Titer method using UV measurement for continuous biological production

Abstract

This invention provides a titer method using UV measurement for continuous biological production. [Solution] This specification discloses a method for determining the loading volume of capture chromatography in a system and method for the continuous production of biological products.

Description

[Technical Field]

[0001] This specification discloses a method for controlling the column loading of proteins for capture chromatography in a system, and a method for the continuous production of biological products (e.g., monoclonal antibodies). Advantageously, the method disclosed herein is automated and does not require external equipment (e.g., HPLC). [Background technology]

[0002] Perfusion cell culture is a highly efficient method for generating therapeutic proteins, using a perfusion membrane to continuously remove cell-free medium as fresh medium is added. The permeate from the bioreactor is then purified through a series of downstream unit operations. A common purification method is multi-column coupled elution chromatography. In multi-column chromatography, the permeate flow is loaded onto two or more columns. When a column reaches its maximum load capacity, the load is switched to another column, and the fully loaded column is washed and eluted for further downstream operations. To determine how much permeate can be loaded onto the columns, it is necessary to determine the concentration of proteins in the permeate. Further complicating matters, the concentration of proteins in the permeate can change over time. In systems consisting of a perfusion bioreactor directly connected to a continuous capture chromatography system, frequent measurement of protein concentration is a critical challenge. To capture changes in titer that occur over time, measurements need to be taken in real time, for example, once every 4-12 to 24 hours. In addition to the burden of measurement, there is also a risk of contamination.

[0003] Various methods and products exist for measuring protein concentration and indicating the load for capture chromatography. A standard method for measuring protein concentration in a composite feed is to use a high-performance liquid chromatography (HPLC)-based titer assay. Protein A HPLC is commonly used to measure the concentration of monoclonal antibodies (mAbs) in materials containing high levels of protein impurities. This method has been shown to be precise and accurate (Fernandez et al. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification. Int Sch Res Notices. (2014) 2014:487101) and is currently the standard method for determining mAb titers in batch-based production processes. However, since HPLC operation requires sampling from the process, there are difficulties in using HPLC to measure titers in continuous processes.

[0004] One method involves manually collecting samples from a sterile sampling manifold and then manually running the HPLC with those samples. This approach is labor-intensive because it requires an operator who can frequently collect samples and run the HPLC. Another option is to use an automated sampling system with an HPLC designed for inline operation, such as those from Waters Patrol. However, this requires the HPLC instrument to be located within a GMP suite close to the sampling location, which is difficult from a compliance standpoint. Furthermore, HPLCs are relatively complex instruments with many moving parts, and maintaining the instrument becomes even more difficult when it is located in a GMP space. Recently, spectroscopy has gained support as a method for measuring protein concentration. International Publication No. 2010151214 describes a spectroscopy method using UV sensors placed in the feed and effluent streams of a protein A capture chromatography system.

[0005] In capture chromatography, where the protein concentration in the feed stream changes over time, there is still a need for a method to determine the protein concentration and indicate the target loading volume. [Overview of the project]

[0006] The technology of the present invention will be specifically described, for example, according to the various viewpoints and embodiments listed below. In one embodiment, a method for optimizing continuous chromatography (e.g., continuous protein A chromatography) in a continuous production system for biological products (e.g., a perfusion bioreactor) is disclosed, the method comprising: (i) obtaining an optical concentration (OD) measurement of the eluate from the chromatography column; and (ii) applying the following formula to the OD measurement. Formula 1: TIFF2026074002000002.tif15157; and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column.

[0007] In another embodiment, a method for optimizing continuous chromatography (e.g., continuous protein A chromatography), optionally performed by a computer, in a continuous production system for biological products (e.g., a perfusion bioreactor) is disclosed, and the method is as follows: (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply the following formula to the OD measurement value. Formula 2: TIFF2026074002000003.tif12164 TIFF2026074002000004.tif11168; and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column. In certain embodiments, the methods disclosed herein enable improvements in one or more characteristics selected from increased productivity, reduced equipment footprint, faster manufacturing processes, and a reduction in overall cost of goods (COG).

[0008] In another embodiment, a method for producing the biological product of interest is provided, and the method is as follows: (I) In cell culture, eukaryotic cells that express the target biological product; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) capturing or purifying the biological product of the target, including continuous chromatography of the fluid feed containing the biological product of the target and one or more impurities or buffering components; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The aforementioned method further (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Applying the formula that is formula 1 or formula 2 to the OD measurement value; and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column.

[0009] As used herein, the term “adjustment” refers to changing or updating the titer value used in Delta V to determine the load requirements. Further advantages of this technology will be readily apparent to those skilled in the art from the following drawings and detailed description. The drawings and description should be considered illustrative and not restrictive. [Brief explanation of the drawing]

[0010] [Figure 1] As an exemplary embodiment of a continuous production system that can utilize the disclosed method, we show a continuous protein production system, such as the iSkid system. [Figure 2A]Shows a chromatogram of a representative Protein A chromatography cycle. The shaded area represents the area under the curve (AUC) of the elution peak and is used to calculate the mass of the product eluted from the column. Volume = 0 is fixed at the start point of eluent collection. Figure 2A shows the chromatogram of the entire chromatography cycle. [Figure 2B] Shows a chromatogram of a representative Protein A chromatography cycle. The shaded area represents the area under the curve (AUC) of the elution peak and is used to calculate the mass of the product eluted from the column. Volume = 0 is fixed at the start point of eluent collection. Figure 2B shows an enlarged view of the elution peak. The elution recovery volume was 3L as indicated by the arrow. [Figure 3] Shows the predicted titer using the "subtract outflow from supply" differential method. Absorbance was measured at 300 nm. Volume was normalized. Eight individual consecutive batches across three different locations are included. Some cycles were excluded from this dataset because inlet UV data was not available for all cycles. [Figure 4] Shows the predicted titer versus the actual titer using the elution UV titer prediction method. Titer prediction was compared to the protein concentration of the actual bioreactor permeate at elution. [Figure 5] Shows the predicted titer versus the actual titer when the linear regression adjustment method is used for titer prediction. [Figure 6A] Shows the predicted load challenge of a Protein A column based on the elution UV titer prediction method. Figure 6A shows the complete dataset. [Figure 6B] Shows the predicted load challenge of a Protein A column based on the elution UV titer prediction method. Figure 6B shows the dataset with runs where column overloading (>65 g / L challenge) led to underprediction removed. [Figure 6C] Shows the predicted load challenge of a Protein A column based on the elution UV titer prediction method. Figure 6C shows the complete dataset with linear regression correction performed. [Figure 6D]Figure 6D shows the predicted load challenge for the protein A column based on the elution UV titer prediction method. Figure 6D shows the linear regression correction method with overload operation removed. [Figure 7] This specification shows a decision tree of methods that can be optionally performed by a computer. [Modes for carrying out the invention]

[0011] As the commercial applicability of continuous biomanufacturing increases, processing large quantities of biomolecules (proteins, antibodies, etc.) from crude solutions (cell culture media, etc.) is becoming increasingly important. Typically, purification is a multi-step process. One of the key steps in a multi-step process is chromatography, which separates the desired biological product from one or more components, such as contaminants. Various strategies and modes of operation for purifying target molecules using chromatography are known in the art.

[0012] This specification discloses methods and systems for continuous downstream processing that enable optimized continuous loading of chromatographic unit operations (e.g., Protein A columns) to allow for the capture / purification of biological products. In particular, the systems and methods described herein address the relatively high and dynamic titers associated with perfusion cell culture processes. Dynamic titer refers to the non-uniform titer, i.e., the change in protein concentration over time. In certain embodiments, the concentration may vary over time by about 5%, about 10%, about 20%, or more than about 25%.

[0013] In certain embodiments, the methods and systems disclosed herein process up to 6 g / L / day (protein g / bioreactor volume L / day). In one embodiment, the methods and systems disclosed herein process about 3 g / L / day, about 4 g / L / day, about 5 g / L / day, or about 6 g / L / day or more. Specifically, the systems and methods of this specification enable the maintenance of the load density within a predetermined target load density range, preventing both underloading (e.g., less than about 20 g / L of resin) and overloading (>90% DBC at a 10% breakthrough, e.g., more than about 65 g / L) of the chromatography column. In some embodiments, the method enables the use of existing equipment, i.e., does not require modification of existing production systems and / or integration of equipment outside of existing production systems.

[0014] The disclosed methods and systems enable one or more improvements selected from increased productivity, reduced equipment footprint, faster manufacturing processes, and a reduction in overall cost of goods (COG). The disclosed methods and systems also reduce the need for human intervention in the handling of biological products, which contributes to cost, error, and contamination. In particular, the methods and systems disclosed herein enable optimized loading density and loading volume with respect to the sample (e.g., feed stream) loaded onto the chromatography column. In certain embodiments, the methods and systems disclosed herein determine the loading amount and then calculate the volume required to achieve the loading amount. In the following detailed description, many specific details are provided to give a complete understanding of the subject art. However, it will be apparent to those skilled in the art that the subject art can be carried out even without some of these specific details. In other examples, well-known structures and techniques are not shown in detail so as not to obscure the subject art. To facilitate understanding of the subject matter of this application, several terms and phrases are defined below.

[0015] I. Definition As used herein, the grammatical articles “one,” “a,” “an,” and “the” are intended to include “at least one” or “one or more,” unless otherwise indicated. Thus, articles are used herein to indicate one or more (i.e., at least one) grammatical objects of the articles. For example, “a component” means one or more components, and therefore, in some cases, multiple components may be expected and employed or used in the implementation of the embodiments described. The term "approximately" generally refers to a small error in measurement and is often expressed as a range of values ​​that includes the true value within a certain confidence level (usually ±1σ at 68% CI). The term "approximately" can be expressed as an integer or a value within ±20% of an integer.

[0016] The term "affinity" refers to the strength of binding of a single molecule (such as a protein) to a ligand. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of biomolecular interactions. The terms “affinity chromatography” and “protein affinity chromatography” are used interchangeably herein and refer to a separation technique in which a target biological product (e.g., an Fc region containing a protein or antibody of interest) specifically binds to a ligand that is specific to the target biological product, i.e., an affinity ligand. In some embodiments, the ligand (e.g., protein A or a functional variant thereof) is covalently bound to the chromatographic solid-phase material, allowing access to the target protein in the solution when the solution comes into contact with the chromatographic solid-phase material. The target biological product generally maintains a specific binding affinity to the ligand during the chromatographic process, while other solutes and / or proteins in the mixture do not bind to the ligand much or specifically. Binding of the target to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatographic medium, while the target biological product remains specifically bound to the immobilized ligand on the solid-phase material. The specifically bound target biological product is then removed in its active form from the immobilized ligand under appropriate conditions (e.g., low pH, high pH, ​​high salinity, competitive ligand, etc.) and passed through a chromatography column with an elution buffer, free from contaminating proteins or protein impurities that have passed through the column previously. Any component can be used as a ligand for purifying each specific binding protein (e.g., antibody). However, in various methods according to the present invention, protein A is used as the ligand for the Fc region containing the target protein. The elution conditions for the target biological product (e.g., the Fc region containing the protein) from the ligand (e.g., protein A) can be easily determined by those skilled in the art. In some embodiments, the ligand is not protein A.

[0017] As used herein, the term "affinity ligand" refers to a molecule that possesses specific non-covalent bonding ability to other molecules. The terms “antibody” and “immunoglobulin” are used interchangeably herein and are understood to include antibody fragments, antibody or antibody fragment fusion proteins, and antibody or antibody fragment complexes. Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies are assigned to various classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Antibodies can be, for example, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, or multispecific antibodies.

[0018] The term "area under a curve" or "AUC" refers to the area between the x-axis and the curve given by the integrand. This is equivalent to the definite integral of the function. As used herein, the term “bioreactor” refers to a device on which a biological reaction or process is carried out. Current bioreactor system options include batch, fed-batch, and continuous (i.e., perfusion) systems. Examples of bioreactors include culture bioreactors and production bioreactors. As used herein, the term “binding” refers to the process by which a resin and an unpurified biological product of a target form a reversible complex (in the case of positive chromatography), or the process by which a resin and impurities form a reversible complex (in the case of negative chromatography). As used herein, the term “binding fragment” refers to Fab, Fab', F(ab')2, scFv, scFab, dsFv, ds-scFv, dimers (e.g., Fc dimers), minibodies, diabodies, and their polymers, multispecific antibody fragments, and domain antibodies.

[0019] As used herein, the term "biologic product" generally refers to a product of interest produced through a biological process or through the chemical or catalytic modification of an existing biological product. Examples of biological processes include cell culture, fermentation, metabolism, and respiration. Examples of biological products of interest include antibodies, antibody fragments, proteins, hormones, vaccines, fragments of natural proteins (fragments of bacterial toxins used as vaccines, e.g., tetanus toxoid), fusion proteins or peptide complexes (e.g., subunit vaccines), and virus-like particles (VLPs). In certain embodiments, the biological product has a UV-Vis absorbance in the range of 190–700 nm. As used herein, the term "buffer" refers to a solution that resists pH changes caused by the action of its acid-base complex components.

[0020] As used herein, the term “capture” refers to the process of partially purifying or isolating (e.g., at least or about 5%, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95% pure weight), concentrating, and stabilizing a protein of interest (e.g., recombinant therapeutic protein) from one or more other components present in a liquid culture medium or diluted liquid culture medium (e.g., culture medium proteins, or one or more other components present in or secreted from mammalian cells (e.g., DNA, RNA, or other proteins)). Capture is typically carried out using a resin that binds to the protein of interest (e.g., through the use of affinity chromatography).

[0021] As used herein, the term “cell culture” refers to cells in a liquid medium. Optionally, the cell culture is contained within a bioreactor. Cells in cell culture may be of any biological origin, such as bacteria, fungi, insects, mammals, or plants. In certain embodiments, cells in cell culture include cells transfected with an expression construct containing nucleic acids encoding a protein of interest (e.g., an antibody). As used herein, the term “cell culture medium” refers to any type of medium used in connection with the culture of cells. Generally, cell culture media include amino acids, at least one carbohydrate as an energy source, trace elements, vitamins, salts, and optionally additional components (for example, to affect cell growth and / or productivity and / or product quality).

[0022] As used herein, the term “chromatography” generally refers to a set of techniques for separating mixtures. A mixture is dissolved in a fluid called the mobile phase, which carries the mixture through a structure that holds other materials called the stationary phase. The stationary phase is sometimes called a “resin.” The various components of the mixture move at different rates to separate. Separation is based on the partition difference between the mobile phase and the stationary phase. Slight differences in the partition coefficients of compounds result in differences in retention in the stationary phase, altering the separation. In column chromatography, the mobile phase or eluent is pumped into a column packed with a stationary chromatography resin, and the components to be separated move through the column at different rates and are collected at the column outlet at different times. Molecules that are more attracted to the stationary phase move through the system more slowly than molecules that are more attracted to the mobile phase. Because the eluent is pumped into the column at a specified flow rate, this also means that molecules elute after different volumes of eluent have passed through the column. This is recorded in a chromatogram, which plots concentration versus time or volume exiting the column. The molecular retention volume (VR) is the volume of the target molecule that passes through the column after being introduced into it.

[0023] The terms “chromatographic resin” or “chromatographic medium” are used interchangeably herein and refer to any type of solid phase that separates an analyte of interest (e.g., an Fc region containing proteins such as immunoglobulins) from other molecules present in a mixture. Typically, the analyte of interest is separated from other molecules as a result of differences in the rates at which individual molecules of the mixture move across the stationary solid phase under the influence of the mobile phase, or differences in binding and elution processes. Non-limiting examples include cation exchange resins, affinity resins, anion exchange resins, anion exchange membranes, hydrophobic interaction resins, and ion exchange monoliths.

[0024] As used herein, the term “contaminant” refers to any undesirable component or compound in a mixture. In cell cultures, cell lysates, or clarified bulk (e.g., clarified cell culture supernatant), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in the cell culture medium. Host cell contaminating proteins include, but are not limited to, proteins produced by the host cell, either naturally or recombinantly, as well as proteins related to or derived from the protein of interest (e.g., proteolytic fragments) and other process-related contaminants. In certain embodiments, the contaminant precipitate is separated from the cell culture using means accepted in the art, such as centrifugation, sterile filtration, deep filtration, and tangential flow filtration.

[0025] As used herein, the term “continuous” has different meanings with respect to unit operations and processes. A unit operation is continuous if it can process a continuous flow input over a long period of time. A continuous unit operation has minimal internal retention. The output is continuous or can be discretized into small packets generated periodically. A process is continuous if it consists of integrated (physically connected) continuous unit operations with zero or minimal intervening retention, and is equipped with appropriate controls to capture process variations. See Konstantinov et al., Journal of Pharmaceutical Sciences 104, no.3 (March 2015):813-20. Ideally, a continuous process is controlled so that all steps or unit operations of the continuous process operate simultaneously and at substantially the same production rate. In this way, cycle time compression is maximized and the shortest possible completion time is achieved.

[0026] As used herein, the term "column" refers to, for example, one or more containers such as tubes in which the separation of compounds occurs. As used herein, the term “column saturation” refers to the point at which a column is close to 100% of its dynamic binding capacity, and loading additional products at the inlet does not result in any further capacity. As used herein, the term "cycle" refers to a multi-step process that begins with equilibrating a chromatography column with a neutral buffer, followed by loading a clarified feed stream onto the column, where the clarified feed stream contains biological products (e.g., antibodies), followed by washing the column to remove loosely bound impurities, and then eluting the target biomolecule from the column. The multi-step process of equilibration, loading, washing, and elution constitutes a cycle or binding and elution cycle. As used herein, the terms “downstream” or “downstream processing” generally refer to some or all of the necessary steps for capturing a biological product from the original solution in which it was produced, purifying a biological product to remove undesirable components and impurities, filtering or inactivating pathogens (e.g., viruses, endotoxins), and for formulation and packaging.

[0027] As used herein, the terms “dynamic coupling capability” or “DBC” refer to the available capability of the stationary phase as a function of load velocity. As used herein, the term “eluate / filtrate” refers to a fluid containing a detectable amount of a target biological product (e.g., a monoclonal antibody) released from a chromatography column or chromatography membrane. As used herein, the term “elution” refers to the process of replacing the resin- and target biological product complex to collect the purified product. As used herein, the term “supply stream” refers to raw materials or raw material solutions originating from a production (upstream) scheme that are sent to the initial unit operation, the raw materials including the biological product of interest (e.g., proteins, polypeptides, antibodies, etc.) and may also include various contaminants (e.g., unwanted proteins, cell fragments, viruses, DNA).

[0028] The terms “holding tank,” “pool tank,” and “intermediate tank,” as used interchangeably herein, refer to any container, tank, or bag that may be used to collect the product of a process step (e.g., eluate from a column). In many conventional processes, one or more intermediate containers are used to adjust the conditions / characteristics of the product from one process step to suit the next process step. For example, it may be necessary to adjust the pH and / or conductivity of the eluate from a chromatography column or step (e.g., containing the protein target and impurities) before loading the pool onto the next chromatography column or step. In various embodiments of the method of the present invention, the need for a holding tank is avoided.

[0029] As used herein, the term “immunoglobulin-binding domain” refers to a domain that can bind to the constant region of an immunoglobulin (e.g., Fc of IgG). As used herein, the term "KD" refers to the koff / kon ratio, the equilibrium dissociation constant between an affinity ligand and a molecule. A lower KD value indicates higher affinity. As used herein in relation to a process, the term "integrated" refers to a process performed using structural elements that work together to achieve a specific outcome (e.g., the production of a therapeutic protein active pharmaceutical ingredient from a liquid culture medium). As used herein, the term “isolated biological product” refers to a product that, when produced by recombinant DNA technology, substantially contains no cell material or culture medium. As used herein, the terms “load density” or “load challenge” refer to the total mass of product loaded onto a column during a loading cycle in a chromatography process or applied to a resin in a batch coupling, and are measured as the unit mass of product per unit volume of resin. As used herein, the term "loading time" refers to the time required to load the column during the loading phase of a chromatography purification process. The term "load volume" refers to the volume of the liquid sample (e.g., feed stream) loaded onto the chromatography column.

[0030] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for variants that may occur during the production of the monoclonal antibody, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is against a single determinant on an antigen. In addition to its specificity, monoclonal antibodies have the advantage of not being contaminated by other immunoglobulins. The modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring any special method of antibody production.

[0031] As used herein, the terms “multi-column chromatography” or “MCC” refer to two or more interconnected or switchable chromatography columns and / or a series of chromatography membranes connected in series or parallel. In certain embodiments, the multi-column chromatography system described herein includes two series of chromatography columns. As used herein, the term “non-uniform” refers to differences or variations in quality or appearance. In one embodiment herein, a column is subjected to a non-uniform flow. As used herein, the term "overload" refers to a mode of chromatography in which a target biological product (e.g., a monoclonal antibody) is loaded beyond the DBC of the chromatographic material for that product, and is therefore called overload. The methods and systems disclosed herein do not reach overload.

[0032] As used herein, the term “perfusion cell culture” refers to a perfusion culture performed by continuously supplying fresh culture medium to a bioreactor and constantly removing used medium that does not contain cells while retaining cells within the reactor. Therefore, because cells are retained within the reactor via a cell retention device, perfusion culture can achieve a higher cell density compared to continuous culture. The perfusion rate varies depending on the requirements of the cell line, the concentration of nutrients in the feed, and the level of toxicity.

[0033] The terms “polypeptide,” “polypeptide product,” “protein,” and “protein product” are used interchangeably herein and refer to molecules consisting of two or more amino acids, for example, at least one chain of amino acids linked via a continuous peptide bond. In one embodiment, the “protein of interest” or “polypeptide of interest” is a protein encoded by an exogenous nucleic acid molecule transformed into a host cell, where the exogenous DNA determines the amino acid sequence. In another embodiment, the “protein of interest” is a protein encoded by an endogenous nucleic acid molecule to the host cell.

[0034] The terms "Protein A" and "ProA" are used interchangeably herein and encompass Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or recombinant techniques), and its variants that retain the ability to bind proteins having CH2 / CH3 domains, such as Fc domains. Protein A is commercially available from Repligen, Pharmacia, and Fermatech. Protein A is typically immobilized on solid-phase support materials. The term "ProA" also refers to affinity chromatography resins or columns containing the chromatography solid support matrix to which Protein A is covalently bound.

[0035] As used herein, the term “purification” refers to the process of isolating a biological product of interest (e.g., a monoclonal antibody) from one or more other impurities (e.g., bulk impurities) or components (e.g., liquid culture medium proteins, or one or more other components present in or secreted from mammalian cells, e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.)) present in the fluid containing the protein of interest. For example, purification can be performed during or after the initial capture step. Purification can be performed using a resin, membrane, or other solid support that binds to either the biological product of interest or the contaminants (e.g., through the use of affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). The protein of interest can be purified from the fluid containing the protein using at least one chromatography column and / or chromatography membrane (e.g., either of the chromatography columns or chromatography membranes described herein).

[0036] As used herein in relation to a process or mode, the term “recycled” refers to an operation in which the resin is cleaned for reuse or subsequent cycles. The term "retention time" refers to the time it takes for half of the solute to elute from the chromatography system. This is determined by the length of the column and the rate at which the solute migrates. As used herein in the context of liquid transfer to and / or from a bioreactor, the term “semi-continuous” means “periodic” or refers to a situation in which a liquid (e.g., culture medium alone and / or culture medium containing cells, cell breeds) is added to and / or removed from a bioreactor once over a long period of time. For example, a burst of liquid is transferred to and / or from a bioreactor for a period of time ranging from a few seconds (e.g., 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, or 60 seconds) to several minutes (e.g., 2 minutes, 5 minutes, 10 minutes, 25 minutes, 50 minutes, 120 minutes, or 240 minutes) once every 1, 2, 5, 10, 15, 30, 45, or 60 minutes, or once every 1 hour, or once every 2-3 hours, or once every 2-3 hours.

[0037] As used herein with respect to chromatography, the term “sequential” refers to a specific sequence of chromatography steps, such as a first chromatography step followed by a second chromatography step, followed by a third chromatography step, and so on. Additional steps may be included between the sequential chromatography steps. As used herein, the term "titer" refers to the total amount of protein produced by cell culture divided by a given volume of culture medium. Essentially, the term "titer" refers to concentration and is generally expressed in milligrams of polypeptide per liter of culture medium. The method of the present invention has the effect of substantially increasing the titer of polypeptide products compared to polypeptide products produced by other cell culture methods known in the art.

[0038] As used herein, the terms “upstream” or “upstream process” generally refer to steps in the production of biopharmaceuticals that involve the generation of active biological products by biological processes or other reactions. Typically, the biological products that are isolated and processed into biopharmaceuticals are the result of fermentation or expression products of recombinantly transformed host cells. Upstream processes, including the generation of biological products in cell culture, are carried out in fermenters or bioreactors and may be batch processes (e.g., batch or fed-batch cell cultures grown in fermenters) or continuous processes (e.g., perfusion cell cultures). As used herein, the term “washing” refers to the process of washing a resin containing a binding product with a washing buffer to remove impurities from the resin (in the case of positive chromatography), or the process of washing a resin containing a binding impurity with a washing buffer to wash away excess (carryover) product from the binding process (in the case of negative chromatography).

[0039] II. Continuous Biological Production Systems In one embodiment, the methods disclosed herein are useful in connection with methods and systems for the serial production of biological products (e.g., monoclonal antibodies). In certain embodiments, the system is an integrated continuous biological production system for producing biological products, comprising a perfusion bioreactor coupled to continuous upstream and downstream processes, more specifically, a continuous capture (purification) function. In other embodiments, the integrated continuous biological production system further comprises one or more additional downstream processes selected from virus inactivation, filtration, formulation, filling, and combinations thereof.

[0040] In one embodiment, the system is a continuous protein production system, such as the iSKID system, which is a fully integrated automated system in which a perfusion bioreactor is connected by hydraulic pressure to several downstream unit operations (2× Protein A column, continuous virus inactivation, flow-through mode anion exchange chromatography, and single-pass tangential flow filtration (SPTFF)). See Figure 1. Within the system, continuous chromatography can be used to capture or purify target biological products (such as monoclonal antibodies). In certain embodiments, purification is provided by one-column continuous chromatography, which consists of a single column integrated with a continuous upstream process. This approach controls the loading and unloading phases of the column by applying a combination of perfusion rate and loading flow rate. The complexity of process control is reduced compared to multi-column operations, where only one column needs to be monitored.

[0041] In one embodiment, purification is provided by multi-column continuous chromatography, which creates a loading region instead of using a single industrial-scale column. Multi-column chromatography typically requires less column volume and less buffer volume compared to standard batch processes, while producing the same amount of biological product. Examples of multi-column approaches include continuous multi-column chromatography (SMCC), three-column periodic chromatography (3C-PCC), and two-column chromatography-capture SMB (2C-PCC). In one embodiment, purification is provided by at least two consecutive chromatography columns. During operation, one of the two columns is always loaded while the other column is processed simultaneously. The basic sequence is washing, elution, regeneration, equilibration, and waiting for the next load.

[0042] The size of the chromatography column may vary. In one embodiment, the chromatography column may have volumes of approximately 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 200 mL, 300 mL, 400 mL, and 500 mL. , about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 25L, about It has a volume of 50L, about 100L, about 200L, 300L, about 400L, about 500L, about 600L, about 700L, about 800L, about 900L, or about 1,000L or more. The type of chromatography column may vary. In certain embodiments, the chromatography resin is an affinity chromatography resin containing an affinity ligand. Any affinity ligand can be used in the system and method, as long as the ligand is a specific binding partner for the target biological product of interest.

[0043] In one embodiment, the affinity ligand has an immunoglobulin domain, such as an Fc-binding domain. The affinity ligand may be a full-length protein or a functional variant of a full-length protein. The affinity ligand may be a monomer, dimer, or polymer of a full-length protein or a functional variant. In one embodiment, the affinity ligand is protein A, protein G, or a functional variant thereof, and the target molecule may be an immunoglobulin or a molecule consisting of the Fc region of an immunoglobulin, or at least a portion of the Fc region of an immunoglobulin. In a particular embodiment, the affinity ligand is a bacterial immunoglobulin-binding protein. In some embodiments, the affinity ligand is protein A or a component thereof. Protein A may be natural (e.g., Staphylococcus aureus) or recombinant protein A conjugated to a natural (agarose or cellulose) or synthetic (polyvinyl ether, polystyrene-divinylbenzene, porous glass, or polymethacrylate) based matrix. In certain embodiments, the affinity ligand is selected from the B, C, A, E, and D domains derived from Staphylococcus protein A or its functional variants. Each of the E, D, A, B, and C domains has a different immunoglobulin binding site. One site is for the Fc (the constant region of IgG class Ig), and the other is for the Fab portion of a specific Ig molecule (the portion of Ig responsible for antigen recognition). Each domain has been reported to contain a Fab binding site. The non-immunoglobulin binding portion is located at the C-terminus and is called the X region or X domain.

[0044] Examples of protein A-based resins that can be used in the method of the present invention include, but are not limited to, PROSEP vA High Capacity, PROSEP A Ultra, PROSEP Ultra Plus (Millipore), Protein A Sepharose FastFlow, rmp Protein A Sepharose FastFlow, MabSelect, MabSelect Xtra, MabSelect SuRe (GE Healthcare), POROS A, POROS MabCapture A (Applied Biosystems), and Sartobind Protein A (Sartorius). In one embodiment, the affinity ligand is protein G. Protein G is an immunoglobulin-binding protein expressed in streptococci. It is similar to protein A but has a different binding specificity. In a specific embodiment, the affinity ligand is the B domain derived from Staphylococcus protein B or a functional variant thereof. Examples of protein G-based resins include, but are not limited to, PROSEP-G (Millipore), Protein G Sepharose® 4 Fast Flow (GE Healthcare), and POROS G (Applied Biosystems). In certain embodiments, the affinity ligand is not a bacterial immunoglobulin-binding protein, but rather an alternative affinity ligand such as a synthetic binding protein, peptide, aptamer, or synthetic small molecule compound. The KD of affinity ligands varies. In one embodiment, the affinity ligand has a KD between about 1 nM and 1 μM, more specifically about 10 nM.

[0045] Capture of the target biological product may include (i) contacting a chromatographic matrix (e.g., composed of affinity chromatography media) with a mixture containing the target biological product under conditions that the target molecule preferentially binds to the chromatographic matrix, and (ii) optionally, eluting the target biological product from the matrix by modifying one or more conditions, for example, by applying an elution buffer. In one embodiment, elution is step elution or gradient elution. The method may optionally include one or more washing steps. Washing steps may be performed, for example, after the target molecule has bound to the matrix and before the target molecule has eluted from the matrix. Additional washing steps may optionally be performed after elution of the target molecule, for example, to wash the matrix of residual binding substances.

[0046] Any suitable buffer can be used. The choice of buffer may depend, for example, on the desired pH, the properties of the biological product of interest (e.g., monoclonal antibody), the chromatographic material, and other variables well known to those skilled in the art. See "A Guide to the Preparation and Use of Buffers in Biological Systems," edited by Gueffroy, D., Calbiochem Corporation (1975). The elution buffer may, for example, have a pH different from the pH of the mixture when applied to the matrix, or it may have a higher salt concentration compared to the original mixture applied to the matrix. In certain embodiments, the capture or purification process comprises loading a fluid sample (e.g., cell culture medium or clarified cell culture medium) containing the target biological product (e.g., monoclonal antibody) onto an affinity chromatography column, washing the column to remove unwanted biological substances (contaminating proteins and / or small molecules, etc.), eluting the target biological product bound to the column, and re-equilibriumating the column. In one embodiment, the capture or purification process includes continuously supplying a fluid (e.g., liquid culture medium) to a first affinity chromatography column, capturing a target biological product (e.g., monoclonal antibody) from the liquid, generating an eluate from the first affinity chromatography column containing the target biological product (e.g., monoclonal antibody), and continuously supplying the eluate to a second affinity chromatography column to subsequently elute the target biological product and produce a purified target biological product.

[0047] Cell culture media can be obtained from a culture of perfused cells (e.g., mammalian cells) (e.g., a perfused bioreactor containing a culture of mammalian cells secreting recombinant proteins). Liquid cell culture media can be filtered or clarified to obtain a liquid culture medium that is substantially free of cells and / or viruses. Liquid cell culture media can be continuously supplied to an affinity chromatography column using various different means (e.g., actively pumping it into the first affinity chromatography column, or using gravity to supply it to the first affinity chromatography column). The "loading" step involves loading the column by passing the fluid (supply) through the inlet so that the feed comes into contact with the adsorbent and a certain amount of the target product binds. Continuous loading allows for integration with a continuous upstream process. To determine the volume of cell culture medium that can be loaded onto the column, it is necessary to determine the concentration of the target biological product (e.g., monoclonal antibody) in the cell culture medium. In batch production, a single, homogeneous permeate is obtained, so only one titer measurement (along with the loading volume) is sufficient to determine the loading mass. However, in continuous production, the permeate is continuously discharged from the perfusion reactor and loaded onto the chromatography column, so the titer of the product may change over the loading period.

[0048] Generally, the user can determine when the loading of a chromatography column ends based on one of two parameters: time or loading density. For time, the system stops loading after a time specified by the operator is reached. For loading density, the total mass of the loaded protein is calculated using the total volume reading from the flow meter, along with the titer-average value derived by the "user". Methods currently known in the art for utilizing the loading density discussed above generally rely on HPLC. In contrast, the method disclosed herein (described in more detail below) includes automated UV curve data acquisition and calculation. Accordingly, the systems disclosed herein include one or more means for detecting the contents of an eluent from a chromatographic medium. The detector may be a light-based detector that relies on multi-wavelength detection or single-wavelength detection. Suitable detectors include spectrophotometers, UV absorption detectors, and fluorescence detectors that can detect wavelengths of visible light. The detector may be a light scattering detector that relies on a laser source, or an electrochemical detector that reacts with either an oxidizing or reducing substance, where the electrical output is a flow of electrons generated by a reaction occurring at the electrode surface.

[0049] III. Method of notifying load volume This specification discloses, but is not limited to, methods for notifying the loading volume of a capture chromatography in systems and methods for the continuous production of biological products. The method disclosed herein provides a simple mathematical means for predicting the average permeate concentration on a currently loaded chromatography column (e.g., a ProA column) and indicating the amount of volume of bioreactor permeate loaded onto the column that falls within a desired loading challenge range. Advantageously, this method is cost-effective and requires minimal development work for implementation. The method described herein can keep the loading challenge within an acceptable range. Specifically, a method is disclosed for controlling the current loading challenge using UV signals from previous elution cycles. The area under the elution curve is integrated and multiplied by a predetermined calibration constant to determine the mass of the product eluted from the chromatography column. By dividing the eluted mass by the volume loaded onto the column, a prediction of the average titer of the product during loading is obtained. In one embodiment, an optional computer-based method for optimizing continuous chromatography (e.g., continuous protein A chromatography) in a continuous production system for biological products (e.g., a perfusion bioreactor) includes (i) obtaining optical concentration (OD) measurements of the eluate from the protein A column, (ii) applying a formula to the OD measurements, and (iii) adjusting the continuous loading of the protein A column to achieve optimization.

[0050] The method of the present invention measures the volume loaded onto a chromatography column and an absorbance detector (e.g., a UV sensor) using a flow meter, and records the amount of ultraviolet or visible light absorbed by the components of the mixture eluted from the chromatography column. UV light is passed through each component of the eluted sample mixture, and the amount of UV light absorbed by each component is measured. The objective of this method is to control the load challenge on the column within a target range. If the system is a continuous protein production system as described herein, no additional equipment is required to carry out the method beyond what is already present in the design, namely, UV sensors in column efflux, etc., for general process control purposes.

[0051] The flow meter can be any suitable flow meter, such as a Levitronix flow meter. The UV sensor can be any suitable fixed-wavelength or tunable-wavelength UV sensor having a diode array detector (DAD or PDA). The UV absorption of the effluent is measured continuously at one or more wavelengths. The wavelength may vary, and in one embodiment, it is about 200 to about 400 nm or about 200 to about 800 nm. In a particular embodiment, the wavelength is about 190 nm to about 700 nm, more specifically 300 nm. In a particular embodiment, the method of the present invention uses a flow meter to measure the volume loaded onto a ProA affinity column and a UV sensor (such as an Optek probe known in the art) and records the optical density of the ProA elution profile at a wavelength of 300 nm. The A300 signal is scaled to match the intensity of the A280 signal.

[0052] As a general principle, the mass of protein eluted from a Protein A column should be proportional to the mass loaded onto the column. The mass of the product eluted from a Protein A column can be determined by integrating the volumetric flow rate with the optical density measured by a post-column UV sensor, calculating the integral with respect to volume, and evaluating the integral of the collected elution volume. The integration is performed using the trapezoidal rule. By dividing the eluted mass by the volume loaded onto the column, the average titer of the product during loading can be predicted. Advantageously, this method allows for obtaining historical titer values ​​without the use of external equipment. In one embodiment, the method of the present invention includes (i) (a) using a UV signal scaled from 300 nm to 280 nm, and (b) integrating the area under the elution curve (OD*liters) by approximating the integral value using the trapezoidal rule; (ii) converting the UV region to elution mass (g) using the molecular mass extinction coefficient εm; (iii) converting the elution mass to loading mass using a calibration parameter α (efficient yield); (iv) calculating the average loading titer (g / L) from the loading mass and loading volume; and (v) combining the formulas corresponding to (i) to (iv) above.

[0053] The mathematical equation corresponding to the integral in (i) above is as follows: The mathematical equation corresponding to the transformation in (ii) above is as follows: The mathematical equation corresponding to the transformation in (iii) above in TIFF2026074002000006.tif1732 is as follows: The mathematical equation corresponding to the calculation in (iv) above is as follows: TIFF2026074002000008.tif1527

[0054] The complete equation used to determine the titer of the product using the elution UV method is as follows: Formula 1: TIFF2026074002000009.tif13150 ·Titer(g / L) = Average permeate titer during column loading ·V l (mL) = Volume of permeate loaded onto the column before elution •i = the number of time points elapsed since the start of eluate collection. n = the number of time points that have passed between the start and end of eluate collection. ·D i = i-th measurement of optical density at 300 nm using an Optek probe Q i = The i-th measured volumetric flow rate during elution. Q is calculated by summing the measured flow rates. ·s is for elution buffer concentrates and WFI dilutions of elution buffers. This is the molecular-specific extinction coefficient at εm = 300 nm, and its unit is g / L / cm². ·k colA and k colB * =A constant specific to each of the two Protein A columns in the continuous protein production system, determined by analysis of previous datasets. This constant is input into the control system of the continuous protein production system before the start of a batch.

[0055] For several reasons, this calculation requires the use of fitted constants rather than applying Beer's Law based on the molecular-specific extinction coefficient. First, although the UV sensor is calibrated so that A300 measurements accurately reflect A280 measurements, calibration errors have been observed in previous datasets. Using fitted constants accounts for persistent calibration offsets. Furthermore, using fitted constants allows for a model that takes into account the fact that column yields are typically less than 100%. Since sensor calibrations often differ between the two columns, each column needs to be calibrated separately. Incorporating separate calibration constants for each column improves the accuracy of model predictions. The value of the fitted constant is determined from historical data from eight separate batches performed across three different locations using three different molecules. In future implementations, the fitted constant will be determined by comparing the eluted mass with the loaded mass using Protein A HPLC at the start of the initial loading cycle. In the historical dataset, time points were spaced at 10-second intervals. Protein A HPLC titer values ​​were used as the calibration dataset. The eluted mass from past runs is calculated for each cycle by multiplying the volumetric flow rate by the optical density at 300 nm, summing this data over eluate collection (when the OD of A280 reached 0.20 and then 3 CV of eluate was collected), and dividing by the corresponding loading volume and mAb extinction coefficient for that cycle. Figure 2 shows a chromatogram of a typical Protein A chromatography cycle, with the area under the elution curve represented as a shaded area.

[0056] There are three methods to predict the titer of a currently loaded column using the titer from previous elutions: (i) single-point method, (ii) linear extrapolation method, and (iii) hybrid method. The single-point method risks (i) over-challenging the column when the titer is increasing very rapidly, and (ii) over-predicting and under-challenging the column when the titer is decreasing. Linear extrapolation works by fitting a line through the previous three titer measurements and using the best-fitting line to estimate the average titer of the next loading cycle. TIFF2026074002000010.tif945m - The slope of the line that best fits the last three measured force values. t - Estimated time to the midpoint of the next load b - The y-intercept of the line that best fits the three force values ​​measured last. If the actual titer is increasing, linear extrapolation is likely to overestimate the titer and result in insufficient challenge to the column. Generally, excessive challenge to the column is more serious than insufficient challenge. Risks of excessive challenge to the column include inaccurate titer from the UV elution signal, potentially affecting product quality, and / or more product being discharged into the drain.

[0057] The hybrid method combines two approaches: (i) positive slope from the last three titers: linear extrapolation, and (ii) negative slope from the last three titers: single-point method, to provide an approach suitable for all titers. Linear regression was performed using the calculated elution mass as the predictor and the mean loading titer as the response. The weighted mean loading titer was determined for each cycle by dividing the total mass of antibody loaded into the column by the total volume of permeate from the bioreactor loaded into the column. The intercept of this regression was forced to zero, and the calibration constant was determined by dividing the slope of the regression by the extinction coefficient of the calibration molecule. The calibration constant needed to be determined once for each UV detector. This method was applied retrospectively to eight batches performed with three separate molecules for three separate continuous protein synthesis systems. Figure 3 shows the predicted titer versus the actual titer. The CV (RMSE) was determined to be 14.1%, indicating high accuracy. Within these datasets, many of the chromatographic cycles for protein A were overloaded, leading to product loss. This product loss affected the size of the elution peak, resulting in some error in concentration prediction. The implementation of the titer prediction method could potentially eliminate all instances of overloading. If these overload cycles are removed from the dataset, the CV (RMSE) will improve to 10.5%. This titer prediction method is expected to adequately control the protein A loading challenge so that overload occurrences are rare, and the system's predicted variability is expected to be close to the 10.5% value obtained when overload cycles are removed from the dataset.

[0058] In operations where the titer value changes rapidly, the accuracy of the elution UV titer prediction method may be low because the titer can change rapidly within a 3-7 hour time difference between the midpoint of the load and the elution cycle. Considering this, a linear regression algorithm that takes into account the rate of titer change was developed. Linear regression was performed to determine the slope and y-intercept of the line that best fit the previous three titer predictions. Linear regression was performed using the following formula. Formula 2: TIFF2026074002000011.tif10150 TIFF2026074002000012.tif9150·m=gradient ·b=y intercept • x = the time point corresponding to the midpoint of the load corresponding to the i-th titer prediction. The midpoint of the load was calculated by determining the total load volume of the cycle and then establishing the time at which half of the total load volume was applied to the column. ·y = Prediction of i-th titer from elution UV method n = number of regression points

[0059] At each subsequent time point, the titer was estimated by multiplying the slope by the elapsed time from the midpoint of the previous load, and then adding the intercept. By selecting the time corresponding to the midpoint of the load, rather than the elution time, as the x-value, the model can be made to account for the potential change in titer between loading and elution. If only one titer prediction occurs in a batch, linear regression is not performed. If three valid titer predictions are obtained, the estimated titer for the next loading cycle is extrapolated using linear regression. To prevent potential over-challenge of the column, if the slope of the linear regression is negative, or if a decrease in titer is predicted, linear regression is not performed, and instead the column is loaded using the last titer measurement of this method.

[0060] In one embodiment, this method makes it possible to control the loading challenge on the protein A column within the target loading challenge range. Using data from batches of seven continuous protein production systems, we determined how the loading challenge of protein A chromatography would be when employing an elution UV titer prediction method with and without linear regression adjustment. The target loading challenge was assumed to be 50 g / L. Figure 6 shows these results with and without linear regression adjustment. Cycles requiring loading times exceeding 12 hours to reach 50 g / L were excluded from the dataset. Clearly, in all datasets, most runs were close to the target of 50 g / L. In the complete dataset without linear regression (Figure 5A), two runs were thought to have had a loading challenge exceeding 65 g / L, and five runs were thought to have had a loading challenge of 60-65 g / L. However, all of these overloaded runs were the result of titer predictions based on overloaded runs. When a cycle is overloaded, the elution yield is lower than expected, leading to an under-prediction of titer. If titer prediction excludes operations based on overload cycles (Figure 6B), operations with loads exceeding 65 g / L would not be performed, and in fact, all operations should have been below a 60 g / L challenge. By employing the elution UV titer method, reliable control of protein A loading is possible, minimizing the possibility of overload and subsequently improving the accuracy of titer prediction. This method is further improved by employing linear regression adjustment, as shown in Figures 6C and 6D. Examining the entire dataset with linear regression (Figure 6C) reveals only one operation with a load challenge >65 g / L and only one additional operation with a load challenge of 60-65 g / L, a significant improvement over the results without linear regression. When predictions based on overload operations are excluded from the linear regression dataset, prediction accuracy improves to the point where there are no operations with challenges exceeding 60 g / L.

[0061] Advantageously, the methods disclosed herein are not severely affected by (i) rapid titer changes; (ii) prolonged stoppages in downstream operations (e.g., 24-48 hours); (iii) changes in the DBC of the chromatography column; or (iv) larger elution UV signals from impurities. Overall, the elution UV titer prediction method is accurate enough to reliably control the protein A loading challenge in most processes. In processes where rapid changes in upstream titer are expected, linear regression can further improve the robustness of the prediction and reduce the possibility of overload. Predictive accuracy is calculated from the ratio of the predicted loading titer to the actual titer. JPEG2026074002000013.jpg1167 The actual load titer (C_HPLC) can be determined by interpolating titer measurements from permeate samples taken periodically throughout the day. The methods disclosed herein also have a favorable yield. The yield of the process can be calculated by the ratio of the eluted mass to the loaded mass in the chromatographic process. The load mass (m_load) is calculated by numerical integration of HPLC titer curves generated from periodic samples collected throughout the day and submitted to QC. In one embodiment, a method is disclosed for capturing a biological product from a feed stream containing the biological product, comprising (i) providing the feed stream, (ii) loading the feed stream onto a chromatography column, and (iii) collecting the purified biological product, wherein the feed stream has a loading density within a target loading density range or a specific loading density. In one embodiment, the feed stream is loaded onto the chromatography column at approximately DBC of the chromatographic material of the biological product.

[0062] In one embodiment, the target load density range is approximately 20 g / L to approximately 90 g / L, more specifically approximately 20 g / L to approximately 80 g / L, approximately 30 g / L to approximately 70 g / L, or approximately 40 to approximately 60 g / L. In one embodiment, the target load density range is approximately 20 g / L to approximately 30 g / L, approximately 30 g / L to approximately 40 g / L, approximately 40 g / L to approximately 50 g / L, approximately 50 g / L to approximately 60 g / L, and approximately 60 g / L to approximately 70 g / L, approximately 70 g / L to approximately 80 g / L, approximately 80 g / L to approximately 90 g / L, or approximately 90 g / L to approximately 100 g / L. In certain embodiments, a particular load density is approximately 20 g / L or more, approximately 25 g / L or more, approximately 30 g / L or more, approximately 35 g / L or more, approximately 40 g / L or more, approximately 45 g / L or more, approximately 50 g / L or more, approximately 55 g / L or more, approximately 60 g / L or more, approximately 65 g / L or more, approximately 70 g / L or more, approximately 75 g / L or more, approximately 80 g / L or more, approximately 85 g / L or more, approximately 90 g / L or more, or approximately 95 g / L or more. In certain embodiments, the specific load density is less than approximately 50 g / L+ / -2. In certain embodiments, the chromatography column is the first column in a multi-column continuous chromatography system. In certain embodiments, the chromatography column is an affinity chromatography column, and more specifically, a ProA chromatography column. In a particular embodiment, the chromatography column is the first chromatography column shown in the system shown in Figure 1.

[0063] In certain embodiments, one or more parameters of the protein A matrix or resin (such as pH, ionic strength, temperature, or addition of other substances) are adjusted before the protein A matrix or resin comes into contact with the sample. In certain embodiments, the feed stream contains intact host cells and / or cell debris. In certain embodiments, the feed stream is treated, for example, by filtration, before loading the chromatography column. Flow rates may vary. In certain embodiments, flow rates are approximately 6 to approximately 20 CV / hour, more specifically approximately 6, approximately 8, approximately 10, approximately 12, approximately 14, approximately 16, approximately 18, or approximately 20 CV / hour or more. The degree of purification obtained by the methods disclosed herein may vary. In one embodiment, the target biological product is purified to an amount of more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 99%. In a particular embodiment, the target biological product is purified to an amount of about 90% to about 99%, more specifically about 92% to about 99%, about 94% to about 99%, about 96% to about 99%, or about 98% to about 99%. In certain embodiments, the productivity of the method disclosed herein is increased compared to a similar method that does not utilize the load optimization strategy disclosed herein. In certain embodiments, the productivity of the method disclosed herein is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 25% or more.

[0064] In certain embodiments, the facility footprint permitted by the methods disclosed herein is reduced compared to similar methods that utilize load optimization strategies, particularly HPLC-based estimation strategies. In certain embodiments, the facility footprint is reduced by approximately 1% to approximately 30%, more specifically by approximately 5% to approximately 20%, and more specifically by approximately 10%. In certain embodiments, the eluate obtained from chromatography-based purification derived from the methods described herein is subsequently subjected to further processing and / or purification, such as further purification, inactivation, formulation, etc. The overall goal of the methods and systems disclosed herein is the production of isolated biological products, such as isolated proteins or antibodies (e.g., monoclonal antibodies). The methods disclosed herein are broadly applicable to any process producing biological products in which a feed stream with continuously changing product concentrations is loaded into a bound-elution capture chromatography step. Furthermore, linear regression adjustments are broadly applicable to all types of processes or bioprocesses having quality attributes. Any suitable capture chromatography method, such as ion exchange, hydrophobic interaction, or mixed-mode chromatography, can be used. The only requirement is that the yield of the chromatography cycle be relatively consistent from run to run (measured by actual yield and predicted yield) to minimize variability in titer prediction.

[0065] The biological product may be any biological product having a UV-Vis absorbance in the range of 190 to 700 nm, and in certain embodiments, it is a monoclonal antibody. As shown in Figures 2 and 3, this method significantly improves the accuracy of the "subtraction of effluent absorbance from feed absorbance" method for determining the titer of the product. Compared to the method of collecting offline HPLC samples, this method requires far less effort because it can be implemented in a fully automated manner through a distributed control system. Compared to the method using in-line HPLC with automated sample collection, this method requires less equipment, less equipment maintenance, and the risk of contamination is reduced because sampling of bioreactor permeates is unnecessary. Compared to the method using Raman or FTIR spectroscopy, this method is easier to calibrate and requires less expensive equipment. Compared to the method of controlling the loading of protein A by continuously loading until a breakthrough of the product is observed (insert citation), this proposed method does not require loading the protein A column until product loss occurs, resulting in improved process yield compared to loading until a breakthrough occurs. Furthermore, the breakthrough method relies on UV absorbance measurements obtained from the bioreactor permeate flow, which contains many impurities that can contaminate the UV sensor. This proposed method relies on UV measurements obtained from protein A eluate, which typically contains much lower levels of impurities than bioreactor permeate flow, making accurate UV absorbance measurements more likely.

[0066] IV. Composition This specification discloses biological products produced by the systems and methods disclosed herein. In one embodiment, the biological product is any biological product having a UV-Vis absorbance in the range of 190 to 700 nm. In one embodiment, the biological product is a polypeptide, protein, or antibody (e.g., a monoclonal antibody), and in particular, a polypeptide, protein, or antibody for administration to a subject (e.g., a human). In a particular embodiment, the antibody is a monoclonal antibody. In a particular embodiment, the biological product is a binding fragment. In certain embodiments, the biological product is incorporated into a pharmaceutical composition suitable for administration to a target. Typically, the pharmaceutical composition includes an antibody and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the biological product purified using the systems and methods disclosed herein can take on a variety of forms, i.e., dosage forms. Methods for producing or generating the biological product of interest known in the art can be used in combination with the systems and methods described herein. For example, those skilled in the art know of methods for producing or generating biological products such as recombinant proteins using fermentation. In certain embodiments, the production of the biological product of interest includes culturing eukaryotic cells expressing the biological product of interest in a cell culture. Culturing eukaryotic cells expressing the biological product of interest in a cell culture may include maintaining the eukaryotic cells in appropriate media and conditions that enable growth and / or protein production / expression. The biological product of interest can be produced by fed-batch cell culture or serial cell culture. Therefore, eukaryotic cells can be cultured in fed-batch cell culture or serial cell culture, preferably in serial cell culture.

[0067] In certain embodiments, the eukaryotic host cell is a yeast cell. In one embodiment, the eukaryotic host cell is a mammalian cell. The mammalian cells used herein are mammalian cell lines suitable for the production of secreted recombinant therapeutic proteins and may therefore also be called “host cells”. In certain embodiments, the mammalian cell is a rodent cell such as a hamster cell. The mammalian cell is an isolated cell or cell line. In certain embodiments, the mammalian cell is a transformed and / or immortalized cell line. In certain embodiments, the mammalian cell is adapted for continuous passage in cell culture and does not contain primary non-transformed cells or cells that are part of an organ structure. In certain embodiments, the mammalian cell is BHK21, BHK TK-, Jarcutt cells, 293 cells, HeLa cells, CV-1 cells, 3T3 cells, CHO, CHO-K1, CHO-DXB11 (also known as CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or a derivative / offspring of any of such cell lines. In certain embodiments, the mammalian cells are CHO cells, e.g., CHO-DG44, CHO-K1, and BHK21, and more preferably CHO-DG44 cells and CHO-K1 cells. In certain embodiments, the mammalian cells are CHO-DG44 cells. Mammalian cells, particularly glutamine synthetase (GS) deficient derivatives of CHO-DG44 and CHO-K1 cells, are also included. In one embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells, e.g., CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHO-S cells, CHO-GS deficient cells, or derivatives thereof.

[0068] In certain embodiments, the host cell may further comprise one or more expression cassettes encoding heterologous proteins, such as therapeutic proteins, for example, recombinant secreted therapeutic proteins. In certain embodiments, the host cell may also be mouse cells, such as mouse myeloma cells, such as NS0 and Sp2 / 0 cells, or derivatives / offspring of any such cell lines. The expression of the target biological product or recombinant protein occurs in cells containing the DNA sequence encoding the target biological product or recombinant protein, which is then transcribed and translated into a protein sequence including post-translational modifications, thereby producing the target biological product or recombinant protein in cell culture.

[0069] This specification discloses a method for producing the biological product of interest, (I) To culture eukaryotic cells that express the desired biological product in cell culture; (II) To collect the target biological product from a cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) capturing or purifying the biological product of interest, including continuous chromatography of the biological product of interest and a fluid feed containing one or more impurities or buffering components; and (IV) Optionally, formulating the biological product of interest into a pharmaceutically acceptable formulation suitable for administration; This method is further (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply a formula to the OD measurement, where the formula is Formula 1; and (iii) Achieve optimization by adjusting the continuous loading of the chromatography column. Includes.

[0070] This specification discloses a method for producing the biological product of interest, (I) To culture eukaryotic cells that express the desired biological product in cell culture; (II) To collect the target biological product from a cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) capturing or purifying the biological product of interest, including continuous chromatography of the biological product of interest and a fluid feed containing one or more impurities or buffering components; and (IV) Optionally, formulating the biological product of interest into a pharmaceutically acceptable formulation suitable for administration; This method is further (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply a formula to the OD measurement, where the formula is formula 2; and (iii) Achieve optimization by adjusting the continuous loading of the chromatography column. Includes. In certain embodiments, the biological product of interest is a recombinant protein. In certain embodiments, the step of culturing eukaryotic cells expressing the biological product of interest in cell culture is performed using fed-batch cell culture. In certain embodiments, the step of culturing eukaryotic cells expressing the biological product of interest in cell culture is performed using serial cell culture. The following examples are provided for illustrative purposes only and are not intended to be limiting. [Examples]

[0071] Example 1: Analytical Method Online ProA titer chromatography was performed using the PATROL UPLC process analysis system at Waters (Milford, Massachusetts), with samples taken directly from the permeate line via an Artesyn valve. Chromatographic separation was performed by injecting 5 μL from a fixed sample loop on a POROS A 20 μm, 2.1 x 30 mm column (Thermo Fisher Scientific, Waltham, Massachusetts) equipped with a 0.5 μm pre-column filter cartridge (IDEX, Lake Forest, Illinois). Mobile phase A was phosphate-buffered saline at pH 7.4, and mobile phase B was phosphate-buffered saline at pH 2.2. Samples were loaded with either 80% mobile phase A and 20% mobile phase B, or 100% mobile phase A. Elution was performed by gradient elution with a final buffer ratio of 100% mobile phase B. Detection was performed by absorbance at 280 nm. Online samples were quantified against a standard curve.

[0072] Example 2: Calculation of the permeate titer in a continuous protein production system by integrating the elution UV signal from protein A chromatography In this example, a procedure for calculating the permeate titer in a continuous protein production system through integration of the elution UV signal from the protein A chromatography step will be described. As a general principle, the mass of protein eluted from the protein A column needs to be proportional to the mass loaded onto the column. The mass of the product eluted from the protein A column can be determined by integrating the area under the curve of the UV signal from the post-column UV sensor during elution. Dividing the elution mass by the volume loaded onto the column gives an estimate of the average titer of the product during loading. The complete mathematical formula used to determine the titer of the product using the elution UV method is as follows. Equation 3: TIFF2026074002000015.tif13150·Titer (g / L) = average permeate titer during the column loading period ·V load (mL) = volume of permeate loaded onto the column before elution ·OD i = the i-th measured value of the optical density at 300 nm by the Optek probe ·Δt (s) = total elapsed time during elution when the product is collected ·k colA and k colB * = constants specific to each of the two protein A columns in the continuous protein production system, determined by analysis of previous data sets. This constant is input into delta V before the start of the batch. ·n = number of time points elapsed between the start and end of collection of the eluate *For several reasons, this calculation requires the use of fitted constants rather than applying Beer's Law based on the molecular-specific extinction coefficient. First, although the UV sensor was calibrated so that A300 measurements accurately reflected A280 measurements, calibration errors were observed in previous datasets. Using fitted constants allows for the consideration of persistent calibration offsets. Furthermore, using fitted constants allows for a model that takes into account the fact that column yields are typically less than 100%. Since sensor calibrations often differ between the two columns, each column needs to be calibrated separately. By using different calibration constants for each column, the accuracy of the model predictions is improved.

[0073] Operation order: a. Before driving ik colA Value and k colB The value is determined by Pfizer / BI through analysis of previous data or from the first elution cycle using offline Protein A HPLC data. ii.k colA Value and k colB The values ​​are programmed into the operating procedure. b. While driving i. Before the system begins loading the Protein A column, manually enter the initial force value into Delta V based on offline Protein A HPLC data. ii. When the first protein A column elution occurs, Delta-V calculates its titer based on Equation 1, and then updates the current titer in Delta-V using the model-based titer. iii. The elution from each Protein A column is used to calculate the new titer, which is then used to update the current titer. iv. The user has the ability to manually overwrite the titer due to the need to manually input the force value into Delta V. If any error occurs that prevents v.DeltaV from accurately calculating the titer based on the UV model, a warning will be displayed to alert the user to the problem.

[0074] Delta V requirements c. Generating a variable to preserve the current titer prediction i. The current force value can be updated manually by user input or automatically by delta-V using an elution UV algorithm. ii. There is an option to stop the automatic updating of titers from the elution UV method. d. Two constant values ​​k are manually entered into Delta-V before the batch starts. colA and k colB Acceptance e. Recording the volume of permeate loaded onto each Protein A column after each cycle. i. The permeate loading totalizer starts when the column begins loading and stops when the load switches to another column or when the buffer begins flowing through the original column. ii. When the system switches to starting a load on another column or when the buffer begins flowing through the original column, the last value of the flow sum device is recorded in a new variable that stores the previous load volume, and the flow sum device is reset. iii. When permeate flow is sent to waste, the flow totalizer stops measuring the flow but does not reset or overwrite the previous load volume variable. f. Recording the duration of collection of protein A eluate. g. Recording absorbance values ​​at 300 nm during eluate collection. i. The measurement interval for absorbance values ​​should not exceed 10 seconds. ii. The time intervals for measuring absorbance values ​​are consistent across batches. h. Determination of the average absorbance at 300 nm across all time points obtained during the collection of protein A eluate. i. Application of Equation 1 to calculate the titer prediction after the collection of eluate is complete. i. Equation 1 is used when using elution data from column A, k colA When using k, and when using elution data from column B, colB It is calculated using [this method]. j. Updating the titer variable using predictions from Equation 1 k. Use of titer variable values ​​to control the loading period to two Protein A columns i. Once the titer variable value is overwritten, calculate the loading period using the new titer value, either manually or by other predictions in the elution UV model. l. After the operation is complete, the user will be able to access the titer prediction data. In each cycle, the user will be able to access the following data: i. Column loading volume ii. Dissolution period iii. Potency prediction

[0075] Error handling If m. elution occurs but the elution UV model cannot be applied properly for any reason, the previous titer prediction is not overwritten, and a warning that the prediction was unsuccessful is provided in Delta V. n. If the eluate collection period is less than 12 minutes, do not overwrite the previous titer prediction, and provide the user with a warning that the titer model prediction was not accurate because the eluate collection period was shorter than expected. o. If the eluate collection volume is determined to be larger than the 4.0 Protein A column volume (CV), the previous titer prediction will not be overwritten, and a warning will be provided to the user indicating that the titer model prediction was unable to accurately predict the titer because the eluate collection volume was larger than expected. The eluate collection volume in i.CV is determined by multiplying the average elution flow rate by the elution period and then dividing by the volume of the Protein A column. p. If the A300 signal at any point during eluate collection exceeds the maximum A300 value of the Optek probe, the system will not overwrite the previous titer prediction and will display a warning to alert the user that the detector has reached saturation.

[0076] Example 3: Comparative Example International Publication No. 2010151214 describes a spectroscopic method using UV sensors placed in the feed and effluent streams of a protein A capture chromatography system. Protein A chromatography exhibits high selectivity for monoclonal antibodies. During column loading, mAbs present in the feed stream bind to the column, while impurities flow through it. Therefore, the effluent stream essentially has the same composition as the feed stream, except for the presence of antibodies. UV sensors placed in the column feed and effluent streams measure the total absorbance at 280 nm. Antibody titer can be determined by subtracting the effluent absorbance from the feed absorbance and dividing by the molecular-specific extinction coefficient. However, there are several practical difficulties in using this method. Absorbance measurements in complex flows are relatively noisy, which tends to reduce the accuracy of titer predictions based on these measurements. Furthermore, UV sensors are susceptible to contamination when exposed to complex feed flows for extended periods. For illustrative purposes, this method was applied to attempt titer prediction across eight separate sequential batches, and the results are shown in Figure 2. Linear regression was used to determine the relationship between the absorbance difference between the supply and outflow streams and the actual concentration measured by Protein A HPLC. Linear interpolation was used to estimate antibody concentrations between Protein A HPLC measurements in order to create a completely continuous time series of Protein A HPLC concentrations for model calibration. Each batch was treated as a separate categorical variable in the linear regression. Model error was measured by calculating the coefficient of variation (CV(RMSE)) of the root mean square error. A CV(RMSE) of 45% was determined, indicating that this method has relatively low predictive power when applied to real-world data.

[0077] According to one embodiment, the method of this specification is implemented by one or more computer devices. Optionally, the computer-implemented program product may include one or more computer-readable storage media having computer-readable program instructions for causing a processor to execute an aspect of the method disclosed herein. In certain embodiments, a warning is incorporated into the code if the elution mass is greater than a set threshold (e.g., ~60 g / L). The elution titer is flagged and may not be considered "effective." If an ineffective titer is used in the prediction, the code may target lower loading challenges until all three previous titers result in "effective elution." The specification has described various embodiments with reference to examples. However, it is clear that various modifications and changes can be made and additional embodiments can be implemented without departing from the broader scope of the exemplary embodiments as described in the claims. The specification and drawings should therefore be considered illustrative, not restrictive.

Claims

1. A method for optimizing continuous chromatography in a continuous production system for biological products, (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply the following formula to the OD measurement value. Formula 1: and (iii) Achieving the optimization by adjusting the continuous loading volume of the chromatography column. The method comprising the above.

2. The method according to claim 1, wherein the biological product is a protein.

3. The method according to claim 1, wherein the biological product is a monoclonal antibody.

4. A method for optimizing continuous chromatography in a continuous production system for biological products, (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply the following formula to the OD measurement value. Formula 4: and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column. The method comprising the above.

5. The method according to claim 4, wherein the biological product is a protein.

6. The method according to claim 4, wherein the biological product is a monoclonal antibody.

7. The method according to any one of claims 1 to 6, wherein high-performance liquid chromatography is not used.

8. The method according to any one of claims 1 to 7, wherein the optimization includes maintaining the load density to keep it at a predetermined target load density or within a predetermined target load density range.

9. The method according to claim 8, wherein the predetermined target load density is less than approximately 65 g / L.

10. The method according to claim 1, which is performed by a computer.

11. The method according to claim 8, wherein the system enables the processing of the biological product at a maximum rate of 6 g / L / day.

12. A method for producing the target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Capturing or purifying the biological product of the target, including continuous chromatography of the fluid feed containing the biological product of the target and one or more impurities or buffering components; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The aforementioned method further (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply the following formula to the OD measurement value. Formula 1: and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column. The manufacturing method comprising the above.

13. The method for producing the biological product of the objective according to claim 12, wherein the biological product of the objective is a recombinant protein.

14. The manufacturing method according to claim 12, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed using fed-batch cell culture.

15. The manufacturing method according to claim 12, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed in continuous cell culture.

16. A method for producing the target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Capturing or purifying the biological product of the target, including continuous chromatography of the fluid feed containing the biological product of the target and one or more impurities or buffering components; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The aforementioned method further (i) Obtain optical concentration (OD) measurements of the eluate from the chromatography column; (ii) Apply the following formula to the OD measurement value. Formula 2: and (iii) Achieving the optimization by adjusting the continuous loading of the chromatography column. The manufacturing method comprising the above.

17. The method for producing the biological product of the objective according to claim 16, wherein the biological product of the objective is a recombinant protein.

18. The manufacturing method according to claim 16, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed using fed-batch cell culture.

19. The manufacturing method according to claim 16, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed in continuous cell culture.

20. The method according to claim 4, which is performed by a computer.

21. The manufacturing method according to claim 12, which is performed by a computer.

22. The manufacturing method according to claim 16, which is performed by a computer.

23. The method according to claim 8, wherein the predetermined target load density is less than approximately 100 g / L.

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