Product quality attribute measurement

Abstract

To provide a system and a method for measuring the product quality attribute of a sample including a recovered sample from a continuous biological manufacturing system.SOLUTION: A system 100 for measuring the product quality attribute of an analyte of a biological sample includes a first flow control device 106, a sample purification device 108, a first sample analyzer 114a, second sample analyzers 114b, 114c, 114d, a second flow control device 110, a detector 116, and a control unit 122. The first sample analyzer 114a includes a first chromatography column. During operation of the system, the control unit 122 adjusts the arrangement of the second flow control device 110 to direct a portion of the biological sample to one of the first and second sample analyzers. The product quality attribute of the analyte of the biological sample is determined on the basis of the analysis of the portion of the biological sample conducted by one of the first and second sample analyzers.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 904,682, filed on September 23, 2019, the entire content of which is incorporated herein by reference.

[0002] This disclosure relates to systems and methods for measuring the quality characteristics of a product generated from a sample containing a recovered sample from a continuous biological manufacturing system.

Background Art

[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 treatments based on such proteins. Monitoring systems are used in biological manufacturing to evaluate various biological products and process conditions.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The integrated continuous biological manufacturing of therapeutic protein substances and other biomolecules can be highly anticipated for the future production 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 various configurations can be used for industrial - scale biological manufacturing. In such systems, the analysis of the eluate from the chromatography system can be used to determine various product quality characteristics and to monitor and adjust a wide variety of bioprocess conditions.

Means for Solving the Problems

[0005] The present disclosure features a method and system for determining one or more product quality characteristics of an analyte in a biological sample, including a sample recovered from a bioreactor and an offline sample introduced into the system in series or in parallel. Various product quality characteristics can be measured, including but not limited to the concentration of the analyte, the charge variant or heterogeneity of the analyte, the aggregation of the analyte, and the integrity or purity of the analyte. The system can include a sample analyzer equipped with different types of chromatography columns specialized for measuring specific product quality characteristics. The measured product quality characteristics can be used to provide feedback and control for parameters and operations related to the biological manufacturing process.

[0006] In one aspect, the present disclosure is a system for measuring the product quality characteristics of an analyte in a biological sample, the system comprising a first flow control device, a sample purification device in fluid communication with the first flow control device, a second flow control device in fluid communication with the first flow control device, the sample purification device, and first and second sample analyzers, wherein the first sample analyzer includes a first chromatography column, a second flow control device, a control unit coupled to the first and second flow control devices, and during operation of the system, the control unit is configured to: (a) adjust the placement of the first flow control device so that a portion of the biological sample is received by the second flow control device and direct a portion of the biological sample from the first flow control device either to the sample purification device or to the second flow control device; (b) adjust the placement of the second flow control device to direct a portion of the biological sample to one of the first and second sample analyzers; and (c) determine the product quality characteristics of the analyte in the biological sample based on the analysis of a portion of the biological sample by one of the first and second sample analyzers. Embodiments of the present system can include any one or more of the following configurations.

[0007] The embodiments of the present system can include any one or more of the following configurations.

[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 chromatography column is 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 chromatography 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, which is different from the first chromatography column and is one of a cation exchange chromatography column, a size exclusion chromatography column, a reverse 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, which is different from the first and second chromatography columns and is 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 that is different from both the first chromatography column and the chromatography columns of the other of the four additional sample analyzers.

[0013] The quality characteristics of the analyte in the produced article are the concentration of the analyte in the biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the charge variant or heterogeneity of the analyte in the biological sample, or a measure of the 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 proteins (such as antibodies) in the biological sample.

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

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

[0017] A portion of the biological sample is the first portion, the generated article quality characteristic is the first generated article quality characteristic, and the control unit, during operation of the system, (d) adjusts the placement of the first flow control device so that a second portion of the biological sample is received by the second flow control device, directing the second portion of the biological sample from the first flow control device either to the sample purification device or to the second flow control device; (e) adjusts the placement 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) is configured to determine a second generated article quality characteristic of the analyte in the biological sample based on the 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 first and second generated article quality characteristics are different, and the first and second generated article quality characteristics can each be selected from the group consisting of the concentration of the analyte in the biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the charge variant or heterogeneity of the analyte in the biological sample, and a measure of the purity or integrity of the analyte in the biological sample.

[0018] A portion of the biological sample is the first portion, the generated article quality characteristic is the first generated article quality characteristic, and the control device can be configured to repeat steps (a)-(c) using another portion of the biological sample to determine two different generated article quality characteristics for the analyte in the biological sample.

[0019] A portion of the biological sample is the first portion, the generated article quality characteristic is the first generated article quality characteristic, and the control device can be configured to repeat steps (a)-(c) using two other portions of the biological sample to determine three different generated article quality characteristics for the analyte in the biological sample.

[0020] A portion of the biological sample is the first portion, the generated article quality characteristic is the first generated article quality characteristic, and the control device can be configured to repeat steps (a)-(c) using three other portions of the biological sample to determine four different generated article quality characteristics for the analyte in the biological sample.

[0021] The generated article quality characteristics can be respectively selected from the group consisting of the concentration of the analyte in the biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the charge variant or heterogeneity of the analyte in the biological sample, and a measure of the purity or integrity of the analyte in the biological sample.

[0022] The system can include a sample collection device connected to a control unit, the sample collection device being configured to receive a biological sample and deliver a portion of the biological sample to a 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 fluid channel configured to receive the biological sample, and the control unit can transmit a signal to the sample collection device during operation of the system to configure the sample collection device to discharge a portion of the biological sample from the fluid channel to the first flow control device.

[0023] The system can include a second flow control device, first and second sample analyzers, and a pump in fluid communication with first and second buffer reservoirs respectively associated with the first and second sample analyzers, and the control unit and the pump are configured such that during operation of the system, when a portion of the biological sample is headed towards 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. That is, it is configured to deliver to one of them.

[0024] This system can include a second flow control device, first, second, and third sample analyzers, and a pump in fluid communication with first, second, and third buffer reservoirs respectively associated with the first, second, and third sample analyzers, wherein the control unit and the pump are configured such that during operation of the system, when a portion of a biological sample is directed towards 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] This system can include a second flow control device, a first sample analyzer, and a pump in fluid communication with a buffer reservoir associated with the first sample analyzer, wherein the first chromatography column is a cation exchange column, and the control unit and the 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 recovered medium extracted from a bioreactor. The biological sample is a solution of an intermediate or product from a biological manufacturing system. The biological sample is a portion of a cell culture.

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

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

[0029] In another aspect, the present disclosure is a system for measuring the generated article quality characteristics of an analyte in 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 a first chromatography column in fluid communication with the second flow control device, a second sample analyzer including a 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: (a) adjusts the placement of the first flow control device so that a portion of the biological sample is received by the second flow control device, directing a first portion of the biological sample from the first flow control device either to the sample purification device or to the second flow control device; (b) adjusts the placement 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) determines a first generated article quality characteristic of the analyte of the biological sample based on the analysis of a portion of the biological sample by one of the first, second, third, and fourth sample analyzers; and (d) repeats steps (a) through (c) using three additional portions of the biological sample, adjusting the placement 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 generated article quality characteristics of the analyte of the biological sample.

[0030] Embodiments of the present system can include any one or more of the following configurations is possible.

[0031] Each of the four generated article quality characteristics can be different. Each of the first, second, and third chromatography columns can be a different type of column. 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 the charge variant or heterogeneity measure of the analyte in the biological sample, the second sample analyzer can determine information about the aggregation measure of the analyte in the biological sample, the third sample analyzer can determine information about the purity or integrity measure 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 generated article quality characteristics can include the charge variant or heterogeneity measure of the analyte in the biological sample, the aggregation measure of the analyte in the biological sample, the purity or integrity measure of the analyte in the biological sample, and the 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 proteins in the biological sample. The proteins can include antibodies in the biological sample.

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

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

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

[0038] In a further aspect, the disclosure is a method for measuring the product quality characteristics of an analyte in a biological sample, the method comprising obtaining a biological sample by extracting the biological sample from an operating bioreactor or a purification device in fluid communication with the operating 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 in 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 in 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 Characterized by a method, wherein at least one is a measure of the charge variant or heterogeneity of an analyte in a biological sample, a measure of the aggregation of the analyte in the biological sample, a measure of the purity or integrity of the analyte in the biological sample, and the concentration of the analyte in the biological sample.

[0039] Unless otherwise specified, embodiments of the method can also include any combination of the configurations disclosed individually in different embodiments, including any of the other configurations described herein.

[0040] Definitions The term "unit operation" is a technical term and means a functional step that can be performed in the process of manufacturing a therapeutic protein drug substance from a liquid culture medium. For example, a unit of operation can be filtering (e.g., removing contaminants such as bacteria, yeast, virus, or mycobacteria, and / or particulate matter from a fluid containing a recombinant therapeutic protein), capturing, removing an epitope tag, purifying, storing or warehousing, polishing, inactivating a virus, adjusting the ionic concentration and / or pH of a fluid containing a recombinant therapeutic protein, and removing unwanted salts.

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

[0042] The term "capturing" means the process of partially purifying or isolating (e.g., at least or about 5% by weight, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95% pure), concentrating, and stabilizing a recombinant therapeutic protein from one or more other components present in a liquid culture medium or a diluted liquid culture medium (e.g., culture medium proteins, or one or more other components (e.g., DNA, RNA, or other proteins) present in or secreted from mammalian cells). Typically, capturing is performed using a resin that binds to the recombinant therapeutic protein (e.g., by use of affinity chromatography). Non-limiting methods for capturing a recombinant therapeutic protein from a liquid culture medium or a diluted liquid culture medium are described herein, and others are known in the art. A recombinant therapeutic protein can be captured from a 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" means the process of isolating 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., culture medium proteins or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.) present in or secreted from mammalian cells). This is done. For example, purification can be carried out during or after the initial capture step. Purification can be performed using a resin, membrane, or any other solid support that binds to either the recombinant therapeutic protein or contaminants (e.g., by using affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). The recombinant therapeutic protein can be purified from a fluid containing the recombinant therapeutic protein using at least one chromatography column and / or chromatography membrane (e.g., any of the chromatography columns or chromatography membranes described herein).

[0044] The term "polishing" is a technical term and refers to the process of removing any remaining trace or small amounts of contaminants or impurities from a fluid containing a recombinant therapeutic protein that is close to the final desired purity. For example, polishing can be carried out by passing a fluid containing the recombinant therapeutic protein through a chromatography column or membrane absorbent that selectively binds to either the target recombinant therapeutic protein or a small amount of 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 absorbent contains the recombinant therapeutic protein.

[0045] The term "filtering" means removing at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of unwanted biological contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses, or mycobacteria) and / or particulate matter (e.g., precipitated protein) 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 technical term and means the fluid discharged from a chromatography column or membrane containing a detectable amount of recombinant therapeutic protein.

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

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

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

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

[0051] The term "biological drug" means any therapeutic substance made or obtained from a living organism or its products, which is used in the prevention, diagnosis, or treatment of medical conditions. Thus, biological drugs or biopharmaceuticals are pharmaceuticals produced using biotechnology for therapeutic or in vivo diagnostic purposes, such as proteins (e.g., recombinant therapeutic proteins) or nucleic acids (DNA, RNA, or antisense oligonucleotides).

[0052] The term "multi-column chromatography system" or "MCCS" means a system of a total of two or more interconnected or switchable chromatography columns and / or chromatography membranes. Non-limiting examples of a multi-column chromatography system are periodic countercurrent chromatography systems (PCCs) containing a total of two or more interconnected or switchable chromatography columns and / or chromatography membranes. Additional examples of multi-column chromatography systems are described herein and are known in the art.

[0053] The term "mammalian cell" means any cell from or derived from any mammal (e.g., human, hamster, mouse, 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" means 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). The cell culture can have a cell density higher than about 0.1×106 cells / mL (e.g., higher than about 1.0×106 cells / mL, higher than about 5.0×106 cells / mL, higher than about 10×106 cells / mL, higher than about 15×106 cells / mL, higher than about 20×106 cells / mL, higher than about 25×106 cells / mL, higher than about 30×106 cells / mL, higher than about 35×106 cells / mL, higher than about 40×106 cells / mL, higher than about 45×106 cells / mL, higher than about 50×106 cells / mL, higher than about 55×106 cells / mL, higher than about 60×106 cells / mL, higher than about 65×106 cells / mL, higher than about 70×106 cells / mL, higher than about 75×106 cells / mL, higher than about 80×106 cells / mL, higher than about 85×106 cells / mL, higher than about 90×106 cells / mL, higher than about 95×106 cells / mL, or higher than about 100×106 cells / mL).

[0055] The term "culturing" or "cell culturing" means 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" means a fluid containing sufficient nutrients to allow mammalian cells to grow in vitro in the medium. For example, the liquid culture medium can contain amino acids ( For example, it can contain one or more of 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, as well as sodium bicarbonate. The liquid culture medium can contain serum derived from mammals. In some examples, the liquid culture medium does not contain serum or another extract derived from mammals (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" means a polypeptide containing 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., variable domain sequence, framework sequence or constant domain sequence). Immunoglobulins can include, for example, light chain immunoglobulins of at least 15 amino acids, such as heavy chain immunoglobulins of at least 15 amino acids. Immunoglobulins can be isolated antibodies (e.g., IgG, IgE, IgD, IgA or IgM). Immunoglobulins can be subclasses of IgG (e.g., IgG1, IgG2, IgG3 or IgG4). Immunoglobulins can be antibody fragments, e.g., Fab fragments, F(ab’)2 fragments or scFv fragments. Immunoglobulins can also be bispecific or trispecific antibodies, or dimeric, trimeric or multimeric antibodies, or diabodies, Affibody® or Nanobody®. Immunoglobulins can also be engineered proteins (e.g., fusion proteins) containing 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, antibodies or antibody fragments, enzymes, engineered proteins, or immunogenic proteins or protein fragments.

[0059] The terms "protein fragment" or "polypeptide fragment" mean 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" means a polypeptide that is not naturally encoded by an endogenous nucleic acid present in an organism (e.g., a mammal). Examples of engineered proteins include enzymes (e.g., one or more amino acid substitutions, deletions, insertions or additions that result in an increase in the stability and / or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bispecific antibodies, trispecific antibodies or diabodies), and antigen-binding proteins that contain at least one recombinant scaffold sequence. Examples include proteins, antibodies (e.g., bispecific antibodies, trispecific antibodies or diabodies), and antigen-binding proteins that contain at least one recombinant scaffold sequence.

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

[0062] The term "perfusion bioreactor" means 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 involves the periodic or continuous removal of the first liquid culture medium and the simultaneous or subsequent addition of a substantially equal volume of a second liquid culture medium to the bioreactor. In some examples, there is a gradual change (e.g., increase or decrease) in the volume of the first liquid culture medium removed and added over a growth period (e.g., a period of about 24 hours, a period from about 1 minute to about 24 hours, or a period longer than 24 hours) during the culturing period (e.g., the daily resupply rate of the culture medium). The proportion of the medium removed and replaced each day can vary depending on the specific cells being cultured, the initial seeding density, and the cell density at a particular time. "RV" or "reactor volume" means the volume of the culture medium present at the start of the culturing process (e.g., the total volume of the culture medium present after seeding).

[0063] The term "fed-batch bioreactor" is a technical term and means 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 involves the periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without substantial or sufficient 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 culturing, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culturing, 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 commonly understood by one of ordinary skill 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 described herein, but the preferred 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, the materials, methods, and examples are illustrative only and 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 configurations and advantages will be apparent from the description, drawings, and claims.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

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

[0068] Introduction Biological manufacturing on an industrial scale can be carried out in two-column and multi-column chromatography systems of various arrangements. In these complex systems, the product yield, quality, and waste rate are functions of parameters and processes related to a number of processes. During the manufacture of therapeutic proteins and other commercially valuable biomolecules, the product outcome is strongly influenced by these parameters and processes. Therefore, appropriate control of such parameters and processes is an important aspect of large-scale manufacturing. The configuration and mode of the biological manufacturing system are disclosed, for example, in International Publication No. WO 2014 / 137903 of the PCT patent application publication, the entire content of which is incorporated herein by reference.

[0069] Appropriate control of biological manufacturing parameters, including automatic control, is facilitated by monitoring the bioreactor harvest, intermediate solution flow, and / or product. Conventional monitoring techniques include, for example, UV absorbance measurement.

[0070] Unfortunately, such methods suffer from drift over a measurement period of several days due to factors such as temperature, humidity, ambient light intensity, and local sample inhomogeneity. Furthermore, such methods may not be able to calculate or otherwise determine multiple quantities. In a complex biological manufacturing environment, multiple quantities are typically evaluated to provide suitable feedback information for the adjustment of process parameters.

[0071] The present disclosure features a system and method that can be used to determine values of a plurality of product quality characteristics. The system can be implemented as a two-dimensional chromatography system. Along a first dimension of the system, a portion of a biological sample can be purified using a sample purification device that 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. Additional portions of the sample can be directed to different sample analyzers to determine different product quality characteristics for the sample.

[0072] Product Quality Characteristic Analysis System FIG. 1 is a schematic diagram showing an example of a measurement system 100 for measuring a plurality of product quality characteristics. The system 100 includes a sample manager 102, a first pump 104 (e.g., a binary pump), a first flow control device 106, a sample purification device 108, a second flow control device 110, a column manager 112, a plurality of sample analyzers 114a-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 connected to the control unit 122 via communication lines 124a-124g.

[0073] During operation, the sample manager 102 receives a biological sample for analysis. The sample manager 102 can be implemented in various ways. In some embodiments Then, for example, the sample manager includes a container reservoir configured to receive a 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 plates, and the container reservoir is configured to receive such plates. 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 Waters H Class Sample Manager with a flow-through needle, and 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 sample collection device, 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 directly recovered from the bioreactor (thus corresponding to a recovered sample of the growth medium), or a solution or medium extracted from another location in the biological manufacturing system.

[0075] FIG. 2 is a schematic diagram of an example of a sample manager 102 configured to directly receive a biological sample from a sample collection device. The sample manager 102 includes an inlet 202, a holding conduit 204, and a gate valve 206 coupled to a 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 within 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 transmits a signal to the gate valve 206. The gate valve 206 opens and a portion of the sample is discharged from the holding conduit 204 into an outlet conduit 208. The discharged portion of the sample is then pumped (e.g., by the first pump 104) into the first flow control device 106.

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

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

[0078] System 100 determines the product quality characteristics for one or more analytes in a biological sample received by sample manager 102. Generally, characteristics can be determined for a wide variety of different types of analytes. For example, in some embodiments, system 100 determines the 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., galactosidase (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 containing at least one multifunctional recombinant protein scaffold (see, e.g., Gebauer et al., Current Opin. Chem. Biol. Vol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, which is incorporated herein by reference in its entirety, for recombinant antigen-binding proteins).

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

[0081] Additional non-limiting examples of recombinant therapeutic proteins that can be analyzed include alglucosidase alpha, laronidase, abatacept, galsulfase, lutropin alpha, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alpha, tenecteplase, etanercept, coagulation factor IX, follicle-stimulating hormone, interferon beta-1a, imiglucerase, dornase alpha, epoetin alpha, insulin or insulin analogs, 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 generated by the present method include acid alpha-glucosidase, alglucosidase alpha (e.g., Myozyme® and Lumizyme®), alpha-L-iduronidase (e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid beta-galactosidase, beta-glucuronidase, N-acetylglucosamine-1-phosphotransferase, alpha-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, beta-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase, alpha-galactosidase-A (e.g., Fabrazyme®), acid beta-galactosidase, beta-gal tosidase, neuraminidase, hexosaminidase A and hexosaminidase B.

[0082] As discussed above, in some embodiments, the analyte is a component of a cell, and the methods and systems described herein can be used for the 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 that grow 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 (Vero) cells and Madin-Darby canine (cocker spaniel) kidney epithelial (MDCK) cells.

[0083] Mammalian cells can contain a recombinant nucleic acid encoding a recombinant therapeutic protein (e.g., a nucleic acid stably integrated into the genome of a mammalian cell). Non-limiting examples of exemplary recombinant nucleic acids encoding recombinant therapeutic proteins can be described below, such as recombinant therapeutic proteins generated using the methods described herein. In some examples, mammalian cells cultured in a bioreactor (e.g., any of the bioreactors described herein) are derived from a large-scale culture.

[0084] Nucleic acids encoding recombinant therapeutic proteins can be incorporated into mammalian cells using a variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentivirus, adenovirus or retrovirus infection) and electroporation. In some examples, the nucleic acid encoding the recombinant therapeutic protein is not stably incorporated into the chromosome of the mammalian cell (transient transfection), while in other examples, the nucleic acid is incorporated. Alternatively, or in addition, the nucleic acid encoding the recombinant therapeutic protein can be present in a plasmid and / or in a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively, or in addition, the nucleic acid can be introduced into cells 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 contain, if desired, a selectable marker (e.g., a gene conferring hygromycin, puromycin or neomycin resistance to mammalian cells).

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

[0086] After the biological sample is received by the sample manager 102, the sample is transported by the first pump 104 through the conduit 126 to the first flow control device 106. The first flow control device 106 is connected to the control unit 122 via the control line 124b. Generally, the biological sample can be delivered by the first flow control device 106 to a plurality of different outputs. In a first arrangement, the first flow control device 106 delivers the biological sample directly to the second flow control device 110 via the conduit 128. In another arrangement, the first flow control device 106 delivers the biological sample to the sample purification device 108 via the conduit 130. The control unit 102 is configured to adjust the arrangement of the first flow control device 106 according to the desired mode of analysis of the biological sample so as to direct the biological sample to any destination.

[0087] The first flow control device 106 can be implemented in various ways. In some embodiments, for example, the first flow control device 106 can be implemented as a multi-way valve. Suitable valves include, for example, the IDEX MX Series II 2-position 6-port UltraLife Switching valve (available from IDEX Corp., Lake Forest, IL). In certain embodiments, the first flow control device 106 can be implemented as a multi-channel fluid device comprising input and / or output manifolds and an electrically controllable flow regulating valve.

[0088] When the biological sample is directed to the sample purification device 108, the biological sample is at least partially purified before the sample is analyzed in the system 100. Purification can occur in various ways, but typically involves removing one or more non-analyte components from the sample. Alternatively, or in addition, purification of the biological sample can include concentration of the analyte in the sample and separation of one analyte from one or more additional analytes in the sample.

[0089] The sample purification device 108 can be implemented in various ways. In some embodiments, for example, the sample purification device 108 is implemented as a chromatography device 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 an analyte molecule (e.g., a recombinant protein analyte) is captured and isolated based on affinity. Affinity chromatography refers to the use of an affinity chromatography resin (e.g., an affinity chromatography resin containing a protein ligand (e.g., Protein A or Protein G)). In some embodiments, affinity chromatography includes a pseudo-affinity chromatography resin. In some embodiments, the affinity chromatography resin includes a cofactor ligand, a substrate ligand, a metal ligand, a product ligand, or an aptamer ligand. Generally, the affinity chromatography resin can include any receptor or ligand having an affinity for any biological analyte, including DNA and oligonucleotides. In some embodiments, the affinity chromatography resin can include a single-domain antibody fragment derived from camelids for the purification of gene therapy vectors. As 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 ~about 60 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 30 amino acids, or about 5 amino acids to about 20 amino acids), a low molecular weight substrate or cofactor of the enzyme, an aptamer, an inhibitor (e.g., a competitive protein inhibitor) or a metal can be mentioned.

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

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

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

[0094] In some embodiments, the 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, the Protein A column can be loaded with 1× PBS at about pH 7.2.

[0095] In some embodiments, the Protein A column is in about 50 mM to about 200 mM citrate phosphate (e.g., about 50 mM to about 190 mM, about 50 mM to about 180 mM, about 50 mM to about 170 mM, about 50 mM to about 160 mM, about 50 mM to about 150 mM, about 50 mM to about 140 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 75 mM to about 200 mM, about 75 mM to about 190 mM, about 75 mM to about 180 mM, about 75 mM to about 170 mM, about 75 mM to about 160 mM, about 75 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) at about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.8, about pH 2 to about pH 3.6, about pH 2 to about pH 3.4, about pH 2 to about pH 3.2, about pH 2 to about pH 3.0, about pH 2 to about pH 2.8, about pH 2 to about pH 2.6, about pH 2 to about 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 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, about 125 mM to about 200 mM, about 125 mM to about 190 mM, about 125 mM to about 180 mM, about 125 mM to about 170 mM, about 125 mM to about 160 mM, about 125 mM to about 150 mM, about 125 mM to about 140 mM, about 125 mM to about 130 mM, about 150 mM to about 200 mM, about 150 mM to about 190 mM, about 150 mM to about 180 mM, about 150 mM to about 170 mM, about 150 mM to about 160 mM, about 175 mM to about 200 mM, about 175 mM to about 190 mM, about 175 mM to about 180 mM, or about 180 mM to about 200 mM), about 50 mM to about 200 mM of 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 180 mM, about 75 mM to about 170 mM, about 75 mM to about 160 mM, about 75 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, about 125 mM to about 200 mM, about 125 mM to about 190 mM, about 125 mM to about 180 mM, about 125 mM to about 170 mM,Eluted with a buffer containing from about 125 mM to about 160 mM, from about 125 mM to about 150 mM, from about 125 mM to about 140 mM, from about 125 mM to about 130 mM, from about 130 mM to about 200 mM, from about 130 mM to about 190 mM, from about 130 mM to about 180 mM, from about 130 mM to about 170 mM, from about 130 mM to about 160 mM, from about 130 mM to about 150 mM, from about 140 mM to about 200 mM, from about 140 mM to about 190 mM, from about 140 mM to about 180 mM, from about 140 mM to about 170 mM, from about 140 mM to about 160 mM, from about 140 mM to about 150 mM, from about 150 mM to about 200 mM, from about 150 mM to about 190 mM, from about 150 mM to about 180 mM, from about 150 mM to about 170 mM, from about 150 mM to about 160 mM, from about 175 mM to about 200 mM, from about 175 mM to about 190 mM, from about 175 mM to about 180 mM, or from about 180 mM to about 200 mM).

[0096] In some embodiments, the protein A column is eluted with sodium acetate at a pH of about 2 to about 4 (e.g., about 2 to about 3.8, about 2 to about 3.75, about 2 to about 3.6, about 2 to about 3.8, about 2 to about 3.6, about 2 to about 3.4, about 2 to about 3.2, about 2 to about 3.0, about 2 to about 2.8, about 2 to about 2.6, about 2 to about 2.4, about 2 to about 2.2, about 3 to about 4, about 3 to about 3.8, about 3 to about 3.75, about 3 to about 3.6, about 3 to about 3.4, or about 3 to about 3.2) and a concentration of 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, about 20 mM to about 100 mM, about 20 mM to about 90 mM, about 20 mM to about 80 mM, about 20 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 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, the sequential chromatography steps of generally loading, washing, eluting, and regenerating the chromatography column. Any of the exemplary flow rates, buffer volumes, and / or lengths of time assigned to each of the sequential chromatography steps described herein can be used in any of these different sequential chromatography steps.

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

[0099] After loading the analyte onto the column, the column is washed with at least one wash buffer. At least one (e.g., 2, 3, or 4) wash buffer means eluting all components that are not the analyte from the column but 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., 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, about 1.0 mL / min to about 15.0 mL / min).

[0101] The volume of the washing buffer used (e.g., when two or more washing buffers are used, the total volume of the washing buffers used) is, for example, about 1× column volume (CV) to about 15× CV (e.g., about 1× CV to about 14× CV, about 1× CV to about 13× CV, about 1× CV to about 12× CV, about 1× CV to about 11× CV, about 2× CV to about 11× CV, about 3× CV to about 11× CV, about 4× CV to about 11× CV, about 5× CV to about 11× CV, or about 5× CV to about 10× CV). The total washing time is, for example, about 2 minutes to about 3 hours (e.g., 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 the elution buffer through the column. The elution buffer can be passed through the column at a flow rate of about 0.2 mL / min to about 25 mL / min (e.g., about 0.1 mL / min to about 20 mL / min, about 0.5 mL / min to about 20 mL / min, about 0.2 mL / min to about 15 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.5 mL / min to about 10 mL / min, about 0.5 mL / min to about 6.0 mL / min, about 1.0 mL / min to about 5.0 mL / min, about 0.5 mL / min to about 14 mL / min, about 1.0 mL / min to about 25.0 mL / min, about 1.0 mL / min to about 15.0 mL / min). For the analyte to be The volume of the elution buffer used to elute the analyte from the column is, for example, about 1× column volume (CV) to about 15× CV (e.g., about 1× CV to about 14× CV, about 1× CV to about 13× CV, about 1× CV to about 12× CV, about 1× CV to about 11× CV, about 2× CV to about 11× CV, about 3× CV to about 11× CV, about 4× CV to about 11× CV, about 5× CV to about 11× CV, or about 5× CV to about 10× CV). The total elution time is, for example, about 0.1 minute 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 minute to about 10 minutes).

[0103] Non-limiting examples of elution buffers that can be used depend on the capture mechanism and / or the analyte. For example, the elution buffer can contain different concentrations of salts (e.g., increasing salt concentration), different pHs (e.g., increasing or decreasing salt concentration), or molecules that compete 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, from about 0.1 mL / min to about 25 mL / min (e.g., from about 0.2 mL / min to about 20 mL / min, from about 0.5 mL / min to about 20 mL / min, from about 0.2 mL / min to about 15 mL / min, from about 0.5 mL / min to about 15 mL / min, from about 0.5 mL / min to about 10 mL / min, from about 0.5 mL / min to about 6.0 mL / min, from about 1.0 mL / min to about 5.0 mL / min, from about 0.5 mL / min to about 14 mL / min, from about 1.0 mL / min to about 25.0 mL / min, from about 5.0 mL / min to about 15.0 mL / min or from 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× column volume (CV) to about 15× CV (e.g., from about 1× CV to about 14× CV, from about 1× CV to about 13× CV, from about 1× CV to about 12× CV, from about 1× CV to about 11× CV, from about 2× CV to about 11× CV, from about 3× CV to about 11× CV, from about 2× CV to about 5× CV, from about 4× CV to about 11× CV, from about 5× CV to about 11× CV, or from about 5× CV to about 10× 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 a plurality of affinity chromatography columns. When multiple columns are used, the columns can be different (e.g., include different chromatography resins), or can be the same. Further, the multiple columns can be loaded and / or eluted with the same solvent and buffer, or with different solvents and / or buffers. Each of the columns in the 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 as described above, or via the sample purification device 108. The second flow control device 110 receives the biological sample and directs it along one of a plurality of flow paths in accordance with a control signal from the control unit 122 transmitted on the control line 124d. The second flow control device 110 can generally be implemented in the same manner as the first flow control device 106 described above.

[0108] Generally, the second flow control device 110 directs the biological sample, as shown in FIG. 1, to one of a plurality of sample analyzers. Generally, the system 100 includes two or more The above sample analyzer (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) can be included. Each of the sample analyzers is related to the measurement of one generated article quality characteristic for the analyte of the biological sample. In FIG. 1, four different sample analyzers 114a-114d are shown as an example. However, it should be understood that the system 100 can include any number of sample analyzers depending on the number of generated article 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 from the second flow control device 110 to the sample analyzer 114a. More specifically, when the generated article quality characteristic related to the sample analyzer 114a is measured, the control unit 122 transmits a control signal to the second flow control device 110, adjusts the arrangement of the second flow control device 110, and delivers the biological sample to the sample analyzer 114a. The generated article quality characteristic related to the sample analyzer 114a is measured for the analyte in the biological sample.

[0110] In a particular embodiment, a particular sample analyzer includes a chromatography column. For example, in FIG. 1, the sample analyzers 114b-114d each include a chromatography column. To deliver the 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, system 100 optionally includes a column manager 112 that receives a biological sample from a second flow control device 110 and directs it towards a sample analyzer. The column manager can be particularly useful in systems that include a plurality of sample analyzers equipped with chromatography columns. As shown in FIG. 1, the column manager 112 can be coupled to the control unit 122 via a control line 124f. To direct the biological sample towards a sample analyzer associated with a particular generated article quality characteristic to be measured, the control unit 122 can send a control signal to the column manager 112 to adjust the placement of the column manager 112 to direct the biological sample into the flow path of the sample analyzer.

[0112] The column manager 112 can optionally be in fluid communication with one or more reservoirs (shown as four reservoirs 120a - 120d in FIG. 1 for illustrative purposes) via a second pump 118, which can also optionally be coupled to the control unit 122 via a control line 124e. When a biological sample is directed towards a particular sample analyzer, the control unit 122 can send a control signal to the second pump 118 to direct a flow of a suitable loading buffer, elution buffer, or other suitable solvent or solution towards the particular sample analyzer. Although four reservoirs are shown by way of example in FIG. 1, it should be understood that generally 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, MA) with additional solvent selection valve modifications.

[0114] After a biological sample is loaded onto the column of a selected chromatographic sample analyzer, the column is developed and eluted, and the eluate is analyzed to provide information about the product quality characteristics associated with the sample analyzer for the analyte of the biological sample.

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

[0116] System 100 generally uses 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, Waters Photodiode Array Detector (Waters Corp., Milford, MA).

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

[0118] In some embodiments, the sample analyzer based on each column can be in fluid communication with different specialized detectors. In certain embodiments, as shown in FIG. 1, a sample analyzer based on two or more columns can be in fluid communication with a common detector. That is, detector 116 can be shared among two or more sample analyzers since system 100 analyzes only a portion of the biological sample at a time.

[0119] In some embodiments, detector 116 functions efficiently as a sample analyzer. For example, in FIG. 1, sample analyzer 114a can include a fluid conduit extending between second flow control device 110 and detector 116. When a sample is delivered to sample analyzer 114a, the sample is simply propagated through the fluid conduit and then directly analyzed by detector 116.

[0120] Generally within system 100, each sample analyzer is specialized for measuring a particular product quality characteristic of an analyte in a biological sample. The particular product quality characteristic to be determined is selected by control unit 122, which adjusts the placement of 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 an analyte in a biological sample.

[0122] Product quality characteristics As discussed above, System 100 can be used to measure multiple product quality characteristics for an analyte in a biological sample. The various product quality characteristics can be measured depending on the nature of the sample analyzers present in System 100.

[0123] (a) Concentration or titer In some embodiments, System 100 includes a sample analyzer that measures the concentration or titer of an analyte in a biological sample. The concentration or titer of the analyte can be measured directly in the biological sample 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 variant or heterogeneity In some embodiments, System 100 includes a sample analyzer that measures the charge variant or heterogeneity for an analyte in a biological sample. The charge variant / heterogeneity can be determined, for example, by a sample analyzer that includes a cation exchange column for performing cation exchange chromatography on the analyte 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] The chromatography cycle using a cation exchange chromatography column (e.g., strong cation exchange chromatography) can include loading a fluid containing the analyte onto the column when the analyte binds to the chromatography resin during the loading step, washing the column to remove unwanted biological material, eluting the analyte bound to the column, and re-equilibrating the column. In certain embodiments, the chromatography cycle using a cation exchange chromatography column can include loading a fluid containing the target protein onto the column when unwanted biological material binds to the chromatography resin during the loading step but the analyte does not, collecting the target recombinant protein in the flow-through, and re-equilibrating the column.

[0127] Any one of the single steps in the chromatography cycle can include a single buffer or multiple buffers (e.g., two or more buffers), and one or more of any one of the single steps in the chromatography cycle can include a buffer gradient. Any combination of various well-known aspects of a single cycle of chromatography can be used in these methods, for example, different chromatography resins, flow rates, buffers, column void volume, column bed volume, volume of buffer used in each step, volume of fluid 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 eluted with sodium acetate at a pH of about 2 to about 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about 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 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) and a concentration of 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, about 20 mM to about 100 mM, about 20 mM to about 90 mM, about 20 mM to about 80 mM, about 20 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 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 in about 10 mM to about 100 mM of 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, 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, about 20 mM to about 100 mM, about 20 mM to about 90 mM at a pH of about 7 to about 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 9 to about pH 9.8, about pH 9.5 to about pH 10, or about pH 9.5 to about pH 9.8). , from about 20 mM to about 80 mM, from about 20 mM to about 70 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from about 20 mM to about 40 mM, from about 20 mM to about 30 mM, from about 30 mM to about 100 mM, from about 30 mM to about 90 mM, from about 30 mM to about 80 mM, from about 30 mM to about 70 mM, from about 30 mM to about 60 mM, from about 30 mM to about 50 mM, from about 30 mM to about 40 mM, from about 40 mM to about 100 mM, from about 40 mM to about 90 mM, from about 40 mM to about 80 mM, from about 40 mM to about 70 mM, from about 40 mM to about 60 mM, from about 40 mM to about 50 mM, from about 50 mM to about 100 mM, from about 50 mM to about 90 mM, from about 50 mM to about 80 mM, from about 50 mM to about 70 mM, from about 50 mM to about 60 mM, from about 60 mM to about 100 mM, from about 60 mM to about 90 mM, from about 60 mM to about 80 mM, from about 60 mM to about 70 mM, from about 70 mM to about 100 mM, from about 70 mM to about 90 mM, from about 70 mM to about 80 mM, from about 80 mM to about 100 mM, from about 80 mM to about 90 mM, or from about 90 mM to about 100 mM), and from about 10 mM to about 100 mM (e.g., from about 10 mM to about 90 mM, from about 10 mM to about 80 mM, from about 10 mM to about 70 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 30 mM, from about 10 mM to about 20 mM, from about 15 mM to about 100 mM, from about 15 mM to about 90 mM, from about 15 mM to about 80 mM, from about 15 mM to about 70 mM, from about 15 mM to about 60 mM, from about 15 mM to about 50 mM, from about 15 mM to about 40 mM, from about 15 mM to about 30 mM, from about 15 mM to about 20 mM, from about 20 mM to about 100 mM, from about 20 mM to about 90 mM, from about 20 mM to about 80 mM, from about 20 mM to about 70 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from about 20 mM to about 40 mM, from about 20 mM to about 30 mM, from about 20 mM to about 25 mM, from about 25 mM to about 100 mM, from about 25 mM to about 90 mM, from about 25 mM to about 80 mM, from about 25 mM to about 70 mM, from about 25 mM to about 60 mM, from about 25 mM to about 50 mM, from about 25 mM to about 40 mM, from about 25 mM to about 30 mM, from about 30 mM to about 100 mM, from about 30 mM to about 90 mM, from about 30 mM to about 80 mM, from about 30 mM to about 70 mM, from about 30 mM to about 60 mM, from about 30 mM to about 50 mM, from about 30 mM to about 40 mM, from about 40 mM to about 100 mM, from about 40 mM to about 90 mM, from about 40 mM to about 80 mM, from about 40 mM to about 70 mM, from about 40 mM to about 60 mM,Eluted with NaCl of 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).

[0131] In some embodiments, the cation exchange column 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), and about 20 mM to about 50 mM of NaCl (e.g., 20 mM, about 25 mM, about 30 mM, about 40 mM, or about 50 mM) at about pH 8 to about pH 10 (e.g., about pH 8 to about 9.8, about 8 to about 9.6, about pH 8 to about pH 9.4, about pH 8 to about pH 9.2, about pH 8 to about pH 9, about pH 8 to about pH 8.8, about pH 8 to about pH 8.6, about pH 8 to about pH 8.4, about pH 8 to about pH 8.2, about pH 9 to about pH 10, about pH 9 to about pH 9.8, about pH 9 to about pH 9.6, about pH 9 to about pH 9.4, about pH 9 to about pH 9.2, or about pH 9.4 to about pH 10).

[0132] (c) Aggregation In some embodiments, system 100 includes a sample analyzer that measures the aggregation of an analyte 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 analyte 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, the size exclusion chromato Using a size exclusion column, protein aggregates, such as protein multimers (e.g., dimers and trimers), are separated. Non-limiting examples of size exclusion chromatography columns include Sephadex G-10, Sephadex G-25, Sephadex G-50, Sephadex G-75, Sephadex G-100, Sephadex G-150, Sephadex G-200, Sepharose 2B, Sepharose 4B, Sepharose 6B, Bio-gel P-300, and Waters BEH SEC 200A.

[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 about pH 7.2.

[0135] (d) Integrity or purity In some embodiments, system 100 includes a sample analyzer that measures the integrity or purity of an analyte in a biological sample. Integrity or purity can be determined, for example, by a sample analyzer that includes a reverse phase column for performing reverse phase chromatography of the analyte 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 and include, for example, Sepax Opalshell-C18. Non-limiting examples of hydrophobic ligands include aliphatic compounds such as C2, C4, C8, C10, C12, C16, and C18, and polyphenyls.

[0137] The chromatography cycle using a reverse-phase chromatography column can include a step of loading a fluid containing the analyte onto the column, a step of washing the column to remove unwanted biological materials, a step of eluting the analyte bound to the column, and a step of re-equilibrating the column.

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

[0139] In some embodiments, the reverse-phase column is from 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 0.0 It is eluted with trifluoroacetic acid (TFA) in water at 6% to approximately 0.2%, approximately 0.06% to approximately 0.1%, approximately 0.08% to approximately 1%, approximately 0.08% to approximately 0.8%, approximately 0.08% to approximately 0.6%, approximately 0.08% to approximately 0.5%, approximately 0.08% to approximately 0.4%, approximately 0.08% to approximately 0.2%, approximately 0.08% to approximately 1%, approximately 0.1% to approximately 1%, approximately 0.1% to approximately 0.8%, approximately 0.1% to approximately 0.6%, approximately 0.1% to approximately 0.5%, approximately 0.1% to approximately 0.4%, approximately 0.1% to approximately 0.2%, approximately 0.2% to approximately 1%, approximately 0.2% to approximately 0.8%, approximately 0.2% to approximately 0.6%, approximately 0.2% to approximately 0.5%, approximately 0.2% to approximately 0.4%, approximately 0.4% to approximately 1%, approximately 0.4% to approximately 0.8%, approximately 0.4% to approximately 0.6%, approximately 0.4% to approximately 0.5%, approximately 0.5% to approximately 1%, approximately 0.5% to approximately 0.8%, approximately 0.5% to approximately 0.6%, approximately 0.6% to approximately 1%, approximately 0.6% to approximately 0.8%, or approximately 0.8% to approximately 1%).

[0140] In some embodiments, the reverse-phase column is eluted with trifluoroacetic acid (TFA) in isopropanol (IPA): acetonitrile (ACN) (e.g., about 1:50, 1:90, 1:100, 1:120, 10:50, 10:90, or 10:120) of about 1:10 to about 10:120 at about 0.01% to about 1% (e.g., about 0.01% to about 0.8%, about 0.01% to about 0.6%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.02% to about 1%, about 0.02% to about 0.8%, about 0.02% to about 0.6%, about 0.02% to about 0.5%, about 0.02% to about 0.4%, about 0.02% to about 0.2%, about 0.02% to about 0.1%, about 0.05% to about 1%, about 0.05% to about 0.8%, about 0.05% to about 0.6%, about 0.05% to about 0.5%, about 0.05% to about 0.4%, about 0.05% to about 0.2%, about 0.05% to about 0.1%, about 0.06% to about 1%, about 0.06% to about 0.8%, about 0.06% to about 0.6%, about 0.06% to about 0.5%, about 0.06% to about 0.4%, about 0.06% to about 0.2%, about 0.06% to about 0.1%, about 0.08% to about 1%, about 0.08% to about 0.8%, about 0.08% to about 0.6%, about 0.08% to about 0.5%, about 0.08% to about 0.4%, about 0.08% to about 0.2%, about 0.08% to about 1%, about 0.1% to about 1%, about 0.1% to about 0.8%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, about 0.1% to about 0.2%, about 0.2% to about 1%, about 0.2% to about 0.8%, about 0.2% to about 0.6%, about 0.2% to about 0.5%, about 0.2% to about 0.4%, about 0.4% to about 1%, about 0.4% to about 0.8%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% to about 1%, about 0.5% to about 0.8%, about 0.5% to about 0.6%, about 0.6% to about 1%, about 0.6% to about 0.8%, or about 0.8% to about 1%).

[0141] In some embodiments, the reverse-phase column is eluted with trifluoroacetic acid (TFA) in isopropanol (IPA): acetonitrile (ACN) of about 10:90 to about 10:80 at 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%).

[0142] In some embodiments, the reverse-phase column is at about pH 2 to about pH 4 (e.g., about pH 2 to about pH 3.8, about pH 2 to about 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 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) and 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 1 It is eluted with sodium acetate (5 mM to about 40 mM, about 15 mM to about 30 mM, about 15 mM to about 20 mM, about 20 mM to about 100 mM, about 20 mM to about 90 mM, about 20 mM to about 80 mM, about 20 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 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, system 100 includes a sample analyzer that measures the reduction of an antibody analyte in a biological sample. In a production platform for biological manufacturing, the reduction degradation of an antibody product is a concern, and the systems described herein can be used to measure antibody reduction for feedback regulation of manufacturing process parameters. Antibody reduction can be determined, for example, by a sample analyzer that includes a reverse-phase column for performing reverse-phase chromatography of the analyte in the sample. Any of the reverse-phase column resins, buffers, pH values, and other operating conditions discussed herein can be used in connection with reverse-phase chromatography in the sample analyzer.

[0144] In some embodiments, a hydrophilic interaction chromatography column can be used to measure the product quality characteristics for a sample analyte. 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, an enzyme chromatography column can be used to perform peptide mapping for the purpose of measuring product quality characteristics. Reverse-phase chromatography separation can be used to separate peptide analytes for analysis.

[0146] The analysis of product quality characteristics using system 100 can generally be performed in substantially real-time to provide timely control feedback for the adjustment of a wide variety of biological manufacturing process conditions and parameters. In some embodiments, for example, the concentration or titer characteristics 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 the initial introduction of a portion of the sample in a suitable sample analyzer to the determination of the value of the characteristic.

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

[0148] In some embodiments, the aggregation characteristics for an analyte of a biological sample are suitable for the sample From the initial introduction of a portion of the sample in the analyzer to the determination of the value of the characteristic, it 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).

[0149] In certain embodiments, the integrity or purity characteristic of an analyte in a biological sample can be determined in 30 minutes or less (e.g., 28 minutes or less, 26 minutes or less, 24 minutes or less, 22 minutes or less, 20 minutes or less, 18 minutes or less, 16 minutes or less, 14 minutes or less, 12 minutes or less, 10 minutes or less) from the initial introduction of a portion of the sample in a suitable sample analyzer to the determination of the value of the characteristic.

[0150] For each of the four aforementioned product quality characteristics whose values are determined for the analyte, the time interval elapsed 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 for the analysis cycle determined for the analyte 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] Figure 3 is a flowchart 300 including an example of a series of steps for determining the product quality characteristics of an analyte in a biological sample. In the first step 302, the sample is received by the 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 sample collection 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. Regardless of whether it is purified, a portion of the biological sample is delivered to the second flow control device 110, from where it is then delivered in step 308 to a sample analyzer related to the desired product quality characteristic.

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

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

[0155] The method by which the biological sample is delivered from the first flow control device 106 to the second flow control device 110, either directly 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. FIGS. 4A - 4C show examples of the delivery of the biological sample between the first and second flow control devices when the first and second flow control devices are implemented as multi - way valves.

[0156] Figure 4A is a schematic diagram of a first flow control device 106 having its configuration adjusted to be in a first position where ports 1 and 2 are connected, ports 3 and 4 are connected, and ports 5 and 6 are connected. The biological sample is injected into port 2 from the sample manager 102. Port 2 is connected to port 1 and then to a second flow control device 110 and a 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] Figures 4B and 4C are schematic diagrams showing the first flow control device 106 and the second flow control device 110 configured to deliver a biological sample to the sample purification device 108. In Figure 4B, the arrangement of the first flow control device is such that the device is in a second position where ports 2 and 3 are connected, ports 4 and 5 are connected, and ports 1 and 6 are connected. The second flow control device is configured in a first position where ports 1 and 2 are connected, ports 3 and 4 are connected, and ports 5 and 6 are connected. In addition, the sample purification device 108 is connected 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 Figure 4B, the biological sample is loaded onto the column of the sample purification device 108, and the loading buffer is introduced through port 1 of the first flow control device.

[0158] When a biological sample is loaded onto the column of the sample purification device, the arrangement of the first and second flow control devices is adjusted as shown in Figure 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 port 2 and port 3 are connected, port 4 and port 5 are connected, and port 1 and port 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 analyte from the column of the sample purification device, the elution buffer is delivered to port 5 of the second flow control device, and the eluted analyte is delivered to the second flow control device and then to one of the sample analyzers in fluid communication with the second flow control device.

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

[0160] In addition, the measurement of product quality characteristics can be used for the validation of spectroscopic models and the validation of ongoing models. The product quality characteristics measured regularly can be used to maintain, for example, a spectroscopic model by verifying the prediction accuracy of chemometric methods using in-line FTIR and / or Raman measurements.

[0161] Using the automated nature of the systems described herein, tedious repetitive analysis steps that would otherwise be performed manually can be eliminated, saving time and reducing the possibility that operator error will affect the values of the characteristics being measured. Similarly, integrated sample purification can be performed in-line and can significantly reduce the time required to obtain a more pure biological sample.

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

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

[0164] Integration and regulation of biological manufacturing systems The systems disclosed herein can be integrated with a biological manufacturing system to provide feedback control to various components and processes in the synthesis and purification processes for various 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 substantially cell-free recombinant therapeutic protein, and then supplying the liquid culture medium to a first multi-column chromatography system (MCCS1). The next steps can include capturing the recombinant therapeutic protein in the liquid culture medium using MCCS1, and then continuously supplying the eluate of MCCS1 containing the recombinant therapeutic protein to a second multi-column chromatography system (MCCS2), and using MCCS2 to purify and polish the protein. The eluate obtained from MCCS2 is considered to be the therapeutic protein drug substance. The process can be integrated and operate continuously from the liquid culture medium to the eluate from MCCS2, which is the therapeutic protein drug substance.

[0166] Biological manufacturing systems are typically used to perform the processes described above. For example, such a system can include MCCS1 that includes an inlet and MCCS2 that includes an outlet. In these systems, the first and second MCCS are in fluid communication with each other. The system is also configured such that fluid enters through the inlet and passes through the first and second MCCS and exits the manufacturing system through the outlet.

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

[0168] Figure 8 is a schematic diagram showing an example of a biological production system. System 1 includes a first MCCS, namely, a 4-column periodic countercurrent chromatography system (PCCS) 2, where 3 of the 4 columns 3, 4, and 5 in the 4-column PCCS 2 perform a unit operation of capturing a recombinant therapeutic protein from a fluid containing the recombinant therapeutic protein (e.g., a liquid culture medium substantially free of mammalian cells), and one of the columns 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 utilizing 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. The inlet 7 is, for example, an opening that allows the inflow of fluid into the PCCS 1.

[0169] System 1 also includes a second MCCS, which is a PCCS 8 including three chromatography columns 9, 10, and 11, and one chromatography membrane 12. In the PCCS 8 the columns 9, 10, and 11 can contain a cation exchange resin. The chromatography membrane 12 in the PCCS 8 can contain a cation exchange resin. The PCCS 8 also has a fluid conduit 13 disposed between the columns 9, 10, and 11 in the PCCS 8 and the chromatography membrane 12 in the PCCS 8. The PCCS 8 also has an in-line buffer adjustment reservoir 14 in fluid communication with the fluid conduit 13, and is configured such that the buffer contained in the in-line buffer adjustment reservoir 14 is introduced into the fluid present in the fluid conduit 13. The PCCS 8 also includes an outlet 15. The outlet 15 is, for example, an opening that allows fluid to exit from the PCCS 8.

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

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

[0172] System 1 can also include a bioreactor 25, as well as a fluid conduit 26 disposed between the bioreactor 25 and the pump 21. A filtration system 27 can be disposed within the fluid conduit 26 for filtering (e.g., removing cells from) the liquid culture medium present in the fluid conduit 26.

[0173] The first MCCS (PCCS2) includes an inlet through which a fluid (e.g., a liquid culture medium substantially free of cells) can pass. The inlet can be of any structure known in the art for such purposes. It can include, for example, threading, rib attachment, or sealing to enable insertion of a fluid conduit such that, after insertion into the inlet of the fluid conduit, the fluid enters the first MCCS through the inlet without significant leakage of fluid from the inlet.

[0174] The first MCCS includes at least two chromatography columns, at least two chromatography membranes, or at least one chromatography column and at least one chromatography membrane, and an inlet. For example, the first MCCS can include, in total, four chromatography columns, or three chromatography columns and one chromatography 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 chromatography membranes present in the first MCCS can contain one or more various resins. For example, one The resin(s) contained therein may be a resin that utilizes a capture mechanism (e.g., Protein A binding capture mechanism, Protein G binding capture mechanism, antibody or antibody fragment binding capture mechanism, substrate binding capture mechanism, cofactor binding capture mechanism, aptamer binding capture mechanism, and / or tag binding capture mechanism). The resin(s) contained in one or more of the chromatography columns and / or chromatography membranes of the first MCCS can be a cation exchange resin, an anion exchange resin, a molecular sieve resin, a hydrophobic interaction resin, 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 chromatography membranes present in the first MCCS. The chromatography columns and / or chromatography membranes present in the first MCCS can contain the same and / or different resins (e.g., any of the resins described herein or resins known in the art for use in recombinant protein purification).

[0176] 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 a recombinant therapeutic protein, or filtering a fluid containing a recombinant therapeutic protein). In a non-limiting example, the first MCCS can perform unit operations of capturing a recombinant therapeutic protein from a fluid (e.g., a liquid culture medium) and inactivating a virus present in the 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 chromatography columns and / or chromatography 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., an automatic pump, e.g., an automatic peristaltic pump). A column switching event can be triggered by detection of the level of a recombinant therapeutic protein in a fluid passing through the first MCCS (e.g., an input to and / or an eluate from one or more of the chromatography columns and / or chromatography membranes in the first MCCS), a specific volume of a liquid (e.g., a buffer), or a specific elapsed time. Column switching generally refers to a mechanism that enables at least two different chromatography columns and / or chromatography membranes in the MCCS (e.g., two or more different chromatography columns and / or chromatography membranes present in the MCCS (e.g., the first or second MCCS)) to pass through different steps (e.g., equilibrating, loading, eluting, or washing) substantially simultaneously during at least a portion of the process.

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

[0179] The column switching event can be triggered by detection of the concentration of a specific protein or other substance in the 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 (e.g., between MCCs1 and MCCs2) in the MCCs. Using the measurement system disclosed herein, the concentration of such a protein can be measured and the concentration information can be transmitted 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 column switching should occur (e.g., based on concentration information derived from infrared spectroscopic measurements, volume of liquid, or elapsed time) and to affect (initiate) the column switching event. The measurement system can optionally be installed at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) inlets of a chromatography column and / or chromatography membrane in the first MCCs and / or at one or more outlets of a chromatography column and / or chromatography membrane 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 adjustment 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 chromatography columns and / or chromatography membranes in the first MCCS. The systems described herein can contain one or more break tanks (e.g., the 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 MCCS or the second MCCS, or do not include a break tank throughout the system. Other examples of the system include a maximum of 1, 2, 3, 4, or 5 break tanks throughout the 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 column 6 described above). The virus inactivation device 6 is configured to inactivate viruses and virus vectors used in biological manufacturing processes. In some embodiments, for example, the virus inactivation device 6 includes a mixer. Alternatively, in certain embodiments, for example, device 6 includes a plug flow inactivation system. Each of these examples of virus inactivation devices helps to eliminate active viruses and virus vectors from the process fluid in the first MCCS.

[0183] The second MCCS includes at least two chromatography columns, at least two cl A chromatography membrane, or at least one chromatography column and at least one chromatography membrane, and an outlet. For example, the second MCCS can include, in total, four chromatography columns, three chromatography columns and one chromatography 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. The chromatography columns and / or chromatography membranes present in the second MCCS can have one or more of any of the shape, size, volume (bed volume) and / or unit operations described herein. The resin contained in one or more of the chromatography columns and / or chromatography membranes present in the second MCCS can be a resin that utilizes a capture mechanism (e.g., a Protein A binding capture mechanism, a Protein G binding capture mechanism, an antibody or antibody fragment binding capture mechanism, a substrate binding capture mechanism, a cofactor binding capture mechanism, a tag binding capture mechanism and / or an 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 chromatography membranes present in the second MCCS can contain the same and / or different resins (e.g., any of the resins described herein or resins known in the art for use in recombinant protein purification).

[0184] The chromatography column and / or chromatography membrane 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 non-limiting examples, the second MCCS can perform unit operations of purifying a recombinant therapeutic protein from a fluid and polishing the recombinant therapeutic protein present in the fluid containing the recombinant therapeutic protein. In other non-limiting examples, the second MCCS can perform unit operations of purifying the recombinant therapeutic protein present in the fluid, polishing the recombinant therapeutic protein present in the fluid, and filtering the fluid containing the recombinant therapeutic protein. In another example, the second MCCS can perform unit operations of purifying the recombinant therapeutic protein present in the fluid, polishing the recombinant therapeutic protein present in the fluid, filtering the fluid containing the recombinant therapeutic protein, and adjusting the ionic concentration and / or pH of the fluid containing the 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 (e.g., 2, 3, 4, or 5) pumps (e.g., an automatic pump, e.g., an automatic peristaltic pump).

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

[0187] The PCCS8 forming the second MCCS can contain three columns that perform unit operations for purifying a recombinant therapeutic protein from a fluid, and a chromatography membrane that performs a unit operation for polishing the recombinant therapeutic protein present in the fluid. For example, the three columns that perform unit operations for purifying a recombinant therapeutic protein from a fluid can contain, for example, a cation exchange resin, and the chromatography membrane that performs a unit operation for polishing can contain a cation exchange resin. The PCCS, which is the second MCCS, 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 can 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. One or more of the 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 includes a controller that executes an operating system that uses 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 the column switching event. In an example 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 a chromatography column and / or chromatography membrane in the second MCCS, and / or at one or more outlets of a chromatography column and / or chromatography membrane 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 adjustment 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 easily pass through one or more chromatography columns and / or chromatography 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 threaded, ribbed, or sealed fitting that allows insertion of a fluid conduit, or a vial designed to contain or store the therapeutic protein drug substance. The outlet can contain a surface that can be used to seal a sterile vial or other such storage container in order to allow the recombinant protein formulation to flow directly into the sterile vial or storage container.

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

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

[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 present system.

[0194] Some examples of the systems provided herein include pump systems. A 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 systems provided herein further include a fluid conduit (e.g., any of the exemplary fluid conduits described herein or known in the art) disposed between a pump and an inlet of a first MCCS. In some examples, this particular fluid conduit can include one or more (e.g., 2, 3, or 4) pumps (e.g., any of the pumps described herein or known in the art) and / or one or more (e.g., 2, 3, or 4) break tanks (e.g., any of the exemplary break tanks described herein), and these 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 a bioreactor and the other end is fluidly connected to the fluid conduit between the pump and the inlet. This 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 foregoing biological manufacturing system enables the continuous production of therapeutic protein drug substances. For example, the systems provided herein enable a percentage yield of recombinant therapeutic protein that is 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% (from starting material, e.g., starting liquid culture medium). The systems described herein can also result in a percentage yield of recombinant therapeutic protein that is from about 80% to about 90%, from about 82% to about 90%, from about 84% to about 90%, from about 84% to about 88%, from about 84% to about 94%, from about 82% to about 92%, or from about 85% to about 95% (from starting material, e.g., starting liquid culture medium). The systems described herein can also result in the production of a therapeutic protein drug substance containing a concentration of recombinant therapeutic protein that is 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.

[0197] The systems described herein can also result in the production of a therapeutic protein drug substance containing a concentration of recombinant therapeutic protein that is 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). The PCCS can include, for example, two or more chromatography columns (e.g., three columns or four columns) that are switched to enable continuous elution of recombinant therapeutic proteins from two or more chromatography columns. The 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 load, wash, elution, and regeneration steps. In a PCCS, multiple columns are used to operate the same steps individually and continuously in a cyclic manner. Since the columns are operated in series, the flow-through and wash solution from one column are captured by another column. This unique configuration of the PCCS allows for loading of resin close to its static binding capacity instead of its dynamic binding capacity, as is typical during batch-mode chromatography.

[0199] An example of three column switching techniques used in a PCCS containing three columns is shown in Figure 9. A cycle is defined as three complete column operations that result in eluate pools from each of the three columns used in the column switching technique. When all steps of the cycle are complete, the cycle is restarted. As a result of continuous cycling and elution, the fluid entering the PCCS is continuously processed, but the elution of recombinant therapeutic proteins from each column is individual and periodic.

[0200] In a PCCS cycle, such as the exemplary cycle shown in FIG. 9, a column switching strategy is used to proceed from one step to another. The column switching method uses two automatic switching operations for each of the three columns in the exemplary PCCS system shown in FIG. 9. The first is related to the appearance of the first product, and the second coincides with column saturation. The decision of when to perform the column switching operation is based on information about the concentration of the recombinant therapeutic protein in the eluate from each chromatographic 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 functions as feedback control for the biological manufacturing system and is sent to the MCCs controller by the control unit 122. After determining that the switch is justified, the column switching is initiated.

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

[0203] As the supply to column 1 continues, column 1 eventually becomes almost saturated with the protein product. At this point, the measured concentration of the protein in the eluate has reached another predetermined value that occurs at time t2. At this point, the MCCs controller switches the inlet supply to column 2.

[0204] The column switching strategy described above enables uniform loading of the columns, regardless of the concentration and volume of the feed product. Similar switching of columns can be implemented based on the level of recombinant protein detected in the eluate from each column. Column switching can also be based on elapsed time, or the amount of fluid (e.g., buffer) that has passed through one or more chromatography columns and / or chromatography 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 processes and operating parameters. One example of such an adjustment is the controlled adjustment of buffer concentration at various stages of the biological manufacturing process.

[0206] Generally, 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 MCCS in any of the processes described herein. As is known in the art, one or more types of buffers used in two or more MCCS used in the processes described herein depend on the resin present in the chromatography columns and / or membranes of the two or more MCCS (e.g., the first and second MCCS), the recombinant therapeutic protein, and the unit operations (e.g., any of the exemplary unit operations described herein) performed by the specific chromatography columns and / or membranes of the two or more MCCS. The volume and type of buffer used during the use of two or more MCCS in any of the processes described herein can also be determined by one of ordinary skill in the art (e.g., as discussed in more detail below). For example, the volume and type of buffer used during the use of two or more MCCS in any of the processes described herein can be selected to optimize one or more of the following in a recombinant protein formulation: the overall yield of the recombinant therapeutic protein, the activity of the recombinant therapeutic protein, the level of purity of the recombinant therapeutic protein, and the removal of biological contaminants from the fluid containing the recombinant therapeutic protein (e.g., the absence of active virus, mycobacteria, yeast, bacteria, or mammalian cells).

[0207] The unit operation of adjusting the ionic concentration and / or pH of the fluid containing the recombinant therapeutic protein can be (e.g., between columns within a single MCCS, or after the last column in the penultimate MCCS (e.g., the first MCCS), and the recombinant therapeutic protein A buffer adjustment reservoir (e.g., an in-line buffer adjustment reservoir) that includes adding a new or additional buffer solution to a fluid containing a recombinant therapeutic protein before the fluid containing it is supplied to the first column of the following MCCS (e.g., the second MCCS), and that can be used with the MCCS (e.g., the first and / or second MCCS) using it. The in-line buffer adjustment reservoir can be of any size (e.g., greater than 100 mL) and can contain any buffering 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 the resin present in at least one chromatographic 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 added to the process fluid is based on concentration or titer information about an analyte in the biological sample. For example, the solute for the purpose of such measurement is a constituent of the buffer solution or a constituent of the 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. The measurement of the concentration information about the constituent is provided to the MCCS controller as feedback information, and the feedback information is used to determine the timing and amount of one or more buffer solutions to discharge into the process fluid. An infrared spectroscopy measurement system can generally be located at any location in the biological manufacturing system for the purpose of measuring the process fluid and providing the MCCS controller with feedback information related to the buffer solution.

[0209] In certain embodiments, antibody concentration information about 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 recovered from the bioreactor, the MCCs controller can adjust the outflow rate of the cell culture into the bioreactor. Adjustment in this manner enables control of the volumetric productivity resulting from the cell density and specific productivity of the bioreactor. For a fixed perfusion rate, such adjustment enables control of the antibody concentration in the process fluid such that the MCCs receives a substantially constant amount of product per unit time. In other words, adjustment of this nature can be used to ensure that the rate of product production within the bioreactor remains substantially constant over a particular period of time.

[0210] In some embodiments, determination of certain quality characteristics associated with the process fluid is 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 characteristics, the acceptable range of values can be established by a calibration procedure. These ranges efficiently establish operating conditions for the system such that the biological product is produced at acceptable rates and purity levels while the yields of by-products and other undesirable species are at acceptably low levels. Operating the system outside one or more of the ranges can reduce the yield and / or purity of the product, increase the rate / amount of generation of undesirable species, increase the reagent consumption rate, and / or result in other undesirable effects or conditions.

[0212] Using the quality characteristics determined for the process fluid at one or more locations within the system to ensure that the system operates within the acceptable ranges of these operating parameters It can be ensured. When the determined values of one or more quality characteristics are outside the established acceptable ranges, the MCCs controller identifies that a potential failure state exists.

[0213] To address the failure state, the MCCs controller (or another system controller connected to the MCCs controller) can adjust any of the operating parameters of the biological manufacturing system to modify its operation, and thereby also adjust the values of the quality characteristics so that they are within the acceptable ranges. This corrective action of this nature ensures that the system can be actively maintained within the established set or range of operating conditions based on the feedback provided by the determined values of the quality characteristics.

[0214] In certain embodiments, when the MCCs controller (or another system controller connected to the MCCs controller) determines that the system is outside the extreme range from its acceptable operating conditions, and as a result, it is difficult or even impossible to return the system to the conditions within the acceptable range or that it will result in other undesirable outcomes, the controller can send a control signal to the bioreactor to interrupt the production and discharge its contents as waste. In such cases, an effective corrective action is not practical or possible, and the production process has deviated extremely from the range of acceptable operating conditions for the system. By simply discharging the contents of the bioreactor, the system can save a significant amount of time by restarting the production process rather than attempting to adjust the ongoing production process that has deviated irreversibly from the range of acceptable conditions.

[0215] Furthermore, the feedback can be provided to the MCCs controller (or another system controller) based on measurements of the media components of one or more bioreactors (e.g., glucose concentration, glutamine concentration, lactate concentration, and ammonium ion concentration), and then used to adjust the reactor conditions to ensure that cell viability, product yield, and other performance metrics are maintained within the target ranges. Any one or more of the process parameters can be adjusted by the controller based on the values of the media components of the bioreactor in a similar manner as the adjustments made based on the values of the product quality characteristics 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 implemented in hardware, software, or a combination of both hardware and software. The control unit 122 typically includes at least one electronic processor, a storage device, an output device (e.g., a display), and a human interface device (e.g., a keyboard, mouse, touchpad, touch sensor display) connected to a memory unit. The control unit 122 receives information as electrical signals from the components of the system along some or all of the control lines shown herein and transmits electrical control signals to the components of the system along the control lines.

[0217] The steps of the methods and control functions described herein can be implemented in a computer program using standard or proprietary programming languages. Such programs are executed by the control unit 122 (e.g., the electronic processor of the control unit) and are designed to cause the control unit to perform the described steps and functions. Each program can be stored on a computer-readable storage medium (e.g., optical, magnetic, solid-state, rewritable, or other persistent storage medium). Examples of suitable programming languages that can be used to provide instructions to the control unit 122 are the Empower® software package software (Waters Corp., Milford, MA).

Example

[0218] The following examples are provided to further illustrate various aspects of the foregoing disclosure and are not intended to limit any configuration of the claims or any aspect of the embodiments other than as explicitly recited.

[0219] Example 1 The system 100 was used to analyze anti-TGFβ from a perfusion bioreactor operating using an enhanced perfusion process. The system included a Process Sample Manager (PSM), a Column Heater (CH), a Column Manager (CM) containing two 1-position 9-port valves, a Binary Solvent Manager (BSM), a Quaternary Solvent Manager (QSM) with a solvent selection valve added on the "D" line, a PDA detector (PDA), a 2-position 10-port column selection valve housing a 5 mL stainless steel sample hold loop (all obtained from Waters Corp., Milford, MA), and two IDEX auxiliary 2-position 6-port switching valves.

[0220] The recovered material from the bioreactor was automatically sampled using a MAST (Modular Automated Sample Technology) (obtained from Lonza, Basel, Switzerland) connected to a liquid handler (obtained from Gilson, Middleton, WI). The MAST system withdrew approximately 40 mL of the recovered material with a positive displacement pump into a clean line and then accumulated 10 mL of the recovered material into a clean 12 mL glass vial on the liquid handler deck. The liquid handler then transferred 7.5 mL of the recovered material to system 100 by reinjecting the material into a PEEK tube transfer line connected to both the liquid handler and aspiration. The sample was transferred directly to a 5 mL sample hold loop that held it until analysis of the sample.

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

[0222] The set of methods operating for this example included a loop load function, two injections in the Protein A (Pro A) column of the sample purification device with two mass loads, two injections in the size exclusion column with two mass loads, separation techniques for common system components (Pro A and size exclusion chromatography (SEC)) and an intermediate water wash method between the final loop wash with 20% methanol, as well as a final zone to ensure that the loop was in the correct position to accept more sample. Note whether a secondary column is mentioned (such as SEC), and this method should be assumed to contain in-line purification by Pro A unless otherwise specified.

[0223] For each injection, 1 mL of the recovered material was withdrawn from the hold loop and transferred to a 50 μL sample injection loop for a full loop injection in the system. To test varying mass loads, both full loop injections were performed using a buffer of 1× phosphate buffered saline (PBS), pH 7.2, similar to an on-line 1:2 dilution.

[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). Details of the gradients 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 at 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 2, Buffer A was 0.1 M phosphate-citrate, 0.14 M sodium chloride at pH 3.2.

[0228] The system was configured so that the purified sample went directly to detector 116 (PDA detector) without passing through any column-based sample analyzer. Separation was monitored at an absorbance of 280 nm. The Protein A column was kept at room temperature and ambient environment. At the time of detection in the PDA detector, the measured chromatograms were integrated and the area under the curve for the peak corresponding to the eluted protein at about 2.5 minutes was calculated. The area of the monoclonal antibody was then quantified by using a calibration curve constructed from a mass load of 50 μg to 2 μg in the column plotted against the area.

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

[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. The 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 and 0.14 M sodium chloride at pH 3.2 and buffer B was 1× Dulbecco's phosphate buffered saline at pH 7.2.

[0234] The column manager was configured so that the sample was directed towards a sample analyzer having a size exclusion chromatography column. Separation of the analyte and other components was monitored at an absorbance of 280 nm. The size exclusion column was maintained at 25 °C in a 30 cm column heater, while the Protein A column was at ambient conditions. Upon detection in the PDA detector 116, the recorded chromatograms were integrated to calculate the areas under the curves for the peaks corresponding to the high molecular weight species (before the main peak), the main species and the low molecular weight species (after the main peak).

[0235] Figure 6A is a graph showing the titer chromatograms for the neat sample and the 1:2 diluted sample, and Figure 6B shows a table with the measured peak information for six different samples at each of the two dilutions. The data show excellent reproducibility between runs, both visually and computationally. Some differences exist in the peak percentages between the mass loads, which are thought to be a result of the column itself.

[0236] Figure 7 is a graph showing the measured chromatograms from experiments where 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 1×PBS at pH 7.2. Both the titer and the aggregate product quality characteristics showed excellent reproducibility among the 12 samples.

[0237] For reverse phase analysis, the separation was performed on a Waters BioResolve Polyphenyl 2.1×100 mm, 2.7 μm, 450 Å column using 0.1% trifluoroacetic acid in water (mobile phase A), 10% isopropanol, 90% acetonitrile with 0.1% trifluoroacetic acid (mobile phase B), and 20 mM sodium acetate, pH 3.75 (Protein A column eluent). For strong cation exchange analysis, the separation was performed on a Thermo MAbPac SCX-10 RS, 2.1×150 mm, 5 μm column. The mobile phases were 20 mM sodium acetate, pH 3.75 (both mobile phase A and Protein A eluent) and 20 mM Tris acetate, 25 mM sodium chloride, pH 9.8 (mobile phase B). So far, this method uses the gradient curvature to create an "S" shaped gradient for optimal peak resolution.

[0238] An offline sample was analyzed using the foregoing method. As discussed above, System 100 can also analyze an offline sample, for example, using a flow-through needle module of a sample manager (available from Waters Corp., Milford, MA). Using a flow-through needle, the sample is injected directly from a vial or well plate. The injection volume can range from 1 μL to 100 μL and can be varied as desired. This flexibility allows for changing the mass load on the column without performing online dilution. Additionally, since all sample volumes are contained within the vial or well plate and multiple injections are possible for each vial, there is no sample hold-up loop in this version of the system.

[0239] Example 2. Application of MIMICS-mPQA to Process Development and At-line Process Monitoring of a Pilot Scale Bioreactor Overview of the Experiment The MIMICS-mPQA platform was applied to the at-scale 100 L operation of an anti-TGFβ monoclonal therapeutic antibody. The harvested samples were run on the MIMICS-mPQA platform to accumulate at-line generated product quality information for three characteristics: titer, aggregation, and purity / completeness.

[0240] The samples analyzed were harvested material (post-ATF) from a 100 L bioreactor and two satellite 3 L bioreactors. For the analysis, a Protein A (Pro A) affinity column was used for titer determination, a size exclusion (SEC) column and an in-line Protein A column were used for aggregation analysis, and a reverse phase (RP) column and an in-line Protein A column were used for purity / completeness information. The tests were conducted over a 4-week period where the samples were run in blocks of approximately 4 harvest days from all reactors within each queue. During the analysis time, the mobile phase of the system was replaced as needed when consumed.

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

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

[0243] The stock used for the standard curve was pre-diluted before operation and kept at -80 °C in sub-aliquots for use in the MIMICS-mP QA campaign.

[0244] Results The recorded data from the Pro A quantification was also compared to two off-line quantitative titrations: the titer by measurement in an Octet using a Pro A biosensor chip and the titer by CEDEX™ Bioanalyzer measurement. Figures 10 - 12 demonstrate the comparability of the MIMICS-mPQA titers compared to the off-line methods for 100 L and 3 L bioreactors.

[0245] Overall, the trends were consistent among the three methods. When comparing MIMICS-mPQA data to the CEDEX™ Bioanalyzer method, the % difference was less than 10% for all runs that were within the acceptable CV for the assay method. The CEDEX™ Bioanalyzer is a method typically used for titer measurements for daily upstream process monitoring. The comparability of MIMICS-mPQA to this technique provided an orthogonal tool for titer determination for mammalian cell culture. An additional advantage of MIMICS-mPQA was the reduction in manual sample handling and potential bias in sample storage if the assay was performed offline.

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

[0247] Overall, the trends were comparable between the offline assay and MIMICS-mPQA. Both methods showed less than 2.5% aggregation levels across the campaign. The MIMICS-mPQA system enabled accumulation of a greater number of data points for this campaign than is typically assayed offline. This increased sampling enabled deeper investigation of the variability of aggregation over the course of the campaign.

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

[0249] As shown in Figure 16, the MIMICS-mPQA system provided an overall lower absolute percentage of purity compared to the offline method. Despite the absolute difference in percentages, the overall trend was 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 enabled an increase in sample collection compared to offline analysis, which in turn enabled a higher process understanding over the course of the campaign. Additionally, the titer and aggregation methods were very comparable to offline analysis, which provided confidence in the in-plant analysis compared to that obtained in the analytical laboratory. The MIMICS-mPQA system also reduced sample consumption and enabled a faster throughput since this step is integrated into the overall MIMICS-mPQA workflow and there is no need for pre-protein A purification prior to analysis. Finally, the system was able to operate over a four-week period with minimal incidents, further strengthening the understanding of the robustness of the equipment and the stability of the columns.

Explanation of symbols

[0251] 1 System 2 Four-column periodic countercurrent chromatography system (PCCS) 3 Column 4 Column 5 Column 6 Column 7 Inlet 8 PCCS 9 Chromatography column 10 Chromatography column 11 Chromatography column 12 Chromatography membrane 13 Fluid conduit 14 In-line buffer conditioning reservoir 15 Outlet 16 Fluid conduit 17 In-line buffer conditioning reservoir 18 Filter 19 Break tank 20 Pump system 21 Pump 22 Fluid conduit 23 Filter 24 Break Tank 25 Bioreactor 26 Fluid Conduit 27 Filtration System 100 System 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 / Buffering Reservoir 120b Solvent / Buffering Reservoir 120c Solvent / Buffering Reservoir 120d Solvent / Buffering 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 Inlet 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 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 the first and second sample analyzers, the first sample analyzer including a first chromatography column; 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 is received by the second flow control device, thereby directing the portion of the biological sample from the first flow control device to either the sample purification device or the second flow control device; (b) adjusting the position of the second flow control device to direct a portion of the biological sample to one of the first and second sample analyzers; 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. 10. The system of claim 1, wherein the first chromatography column is a cation exchange chromatography column.

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 comprises 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. 11. The system of claim 10, 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.

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 the pump are configured such that, during operation of the system, the pump delivers an acetate buffer to the first chromatographic column to propagate a portion of the biological sample along the first chromatographic 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 in fluid communication with a 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 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 a first flow control device to direct a first 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 of the biological sample is received by the second flow control device; (b) adjusting a configuration of a second flow control device to direct a 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 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 and adjusting the 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 attributes of the analytes 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. 43. The system of claim 42, 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.

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.

56. 1. A method for measuring a product quality attribute of an analyte of a biological sample, the method comprising: obtaining a biological sample by extracting the biological sample from the operating bioreactor or a purification device in fluid communication with the operating 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 an 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 an analyte of the biological sample. Including, the first and second product quality attributes are different; at least one of the first and second product quality characteristics comprises 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 method.

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