Measurement of Generated Substance Quality Characteristics

Abstract

A system for measuring product quality characteristics of analytes of a biological sample includes a first flow control device, a sample purification device, a second flow control device in fluid communication with first and second sample analyzers, the first sample analyzer including a first chromatography column, and a control unit configured, during operation of the system, to adjust the positioning of the second flow control device to direct a portion of the biological sample to one of the first and second sample analyzers and determine a product quality characteristic of the analyte of the biological sample based on analysis of the portion of the biological sample by one of the first and second sample analyzers.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 904,682, filed September 23, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to systems and methods for measuring product quality attributes on samples, including harvest samples, from continuous biological manufacturing systems. [Background technology]

[0003] Mammalian cells containing nucleic acids encoding recombinant proteins are often used to produce therapeutically or commercially important proteins. Integrated, continuous biological manufacturing is an important aspect of reducing the costs associated with such protein-based therapeutics. Monitoring systems are used in biological manufacturing to evaluate various biological products and process conditions. Summary of the Invention [Problem to be solved by the invention]

[0004] Integrated, continuous biological manufacturing of therapeutic protein substances and other biomolecules holds great promise for the future generation of life-saving drugs and for enhancing the widespread adoption of treatments that rely on the availability of such biomolecules. Two-column and multi-column chromatography systems in a variety of configurations can be used for industrial-scale biological manufacturing. In such systems, analysis of the eluate from the chromatography system can be used to determine various product quality attributes to monitor and regulate a wide variety of bioprocess conditions. [Means for solving the problem]

[0005] The present disclosure features methods and systems for determining one or more product quality attributes for analytes in biological samples, including samples harvested from a bioreactor and offline samples introduced into the system in series or parallel. A variety of product quality attributes can be measured, including, but not limited to, analyte concentration, analyte charge variant or heterogeneity, analyte aggregation, and analyte integrity or purity. The system can include a sample analyzer with different types of chromatographic columns dedicated to measuring specific product quality attributes. The measured product quality attributes can be used to provide feedback and control to parameters and operations associated with a biological manufacturing process.

[0006] In one embodiment, the disclosure provides a system for measuring a product quality attribute of an analyte of 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 a first and a second sample analyzer, the first sample analyzer comprising a first chromatography column, a control unit coupled to the first and second flow control devices, wherein during operation of the system, the control unit controls: (a) a flow rate of an analyte of the biological sample; The system features a control unit configured to: (a) adjust a position of the first flow control device to direct a portion of the biological sample from the first flow control device to either a sample purification device or a second flow control device such that a portion is received by the second flow control device; (b) adjust a position 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) determine a product quality characteristic of an analyte of the biological sample based on analysis of the portion of the biological sample by one of the first and second sample analyzers.

[0007] Embodiments of the system may include any one or more of the following features.

[0008] The first chromatography column is a cation exchange chromatography column, a size exclusion chromatography column or a reverse phase chromatography column. The sample purification device can include an affinity chromatography column.

[0009] The second sample analyzer can include a quantitative detector configured to generate an electrical signal representative of the amount of analyte in the biological sample. The first chromatographic column can be in fluid communication with the quantitative detector, and the quantitative detector can be configured to generate an electrical signal representative of the amount of analyte in the eluate stream from the first chromatographic column.

[0010] The first sample analyzer can include a quantitative detector in fluid communication with the first chromatography column and configured to generate an electrical signal representative of the amount of analyte in the eluate stream from the first chromatography column. The second sample analyzer can include a second chromatography column, the second chromatography column being different from the first chromatography column and being one of a cation exchange chromatography column, a size exclusion chromatography column, a reversed phase chromatography column, and a hydrophilic interaction chromatography column.

[0011] The second flow control device can be in fluid communication with a third sample analyzer that includes a third chromatography column, the third chromatography column being different from the first and second chromatography columns and being one of a cation exchange chromatography column, a size exclusion chromatography column, a reverse phase chromatography column, and a hydrophilic interaction chromatography column.

[0012] The second flow control device can be in fluid communication with four additional sample analyzers, each of the four additional sample analyzers featuring a chromatography column different from both the first chromatography column and the chromatography columns of the others of the four additional sample analyzers.

[0013] The product quality characteristic of the analyte is the concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variants or heterogeneity of the analyte in the biological sample, or a measure of purity or integrity of the analyte in the biological sample.

[0014] The affinity chromatography column is one of a Protein A chromatography column, a Protein G chromatography column, and a receptor binding column. The analyte can include a protein (e.g., an antibody) in a biological sample.

[0015] The system can include a column manager in fluid communication with the first and second sample analyzers and the second flow control device and coupled to a control unit, the control unit configured to adjust a position of the column manager to direct a portion of the biological sample to one of the first and second sample analyzers.

[0016] The system can include a column manager in fluid communication with the first, second and third sample analyzers and the second flow control device and coupled to a control unit, and the control unit can be configured to adjust a position of the column manager to direct a portion of the biological sample to one of the first, second, third and fourth sample analyzers.

[0017] The portion of the biological sample is a first portion, the product quality characteristic is a first product quality characteristic, and the control unit can be configured, during operation of the system, to: (d) adjust a positioning of the first flow control device such that a second portion of the biological sample is received by the second flow control device to direct the second portion of the biological sample from the first flow control device to either the sample purification device or the second flow control device; (e) adjust a positioning 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) determine a second product quality characteristic of an 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 characteristics are different, and the first and second product quality characteristics can each be selected from the group consisting of a concentration of an analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variants or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

[0018] The portion of the biological sample may be a first portion, the product quality characteristic may be a first product quality characteristic, and the control device may be configured to repeat steps (a)-(c) with another portion of the biological sample to determine two different product quality characteristics for the analytes of the biological sample.

[0019] The portion of the biological sample may be a first portion, the product quality characteristic may be a first product quality characteristic, and the control device may be configured to repeat steps (a)-(c) with two other portions of the biological sample to determine three different product quality characteristics for analytes of the biological sample.

[0020] The portion of the biological sample may be a first portion, the product quality characteristic may be a first product quality characteristic, and the control device may be configured to repeat steps (a)-(c) with three other portions of the biological sample to determine four different product quality characteristics for analytes in the biological sample.

[0021] The product quality characteristic 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 variants or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

[0022] The system can include a sample collection device coupled to the control unit, the sample collection device configured to receive a biological sample and deliver a portion of the biological sample to the first fluid control device. The sample collection device can include a container interface configured to receive the biological sample in a container. The sample collection device can include a fluidic channel configured to receive the biological sample, and the control unit can be configured to send a signal to the sample collection device during operation of the system to cause the sample collection device to expel a portion of the biological sample from the fluidic channel to the first flow control device.

[0023] The system can include 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, and the control unit and pump are configured such that during operation of the system, when a portion of the biological sample is directed to 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.

[0024] The system may include 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, and the control unit and pump are configured such that during operation of the system, when a portion of the biological sample is directed to one of the first, second and third sample analyzers, the pump delivers a buffer solution from the corresponding associated buffer reservoir to one of the first, second and third sample analyzers.

[0025] The system can 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, the first chromatography column being a cation exchange column, and the control unit and pump are configured such that during operation of the system, the pump delivers an acetate buffer to the first chromatography column to propagate a portion of the biological sample along the first chromatography column. The acetate buffer can have a pH of 4.0 or less.

[0026] The biological sample is a harvest medium extracted from a bioreactor. The biological sample is an intermediate or product solution from a biological manufacturing system. The biological sample is a portion of a cell culture.

[0027] The quantitative detector may include a diode array detector, a spectrometric detector configured to measure absorbance information about a portion of the biological sample, a fluorescence detector, and / or a mass spectrometry detector.

[0028] Embodiments of the system may also include any of the other configurations described herein, including any combination of the configurations disclosed individually in different embodiments, unless otherwise stated.

[0029] In another aspect, the disclosure provides a system for measuring a product quality attribute for an analyte of a biological sample, the system comprising a first flow control device, a sample purification device including 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 including the first chromatography column in fluid communication with the second flow control device, a second sample analyzer including the second chromatography column in fluid communication with the second flow control device, a third sample analyzer including a third chromatography column in fluid communication with the second flow control device, a fourth sample analyzer including a quantitative detector, and a control unit coupled to the first and second flow control devices, wherein during operation of the system, the control unit controls: (a) a portion of the biological sample to flow through the second flow control device; (b) adjusting a configuration of the first flow control device to direct a first portion of the biological sample from the first flow control device to either the sample purification device or the second flow control device so as to be received by the quantity control device; (b) adjusting a 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 characteristic 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) with three additional portions of the biological sample and adjusting a configuration of the second flow control device to direct each portion of the biological sample to a different one of the sample analyzers to determine a total of four product quality characteristics of the analyte of the biological sample.

[0030] Embodiments of the system may include any one or more of the following features.

[0031] Each of the four product quality attributes can be different. Each of the first, second and third chromatography columns can be different types of columns. The first chromatography column can be a cation exchange column, the second chromatography column can be a size exclusion column, and the third chromatography column can be a reverse phase column or a hydrophilic interaction column.

[0032] The first sample analyzer can determine information about a measure of charge variants or heterogeneity of the analyte in the biological sample, the second sample analyzer can determine information about a measure of aggregation of the analyte in the biological sample, the third sample analyzer can determine information about a measure of purity or integrity of the analyte in the biological sample, and the fourth sample analyzer can determine information about the concentration of the analyte in the biological sample.

[0033] The four product quality attributes may include a measure of charge variants 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 chromatography column is a cation exchange chromatography column, the second chromatography column is a size exclusion chromatography column, and the third chromatography column is a reverse phase chromatography column.

[0034] The sample purification device can include an affinity chromatography column. The analyte can include a protein in the biological sample. The protein can include an antibody in the biological sample.

[0035] The system can include a column manager in fluid communication with the first, second and third sample analyzers and the second flow control device and coupled to a control unit, the control unit configured to adjust a configuration of the column manager to direct a portion of the biological sample to one of the first, second and third sample analyzers. The system can include a sample collection device coupled to the control unit, the sample collection device configured to receive the biological sample and deliver a portion of the biological sample to the first fluid control device.

[0036] The quantitative detector can include one of a diode array detector, a spectrometric detector, a fluorescence detector, and a mass spectrometry detector configured to measure absorbance information about a portion of the biological sample.

[0037] Embodiments of the system may also include any of the other configurations described herein, including any combination of the configurations disclosed individually in different embodiments, unless otherwise stated.

[0038] In a further aspect, the disclosure features a method for measuring a product quality characteristic of an analyte of a biological sample, the method including obtaining the biological sample by extracting the biological sample from an operational bioreactor or a purification device in fluid communication with the operational bioreactor, directing a first portion of the biological sample to a first sample analyzer, and analyzing the first portion of the biological sample in the first sample analyzer to obtain information about a first product quality characteristic of the analyte of the biological sample, directing a second portion of the biological sample to a second sample analyzer, and analyzing the second portion of the biological sample in the second sample analyzer to obtain information about a second product quality characteristic of the analyte of the biological sample, wherein the first and second product quality characteristics are different, and at least one of the first and second product quality characteristics includes a measure of charge variants 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.

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

[0040] definition The term "unit operation" is a term of art and refers to a functional step that can be performed in the process of producing a therapeutic protein drug substance from a liquid culture medium. For example, units of operation are filtering (e.g., removing contaminating bacteria, yeast viruses or mycobacteria, and / or particulate matter from a fluid containing a recombinant therapeutic protein), capturing, removing epitope tags, purifying, storing or storing, polishing, inactivating viruses, adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein, and removing undesirable salts.

[0041] The term "chromatographic cycle" or "chromatographic cycle" is a technical term and refers to all steps performed in a single round of chromatography using a single chromatographic column. For example, a chromatographic cycle can include the steps of equilibrating the chromatographic column with a buffer, passing a sample containing a recombinant protein through the chromatographic column, eluting the recombinant protein from the chromatographic column, and washing the chromatographic column by passing a denaturing buffer through the column. Additional examples of steps performed in a chromatographic cycle are described herein. Further examples of steps performed in a chromatographic cycle are also well known in the art.

[0042] The term "capturing" refers to a process performed to partially purify or isolate (e.g., at least or about 5%, 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 by weight), concentrate, and stabilize a recombinant therapeutic protein from one or more other components present in liquid culture medium or diluted liquid culture medium (e.g., culture medium proteins, or one or more other components (e.g., DNA, RNA or other proteins) present in or secreted from a mammalian cell). Typically, capturing is performed using a resin that binds the recombinant therapeutic protein (e.g., by use of affinity chromatography). Non-limiting methods for capturing recombinant therapeutic proteins from liquid culture medium or diluted liquid culture medium are described herein and others are known in the art. The recombinant therapeutic protein can be captured from the liquid culture medium using at least one chromatography column and / or chromatography membrane (e.g., any of the chromatography columns and / or chromatography membranes described herein).

[0043] The term "purifying" refers to a step performed to isolate a recombinant therapeutic protein from one or more other impurities (e.g., bulk impurities) or components present in a fluid containing the recombinant therapeutic protein (e.g., proteins of a liquid culture medium or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.) present in or secreted from mammalian cells). For example, purifying 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 either the recombinant therapeutic protein or the 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 chromatography column and / or chromatographic membrane (e.g., any of the chromatography columns or chromatographic membranes described herein).

[0044] The term "polishing" is a term of art and refers to a process carried out to remove remaining trace or minor contaminants or impurities from a fluid containing a recombinant therapeutic protein close to the final desired purity. For example, polishing can be carried out by passing the fluid containing the recombinant therapeutic protein through a chromatography column or membrane absorber that selectively binds either the target recombinant therapeutic protein or minor contaminants or impurities present in the fluid containing the recombinant therapeutic protein. In such an example, the eluate / filtrate of the chromatography column or membrane absorber contains the recombinant therapeutic protein.

[0045] The term "filtering" refers to the removal of 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 present in any of the systems or processes described herein).

[0046] The term "eluate / filtrate" is a term of art and refers to the fluid exiting a chromatography column or membrane that contains a detectable amount of recombinant therapeutic protein.

[0047] In certain contexts, the term "isolate" or "isolating" refers to at least partially purifying or purifying a recombinant protein (e.g., at least or about 5%, 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 by weight) from one or more other components present in the filtrate (e.g., a filtrate generated using the methods described herein), such as one or more of the DNA, RNA and / or other protein components present in the filtrate. Non-limiting methods for isolating proteins from filtrates are described herein and others are known in the art.

[0048] The term "integrated process" refers to a process carried out using structural elements that function cooperatively to achieve a particular result (e.g., production of a therapeutic protein drug substance from a liquid culture medium).

[0049] The term "continuous process" refers to a process in which a fluid is continuously supplied by at least a portion of the system. For example, in any of the exemplary continuous biological manufacturing systems described herein, liquid culture medium containing a recombinant therapeutic protein is continuously supplied to the system while the system is in operation, and therapeutic protein drug substance is pumped out of the system. In another example, a continuous process is a process in which a first MCCS continuously supplies liquid culture medium containing a recombinant therapeutic protein from a bioreactor. Another example of a continuous process is a process in which a first and a second MCCS continuously supplies liquid culture medium containing a recombinant therapeutic protein from a bioreactor. Additional examples include a process in which a first MCCS continuously supplies liquid culture medium containing a recombinant therapeutic protein, a first and a second MCCS continuously supplies liquid culture medium containing a recombinant therapeutic protein, or a second MCCS continuously supplies a fluid containing a recombinant therapeutic protein.

[0050] The term "biological manufacturing system" or "biological production system" refers to a system for producing a biological drug.

[0051] The term "biological drug" refers to any therapeutic substance made or obtained from a living organism or its products, which is used in the prevention, diagnosis, or treatment of a disease state. Thus, a biological drug or biopharmaceutical is a pharmaceutical produced using biotechnology, such as a protein (e.g., a recombinant therapeutic protein) or a nucleic acid (DNA, RNA, or antisense oligonucleotide), used for therapeutic or in vivo diagnostic purposes.

[0052] The term "multi-column chromatography system" or "MCCS" refers to a system of 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 contains a total of two or more interconnected or switched chromatographic columns and / or chromatographic membranes. Additional examples of multi-column chromatography systems are described herein and known in the art.

[0053] The term "mammalian cell" refers to any cell derived from or from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). In some embodiments, the mammalian cell is, for example, an immortalized cell, a differentiated cell, or an undifferentiated cell.

[0054] The term "cell culture" refers to a plurality of mammalian cells (e.g., any of the mammalian cells described herein) suspended in a liquid culture medium (e.g., any of the liquid culture media described herein). A cell culture can be 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 ... The cell density may be greater than about 55×106 cells / mL, greater than about 60×106 cells / mL, greater than about 65×106 cells / mL, greater than about 70×106 cells / mL, greater than about 75×106 cells / mL, greater than about 80×106 cells / mL, greater than about 85×106 cells / mL, greater than about 90×106 cells / mL, greater than about 95×106 cells / mL, or greater than about 100×106 cells / mL.

[0055] The term "culturing" or "cell culturing" refers to the maintenance or growth of mammalian cells in a liquid culture medium under a controlled set of physical conditions.

[0056] The term "liquid culture medium" refers to a fluid containing sufficient nutrients to allow mammalian cells to grow in the medium in vitro. For example, the liquid culture medium can contain one or more of amino acids (e.g., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, ions, copper, zinc, selenium, and other necessary trace metals, and sodium bicarbonate. The liquid culture medium can contain serum from a mammal. In some examples, the liquid culture medium does not contain serum or another extract from a mammal (defined liquid culture medium). The liquid culture medium can contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Non-limiting examples of liquid culture media are described herein, and additional examples are known in the art and commercially available.

[0057] The term "immunoglobulin" refers to a polypeptide that contains an amino acid sequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., a variable domain sequence, a framework sequence, or a constant domain sequence). The immunoglobulin can, for example, comprise a light chain immunoglobulin of at least 15 amino acids, e.g., a heavy chain immunoglobulin of at least 15 amino acids. The immunoglobulin can be an isolated antibody (e.g., IgG, IgE, IgD, IgA, or IgM). The immunoglobulin can be a subclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The immunoglobulin can be an antibody fragment, e.g., a Fab fragment, a F(ab')2 fragment, or a scFv fragment. The immunoglobulin can also be a bispecific or trispecific antibody, or a dimeric, trimeric, or multimeric antibody, or a diabody, Affibody®, or Nanobody®. An immunoglobulin can also be an engineered protein (e.g., a fusion protein) that contains at least one immunoglobulin domain. Non-limiting examples of immunoglobulins are described herein, and additional examples of immunoglobulins are known in the art.

[0058] The term "recombinant therapeutic protein" or "recombinant protein" refers to any therapeutic protein obtained using recombinant DNA technology. As used herein, "recombinant therapeutic protein" includes, for example, an antibody or antibody fragment, an enzyme, an engineered protein, or an immunogenic protein or protein fragment.

[0059] The term "protein fragment" or "polypeptide fragment" refers to 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 longer than 20 amino acids. Recombinant protein fragments can be generated using any of the processes described herein.

[0060] The term "engineered protein" refers to 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., substitution, deletion, insertion, or addition of one or more amino acids that result in increased stability and / or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins that contain at least one recombinant scaffold sequence.

[0061] The term "secreted protein" or "secreted recombinant protein" means that in a mammalian cell, a protein (e.g., a recombinant protein) that originally contains at least one secretory signal sequence is secreted, at least in part, into the extracellular space (e.g., liquid culture medium) when it is translated within the mammalian cell and, at least in part, by enzymatic cleavage of the secretory signal sequence. A "secreted" protein need not be completely dissociated from the cell to be considered a secreted protein.

[0062] The term "perfusion bioreactor" refers to a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where culturing the cells present in the bioreactor comprises periodic or continuous removal of the first liquid culture medium and simultaneously or immediately thereafter adding a substantially equal volume of a second liquid culture medium to the bioreactor. In some instances, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium that is removed and added over an incremental period during the culturing period (e.g., about a 24 hour period, about a 1 minute to about 24 hour period, or a period longer than 24 hours) (e.g., a daily refeed rate of the culture medium). The rate of medium removed and replaced each day can vary depending on the particular cells being cultured, the initial seeding density, and the cell density at a particular time. "RV" or "reactor volume" refers to the volume of culture medium present at the beginning of the culturing process (e.g., the total volume of culture medium present after seeding).

[0063] The term "fed-batch bioreactor" is a term of art and refers to a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, where culturing the cells present in the bioreactor includes periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without substantial or substantial removal of the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium can be the same as the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is added as a dry powder.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter herein, but suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0065] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description, drawings, and claims. [Brief description of the drawings]

[0066] [Figure 1] FIG. 1 is a schematic diagram of an example system for determining a product quality attribute for an analyte of a biological sample. [Diagram 2] FIG. 2 is a schematic diagram of an example sample manager. [Diagram 3] FIG. 3 is a flow chart illustrating an example set of steps for determining an analyte product quality attribute for a biological sample. [Figure 4-1] 4A to 4C are schematic diagrams showing examples of the arrangement of flow control devices. [Figure 4-2] Continued from Figure 4-1. [Figure 5A] FIG. 5A is a graph showing titer chromatograms for multiple samples. [Figure 5B] FIG. 5B is a table showing the calculated mass loadings and concentrations for several samples. [Figure 6A] FIG. 6A is a graph showing titer chromatograms for multiple samples. [Figure 6B]FIG. 6B is a set of tables with the measured peak information for different samples. [Figure 7] FIG. 7 is a graph showing the chromatograms measured for a set of samples. [Figure 8] FIG. 8 is a schematic diagram illustrating an example of a biological manufacturing system. [Figure 9] FIG. 9 is a schematic diagram showing examples of three column switching techniques. [Figure 10] Figure 10 is a graph comparing three titration methods (MIMICS-mPQA-blue; CEDEX™ Bioanalyzer-red; Octet-green) for a 100 L bioreactor of therapeutic monoclonal antibodies. All plotted data points in MIMICs-mPQA were frozen harvests. Gaps in the data represent samples that were not analyzed. [Figure 11] FIG. 11 is a graph comparing three titration methods (MIMICS-mPQA-blue; CEDEX™ Bioanalyzer-red; Octet-green) for a 3L bioreactor (V09) of therapeutic monoclonal antibodies. All plotted data points in MIMICs-mPQA were frozen harvests. Gaps in the data represent samples that were not analyzed. [Figure 12] Figure 12 is a graph comparing three titration methods (MIMICS-mPQA-blue; CEDEX™ Bioanalyzer-red; Octet-green) for a 3L bioreactor (V12) of a therapeutic monoclonal antibody. All plotted data points in MIMICs-mPQA were frozen harvests. Gaps in the data represent samples that were not analyzed. [Figure 13]Figure 13 is a graph comparing two aggregation methods (MIMICS-mPQA and offline method) for a 100 L bioreactor of therapeutic monoclonal antibodies. The graph compares the output of the two assays: percentage of monomer and percentage of high molecular weight (HMW). All plotted data points in MIMICS-mPQA were from frozen harvests. Gaps in the data represent samples that were not analyzed. [Figure 14] Figure 14 is a graph comparing two aggregation methods (MIMICS-mPQA and offline method) for a 3L bioreactor (V09) of therapeutic monoclonal antibodies. The graph compares the output of the two assays: percentage of monomer and percentage of high molecular weight (HMW). All plotted data points in MIMICS-mPQA were from frozen harvests. Gaps in the data represent samples that were not analyzed. [Figure 15] Figure 15 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 the output of the two assays: percentage of monomer and percentage of high molecular weight (HMW). All plotted data points in MIMICS-mPQA were from frozen harvests. Gaps in the data represent samples not analyzed. [Figure 16] Figure 16 is a graph comparing the two purity methods (MIMICS-mPQA and offline method) for all bioreactors (100L, 3L V09, 3L V12) from a therapeutic monoclonal antibody for overall purity %. All plotted data points in MIMICS-mPQA were from frozen harvests. Gaps in the data represent samples that were not analyzed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Like symbols in the drawings indicate like elements.

[0068] Introduction Industrial-scale biological production can be carried out in two-column and multi-column chromatography systems in various configurations. In these complex systems, product yield, quality and waste rate are functions of numerous process-related parameters and steps. During the production of therapeutic proteins and other commercially valuable biomolecules, product outcomes are strongly influenced by these parameters and steps. Thus, proper control over such parameters and steps is an important aspect of large-scale production. Configurations and aspects of biological production systems are disclosed, for example, in PCT Patent Application Publication WO 2014 / 137903, the entire contents of which are incorporated herein by reference.

[0069] Providing adequate control over biological production parameters, including automated control, is facilitated by monitoring bioreactor harvest, intermediate solution streams and / or products. Conventional monitoring techniques include, for example, UV absorbance measurements.

[0070] Unfortunately, such methods are subject to drift over measurement periods of several days due to factors such as temperature, humidity, ambient light intensity and local sample non-uniformity. Furthermore, such methods may not be capable of calculating or otherwise determining multiple quantities. In complex biological manufacturing environments, multiple quantities are typically evaluated to provide suitable feedback information for adjustment of process parameters.

[0071] The present disclosure features systems and methods that can be used to determine values ​​of multiple product quality characteristics. The system can be implemented as a two-dimensional chromatography system. Along a first dimension of the system, a portion of the biological sample can be purified using a sample purification device, which can optionally include a chromatography column. Along a second dimension of the system, a portion of the sample can then be directed to one of a plurality of different sample analyzers to determine a product quality characteristic for the sample. An additional portion of the sample can be directed to a different sample analyzer to determine a different product quality characteristic for the sample.

[0072] Product quality characteristic analysis system 1 is a schematic diagram illustrating 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, multiple sample analyzers 114a-114b, a detector 116, a second pump 118 (e.g., a quaternary pump), solvent / buffer reservoirs 120a-120d, and a control unit 112. 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 coupled to a control unit 122 via communication lines 124a-124g.

[0073] During operation, the sample manager 102 accepts a biological sample for analysis. The sample manager 102 can be implemented in a variety of ways. In some embodiments, for example, the sample manager includes a container reservoir configured to accept the sample in a container. Suitable containers include, for example, vials, tubes, and other sealed or unsealed vessels. In certain embodiments, the sample can be carried by a single or multiple well plate, and the container reservoir is configured to accept such a plate. The sample manager 102 can optionally include a transfer mechanism for transferring a portion of the biological sample to the first flow control device 106. Suitable transfer mechanisms include, but are not limited to, syringe-based sample injection devices, and single or multiple channel fluid transfer devices. Examples of suitable sample managers include the Waters H Class Sample Manager with a flow-through needle, and the Waters Process Sample Manager (both available from Waters Corp., Milford, MA).

[0074] In certain embodiments, the sample manager 102 receives the biological sample from a sampling device, a fluid conduit, or another component of the biological manufacturing system that is in fluid communication with the bioreactor. For example, the biological sample can be collected directly from the bioreactor (and thus represents a collected sample of growth medium) or it can be a solution or medium extracted from elsewhere in the biological manufacturing system.

[0075] 2 is a schematic diagram of an example of a sample manager 102 configured to accept a biological sample directly from a sample collection device. The sample manager 102 includes an inlet 202, a holding conduit 204, and a gate valve 206 coupled to the control unit 112 via a control line 124c. The biological sample is introduced into the sample manager 102 through the inlet 202, and the sample is maintained in the holding conduit 204 before being delivered to the first flow control device 106. 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 and a portion of the sample is ejected from the holding conduit 204 to the outlet conduit 208. The ejected portion of the sample is then pumped (e.g., by the first pump 104) to the first flow control device 106.

[0076] In general, a wide variety of different biological samples can be accepted by the sample manager 102. In some embodiments, the biological sample corresponds to a harvested portion of the growth medium from a bioreactor, as described above. In certain embodiments, the biological sample corresponds to a process fluid or medium extracted from another location in a biological manufacturing system, for example, a product- or intermediate-containing solution sampled before or after a purification step in a biological manufacturing system.

[0077] In certain embodiments, system 100 can be used to determine product quality characteristics for cell line development, and the biological sample represents a portion of a cell culture, cell culture medium, a fluid suspension of cells, or another type of sample in which cells, cell degradation products, intracellular metabolites, and / or cell culture impurities are present.

[0078] The system 100 determines product quality characteristics for one or more analytes in a biological sample received by the sample manager 102. In general, characteristics can be determined for a wide variety of different types of analytes. For example, in some embodiments, the system 100 determines product quality characteristics for protein analytes including, but not limited to, antibodies (monospecific, bispecific and trispecific antibodies), non-antibody proteins, fusion proteins and / or Fab fragments.

[0079] 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 of the antibody fragments described herein)), enzymes (e.g., galactosidases (e.g., alpha-galactosidase), myozyme or cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF) or interferon alpha or beta), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in vaccines). The recombinant therapeutic protein can be an engineered antigen-binding polypeptide that contains at least one multifunctional recombinant protein scaffold (see, e.g., the recombinant antigen-binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, the entire contents of which are incorporated herein by reference).

[0080] Non-limiting examples of recombinant therapeutic proteins that are antibodies include panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab, tocilizumab, basiliximab, bectumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, bicilomab, canakinumab, certolizumab, cetuximab, cizutumumab, daclizumab, These include denosumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab, imagatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab.

[0081] Additional non-limiting examples of recombinant therapeutic proteins that can be analyzed include alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic 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, These include mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, interleukin-11, laronidase, idursulfase, galsulfase, alpha-1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alpha (e.g., Thyrogen®), and alteplase. Additional examples of recombinant proteins that can be produced by the present methods include acid α-glucosidase, alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase (e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, and β-glucuronidase. Examples of enzymes that may be used include lysosomal acid ceramidase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase, α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A and hexosaminidase B.

[0082] As discussed above, in some embodiments, the analyte is a cellular component, and the methods and systems described herein can be used for process development of cell lines. 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 growing in suspension or adherent cells. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells or CHO-K1s 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) cells.

[0083] The mammalian cell can contain a recombinant nucleic acid (e.g., a nucleic acid that is stably integrated into the genome of the mammalian cell) that encodes a recombinant therapeutic protein. Non-limiting examples of recombinant nucleic acids that encode exemplary recombinant therapeutic proteins are described below, as are recombinant therapeutic proteins that can be produced using the methods described herein. In some examples, the mammalian cells cultured in a bioreactor (e.g., any of the bioreactors described herein) were derived from a larger culture.

[0084] Nucleic acids encoding recombinant therapeutic proteins can be incorporated into mammalian cells using a wide variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentiviral, adenoviral or retroviral infection) and electroporation. In some instances, the nucleic acid encoding the recombinant therapeutic protein is not stably integrated into the chromosome of the mammalian cell (transient transfection), while in other instances, the nucleic acid is integrated. Alternatively, or in addition, the nucleic acid encoding the recombinant therapeutic protein can be present in a plasmid and / or a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively, or in addition, the nucleic acid can be introduced into the cell using a viral vector (e.g., a lentiviral, retroviral or adenoviral vector). The nucleic acid can be operably linked to a promoter sequence (e.g., a strong promoter such as the β-actin promoter and the CMV promoter, or an inducible promoter). The vector containing the nucleic acid can also optionally contain a selectable marker, such as a gene that confers hygromycin, puromycin or neomycin resistance to mammalian cells.

[0085] In some embodiments, the recombinant therapeutic protein is a secreted protein and is released by mammalian cells into the extracellular medium. For example, the nucleic acid sequence encoding a soluble recombinant therapeutic protein can contain a sequence encoding a secretory signal peptide at the N- or C-terminus of the recombinant therapeutic protein, which is cleaved by an enzyme present in mammalian cells and then released into the extracellular medium.

[0086] After the biological sample is accepted by the sample manager 102, the sample is transported by the first pump 104 through a conduit 126 to the first flow control device 106. The first flow control device 106 is connected to the control unit 122 via a control line 124b. In general, the biological sample can be delivered by the first flow control device 106 to a number of different outputs. In a first configuration, the first flow control device 106 delivers the biological sample directly to the second flow control device 110 via a conduit 128. In another configuration, the first flow control device 106 delivers the biological sample to the sample purification device 108 via a conduit 130. The control unit 102 is configured to adjust the configuration of the first flow control device 106 to direct the biological sample to either destination depending on the desired mode of analysis of the biological sample.

[0087] The first flow control device 106 can be implemented in a variety of 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, IDEX MX Series II 2-position 6-port UltraLife Switching Valves (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 with input and / or output manifolds and electrically controllable flow regulators.

[0088] When a 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 occur in a variety of ways, but typically involves removing one or more non-analyte components from the sample. Alternatively, or in addition, purification of a biological sample can include concentration of an analyte in the sample, as well as separation of an analyte from one or more additional analytes in the sample.

[0089] The sample purification device 108 can be implemented in a variety of ways. In some embodiments, for example, the sample purification device 108 is implemented as a chromatography apparatus and features one or more chromatography columns. Suitable chromatography columns for use in the sample purification device 108 include, for example, affinity chromatography columns. The term "affinity chromatography" refers to a type of chromatography in which analyte molecules (e.g., recombinant protein analytes) are captured and isolated based on affinity. Affinity chromatography refers to the use of affinity chromatography resins (e.g., affinity chromatography resins that include a protein ligand (e.g., Protein A or Protein G)). In some embodiments, affinity chromatography includes pseudoaffinity chromatography resins. In some embodiments, affinity chromatography resins include cofactor ligands, substrate ligands, metal ligands, product ligands, or aptamer ligands. In general, affinity chromatography resins can include any receptor or ligand that has affinity for any biological analyte, including DNA, oligonucleotides. In some embodiments, affinity chromatography resins can include single domain antibody fragments from Camelidae for the purification of gene therapy vectors. In another example, the affinity chromatography column is an adeno-associated virus (AAV) affinity chromatography column.

[0090] Non-limiting examples of affinity chromatography resins include protein or peptide ligands (e.g., from about 5 amino acids to about 100 amino acids, from about 5 amino acids to about 90 amino acids, from about 5 amino acids to about 80 amino acids, from about 5 amino acids to about 70 amino acids, from about 5 amino acids to about 60 amino acids, from about 5 amino acids to about 50 amino acids, from about 5 amino acids to about 40 amino acids, from about 5 amino acids to about 30 amino acids, or from about 5 amino acids to about 20 amino acids), small molecule substrates or cofactors for enzymes, aptamers, inhibitors (e.g., competitive protein inhibitors), or metals.

[0091] Non-limiting examples of Protein A affinity chromatography resins are GE MabSelect SuRe™ (highly cross-linked agarose resin with a particle size of 85 μm and epoxy functional groups connecting Protein A to the agarose), JSR LifeSciences Amsphere ProA JWT203 (a porous poly-methacrylate resin with a particle size of approximately 50 μm and epoxy functional groups connecting Protein A to the poly-methacrylate), and Kaneka KanCap A (highly cross-linked cellulose with a particle size of 65-85 μm and Protein A linked to the cellulose by reductive amination).

[0092] For an affinity chromatography column, e.g., a Protein A column, the steps in a cycle of affinity chromatography can include loading an analyte-containing fluid onto the affinity column, e.g., a Protein A chromatography column, washing the column to remove undesired biomaterial (e.g., contaminating proteins and / or small molecules), eluting the target recombinant protein bound to the column, and re-equilibrating the column.

[0093] Any of the single steps in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any of the single steps in a chromatography cycle can include a buffer gradient. Any of a variety of well-known combinations of aspects of a single cycle of chromatography can be used in these methods, for example, different chromatography resins, flow rates, buffers, void volumes of the columns, bed volumes of the columns, volumes of buffers used in each step, volumes of fluids containing the target protein, and the number and types of buffers used in each step, in any combination.

[0094] In some embodiments, a Protein A column can be loaded with 1× phosphate buffered saline (PBS) at a pH of about 7 (e.g., about pH 7.0, 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, a Protein A column can be loaded with 1× PBS at a pH of about 7.2.

[0095] In the embodiment of this invention, the process A-label is about pH 2 to about pH 4 (for example, 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), about 50 mM to about 200 mM. Benzyl pyrrolidone (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 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 75m The present invention relates to a polymer comprising: a molecular weight of about 100 mM to about 150 mM, about 75 mM to about 140 mM, about 75 mM to about 130 mM, about 75 mM to about 120 mM, about 75 mM to about 110 mM, about 75 mM to about 100 mM, about 75 mM to about 90 mM, about 75 mM to about 80 mM, about 100 mM to about 200 mM, about 100 mM to about 190 mM, about 100 mM to about 180 mM, about 100 mM to about 170 mM, about 100 mM to about 160 mM, about 100 mM to about 150 mM, about 100 mM to about 140 mM, about 100 mM to about 130 mM, about 100 mM to about 120 mM, about 100 mM to about 110 mM 10mM, 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, generally about 180mM to about 200mM),about 50 mM to about 200 mM NaCl (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 75 mM to about 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 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 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 about 150mM, about 140mM to about 200mM, about 140mM to about 190mM, about 140mM to about 180mM, about 140mM to about 170mM, about 140mM to about 160mM, about 140mM to about 150mM, about 150mM to about 200mM, about 150mM to about 190 mM, 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, and about 180mM to about 200mM).

[0096] In some embodiments, the Protein A column is at 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), 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 60 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 2 0mM, approximately 20mM to approximately 100mM, approximately 20mM to approximately 90mM, approximately 20mM to approximately 80mM, approximately 20mM to approximately 70mM, approximately 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 ~approx. 100mM, approx. 30mM ~ approx. 90mM, approx. 30mM ~ approx. 80mM, approx. 30mM ~ approx. 70mM, approx. 30mM ~ approx. 60mM, approx. 30 mM to approx. 50mM, approx. 30mM to approx. 40mM, approx. 40mM to approx. 100mM, approx. 40mM to approx. 90mM, approx. 40mM to approx. 80mM, approx. 4 The sample is eluted with sodium acetate (0 mM to about 70 mM, about 40 mM to about 60 mM, about 40 mM to about 50 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 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).

[0097] Chromatography performed using this type of chromatography column can include, for example, sequential chromatography steps of loading, washing, eluting, and regenerating the chromatography column as typically performed. Any of the exemplary flow rates, buffer volumes, and / or lengths of time assigned for each sequential chromatography step described herein can be used in any of these different sequential chromatography steps.

[0098] In some embodiments, a single chromatographic column or a single chromatographic membrane containing a resin capable of capturing an analyte is loaded in, for example, about 5 minutes to about 90 minutes (e.g., about 10 minutes to about 90 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 80 minutes, about 30 minutes to about 80 minutes, about 40 minutes to about 80 minutes, and about 50 minutes to about 80 minutes).

[0099] After loading of the analyte onto the column, the column is washed with at least one wash buffer, which is meant to elute all components that are not the analyte from the column but that do not interfere with the interaction of the analyte with the resin.

[0100] The wash buffer can be passed through the column at a flow rate of about 0.1 mL / min to about 25 mL / min (e.g., a flow rate of about 0.2 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 14 mL / min, about 1.0 mL / min to about 25.0 mL / min, or about 1.0 mL / min to about 15.0 mL / min).

[0101] The volume of the washing buffer used (for example, when two or more washing buffers are used, the total volume of the washing buffers used) is, for example, about 1 x column volume (CV) to about 15 x CV (for example, about 1 x CV to about 14 x CV, about 1 x CV to about 13 x CV, about 1 x CV to about 12 x CV, about 1 x CV to about 11 x CV, about 2 x CV to about 11 x CV, about 3 x CV to about 11 x CV, about 4 x CV to about 11 x CV, about 5 x CV to about 11 x CV, or about 5 x CV to about 10 x CV). The total time of washing is, for example, about 2 minutes to about 3 hours (for example, about 2 minutes to about 2.5 hours, about 2 minutes to about 2.0 hours, about 5 minutes to about 1.5 hours, about 10 minutes to about 1.5 hours, about 10 minutes to about 1.25 hours, about 20 minutes to about 1.25 hours, or about 30 minutes to about 1 hour).

[0102] After washing the column, the analyte is eluted from the column by passing an 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 is, for example, from about 1 x column volume (CV) to about 15 x CV (e.g., from about 1 x CV to about 14 x CV, from about 1 x CV to about 13 x CV, from about 1 x CV to about 12 x CV, from about 1 x CV to about 11 x CV, from about 2 x CV to about 11 x CV, from about 3 x CV to about 11 x CV, from about 4 x CV to about 11 x CV, from about 5 x CV to about 11 x CV, or from about 5 x CV to about 10 x CV). The total elution time is, for example, about 0.1 minutes to about 3 hours (e.g., about 2 minutes to about 2.5 hours, about 2 minutes to about 2.0 hours, about 2 minutes to about 1.5 hours, about 2 minutes to about 1.5 hours, about 2 minutes to about 1.25 hours, about 2 minutes to about 1.25 hours, about 2 minutes to about 1 hour, about 2 minutes to about 40 minutes, about 10 minutes to about 40 minutes, about 20 minutes to about 40 minutes, about 0.1 minutes to about 10 minutes).

[0103] Non-limiting examples of elution buffers that can be used depend on the capture mechanism and / or analyte. For example, the elution buffer can contain a different concentration of salt (e.g., increased salt concentration), a different pH (e.g., increased or decreased salt concentration), or a molecule that competes with the analyte for binding to the resin. Examples of such elution buffers are described above.

[0104] After elution of the analyte from the column, the column can be equilibrated using a regeneration buffer. The regeneration buffer can be passed through the column at a flow rate of, for example, about 0.1 mL / min to about 25 mL / min (e.g., about 0.2 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 5.0 mL / min to about 15.0 mL / min, or about 1.0 mL / min to about 15.0 mL / min).

[0105] The volume of the regeneration buffer used to equilibrate the column is, for example, from about 1 x column volume (CV) to about 15 x CV (e.g., from about 1 x CV to about 14 x CV, from about 1 x CV to about 13 x CV, from about 1 x CV to about 12 x CV, from about 1 x CV to about 11 x CV, from about 2 x CV to about 11 x CV, from about 3 x CV to about 11 x CV, from about 2 x CV to about 5 x CV, from about 4 x CV to about 11 x CV, from about 5 x CV to about 11 x CV, or from about 5 x CV to about 10 x CV).

[0106] 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., include different chromatography resins) or can be the same. Furthermore, the multiple columns can be loaded and / or eluted with the same solvents and buffers or with different solvents and / or buffers. Each of the columns in a multi-column sample purification device can include any one or more of the chromatography resins described herein and can be loaded and / or eluted with any one or more of the different solvents and buffers described herein.

[0107] The biological sample is delivered to the second flow control device 110, either directly from the first flow control device 106 or via the sample purification device 108, as described above. The second flow control device 110 accepts the biological sample and directs it along one of a number of flow paths according to a control signal from the control unit 122 transmitted on control line 124d. The second flow control device 110 can generally be performed in the same manner as the first flow control device 106 described above.

[0108] Generally, the second flow control device 110 directs the biological sample to one of a number of sample analyzers, as shown in FIG. 1. Generally, the system 100 can include two or more sample analyzers (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, ten or more, fifteen or more, or even more). Each of the sample analyzers is associated with measuring one product quality characteristic for an analyte of the biological sample. In FIG. 1, four different sample analyzers 114a-114d are shown as examples. However, it should be understood that the system 100 can include any number of sample analyzers depending on the number of product quality characteristics to be measured.

[0109] In some embodiments, a particular sample analyzer does not include a chromatography column. For example, in FIG. 1, the sample analyzer 114a does not include a chromatography column. The biological sample can be delivered directly to the sample analyzer 114a from the second flow control device 110. More specifically, when a product quality characteristic associated with the sample analyzer 114a is measured, the control unit 122 sends a control signal to the second flow control device 110 to adjust the configuration of the second flow control device 110 to deliver the biological sample to the sample analyzer 114a. The product quality characteristic associated with the sample analyzer 114a is measured for an analyte in the biological sample.

[0110] In certain embodiments, certain sample analyzers include a chromatography column. For example, in FIG. 1, sample analyzers 114b-114d each include a chromatography column. To deliver a biological sample to such a sample analyzer, the sample can be delivered directly from the second flow control device 110 under the control of the control unit 122, as described above.

[0111] Alternatively, in some embodiments, the system 100 optionally includes a column manager 112 that accepts the biological sample from the second flow control device 110 and directs it to the sample analyzer. The column manager can be particularly useful in systems that include multiple sample analyzers with chromatographic columns. As shown in FIG. 1, the column manager 112 can be coupled to the control unit 122 via control line 124f. To direct the biological sample to the sample analyzer associated with the particular product quality characteristic being 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 direct the biological sample to the flow path of the sample analyzer.

[0112] The column manager 112 can be in fluid communication with one or more reservoirs (shown as four reservoirs 120a-120d in FIG. 1 for illustrative purposes) optionally via a second pump 118, which can also be optionally coupled to a control unit 122 via control line 124e. Once the biological sample is directed to a particular sample analyzer, the control unit 122 sends a control signal to the second pump 118, causing the second pump 118 to direct a flow of a suitable load buffer, elution buffer, or other suitable solvent or solution to the particular sample analyzer. Although four reservoirs are shown by way of example in FIG. 1, it should be understood that in general the second pump 118 can be in fluid communication with two or more reservoirs (e.g., three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, twenty or more, or even more).

[0113] 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, Mass.) with additional solvent selection valve modifications.

[0114] After the biological sample is loaded onto a column of a selected chromatography 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 for the analytes of the biological sample.

[0115] Analysis of the eluate can be performed in a variety of ways. In some embodiments, for example, system 100 includes a detector 116 in fluid communication with the outlet of a chromatography column of the sample analyzer. Detector 116 detects analytes in the eluate and provides measurement information to control unit 122 via control line 124g. Control unit 122 uses the measurement information (which generally corresponds to chromatographic and / or chromatographic information such as height, area and time of detected peaks) to determine values ​​of certain product quality attributes for the analytes of the biological sample.

[0116] System 100 generally employs a wide variety of detectors. In some embodiments, detector 116 corresponds to a photodiode array detector. Suitable diode array detectors for use in system 100 include, but are not limited to, the Waters Photodiode Array Detector (Waters Corp., Milford, Mass.).

[0117] Other types of detectors can also be used. For example, in some embodiments, the detector 116 corresponds to a spectrophotometer that measures the absorbance of the biological sample (e.g., in at least one of the ultraviolet, visible, and infrared regions of the electromagnetic spectrum). In certain embodiments, the detector 116 can be implemented as a fluorescence detector and includes a light source for directing illumination light to the biological sample and a detection element for measuring fluorescent emissions from the sample. In some embodiments, the detector 116 can be implemented as a mass spectrometry detector in which the biological sample is ionized and the distribution of ions is resolved by mass to determine abundance information about the biological sample. In certain embodiments, the detector 116 can be implemented as a multi-angle light scattering detector, or a refractive angle detector.

[0118] In some embodiments, each column-based sample analyzer can be in fluid communication with a different specialized detector. In certain embodiments, as shown in FIG. 1, 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, since system 100 analyzes only a portion of the biological sample at a time.

[0119] In some embodiments, the detector 116 effectively functions as a sample analyzer. For example, in FIG. 1, the sample analyzer 114a can 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 directly analyzed by the detector 116.

[0120] Within system 100, typically each sample analyzer is specialized to measure a particular product quality characteristic for an analyte in a biological sample. The particular product quality characteristic to be determined is selected by the control unit 122, which adjusts the configuration of the second flow control device 110 to direct a portion of the biological sample to one of the sample analyzers.

[0121] System 100 can generally be configured to measure any number of product quality characteristics depending on the number of sample analyzers present in the system. For example, in certain embodiments, system 100 can measure two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, ten or more, twelve or more, fifteen or more, twenty or more, or even more) product quality characteristics for analytes in a biological sample.

[0122] Product quality characteristics As discussed above, multiple product quality attributes for analytes in a biological sample can be measured using system 100. Various product quality attributes can be measured depending on the nature of the sample analyzer present in system 100.

[0123] (a) Concentration or titer In some embodiments, the system 100 includes a sample analyzer that measures the concentration or titer of an analyte in a biological sample. The concentration or titer of an analyte can be measured directly in the biological sample by any of the different types of detectors described above. Thus, for example, to measure the concentration or titer for the biological sample in FIG. 1, a portion of the biological sample can be delivered directly from the second flow control device 110 to the detector 116, i.e., the sample analyzer can effectively be a fluid conduit extending between the second flow control device 110 and the detector 116.

[0124] (b) Charge variants or heterogeneity In some embodiments, the system 100 includes a sample analyzer that measures charge variants or heterogeneity for analytes in a biological sample. The charge variants / heterogeneity can be determined, for example, by a sample analyzer that includes a cation exchange column for performing cation exchange chromatography of the analytes in the sample.

[0125] The term "cation exchange chromatography" refers to a type of ion exchange chromatography that uses a negatively charged ion exchange resin to separate molecules based on differences in charge. 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.

[0126] A cycle of chromatography using a cation exchange chromatography column (e.g., strong cation exchange chromatography) can include the steps of loading a fluid containing the analyte onto the column if the analyte binds to the chromatography resin during the loading step, washing the column to remove undesired biomaterial, eluting the analyte bound to the column, and re-equilibrating the column. In certain embodiments, a cycle of chromatography using a cation exchange chromatography column can include the steps of loading a fluid containing the target protein onto the column if undesired biomaterial but not the analyte binds to the chromatography resin during the loading step, collecting the target recombinant protein in the flow-through, and re-equilibrating the column.

[0127] Any of the single steps in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any of the single steps in a chromatography cycle can include a buffer gradient. Any of a variety of well-known combinations of aspects of a single cycle of chromatography can be used in these methods, for example, different chromatography resins, flow rates, buffers, void volumes of the columns, bed volumes of the columns, volumes of buffers used in each step, volumes of fluids containing the target protein, and the number and types of buffers used in each step, in any combination.

[0128] 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), at about pH 3 to 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).

[0129] In some embodiments, the Protein A column is at 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), 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 60 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 2 0mM, approximately 20mM to approximately 100mM, approximately 20mM to approximately 90mM, approximately 20mM to approximately 80mM, approximately 20mM to approximately 70mM, approximately 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 ~approx. 100mM, approx. 30mM ~ approx. 90mM, approx. 30mM ~ approx. 80mM, approx. 30mM ~ approx. 70mM, approx. 30mM ~ approx. 60mM, approx. 30 mM to approx. 50mM, approx. 30mM to approx. 40mM, approx. 40mM to approx. 100mM, approx. 40mM to approx. 90mM, approx. 40mM to approx. 80mM, approx. 4 The sample is eluted with sodium acetate (0 mM to about 70 mM, about 40 mM to about 60 mM, about 40 mM to about 50 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 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).

[0130] In some embodiments, the cation exchange column is 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 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 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), about 10 mM to about 100 mM Tris acetate (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, Approximately 10mM to approximately 50mM, approximately 10mM to approximately 40mM, approximately 10mM to approximately 30mM, approximately 10mM to approximately 20mM, approximately 15mM to approximately 100mM, approximately 15mM to about 90mM, about 15mM to about 80mM, about 15mM to about 70mM, about 15mM to about 60mM, about 15mM to about 50mM, about 15 mM~about 40mM, about 15mM~about 30mM, about 15mM~about 20mM, about 20mM~about 100mM, about 20mM~about 90mM, about 20m M ~ about 80mM, about 20mM - about 70mM, about 20mM - about 60mM, about 20mM - about 50mM, about 20mM - about 40mM, about 20mM - 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 6 0mM, about 30mM to about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80 mM, about 40mM to about 70mM, about 40mM to about 60mM, about 40mM to about 50mM, about 50mM to about 100mM, about 50mM to about 90m M, 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), or about 10mM to about 100mM (e.g., about 10mM to about 90mM, about 10mM to about 80mM, about 10mM to about 70mM, about 10mM to about 60mM, about 10mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 20mM, about 1 5mM 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 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 25mM to about 10 0mM, 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 40 mM 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, (may be about 90mM to about 100mM) of NaCl.

[0131] In some embodiments, the cation exchange column is at a pH of about 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 and is eluted 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) NaCl (e.g., 20 mM, about 25 mM, about 30 mM, about 40 mM, or about 50 mM).

[0132] (c) agglomeration In some embodiments, the system 100 includes a sample analyzer that measures aggregation of analytes in a biological sample. The degree of aggregation can be determined, for example, by a sample analyzer that includes a size exclusion column for performing size exclusion chromatography of the analytes in the sample.

[0133] The term "size-exclusion chromatography column" or "molecular sieve chromatography" refers to a chromatography column in which analytes and other components are separated by size and / or molecular weight. In some embodiments, size-exclusion chromatography columns are used to separate protein aggregates, such as protein multimers (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, Sephadex 2B, Sephadex 4B, Sephadex 6B, Bio-gel P-300, and Waters BEH SEC 200A.

[0134] In some embodiments, the size exclusion column is loaded with 1× phosphate buffered saline (PBS) at a pH of about 7 (e.g., about pH 7.0, 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 1× PBS at a pH of about 7.2.

[0135] (d) Integrity or Purity In some embodiments, the 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 reversed-phase column for performing reversed-phase chromatography of the analytes in the sample.

[0136] 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, such as Sepax Opalshell-C18. Non-limiting examples of hydrophobic ligands include aliphatic compounds, such as C2, C4, C8, C10, C12, C16 and C18, and polyphenyl.

[0137] A chromatography cycle using a reversed-phase chromatography column can include the steps of loading the column with a fluid containing the analyte, washing the column to remove undesired biomaterial, eluting the analyte bound to the column, and re-equilibrating the column.

[0138] Any of the single steps in a chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any of the single steps in a chromatography cycle can include a buffer gradient. Any of a variety of well-known combinations of aspects of a single cycle of chromatography can be used in these methods, for example, different chromatography resins, flow rates, buffers, void volumes of the columns, bed volumes of the columns, volumes of buffers used in each step, volumes of fluids containing the target protein, and the number and types of buffers used in each step, in any combination.

[0139] In some embodiments, the reverse phase column is at a concentration of 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% ~ 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. Eluted with trifluoroacetic acid (TFA) in water at 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%).

[0140] In some embodiments, the reverse phase column is at a concentration of 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%, Eluted with 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% trifluoroacetic acid (TFA) 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).

[0141] 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 10:80 isopropanol (IPA):acetonitrile (ACN).

[0142] In some embodiments, the reverse phase column has 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), 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 60 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 20 mM M, about 20mM to about 100mM, about 20mM to about 90mM, about 20mM to about 80mM, about 20mM to about 70mM, about 20mM to about 6 0mM, 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 ~about 50mM, about 30mM to about 40mM, about 40mM to about 100mM, about 40mM to about 90mM, about 40mM to about 80mM, about 40 The sample is eluted with sodium acetate (mM to about 70 mM, about 40 mM to about 60 mM, about 40 mM to about 50 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 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).

[0143] (e) Antibody Reduction In some embodiments, the system 100 includes a sample analyzer that measures the reduction of antibody analytes in a biological sample. In biological manufacturing production platforms, reductive degradation of antibody products is a concern, and the systems described herein can be used to measure antibody reduction for feedback regulation of manufacturing process parameters. The reduction of antibodies can be determined, for example, by a sample analyzer that includes a reversed-phase column for performing reversed-phase chromatography of analytes in a sample. Any of the reversed-phase column resins, buffers, pH values, and other operating conditions discussed herein can be used in connection with reversed-phase chromatography in the sample analyzer.

[0144] In some embodiments, a hydrophilic interaction chromatography column can be used to measure product quality characteristics for sample analytes. In certain embodiments, a hydrophobic interaction chromatography column can be used to measure product quality characteristics such as oxidation analysis. In some embodiments, a lectin column can be used to obtain product quality characteristics related to glycosylation information.

[0145] In certain embodiments, enzymatic chromatography columns can be used to perform peptide mapping for the purposes of measuring product quality attributes. Reverse phase chromatographic separation can be used to separate peptide analytes for analysis.

[0146] Analysis of product quality attributes using system 100 can generally be performed in near real-time to provide timely control feedback for adjustment of conditions and parameters of a wide variety of biological manufacturing processes. In some embodiments, for example, a concentration or titer attribute for 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 in a suitable sample analyzer to determination of the value of the attribute.

[0147] In certain embodiments, a charge variant or heterogeneity characteristic for 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 initial introduction of a portion of the sample in a suitable sample analyzer to determination of the value of the characteristic.

[0148] In some embodiments, an agglutination characteristic for 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 initial introduction of a portion of the sample in a suitable sample analyzer to determination of the value of the characteristic.

[0149] In certain embodiments, an integrity or purity characteristic for 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 initial introduction of a portion of the sample into a suitable sample analyzer to determination of the value of the characteristic.

[0150] For an analysis cycle in which values ​​of each of the four aforementioned product quality characteristics are determined for an analyte, the time interval that elapses from the initial introduction of the first portion of the biological sample into the first sample analyzer to the determination of the values ​​of the four characteristics is 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).

[0151] 3 is a flow chart 300 including an example series of steps for determining product quality attributes for analytes of a biological sample. In a first step 302, a sample is received into 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 at-line sampling device and held in a fluid loop or channel from which a portion of the biological sample is injected into the first flow control device 106.

[0152] Next, in step 304, a portion of the biological sample is injected into the first flow control device 106. The injected portion of the sample can 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, from which it is then delivered in step 308 to a sample analyzer associated with the product quality characteristic of interest.

[0153] Within the sample analyzer, a portion of the biological sample is analyzed to determine values ​​of relevant product quality attributes for the analytes in the sample, in step 310. After the product quality attribute values ​​have been determined, if values ​​of all product quality attributes for the sample analysis cycle have not yet been determined (see step 312), then control returns to step 304 and another portion of the biological sample is injected into the first flow control device 106.

[0154] Alternatively, once all product quality attribute values ​​have been determined in step 312, the attribute values ​​can then optionally be transmitted by the control unit 122 to a master controller for adjustment of one or more biological manufacturing process parameters. The analysis cycle then ends in step 316.

[0155] The manner in which the biological sample is delivered from the first flow control device 106, either directly to the second flow control device 110, or through the sample purification device 108 to the second flow control device 110, depends, among other things, on the nature of the flow control devices. Figures 4A-4C show examples of delivery of a biological sample between the first and second flow control devices when the first and second flow control devices are implemented as a multi-way valve.

[0156] 4A is a schematic diagram of the first flow control device 106 with its configuration adjusted to be in a first position, with ports 1 and 2 connected, ports 3 and 4 connected, and ports 5 and 6 connected. A biological sample is injected from the sample manager 102 into port 2. Port 2 is connected to port 1, which is then 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.

[0157] 4B and 4C are schematic diagrams showing a first flow control device 106 and a second flow control device 110 configured to deliver a biological sample to a sample purification device 108. In FIG. 4B, the arrangement of the first flow control device is such that the device is in a second position with ports 2 and 3 connected, ports 4 and 5 connected, and ports 1 and 6 connected. The second flow control device is configured in a first position with ports 1 and 2 connected, ports 3 and 4 connected, and ports 5 and 6 connected. In addition, the sample purification device 108 is connected across ports 1 and 4, port 3 is connected to a waste reservoir, and port 2 is connected to port 3 of the first flow control device. Using the arrangement shown in FIG. 4B, a biological sample is loaded into the column of the sample purification device 108 and a loading buffer is introduced through port 1 of the first flow control device.

[0158] Once the biological sample is loaded into the column of the sample purification device, the arrangement of the first and second flow control devices is adjusted as shown in FIG. 4C to elute the biological sample from the column of the sample purification device. In this arrangement, the second flow control device is adjusted to a different arrangement in which 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. To elute the analytes from the column of the sample purification device, an elution buffer is delivered to port 5 of the second flow control device, and the eluted analytes are delivered to the second flow control device and then to one of the sample analyzers in fluid communication with the second flow control device.

[0159] Analyzing biological samples to determine the value of a product quality attribute according to the systems and methods described herein can be advantageous in a variety of ways. For example, the described systems can serve as a single collection platform for many different product quality attributes throughout process development (e.g., cell line development to formulation development), maintaining data continuity and consistency in collection methodology.

[0160] In addition, measurements of product quality attributes can be used for spectroscopic model validation and ongoing model validation. Periodically measured product quality attributes can be used, for example, to maintain spectroscopic models by validating the predictive accuracy of chemometric methods using in-line FTIR and / or Raman measurements.

[0161] The automated nature of the systems described herein can be used to eliminate tedious, repetitive analytical steps that would otherwise be performed manually, saving time and reducing the chance that operator error will affect the value of the measured property. Similarly, integrated sample purification can be performed in-line, significantly reducing the time required to obtain a purer biological sample.

[0162] Using the systems and methods described herein, harvested media can be analyzed directly in a location close to the bioreactor in the manufacturing system. Feedback of information about measured product quality attributes (e.g., by control unit 122) can, in some embodiments, provide more direct and timely control over manufacturing process parameters that can be adjusted to increase product yields, reduce waste rates, and otherwise improve the efficiency of the biological manufacturing process.

[0163] In addition to applications including direct assessment of product quality attributes for bioreactor management, the attribute values ​​can be used in cell line process development. For example, the methods described herein can be applied to determine product quality attributes for monoclonal antibody therapeutics for analytes and comparison of different clones.

[0164] Integration and regulation of biological manufacturing systems. The systems disclosed herein can be integrated with biological manufacturing systems to provide feedback control to various components and steps in the synthesis and purification processes for a variety of biological products.

[0165] An integrated, fully continuous process for manufacturing therapeutic protein drugs and other substances can include, for example, providing a liquid culture medium containing a recombinant therapeutic protein that is substantially cell-free, and then feeding the liquid culture medium to a first multi-column chromatography system (MCCS1). The next step includes capturing the recombinant therapeutic protein in the liquid culture medium using MCCS1, and then continuously feeding the eluate of MCCS1 containing the recombinant therapeutic protein to a second multi-column chromatography system (MCCS2), and purifying and polishing the protein using MCCS2. The eluate obtained from MCCS2 is considered the therapeutic protein drug substance. The process can be integrated and run continuously from the liquid culture medium to the eluate from MCCS2, which is the therapeutic protein drug substance.

[0166] A biological manufacturing system is typically used to carry out the above process. For example, such a system may include an MCCS1 including an inlet, and an MCCS2 including an outlet. In these systems, the first and second MCCSs are in fluid communication with each other. The system is also configured such that a fluid enters the inlet through the first and second MCCSs and exits the manufacturing system through the outlet.

[0167] Such a system can provide continuous, time-efficient production of therapeutic drug substance from liquid culture medium, for example, the elapsed time between feeding a fluid (e.g., liquid culture medium) containing a therapeutic protein into a first MCCS and eluting the therapeutic protein drug substance (containing the therapeutic protein) from the outlet of a second MCCS is, for example, about 4 hours to about 48 hours.

[0168] FIG. 8 is a schematic diagram showing an example of a biological manufacturing system. System 1 includes a first MCCS, i.e., a four-column periodic countercurrent chromatography system (PCCS) 2, where three of the four columns in the four-column PCCS 2, columns 3, 4, and 5, perform a unit operation of capturing a recombinant therapeutic protein from a fluid (e.g., liquid culture medium substantially free of mammalian cells) containing the recombinant therapeutic protein, and one of the columns, column 6, in the PCCS 2 performs a unit operation of inactivating viruses present in the eluate from columns 3, 4, and 5 in the PCCS 2 containing the recombinant therapeutic protein. Columns 3, 4, and 5 can contain a resin that utilizes a Protein A binding capture mechanism. Column 6 can hold the fluid at a pH of about 3.75 for about 1 hour. PCCS 1 also has an inlet 7. Inlet 7 is, for example, an opening that allows fluid to enter PCCS 1.

[0169] The system 1 also includes a second MCCS, which is a PCCS8, including three chromatographic columns 9, 10, and 11, and one chromatographic membrane 12. The columns 9, 10, and 11 in the PCCS8 can contain a cation exchange resin. The chromatographic membrane 12 in the PCCS8 can contain a cation exchange resin. The PCCS8 also has a fluid conduit 13 disposed between the columns 9, 10, and 11 in the PCCS8, and the chromatographic membrane 12 in the PCCS8. The PCCS8 also has an in-line buffer adjustment reservoir 14 in fluid communication with the fluid conduit 13, and is configured such that a buffer contained within the in-line buffer adjustment reservoir 14 is introduced into the fluid present in the fluid conduit 13. The PCCS8 also includes an outlet 15. The outlet 15 is, for example, an opening that allows fluid to exit the PCCS8.

[0170] System 1 may further include a fluid conduit 16 disposed between PCCS 2 and PCCS 8. System 1 may also include an in-line buffer adjustment reservoir 17 in fluid communication with fluid conduit 16 configured such that a buffer contained within in-line buffer adjustment reservoir 17 is introduced to the fluid present in fluid conduit 16. System 1 may include a filter 18 disposed in fluid conduit 16 for filtering the fluid present in fluid conduit 16. System 1 may also include a break tank 19 disposed in fluid conduit 16 and configured to hold any fluid in fluid conduit 16 that cannot be readily provided to PCCS 8.

[0171] System 1 may further include a pump system 20 in fluid communication with inlet 7. Pump system 20 may include a pump 21 for pushing fluid into inlet 7. System 1 may also include a fluid conduit 22 disposed between pump 21 and inlet 7. System 1 may also include a filter 23 disposed in fluid conduit 22 for filtering fluid (e.g., liquid culture medium) present in fluid conduit 22. System 1 may also include a break tank 24 disposed in fluid conduit 22, in fluid communication with fluid conduit 22 and configured to store any fluid present in fluid conduit 22 that cannot enter inlet 7.

[0172] System 1 may also include a bioreactor 25 and a fluid conduit 26 disposed between bioreactor 25 and pump 21. A filtration system 27 may be disposed in fluid conduit 26 to filter (e.g., remove cells therefrom) the liquid culture medium present in fluid conduit 26.

[0173] The first MCCS (PCCS2) includes an inlet through which fluid (e.g., liquid culture medium substantially free of cells) can pass through the first MCCS. The inlet can be any structure known in the art for such purpose. It can include, for example, threads, ribs, or seals that allow a fluid conduit to be inserted such that after insertion of the fluid conduit into the inlet, the fluid passes through the inlet into the first MCCS without significant leakage of the fluid from the inlet.

[0174] 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 can include a total of four chromatographic columns, or three chromatographic columns and one chromatographic membrane, or any of the other exemplary MCCSs described herein, or can have one or more of any of the exemplary configurations of the MCCSs (in any combination) described herein.

[0175] The chromatography columns and / or chromatographic membranes present in the first MCCS can contain one or more of various resins. For example, the resins contained in one or more of the chromatography columns and / or chromatographic membranes present in the first MCCS can be resins that utilize 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, aptamer-binding capture mechanism, and / or tag-binding capture mechanism). The resins contained in one or more of the chromatography columns and / or chromatographic membranes of the first MCCS can be cation exchange resins, anion exchange resins, molecular sieve resins, or hydrophobic interaction resins, or any combination thereof. Additional examples of resins that can be used to purify recombinant therapeutic proteins are known in the art and can be contained in one or more of the chromatography columns and / or chromatographic membranes present in the first MCCS. The chromatography columns and / or chromatographic membranes present in the first MCCS can contain the same and / or different resins (e.g., any of the resins described herein or known in the art for use in recombinant protein purification).

[0176] 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 a recombinant therapeutic protein, purifying a recombinant therapeutic protein, polishing a recombinant therapeutic protein, inactivating a virus, adjusting the ionic concentration and / or pH of a fluid containing the recombinant therapeutic protein, or filtering a fluid containing the recombinant therapeutic protein). In a non-limiting example, the first MCCS can perform the unit operations of capturing a recombinant therapeutic protein from a fluid (e.g., a liquid culture medium) and inactivating a virus present in a fluid containing the recombinant therapeutic protein. The first MCCS can perform any combination of two or more unit operations described herein or known in the art.

[0177] The chromatographic columns and / or 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 can also include one or more (e.g., 2, 3, 4, or 5) pumps (e.g., automated pumps, e.g., automated peristaltic pumps). A column switching event can be triggered by detection of a level of recombinant therapeutic protein in a fluid passing through the first MCCS (e.g., input to and / or elution from one or more of the chromatographic columns and / or chromatographic membranes in the first MCCS), a specific volume of liquid (e.g., buffer), or a specific elapsed time. Column switching generally refers to a mechanism that allows at least two different chromatographic columns and / or chromatographic membranes in the MCCS (e.g., two or more different chromatographic columns and / or chromatographic membranes present in the MCCS (e.g., the 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.

[0178] The first MCCS, PCCS2, can include four chromatography columns, with the first three columns performing a unit operation of capturing recombinant therapeutic proteins from a fluid (e.g., liquid culture medium), and the fourth column of the PCCS performing a unit operation of inactivating viruses in the fluid containing the recombinant therapeutic proteins. The first MCCS, PCCS, can utilize a column switching mechanism. The PCC system can utilize a modified AKTA system (GE Healthcare, Piscataway, NJ) that can operate up to, for example, 4, 5, 6, 7, or 8 columns, or more.

[0179] A column switching event can be triggered by detection of the concentration of a particular protein or other substance in a fluid eluting from one of the columns of PCCS2 or PCCS8, flow through a filter in the MCCS contained in the break tank of the MCCS, or flow through a conduit in the MCCS (e.g., between MCCS1 and MCCS2). Using the measurement systems disclosed herein, the concentration of such proteins can be measured and the concentration information can be sent to a controller in system 1 that initiates events such as column switching, filtration and fluid transport in system 1.

[0180] The first MCCS can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) measurement systems (e.g., system 100) configured to obtain infrared spectroscopic information about the process fluid, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. The first MCCS can also include a controller that executes an operating system that utilizes software (e.g., software based on Unicorn, GE Healthcare, Piscataway, NJ, or other software implementing similar functionality) to determine when a column switch should occur (e.g., based on concentration information derived from infrared spectroscopic measurements, volume of liquid, or elapsed time) and to affect (trigger) a column switching event. The measurement system can optionally be installed at the inlet of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) chromatographic columns and / or chromatographic membranes in the first MCCS and / or at the outlet of one or more chromatographic columns and / or chromatographic membranes in the first MCCS.

[0181] The first MCCS can further include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) in-line buffer conditioning reservoirs and / or buffer reservoirs. In other examples, the first MCCS can include one or more (e.g., 2, 3, 4, 5, or 6) break tanks that can hold fluids that cannot easily pass through one or more chromatographic columns and / or chromatographic membranes in the first MCCS. The systems described herein can contain one or more break tanks (e.g., break tanks described herein) in the first and / or second MCCS. Other examples of the systems described herein do not include a break tank in the first or second MCCS or do not include a break tank in the entire system. Other examples of the systems include up to 1, 2, 3, 4, or 5 break tanks in the entire system.

[0182] In some embodiments, the first MCCS can include a virus inactivation device. For example, referring to FIG. 8, in certain embodiments, the first MCCS includes a virus inactivation device 6 (i.e., instead of the column 6 described above). The virus inactivation device 6 is configured to inactivate viruses and viral vectors used in the biological manufacturing process. In some embodiments, for example, the virus inactivation device 6 includes a mixer. Alternatively, in certain embodiments, for example, the device 6 includes a plug flow inactivation system. Each of these examples of virus inactivation devices helps eliminate active viruses and viral vectors from the process fluid in the first MCCS.

[0183] The second MCCS comprises 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 can comprise a total of four chromatographic columns, three chromatographic columns and one chromatographic membrane, or any of the other exemplary MCCSs described herein, or can have one or more of any of the exemplary configurations of MCCSs (in any combination) described herein. The chromatographic columns and / or chromatographic membranes present in the second MCCS can have one or more of any of the shapes, sizes, volumes (bed volumes) and / or unit operations described herein. The resins contained in one or more of the chromatographic columns and / or chromatographic membranes present in the second MCCS can be resins that utilize 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, tag-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 chromatography columns and / or chromatographic membranes present in the second MCCS can contain the same and / or different resins (e.g., any of the resins described herein or known in the art for use in recombinant protein purification).

[0184] The chromatographic columns and / or chromatographic membranes present in the second MCCS can perform one or more unit operations (e.g., any of the unit operations described herein, or any combination of the unit operations described herein). In a non-limiting example, the second MCCS can perform the unit operations of purifying a recombinant therapeutic protein from a fluid, and polishing a recombinant therapeutic protein present in a fluid containing the recombinant therapeutic protein. In another non-limiting example, the second MCCS can perform the unit operations of purifying a recombinant therapeutic protein present in a fluid, polishing a recombinant therapeutic protein present in a fluid, and filtering a fluid containing a recombinant therapeutic protein. In another example, the second MCCS can perform the unit operations of purifying a recombinant therapeutic protein present in a fluid, polishing a recombinant therapeutic protein present in a fluid, filtering a fluid containing a recombinant therapeutic protein, and adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein. The second MCCS can perform any combination of two or more unit operations described herein or known in the art.

[0185] The second MCCS can also include one or more (eg, 2, 3, 4, or 5) pumps (eg, automated pumps, eg, automated peristaltic pumps).

[0186] The chromatography columns and / or chromatographic membranes present in the second MCCS can be connected or moved relative to one another by a switching mechanism (e.g., a column switching mechanism). A column switching event can be triggered by detection of a level of a recombinant therapeutic protein or other substance by analysis thereof using infrared spectroscopy and chemometric models, a specific volume of liquid (e.g., buffer), or a specific elapsed time to determine the level of the recombinant therapeutic protein in a fluid passing through the second MCCS (e.g., input to and / or elution from one or more of the chromatography columns and / or chromatographic membranes in the second MCCS), as discussed above.

[0187] The PCCS8 forming the second MCCS can contain three columns performing the unit operation of purifying recombinant therapeutic protein from a fluid, and a chromatographic membrane performing the unit operation of polishing recombinant therapeutic protein present in the fluid. For example, the three columns performing the unit operation of purifying recombinant therapeutic protein from a fluid can contain, for example, a cation exchange resin, and the chromatographic membrane performing the unit operation of polishing can contain a cation exchange resin. The second MCCS, PCCS, can utilize a column switching mechanism. For example, the PCCS can utilize a modified AKTA system (GE Healthcare, Piscataway, NJ) that can operate up to, for example, 4, 5, 6, 7 or 8 columns, or more.

[0188] Similar to the first MCCS, the second MCCS may also include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) infrared spectroscopy measurement systems, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) valves, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pH meters, and / or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) conductivity meters. The one or more measurement systems send concentration information about proteins or other substances in the fluid being measured to a controller that uses the concentration information to determine whether to trigger a column switching event. The second MCCS can include a controller that executes an operating system executed by the controller that receives the concentration information, utilizing software (e.g., software based on Unicorn, GE Healthcare, Piscataway, NJ) to determine when a column switching event should occur (e.g., based on infrared spectroscopy measurements, volume of liquid, or elapsed time) and to initiate a column switching event. In examples where the second MCCS includes one or more infrared spectroscopy measurement systems, the measurement systems can optionally be installed at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) inlets of the chromatographic columns and / or chromatographic membranes in the second MCCS and / or at one or more outlets of the chromatographic columns and / or chromatographic membranes in the second MCCS.

[0189] The second MCCS can further include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) in-line buffer conditioning reservoirs and / or buffer reservoirs. In other examples, the second MCCS can include one or more (e.g., 2, 3, 4, 5, or 6) break tanks (e.g., any of the break tanks described herein) that can hold fluids that cannot readily pass through one or more chromatographic columns and / or chromatographic membranes in the second MCCS.

[0190] The second MCCS includes an outlet through which the therapeutic protein drug substance can exit the system. The outlet can include, for example, a thread, rib or seal that allows a fluid conduit to be inserted, or a vial designed to contain or store the therapeutic protein drug substance. The outlet can include a surface at the outlet that can be used to seal a sterile vial or other such storage container to allow the recombinant protein formulation to flow directly into the sterile vial or storage container.

[0191] Any of the fluid conduits described herein are, for example, tubes made of, for example, polyethylene, polycarbonate, or plastic. The fluid conduits disposed between the first and second MCCSs can further include one or more of the following, in any combination: one or more in-line buffer adjustment reservoirs in fluid communication with the fluid conduits and positioned such that buffer stored in the in-line buffer adjustment reservoirs is added to the fluid present in the fluid conduits; a break tank (e.g., any of the break tanks described herein) in fluid communication with the fluid conduits and positioned to hold any excess fluid present in the fluid conduits that cannot be readily supplied to the second MCCS; and one or more filters disposed in the fluid conduits to filter (e.g., remove bacteria) the fluid present in the fluid conduits. Any of the in-line buffer adjustment reservoirs can contain, for example, a volume of about 0.5 L to 50 L of buffer (e.g., at 50° C. or lower, 37° C., 25° C., 15° C., or 10° C.).

[0192] The systems described herein may optionally include a fluid conduit disposed between the final chromatography column or chromatographic membrane in the second MCCS and the outlet. The systems described herein may further include one or more filters in fluid communication with the fluid conduit disposed between the final chromatography column or chromatographic membrane in the second MCCS and the outlet, such that the filters can remove, for example, precipitated matter, particulate matter, or bacteria from the fluid present in the fluid conduit disposed between the final chromatography column or chromatographic membrane in the second MCCS and the outlet.

[0193] Some examples of the systems provided herein also include a bioreactor that is in fluid communication with the inlet of the first MCCS. Any of the exemplary bioreactors described herein or known in the art can be used in the systems.

[0194] Some examples of the system provided herein also include a pump system. The pump system can include one or more of the following: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) pumps (e.g., any of the pumps described herein or known in the art), one or more (e.g., 2, 3, 4, or 5) filters (e.g., any of the filters described herein or known in the art), one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) UV detectors, and one or more (e.g., 2, 3, 4, or 5) break tanks (e.g., any of the break tanks described herein). Some examples of the system provided herein further include a fluid conduit (e.g., any of the exemplary fluid conduits 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 can include one or more (e.g., two, three, or four) pumps (e.g., any of the pumps described herein or known in the art) and / or one or more (e.g., two, three, or four) break tanks (e.g., any of the exemplary break tanks described herein), which pumps and / or break tanks are in fluid communication with the fluid present in the liquid conduit.

[0195] Some examples of the systems described herein further include an additional fluid conduit connected to the fluid conduit between the pump and the inlet, where one end of the additional fluid conduit is fluidly connected to the bioreactor and the other end is fluidly connected to the fluid conduit between the pump and the inlet. The additional fluid conduit can include a filter (e.g., an ATF cell retention system) capable of removing cells from the liquid culture medium removed from the bioreactor.

[0196] The aforementioned biological manufacturing systems allow for the continuous production of therapeutic protein drug substances. For example, the systems provided herein allow for a percentage yield of recombinant therapeutic protein (from starting material, e.g., starting liquid culture medium) of 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 systems described herein can also result in a percentage yield of recombinant therapeutic protein (from starting material, e.g., starting liquid culture medium) of about 80% to about 90%, about 82% to about 90%, about 84% to about 90%, about 84% to about 88%, about 84% to about 94%, about 82% to about 92%, or about 85% to about 95%.

[0197] The systems described herein can also result in the production of therapeutic protein drug substance containing a concentration of recombinant therapeutic protein greater than about 1.0 mg / mL, e.g., 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.

[0198] As discussed above, in some embodiments, the first and / or second MCCS is a periodic countercurrent chromatography system (PCCS). A PCCS can include two or more chromatography columns (e.g., three or four columns) that can be switched to allow, for example, sequential elution of recombinant therapeutic proteins from two or more chromatography columns. A PCCS can include two or more chromatography columns, two or more chromatography membranes, or at least one chromatography column and at least one chromatography membrane. Column operation generally consists of loading, washing, elution, and regeneration steps. In a PCCS, multiple columns are used to operate the same steps individually and consecutively in a cyclic manner. Because the columns are operated in series, the flow-through and wash from one column is captured by another column. This unique configuration of the PCCS allows loading of the resin close to its static binding capacity, instead of at its dynamic binding capacity, as is typical during batch mode chromatography.

[0199] An example of a three column switching technique used in a PCCS containing three columns is shown in Figure 9. A cycle is defined as three complete column operations resulting in an eluate pool from each of the three columns used in the column switching technique. Once all steps of a cycle are completed, the cycle is restarted. As a result of the successive cycles and elutions, the fluid entering the PCCS is processed continuously, but the recombinant therapeutic protein elution from each column is individual and periodic.

[0200] A column switching strategy is used to proceed from one step to another in a PCCS cycle, such as the exemplary cycle shown in Figure 9. The column switching method uses two automated switching operations per column for the three columns in the exemplary PCCS system shown in Figure 9, the first of which is related to the appearance of the first product and the second coincides with column saturation. The decision of when a column switching operation should be performed is based on information about the recombinant therapeutic protein concentration in the eluate from each chromatography column in the PCCS.

[0201] As discussed 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 serves as a feedback control for the biological manufacturing system and is sent by the control unit 122 to the MCCS controller to initiate a column switch after determining that a switch is warranted.

[0202] As an example, during column load, the PCC control system can use the infrared spectroscopy measurement system discussed above to determine a baseline concentration of therapeutic protein material eluting from the column (typically zero concentration). During active elution, as protein material appears, the measured protein concentration increases (e.g., above the baseline concentration). The system continues to monitor the increasing protein concentration, and once the concentration reaches a predetermined threshold, the flow-through from column 1 is directed to column 2 instead of to waste. Nominally, this occurs at time t1.

[0203] As the feed to column 1 continues, column 1 eventually becomes nearly saturated with protein product. At this point, the measured concentration of protein in the eluate reaches another predetermined value, which occurs at time t2. At this point, the MCCS controller switches the inlet feed to column 2.

[0204] The above column switching strategy allows for uniform loading of columns regardless of the concentration and volume of the feed product. Similar column switching based on the level of recombinant protein detected in the eluate from each column can be implemented. Column switching can also be based on the elapsed time or the amount of fluid (e.g., buffer) passed through one or more chromatographic columns and / or chromatographic membranes in the first or second MCCS.

[0205] In addition to providing feedback information for controlling column switching events, the measurement systems disclosed herein can also provide feedback information for the adjustment of various other biological manufacturing process and operating parameters. One example of such an adjustment is the controlled adjustment of buffer concentrations at various stages of a biological manufacturing process.

[0206] In general, one or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) different types of buffers can be used during the use of two or more MCCSs in any of the processes described herein. As is known in the art, the one or more types of buffers used in the two or more MCCSs used in the processes described herein depend on the resins present in the chromatographic columns and / or chromatographic membranes of the two or more MCCSs (e.g., the first and second MCCSs), the recombinant therapeutic protein, and the unit operations (e.g., any of the exemplary unit operations described herein) performed by the particular chromatographic columns and / or chromatographic membranes of the two or more MCCSs. The volumes and types of buffers used during the use of two or more MCCSs in any of the processes described herein can also be determined by one of skill in the art (e.g., as discussed in more detail below). For example, the volumes and types of buffers used during the use of two or more MCCSs in any of the processes described herein can be selected to optimize one or more of the following in the recombinant protein preparation: overall yield of recombinant therapeutic protein, activity of the recombinant therapeutic protein, level of purity of the recombinant therapeutic protein, and removal of biological contaminants from fluids containing the recombinant therapeutic protein (e.g., absence of active viruses, mycobacteria, yeast, bacteria or mammalian cells).

[0207] Unit operations that adjust the ionic concentration and / or pH of a fluid containing a recombinant therapeutic protein can be performed using an MCCS (e.g., the first and / or second MCCS) that includes and employs a buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) that adds 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 penultimate MCCS (e.g., the first MCCS) and before the fluid containing the recombinant therapeutic protein is fed to the first column of the next MCCS (e.g., the second MCCS)). The in-line buffer conditioning reservoir can be of any size (e.g., greater than 100 mL) and contain any buffered solution (e.g., a buffer solution having one or more of an increased or decreased pH compared to the fluid containing the recombinant therapeutic protein, an increased or decreased ion (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 chromatography column or at least one chromatographic membrane of the MCCS (e.g., the first or second MSCCS).

[0208] In some embodiments, the determination by the MCCS controller of the amount of buffer solution to be added to the process fluid is based on concentration or titer information for an analyte in a biological sample. For example, a solute for purposes of such measurement is a component of a buffer solution or a component of a process fluid whose concentration is related to the buffer composition of the fluid, the pH of the process fluid, and / or the ionic strength of the process fluid. Measurement of the concentration information for the component is provided as feedback information to the MCCS controller, which uses the feedback information to determine when and how much of one or more buffer solutions to drain into the process fluid. The infrared spectroscopy measurement system can generally be located anywhere in the biological manufacturing system for the purposes of measuring the process fluid and providing feedback information related to the buffer to the MCCS controller.

[0209] In certain embodiments, antibody concentration information for the process fluid can be used to control the rate at which the cell culture is introduced into the bioreactor. In particular, by determining the antibody concentration value in the process fluid withdrawn from the bioreactor, the MCCS controller can adjust the outflow rate of the cell culture into the bioreactor. Adjustment in this manner allows for control of the volumetric productivity derived from the cell density and specific productivity of the bioreactor. For a fixed perfusion rate, such adjustment allows for control of the antibody concentration in the process fluid so that the MCCS1 receives an approximately constant amount of product per unit time. In other words, adjustment of this nature can be used to ensure that the rate of production of the product in the bioreactor remains approximately constant over a certain period of time.

[0210] In some embodiments, the determination of certain quality attributes associated with the process fluid can be used by the MCCS controller to determine whether the biological manufacturing system is operating within an acceptable range of parameters or, during operation, whether the system is outside one or more of the acceptable parameter ranges.

[0211] For each of one or more quality attributes, a range of acceptable values ​​can be established by a calibration procedure. These ranges effectively establish operating conditions for the system under which the biological product is produced at an acceptable rate and level of purity, while the yield of by-products and other undesirable species is at an acceptably low level. If the system is operated outside one or more ranges, the product yield and / or purity may decrease, the rate / amount of production of undesirable species may increase, the rate of consumption of reagents may increase, and / or other undesirable effects or conditions may result.

[0212] The determined quality characteristics of the process fluid at one or more locations in the system can be used to ensure that the system is operating within acceptable ranges of these operating parameters. If the determined values ​​of one or more quality characteristics are outside the established acceptable ranges, the MCCS controller identifies that a potential fault condition exists.

[0213] To address a fault condition, the MCCS controller (or another system controller connected to the MCCS controller) can adjust any of the operational parameters of the biological manufacturing system to alter its operation, and thereby also adjust the values ​​of the quality attributes so that they are within acceptable ranges. Corrective actions of this nature ensure that the system can proactively maintain within an established set or range of operating conditions based on feedback provided by the determined values ​​of the quality attributes.

[0214] In certain embodiments, when the MCCS controller (or another system controller connected to the MCCS controller) determines that the system is too far outside its range of acceptable operating conditions, such that returning the system to the acceptable range of conditions will be difficult or even impossible, or will result in other undesirable results, the controller can send a control signal to the bioreactor to abort production and drain its contents to waste. In such cases, effective corrective action is impractical or impossible, and the production process has deviated too far from the range of acceptable operating conditions for the system. By simply draining the bioreactor contents, the system can save significant time by restarting the production process, rather than attempting to regulate an ongoing production process that has irrecoverably deviated from the range of acceptable conditions.

[0215] Additionally, feedback can be provided to the MCCS controller (or another system controller) based on measurements of one or more bioreactor medium constituents (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 metrics are maintained within target ranges. Any one or more process parameters can be adjusted by the controller based on values ​​of bioreactor medium constituents in a manner similar to adjustments made based on values ​​of product quality attributes and other measured quantities.

[0216] Hardware and Software Implementation The control unit 122 can be configured to perform any of the control functions described herein and can be implemented in hardware, software, or a combination of both 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 interface device (e.g., a keyboard, a mouse, a touchpad, a touch-sensitive display). The control unit 122 receives information as electrical signals from and transmits electrical control signals to the components of the system along some or all of the control lines shown herein.

[0217] The steps and control functions of the methods described herein can 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., the electronic processor of the control unit) to cause the control unit to perform the steps and functions described. Each program can be stored in a computer-readable storage medium (e.g., optical, magnetic, solid-state, rewritable, or other persistent storage medium). An example of a suitable programming language that can be used to provide instructions to the control unit 122 is the Empower® software suite (Waters Corp., Milford, MA). EXAMPLES

[0218] The following examples are provided to further illustrate various aspects of the foregoing disclosure and are not intended to otherwise limit any configuration of the claims or to limit any aspect of the embodiments, unless expressly stated.

[0219] Example 1 System 100 was used to analyze anti-TGF-β from a perfusion bioreactor operating with an enhanced perfusion process. The system included a process sample manager (PSM), a column heater (CH), a column manager (CM) containing two one-position nine-port valves, a binary pump (BSM), a quaternary pump (QSM) with a solvent selection valve added on the "D" line, a PDA detector (PDA), a two-position ten-port column selection valve housing a 5 mL stainless steel sample holding loop (all obtained from Waters Corp., Milford, Mass.), and two IDEX auxiliary two-position six-port switching valves.

[0220] The harvest from the bioreactor was sampled automatically using MAST (Modular Automated Sampling Technology) (obtained from Lonza, Basel, Switzerland) connected to a liquid handler (obtained from Gilson, Middleton, WI). The MAST system drew approximately 40 mL of harvest into a clean line by a positive displacement pump, after which 10 mL of harvest accumulated in a clean 12 mL glass vial on the liquid handler deck. The liquid handler then transferred 7.5 mL of harvest to the system 100 by reinjection of material into a PEEK tubing transfer line connected to both the liquid handler and the suction. The sample was transferred directly to a 5 mL sample holding loop that held it until analysis of the sample.

[0221] Once sample transfer was complete, the MAST computer sent a signal to the control unit 122 to initiate the method set specified in the Empower® 3 software.

[0222] The set of methods run for this example included loop load function, 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 two mass loads, intermediate water washes between the common system component separation techniques (Pro A and size exclusion chromatography (SEC)) and a final loop wash with 20% methanol, as well as a final section to ensure that the loop is in the correct position to accept more sample. It should be noted if a secondary column is mentioned (such as SEC) and it should be assumed that the method contains an in-line purification with Pro A unless otherwise stated.

[0223] For each injection, 1 mL of recovery was drawn from the holding loop and transferred to a 50 μL sample injection loop for full loop injection in the system. To test varying mass loading, both full loop injections were performed as well as an online 1:2 dilution using 1× phosphate buffered saline (PBS), pH 7.2 buffer.

[0224] Sample purification for titer analysis was performed on a Thermo POROS A 2.1×30 mm, 20 μm column (i.e., a Protein A affinity column). Gradient details for the first and second pumps are shown in Tables 1 and 2.

[0225] [Table 1]

[0226] [Table 2]

[0227] For the method shown in Table 1, Buffer A was 1× 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.

[0228] The system was configured such that the purified sample went directly to the detector 116 (PDA detector) without passing through any column-based sample analyzer. Separation was monitored by absorbance at 280 nm. The Protein A column was kept at room temperature and ambient. Upon detection in the PDA detector, the measured chromatogram was integrated to calculate the area under the curve for the peak corresponding to the eluted protein at approximately 2.5 minutes. The area of ​​the monoclonal antibody was then quantified by use of a calibration curve constructed from mass loadings of 50 μg to 2 μg on the column plotted against area.

[0229] Figure 5A is a graph showing the titer chromatograms for the neat and 1:2 diluted samples, and Figure 5B is a table showing the calculated mass loadings and concentrations from six different runs at each dilution ratio. The data show excellent reproducibility between runs, both visually and between the calculated results.

[0230] Aggregation analysis was performed using size exclusion separation on a Waters UPLC BEH SEC 4.6×300 mm, 1.7 μm and 200 Å column. Gradient details for the first and second pumps are shown in Tables 3 and 4.

[0231] [Table 3]

[0232] [Table 4]

[0233] For the method shown in Table 3, Buffer A was 1× Dulbecco's Phosphate Buffered Saline, pH 7.2, and Buffer B was 20 mM sodium phosphate, 1 M sodium chloride, and 7.5% isopropanol at pH 8.0. For the method shown in Table 4, Buffer A was 0.1 M phosphate-citrate, pH 3.2, and 0.14 M sodium chloride, and Buffer B was 1× Dulbecco's Phosphate Buffered Saline, pH 7.2.

[0234] The column manager was configured to direct the sample to a sample analyzer with a size-exclusion chromatography column. Separation of the analytes and other components was monitored by absorbance at 280 nm. The size-exclusion column was kept at 25° C. in a 30 cm column heater, while the Protein A column was in ambient environment. Upon detection in the PDA detector 116, the recorded chromatograms were integrated to calculate the areas under the curve for peaks corresponding to high molecular weight species (before the main peak), major species, and low molecular weight species (after the main peak).

[0235] Figure 6A is a graph showing the titer chromatograms for the neat and 1:2 diluted samples, and Figure 6B shows a table with the measured peak information for six different samples at each of the two dilutions. The data shows excellent reproducibility between runs, both visually and between the calculated results. There is some discrepancy in peak percentages between mass loads that can be attributed to the column itself.

[0236] Figure 7 is a graph showing measured chromatograms from experiments in which samples were obtained every 1.5 hours for a total of 12 samples. For these experiments, only one mass load was delivered to both the Protein A and size exclusion chromatography columns: 1:2 online dilution in the sample manager with 1x PBS at pH 7.2. Both product quality attributes of titer and aggregation showed excellent reproducibility among the 12 samples.

[0237] For reversed-phase analysis, separations were performed on a Waters BioResolve Polyphenyl 2.1 x 100 mm, 2.7 μm, 450 Å column using 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). For strong cation exchange analysis, separations were performed on a Thermo MAbPac SCX-10 RS, 2.1 x 150 mm, 5 μm column. The mobile phases were 20 mM sodium acetate, pH 3.75 (both mobile phase A and Protein A eluate) and 20 mM Tris acetate, 25 mM sodium chloride, pH 9.8 (mobile phase B). Thus far, the method employs the use of gradient curvature to create an "S" shaped gradient for optimal peak resolution.

[0238] At-line samples were analyzed using the methods described above. As discussed above, system 100 can also analyze offline samples, for example, using the Sample Manager's Flow-Through Needle Module (available from Waters Corp., Milford, MA). With the flow-through needle, samples are injected directly from a vial or well plate. Injection volumes can range from 1 μL to 100 μL and can be varied as desired. This flexibility allows for varying the mass load on the column without performing online dilutions. In addition, there is no sample holding loop in this version of the system, since all sample volumes are contained within the vial or well plate and multiple injections are possible for each vial.

[0239] Example 2. Application of MIMICS-mPQA to process development and at-line process monitoring of a pilot-scale bioreactor Experimental Overview The MIMICS-mPQA platform was applied to an attoscale 100 L run of an anti-TGFβ monoclonal therapeutic antibody. Harvested samples were run in the MIMICS-mPQA platform to accumulate at-line product quality information for three attributes: potency, aggregation, and purity / integrity.

[0240] Samples analyzed were harvested material (post-ATF) from the 100L bioreactor and two satellite 3L bioreactors. For analysis, a Protein A (Pro A) affinity column was used for titer determination, a size exclusion (SEC) column with an in-line Protein A column was used for aggregation analysis, and a reversed phase (RP) column with an in-line Protein A column was used for purity / integrity information. The study was conducted over the course of a four week period during which samples were run in blocks of approximately four harvest days from every reactor in each queue. During the analysis period, the mobile phase of the system was replaced as required as it was consumed.

[0241] For each harvest date tested from each bioreactor, 0.75 mL of material was transferred to an autosampler vial and placed in the MIMICS-mPQA system. Each sample was injected for one cycle of analysis on the system to quantify the three target attributes. The injection volume was kept constant at 20 μL, but the mass load on each column was varied by applying a variable online dilution factor ranging from 1:2 to 1:10 to target the linear range of each method based on previous development experience. Briefly, the load on the Pro A column was targeted to be 10-25 μg, on the SEC column to be 20-50 μg, and on the RP column to be 3-7 μg.

[0242] For the Pro A titration method, a standard curve was run at the beginning of the entire campaign and again for the entire campaign at a second point during Pro A buffer exchange for a total of two standard curves. The standard curve was constructed by various online dilutions of 2.5 mg / mL stock anti-TGFβ DS to generate a curve from 2 to 50 μg on column.

[0243] Stocks used for the standard curve were pre-diluted prior to the run and kept in sub-aliquots at -80°C for use in the MIMICS-mPQA campaign.

[0244] result The recorded data from Pro A quantification was also compared to two offline quantitative titer measurements: titer measured on an Octet using a Pro A biosensor chip, and titer measured on a CEDEX™ Bioanalyzer. Figures 10-12 demonstrate the comparability of the titer of MIMICS-mPQA compared to the offline method for 100 L and 3 L bioreactors.

[0245] Overall, trends were consistent between the three methods. When comparing the MIMICS-mPQA data to the CEDEX™ Bioanalyzer method, the % difference was less than 10% for all runs, which is an acceptable CV for the analytical method. The CEDEX™ Bioanalyzer is the method typically used for titer determinations for daily upstream process monitoring. The comparability of MIMICS-mPQA to this technique provided an orthogonal tool for titer determinations for mammalian culture. An additional benefit of MIMICS-mPQA was the reduction in manual handling of samples and the reduction in potential bias of sample storage when testing is performed offline.

[0246] The aggregation data recorded in this experiment was compared to the operation of an offline SEC method for routine process monitoring. The data for each bioreactor is presented in Figures 13-15.

[0247] Overall, the trends are comparable between the offline analytical method and MIMICS-mPQA. Both methods showed aggregation levels below 2.5% throughout the entire campaign. The MIMICS-mPQA system allowed a larger number of data points to be accumulated for this campaign than would normally be tested in offline analysis. This increased sampling allows for a deeper look into the variability in aggregation over the course of the campaign.

[0248] Purity data recorded from the MIMICS-mPQA analysis was also compared to the operation of an offline purity method for process monitoring shown in Figure 16. The offline method was based on CE-SDS methodology that is orthogonal to the purity measurements made using MIMICS-mPQA.

[0249] As shown in Figure 16, the MIMICS-mPQA system provides lower absolute percentages of purity overall compared to the offline method. Despite the absolute differences in percentages, the overall trends were consistent between both methods.

[0250] Overall, the MIMICS-mPQA system was successfully applied in an at-line monitoring mode to a 100 L pilot-scale bioreactor campaign from anti-TGFβ. The system allowed for increased sample collection compared to offline analysis, which further allowed for a higher process understanding over the course of the campaign. In addition, titers and aggregation rates were highly comparable with the offline analysis providing confidence in the analysis at the worksite compared to those obtained in the analytical laboratory. The MIMICS-mPQA system also reduced sample consumption and allowed for more rapid throughput, as there was no need for prior Protein A purification before analysis, as this step was incorporated into the overall MIMICS-mPQA workflow. Finally, the system was capable of operating over the course of a four week period with minimal incidents, further enhancing our understanding of the instrument robustness and column stability. [Explanation of symbols]

[0251] 1 System 2. Four-column Periodic Countercurrent Chromatography System (PCCS) 3 Columns 4 Columns 5 Columns 6 Columns 7 Entrance 8 PCCS 9 Chromatography Columns 10 Chromatography columns 11 Chromatography columns 12 Chromatography membrane 13 Fluid conduit 14 In-line Buffer Regulation Reservoir 15 Exit 16 Fluid conduit 17 In-line Buffer Regulation Reservoir 18 Filters 19 Break Tank 20 Pump System 21 Pump 22 Fluid conduit 23 Filters 24 Break Tank 25 Bioreactor 26 Fluid conduit 27 Filtration System 100 Systems 102 Sample Manager 104 First pump 104 106 First flow control device 108 Sample Purification Device 110 Second flow control device 112 Column Manager 112 114a Sample analyzer 114b Sample analyzer 114c Sample analyzer 114d Sample analyzer 116 Detector 118 Second Pump 120a Solvent / Buffer Reservoir 120b Solvent / Buffer Reservoir 120c Solvent / Buffer Reservoir 120d Solvent / Buffer Reservoir 122 Control Unit 124a communication line 124b communication line 124c communication line 124d Communication Line 124e communication line 124f Communication Line 124g communication line 202 Entrance 204 Holding Conduit 206 Gate valve 208 Outlet conduit

Claims

1. 1. A system for measuring a product quality attribute of an analyte of a biological sample, the system comprising: A first flow control device; a sample purification device downstream of the first flow control device and in fluid communication with the first flow control device; a second flow control device downstream of the first flow control device and in fluid communication with the first flow control device, the sample purification device, and the first and second sample analyzers, the first sample analyzer including a first chromatography column; wherein the first flow control device is configured to switch between directing a portion of the biological sample containing the analyte to the sample purification device and directing a portion of the biological sample containing the analyte to the second flow control device; and wherein the second flow control device is configured to receive the portion of the biological sample directly from the first flow control device or from the sample purification device; a control unit coupled to the first and second flow control devices, the control unit controlling, during operation of the system, (a) adjusting the configuration of the first flow control device such that a portion of the biological sample containing the analyte is received by the second flow control device either directly from the first flow control device or from the sample purification device, thereby directing a portion of the biological sample containing the analyte from the first flow control device to either the sample purification device or the second flow control device; (b) adjusting the configuration of the second flow control device to direct a portion of the biological sample containing the analyte to one of the first and second sample analyzers, where each of the first and second sample analyzers is configured to determine a different product quality attribute of the analyte; and (c) determining a product quality characteristic of an analyte of the biological sample based on an analysis of the portion of the biological sample by one of the first and second sample analyzers. A control unit configured to The system comprising:

2. the first chromatography column is a cation exchange chromatography column; The system of claim 1 .

3. 2. The system of claim 1, wherein the first chromatography column is a size exclusion chromatography column.

4. 2. The system of claim 1, wherein the first chromatography column is a reverse phase chromatography column.

5. The system of claim 1 , wherein the sample purification device comprises an affinity chromatography column.

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

8. 2. The system of 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 eluate stream from the first chromatographic column.

9. 2. The system of claim 1, wherein the second sample analyzer includes a second chromatography column, the second chromatography column being different from the first chromatography column and being one of a cation exchange chromatography column, a size exclusion chromatography column, a reverse phase chromatography column, and a hydrophilic interaction chromatography column.

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

11. 2. The system of claim 1, wherein the second flow control device is in fluid communication with four additional sample analyzers, each of the four additional sample analyzers including a chromatography column that is different from both the first chromatography column and the chromatography columns of others of the four additional sample analyzers.

12. The system of claim 1 , wherein the product quality characteristic of the analyte is a concentration of the analyte in the biological sample.

13. The system of claim 1 , wherein the product quality characteristic of the analyte is a measure of aggregation of the analyte in the biological sample.

14. 10. The system of claim 1, wherein the product quality characteristic of the analyte is a measure of charge variants or heterogeneity of the analyte in the biological sample.

15. 10. The system of claim 1, wherein the product quality characteristic of the analyte is a measure of the purity or integrity of the analyte in the biological sample.

16. 6. 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.

17. The system of claim 1 , wherein the analyte comprises a protein in a biological sample.

18. The system of claim 1 , wherein the protein comprises an antibody in the biological sample.

19. 10. The system of claim 9, further comprising 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, the control unit configured to adjust a position of the column manager to direct a portion of the biological sample to one of the first and second sample analyzers.

20. 11. The system of claim 10, further comprising 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, the control unit configured to adjust a position of the column manager to direct a portion of the biological sample to one of the first, second, third and fourth sample analyzers.

21. The portion of the biological sample is a first portion, the product quality characteristic is a first product quality characteristic, and the control unit, during operation of the system, (d) adjusting the positioning of the first flow control device such that the second portion of the biological sample is received by the second flow control device to direct the second portion of the biological sample from the first flow control device to either the sample purification device or the second flow control device; (e) adjusting the position 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) determining a second product quality characteristic of an analyte of the biological sample based on an analysis of the second portion of the biological sample by one of the first and second sample analyzers that received the second portion of the biological sample. The system of claim 1 , configured as follows:

22. 22. The system of claim 21 , wherein the first and second product quality characteristics are different, and the first and second product quality characteristics are each selected from the group consisting of a concentration of an analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variants or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

23. 10. The system of claim 9, wherein the portion of the biological sample is a first portion and the product quality characteristic is a first product quality characteristic, and the control device is configured to repeat steps (a)-(c) with another portion of the biological sample to determine two different product quality characteristics for an analyte of the biological sample.

24. 10. The system of claim 9, wherein the portion of the biological sample is a first portion and the product quality characteristic is a first product quality characteristic, and the control device is configured to repeat steps (a)-(c) with two other portions of the biological sample to determine three different product quality characteristics for analytes of the biological sample.

25. The portion of the biological sample is a first portion, the product quality characteristic is a first product quality characteristic, and the control device repeats steps (a)-(c) with another portion of the biological sample to produce a product quality characteristic. The system of claim 10 , configured to determine two different product quality attributes for an analyte of a body sample.

26. 11. The system of claim 10, wherein the portion of the biological sample is a first portion, the product quality characteristic is a first product quality characteristic, and the control device is configured to repeat steps (a)-(c) with three other portions of the biological sample to determine four different product quality characteristics for analytes of the biological sample.

27. 27. The system of any one of claims 23 to 26, wherein the product quality characteristics are each 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 variants or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

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

29. 30. The system of claim 28, wherein the sample collection device includes a container interface configured to receive a biological sample in the container.

30. 30. The system of claim 28, wherein the sample collection device includes a fluid channel configured to receive a biological sample, and the control unit is configured, during operation of the system, to send a signal to the sample collection device to cause the sample collection device to expel a portion of the biological sample from the fluid channel to the first flow control device.

31. 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; Further comprising: the control unit and the pump are configured such that, during operation of the system, when a portion of the biological sample is directed to one of the first and second sample analyzers, the pump delivers a buffer solution from a corresponding associated buffer reservoir to one of the first and second sample analyzers; The system of claim 9.

32. 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; Further comprising: the control unit and the pump are configured such that, during operation of the system, when a portion of the biological sample is directed to one of the first, second and third sample analyzers, the pump delivers a buffer solution from a corresponding associated buffer reservoir to one of the first, second and third sample analyzers; The system of claim 10.

33. 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; Further comprising: the first chromatography column is a cation exchange column; The control unit and pump are configured to control the operation of the system such that the pump pumps acetate buffer into the first a first chromatography column configured to deliver the biological sample to the first chromatography column and propagate a portion of the biological sample along the first chromatography column; The system of claim 1 .

34. 34. The system of claim 33, wherein the acetate buffer has a pH of 4.0 or less.

35. The system of claim 1 , wherein the biological sample is a harvest medium extracted from a bioreactor.

36. The system of claim 1 , wherein the biological sample is an intermediate or product solution from a biological manufacturing system.

37. The system of claim 1 , wherein the biological sample is part of a cell culture.

38. The system of claim 6 , wherein the quantitative detector comprises a diode array detector.

39. The system of claim 6 , wherein the quantitative detector comprises a spectrophotometric detector configured to measure absorbance information for a portion of the biological sample.

40. The system of claim 6 , wherein the quantitative detector comprises a fluorescence detector.

41. The system of claim 6 , wherein the quantitative detector comprises a mass spectrometry detector.

42. 1. A system for measuring a product quality attribute for an analyte of a biological sample, the system comprising: A first flow control device; a sample purification device including a purification chromatography column downstream of the first flow control device and in fluid communication with the first flow control device; a second flow control device downstream of the first flow control device and in fluid communication with the first flow control device and the sample purification device; wherein the first flow control device is configured to switch between directing a portion of the biological sample containing the analyte to the sample purification device and directing a portion of the biological sample containing the analyte to the second flow control device; and wherein the second flow control device is configured to receive the portion of the biological sample directly from the first flow control device or from the sample purification device; a first sample analyzer including a first chromatography column in fluid communication with a second flow control device; a second sample analyzer including a second chromatography column in fluid communication with a second flow control device; a third sample analyzer including a third chromatography column in fluid communication with the second flow control device; a fourth sample analyzer including a quantitative detector; and a control unit coupled to the first and second flow control devices, the control unit controlling, during operation of the system, (a) adjusting the configuration of the first flow control device so that a portion of the biological sample containing the analyte is received by the second flow control device either directly from the first flow control device or from the sample purification device, directing a first portion of the biological sample containing the analyte from the first flow control device to either the sample purification device or the second flow control device; (b) adjusting a configuration of a second flow control device to direct a first portion of the biological sample containing the analyte to one of the first, second, third, and fourth sample analyzers; (c) determining a first product quality characteristic of an analyte of the biological sample based on an analysis of the portion of the biological sample by one of the first, second, third, and fourth sample analyzers; and (d) repeating steps (a)-(c) with three additional portions of the biological sample containing the analyte, adjusting the configuration of the second flow control device to direct each portion of the biological sample containing the analyte to a different one of the sample analyzers to determine a total of four different product quality attributes of the analyte of the biological sample. A control unit configured to The system comprising:

43. 43. The system of claim 42, wherein each of the four product quality attributes is different.

44. 43. The system of claim 42, wherein each of the first, second and third chromatography columns is a different type of column.

45. 45. The system of claim 44, wherein the first chromatography column is a cation exchange column, the second chromatography column is a size exclusion column, and the third chromatography column is a reverse phase column or a hydrophilic interaction column.

46. 43. The system of claim 42, wherein a first sample analyzer determines information about a measure of charge variants or heterogeneity of an analyte in the biological sample, a second sample analyzer determines information about a measure of aggregation of the analyte in the biological sample, a third sample analyzer determines information about a measure of purity or integrity of the analyte in the biological sample, and a fourth sample analyzer determines information about a concentration of the analyte in the biological sample.

47. 43. The system of claim 42, wherein the four quality attributes include a measure of charge variants 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.

48. 43. The system of claim 42, wherein the first chromatography column is a cation exchange chromatography column, the second chromatography column is a size exclusion chromatography column, and the third chromatography column is a reverse phase chromatography column.

49. 43. The system of claim 42, wherein the sample purification device comprises an affinity chromatography column.

50. 43. The system of claim 42, wherein the four product quality characteristics include a concentration of the analyte in the biological sample, a measure of aggregation of the analyte in the biological sample, a measure of charge variants or heterogeneity of the analyte in the biological sample, and a measure of purity or integrity of the analyte in the biological sample.

51. 43. The system of claim 42, wherein the analyte comprises a protein in a biological sample.

52. The system of claim 51 , wherein the protein comprises an antibody in the biological sample.

53. 43. The system of claim 42, further comprising 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, the control unit configured to adjust a position of the column manager to direct a portion of the biological sample to one of the first, second and third sample analyzers.

54. a sample collection device coupled to the control unit for receiving a biological sample and 43. The system of claim 42, further comprising a sampling device configured to deliver a portion of the first fluid control device.

55. 43. The system of claim 42, wherein the quantitative detector comprises one of a diode array detector, a spectrometric detector, a fluorescence detector, and a mass spectrometry detector configured to measure absorbance information about a portion of the biological sample.

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