to determine four different product quality attributes of the analyte of the biological sample.

[0339] 27. A system according to any one of items 23-26, wherein the product quality attributes are each selected from the concentration of the analyte in the biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the charge variation or heterogeneity of the analyte in the biological sample, and a measure of the purity or integrity of the analyte in the biological sample.

[0340] 28. The system of claim 1, further comprising a sampling device coupled to the control unit and configured to receive the biological sample and deliver the portion of the biological sample to the first fluid control device.

[0341] 29. A system according to claim 28, wherein the sampling device includes a container interface, which is configured to receive the biological sample in a container.

[0342] 30. A system according to claim 28, wherein the sampling device comprises a fluid channel configured to receive the biological sample, and wherein the control unit is configured so that during operation of the system, the control unit sends a signal to the sampling device to cause the sampling device to transfer the biological sample to the sampling device.

[0343] The portion of the biological sample is discharged from the fluid channel into the first flow control device.

[0344] 31. The system according to item 9, further comprising:

[0345] a pump in fluid communication with the second flow control device, the first and second sample analyzers, and first and second buffer reservoirs associated with the first and second sample analyzers, respectively,

[0346] wherein the control unit and pump are configured such that during operation of the system, when the portion of the biological sample is directed into one of the first and second sample analyzers, the pump delivers a buffer solution from a corresponding associated buffer reservoir to the one of the first and second sample analyzers.

[0347] 32. The system according to item 10, further comprising:

[0348] a pump in fluid communication with the second flow control device, the first, second, and third sample analyzers, and first, second, and third buffer reservoirs associated with the first, second, and third sample analyzers, respectively,

[0349] wherein the control unit and the pump are configured such that during operation of the system, when the portion of the biological sample is directed into one of the first, second, and third sample analyzers, the pump delivers a buffer solution from a corresponding associated buffer reservoir to the one of the first, second, and third sample analyzers.

[0350] 33. The system according to claim 1, further comprising:

[0351] a pump in fluid communication with the second flow control device, the first sample analyzer, and a buffer reservoir associated with the first sample analyzer,

[0352] wherein the first chromatographic column is a cation exchange column; and

[0353] Wherein the control unit and the pump are configured such that during operation of the system, the pump delivers acetate buffer to the first chromatography column to propagate the portion of the biological sample along the first chromatography column.

[0354] 34. A system according to claim 33, wherein the acetate buffer has a pH of 4.0 or less.

[0355] 35. A system according to claim 1, wherein the biological sample is harvested culture medium extracted from a bioreactor.

[0356] 36. A system according to claim 1, wherein the biological sample is an intermediate solution or a product solution from a biomanufacturing system.

[0357] 37. A system according to claim 1, wherein the biological sample is part of a cell culture.

[0358] 38. A system according to item 6, wherein the quantization detector comprises a diode array detector.

[0359] 39. A system according to claim 6, wherein the quantitative detector includes a spectral detector configured to measure absorbance information of the portion of the biological sample.

[0360] 40. A system according to claim 6, wherein the quantitative detector comprises a fluorescence detector.

[0361] 41. A system according to claim 6, wherein the quantitative detector comprises a mass spectrometer detector.

[0362] 42. A system for measuring a plurality of product quality attributes of an analyte of a biological sample, the system comprising:

[0363] a first flow control device;

[0364] a sample purification device comprising a purification chromatography column in fluid communication with the first flow control device;

[0365] a second flow control device in fluid communication with the first flow control device and the sample purification device;

[0366] a first sample analyzer comprising a first chromatography column in fluid communication with the second flow control device;

[0367] a second sample analyzer comprising a second chromatography column in fluid communication with the second flow control device;

[0368] a third sample analyzer comprising a third chromatography column in fluid communication with the second flow control device;

[0369] a fourth sample analyzer comprising a quantitative detector; and

[0370] a control unit coupled to the first and second flow control devices and configured to cause

[0371] During operation of the system, the control unit:

[0372] (a) adjusting the configuration of the first flow control device to direct a first portion of the biological sample from the first flow control device to the sample purification device or the second flow control device, so that the portion of the biological sample is purified by the second flow control device;

[0373] Set to receive;

[0374] (b) adjusting the configuration of the second flow control device to direct the first portion of the biological sample to one of the first, second, third, and fourth sample analyzers;

[0375] (c) determining a first product quality attribute of an analyte of the biological sample based on analysis of the portion of the biological sample by said one of the first, second, third, and fourth sample analyzers; and

[0376] (d) repeating steps (a)-(c) for three additional portions of the biological sample, adjusting the configuration of the second flow control device so that each portion of the biological sample is directed to a different one of the sample analyzers to determine a total of four product quality attributes of the analytes of the biological sample.

[0377] 43. A system according to item 42, wherein the four product quality attributes are different.

[0378] 44. A system according to item 42, wherein the first, second and third chromatographic columns are different types of columns.

[0379] 45. A system according to item 44, wherein the first chromatographic column is a cation exchange column, the second chromatographic column is a size exclusion column, and the third chromatographic column is a reverse phase column or a hydrophilic interaction column.

[0380] 46. ​​A system according to claim 42, wherein the first sample analyzer determines information about a measure of charge variation or heterogeneity of the analyte in the biological sample, wherein the second sample analyzer determines information about a measure of aggregation of the analyte in the biological sample, wherein the third sample analyzer determines information about a measure of purity or integrity of the analyte in the biological sample, and wherein the fourth sample analyzer determines information about the concentration of the analyte in the biological sample.

[0381] 47. A system according to claim 42, wherein the four quality attributes include a measure of charge variation or heterogeneity of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of purity or integrity of the analyte in the biological sample, and the concentration of the analyte in the biological sample.

[0382] 48. A system according to claim 42, wherein the first chromatographic column is a cation exchange chromatographic column, the second chromatographic column is a size exclusion chromatographic column, and the third chromatographic column is a reverse phase chromatographic column.

[0383] 49. A system according to claim 42, wherein the sample purification device comprises an affinity chromatography column.

[0384] 50. A system according to claim 42, wherein the four product quality attributes include the concentration of the analyte in the biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the charge variation or heterogeneity of the analyte in the biological sample, and a measure of the purity or integrity of the analyte in the biological sample.

[0385] 51. A system according to claim 42, wherein the analyte comprises a protein in the biological sample.

[0386] 52. A system according to item 51, wherein the protein includes antibodies in the biological sample.

[0387] 53. A system according to claim 42, further comprising a column manager which is fluidly connected to the first, second, and third sample analyzers and the second flow control device, and is connected to the control unit, wherein the control unit is configured to adjust the configuration of the column manager to direct the portion of the biological sample into one of the first, second, and third sample analyzers.

[0388] 54. The system of claim 42, further comprising a sampling device coupled to the control unit and configured to receive the biological sample and deliver the portion of the biological sample to the first fluid control device.

[0389] 55. A system according to claim 42, wherein the quantitative detector comprises one of a diode array detector, a spectral detector configured to measure absorbance information of the portion of the biological sample, a fluorescence detector, and a mass spectrometer detector.

[0390] 56. A method for measuring a plurality of product quality attributes of an analyte of a biological sample, the method comprising:

[0391] obtaining the biological sample by extracting the biological sample from an operating bioreactor or from a purification device in fluid communication with the operating bioreactor;

[0392] directing a first portion of the biological sample to a first sample analyzer and obtaining information about a first product quality attribute of an analyte of the biological sample by analyzing the first portion of the biological sample in the first sample analyzer;

[0393] directing a second portion of the biological sample to a second sample analyzer and obtaining information about a second product quality attribute of an analyte of the biological sample by analyzing the second portion of the biological sample in the second sample analyzer,

[0394] wherein the first and second product quality attributes are different; and

[0395] Wherein at least one of the first and second product quality attributes comprises a measure of charge variation or heterogeneity of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of purity or integrity of the analyte in the biological sample, and a concentration of the analyte in the biological sample.

Claims

1. A system for measuring product quality attributes of an analyte in a biological sample, the system comprising: a first flow control device; a sample purification device in fluid communication with the first flow control device; a second flow control device in fluid communication with the first flow control device, the sample purification device, and first and second sample analyzers, wherein the first sample analyzer includes a first chromatographic column; and a control unit coupled to the first and second flow control devices and configured such that during operation of the system, the control unit: (a) adjusts the configuration of the first flow control device to direct a portion of the biological sample from the first flow control device into the sample purification device or into the second flow control device such that the portion of the biological sample is received by the second flow control device; (b) adjusts the configuration of the second flow control device to direct the portion of the biological sample to one of the first and second sample analyzers; and (c) determines the product quality attributes of the analyte in the biological sample based on the analysis of the portion of the biological sample by one of the first and second sample analyzers.

2. The system according to claim 1, wherein the first chromatographic column is a cation exchange chromatographic column.

3. The system according to claim 1, wherein the first chromatographic column is a size exclusion chromatographic column.

4. The system according to claim 1, wherein the first chromatographic column is a reversed-phase chromatographic column.

5. The system according to claim 1, wherein the sample purification device includes an affinity chromatographic column.

6. The system according to claim 1, wherein the second sample analyzer includes a quantitative detector configured to generate an electrical signal representative of the amount of analyte in the biological sample.

7. The system according to claim 6, wherein the first chromatographic column is in fluid communication with the quantitative detector, and wherein the quantitative detector is configured to generate an electrical signal representative of the amount of analyte in the eluent stream from the first chromatographic column.

8. The system according to claim 1, wherein the first sample analyzer includes a quantitative detector in fluid communication with the first chromatographic column and configured to generate an electrical signal representative of the amount of analyte in the eluent stream from the first chromatographic column.

9. The system according to claim 1, wherein the second sample analyzer includes a second chromatographic column, and wherein the second chromatographic column is different from the first chromatographic column and is one of a cation exchange chromatographic column, a size exclusion chromatographic column, a reversed-phase chromatographic column, and a hydrophilic interaction chromatographic column.

10. The system according to claim 9, wherein the second flow control device is in fluid communication with a third sample analyzer including a third chromatographic column, and wherein the third chromatographic column is different from the first and second chromatographic columns and is one of a cation exchange chromatographic column, a size exclusion chromatographic column, a reversed-phase chromatographic column, and a hydrophilic interaction chromatographic column.

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