System and method for detecting failure mode in processing chromatography

Abstract

To provide a system and a method which are useful for predicting or detecting in real time, a functional failure in chromatography processing.SOLUTION: In some embodiments, the disclosure provides a system and a method for detecting an atypical profile in processing chromatogram in ion exchange chromatography of biological products.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Patent Application No. 62 / 631,167, filed February 15, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to methods for purifying biological products, including real-time detection of errors in chromatographic processes. [Background technology]

[0003] Successful and timely identification of failure modes in protein chromatography operations can be difficult not only because the failure modes are specific to each unit operation associated with the program, but also because automated detection methods are not readily available. This is especially true in large-scale manufacturing. Thus, there exists a long-felt and unmet need for in-process, scalable failure mode detection systems and methods. The present disclosure provides such novel systems and methods to address and solve this need. Summary of the Invention

[0004] The present disclosure provides systems and methods for monitoring process chromatograms in an automated or semi-automated manner. In some embodiments of the disclosed methods, the measured absorbance intensity within an integral volume window is compared to a maximum absorbance intensity estimated from at least one reference chromatogram, and the measured absorbance intensity is used to identify instabilities in the chromatographic matrix and predict and / or identify impending failures, thereby improving process performance at any scale. Thus, the present disclosure provides methods and devices useful for failure mode detection in chromatographic processes.

[0005] The present disclosure provides a method for predicting or detecting malfunctions in a chromatography process, the method including: (a) generating a reference chromatogram from a chromatography column; (b) generating a process chromatogram from the chromatography column; and (c) detecting an atypical profile in the process chromatogram, wherein the atypical profile is indicative of a potential or actual malfunction of the chromatography process.

[0006] In some embodiments of the methods of the present disclosure, the method further comprises (d) generating a warning signal.

[0007] In some embodiments of the disclosed methods, the method further comprises (e) repacking the chromatography column.

[0008] In some embodiments of the disclosed methods, the chromatography column is suitable for use in ion exchange chromatography.

[0009] In some embodiments of the disclosed methods, the chromatography column is suitable for use in affinity chromatography.

[0010] In some embodiments of the disclosed methods, the chromatography column is suitable for use in hydrophobic interaction chromatography.

[0011] In some embodiments of the disclosed methods, the potential or actual malfunction of the chromatography process is caused by column degradation, hi some embodiments, the column degradation comprises a broken column bed.

[0012] In some embodiments of the disclosed methods, the atypical profile comprises an additional peak on the process chromatogram when compared to an equivalent position on the reference chromatogram. In some embodiments, the additional peak occurs during a wash step in the chromatographic process. In some embodiments, the wash step precedes an elution step in the chromatographic process. In some embodiments, the additional peak occurs during a wash step and precedes an elution step in the chromatographic process.

[0013] In some embodiments of the disclosed methods, such as those in which the atypical profile comprises an additional peak on the process chromatogram compared to a comparable position on the reference chromatogram, the magnitude of the additional peak indicates the severity of a potential or actual malfunction of the chromatographic process. In some embodiments, an increase in the magnitude of the additional peak indicates an increase in the severity of a potential or actual malfunction of the chromatographic process. In some embodiments, the malfunction of the chromatographic process induces a loss of separation capacity of the chromatographic column.

[0014] In some embodiments of the disclosed methods, such as those in which the atypical profile comprises an additional peak on the process chromatogram compared to an equivalent position on the reference chromatogram, the atypical profile comprises at least one peak or at least one dip on the process chromatogram that has a reduced sharpness or a reduced magnitude compared to the peak or dip at the equivalent position on the reference chromatogram. In some embodiments, the at least one peak or at least one dip on the process chromatogram that has a reduced sharpness or a reduced magnitude occurs during a wash step. In some embodiments, the at least one peak or at least one dip on the process chromatogram that has a reduced sharpness or a reduced magnitude occurs after an elution step.

[0015] In some embodiments of the disclosed methods, such as those in which the atypical profile comprises an additional peak on the process chromatogram compared to an equivalent position on the reference chromatogram, the atypical profile comprises a first peak or a first dip having a reduced sharpness or a reduced magnitude on the process chromatogram compared to a peak or a dip at an equivalent first position on the reference chromatogram, and a second peak or a second dip having a reduced sharpness or a reduced magnitude on the process chromatogram compared to a peak or a dip at an equivalent second position on the reference chromatogram. In some embodiments, the first peak or the first dip occurs after the elution step. In some embodiments, the first peak or the first dip corresponds to a dip occurring at the beginning of the collection step. In some embodiments, the second peak or the second dip occurs after the collection step. In some embodiments, the second peak or the second dip comprises a strip peak.

[0016] In some embodiments of the disclosed methods, the process chromatogram includes a measured absorbance intensity, a maximum absorbance intensity, and a global volume window. In some embodiments, the atypical profile includes a measured absorbance intensity that exceeds a maximum absorbance intensity during the global volume window. In some embodiments, the measured absorbance intensity is determined at a wavelength corresponding to ultraviolet (UV) light. In some embodiments, the measured absorbance intensity is determined at wavelengths between 260 nanometers (nm) and 280 nm, inclusive. In some embodiments, the measured absorbance intensity is determined at a wavelength of 260 nm, and A 260 In some embodiments, the measured absorbance intensity is determined at a wavelength of 280 nm to provide an A 280 In some embodiments, the atypical profile comprises a measured absorbance intensity that exceeds a maximum absorbance intensity during the entire volume window, and the maximum absorbance intensity is greater than an A of at least 0.05. 260 Value or A 280In some embodiments of the disclosed methods, the standard profile includes a measured absorbance intensity that does not exceed a maximum absorbance intensity during the entire volume window, and the maximum absorbance intensity has an A value of 0.05 or less. 260 Value or A 280 It has a value.

[0017] In some embodiments of the disclosed methods, absorbance intensity values ​​of the atypical profile and / or the typical profile are measured during the elution step of a preceding column process. In some embodiments, the absorbance intensity values ​​of the atypical profile and / or the typical profile are determined at wavelengths corresponding to ultraviolet (UV) light. In some embodiments, the measured absorbance intensity is determined at wavelengths between 260 nanometers (nm) and 280 nm, inclusive. In some embodiments, the measured absorbance intensity is determined at a wavelength of 260 nm, and A 260 In some embodiments, the measured absorbance intensity is determined at a wavelength of 280 nm to provide an A 280 In some embodiments, a signal is sent after a value indicative of an atypical profile indicative of column degradation.

[0018] In some embodiments of the disclosed methods, the absorbance intensity values ​​of the atypical and / or typical profile are measured during the elution step of a preceding column process. In some embodiments, the absorbance intensity values ​​of the atypical and / or typical profile are determined by assessing the pH level of the eluate during the elution step. In some embodiments, the pH is indicative of an atypical profile because the pH value is higher than expected from a typical profile. In some embodiments, the pH is indicative of an atypical profile because the pH value is lower than expected from a typical profile. In some embodiments, a signal is transmitted after a value indicative of an atypical profile, which is indicative of column degradation.

[0019] In some embodiments of the disclosed methods, the overall volume window occurs between 1 L and 5,000 L, inclusive. In some embodiments of the disclosed methods, the overall volume window occurs between 1000 L and 3,000 L, inclusive. In some embodiments of the disclosed methods, the overall volume window occurs between 1000 L and 2500 L, inclusive. In some embodiments, the overall volume window occurs between 1250 L and 2250 L, inclusive. In some embodiments, the overall volume window occurs between 1600 L and 1800 L, inclusive. In some embodiments of the disclosed methods, the overall volume window occurs over a volume of 1 L, 10 L, 100 L, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 4000 L, 5000 L, or any number of liters therebetween.

[0020] In some embodiments of the disclosed methods, the chromatography column comprises Q Sepharose anion exchange resin.

[0021] In some embodiments of the disclosed method, the step of (a) generating a reference chromatogram includes: (1) contacting a starting composition comprising a plurality of predetermined analytes and a separation composition comprising a matrix under conditions sufficient for at least one analyte of the starting composition to reversibly bind to the matrix, whereby a chromatography column comprises the separation composition, producing an analyte-binding column and a first waste composition; (2) contacting the analyte-binding column of (1) with a wash composition, whereby only at least one analyte of the starting composition remains bound to the matrix, producing a purified analyte-binding column and a second waste composition; and (3) contacting the purified analyte-binding column of (2) with an elution composition, whereby at least one analyte of the starting composition is reversibly bound to the matrix in the elution composition. (4) recovering the eluted analyte composition; (5) simultaneously with each of steps (1) through (4), contacting each of the first waste composition, the second waste composition, and the eluted analyte composition with ultraviolet (UV) light; (6) measuring the absorbance of UV light for each of the first waste composition, the second waste composition, and the eluted analyte composition to generate an absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition; and (7) generating a chromatogram by plotting the absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition as a function of the volume of liquid passed through the chromatography column.

[0022] In some embodiments of the disclosed method, (b) generating a process chromatogram includes: (1) contacting a starting composition comprising a plurality of test analytes and a separation composition comprising a matrix under conditions sufficient for at least one analyte of the starting composition to reversibly bind to the matrix, whereby a chromatography column comprises the separation composition, producing an analyte-binding column and a first waste composition; (2) contacting the analyte-binding column of (1) with a wash composition, whereby only at least one analyte of the starting composition remains bound to the matrix, producing a purified analyte-binding column and a second waste composition; and (3) contacting the purified analyte-binding column of (2) with an elution composition, whereby at least one analyte of the starting composition is released from the matrix into the elution composition. (4) recovering the eluted analyte composition; (5) simultaneously with each of steps (1) through (4), contacting each of the first waste composition, the second waste composition, and the eluted analyte composition with ultraviolet (UV) light; (6) measuring the absorbance of UV light for each of the first waste composition, the second waste composition, and the eluted analyte composition to generate an absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition; and (7) generating a chromatogram by plotting the absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition as a function of the volume of liquid passed through the chromatography column.

[0023] In some embodiments of the disclosed methods, step (a) or (b) of generating a chromatogram comprises a contacting step (5) and a measuring step (6), and further, the contacting step (5) and the measuring step (6) are sequential. In some embodiments, the contacting step (5) and the measuring step (6) are simultaneous, sequential, or alternating and sequential.

[0024] In some embodiments of the disclosed methods, the chromatography column comprises a matrix, and the matrix comprises a resin.

[0025] In some embodiments of the methods of the present disclosure, particularly with respect to step (a) or (b) of generating a chromatogram, the test analyte is a biological composition. In some embodiments, the biological composition is a therapeutic composition for use in a method of treating a human subject. In some embodiments, the biological composition comprises a therapeutic protein.

[0026] In some embodiments of the disclosed methods, the warning signal is electronically transmitted to a processor, and the processor is operably linked to the chromatography column. In some embodiments, the processor receives the warning signal and halts the chromatography process or prevents the initiation of further chromatography processes until the potential or actual malfunction of the chromatography column is resolved.

[0027] Additional aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the reference chromatogram, which is the historical standard. [Figure 2] FIG. 2 shows another reference chromatogram. [Figure 3] FIG. 3 depicts a chromatogram showing an irregular wash 2 peak. [Figure 4] FIG. 4 depicts a chromatogram showing an erratic wash 2 peak, an erratic elution peak, and loss of sharpness in the strip peak. [Figure 5] Figure 5 shows the failure chromatogram. The Wash 2 peak is present. [Figure 6] Figure 6 shows a comparison of typical and atypical VEGF-Trap Q chromatograms. The majority of the atypical UV traces have an absorbance > 0.05 au at 635 liters into wash 2. [Figure 7]FIG. 7 depicts an example of an additional atypical VEGF-Trap Q chromatogram, namely, the Wash 2 peak. [Figure 8] Figure 8 shows an additional typical VEGF-Trap Q chromatogram. The UV trace does not exceed an absorbance of 0.05 in 635 liters into wash 2. DETAILED DESCRIPTION OF THE INVENTION

[0029] Successful and timely identification of failure modes in protein chromatography operations can be difficult, especially in large-scale manufacturing, not only because the failure modes are specific to each unit operation associated with the program, but also because, in the absence of the compositions and methods of the present disclosure, automated detection methods are not readily available.

[0030] The present disclosure provides a novel watch system and method for failure mode detection. This new watch system and method for chromatography column monitoring is intended to alert an operator when a column is not performing its intended function. The disclosed watch system and method automatically monitors system signals at global time points to determine whether the column is functioning optimally. If the system does not meet acceptance criteria, a message is displayed to the operator instructing them to add the event to a quality control system for further investigation and potentially remediation. The monitored signal, significant signal intensity, and overall time window are all empirically determined for each specific chromatography unit operation to be monitored.

[0031] Chromatography The disclosed systems and methods may be used for preparative or analytical chromatography. In some embodiments, the disclosed methods are used to purify proteins that can be used in compositions for treating diseases. For methods involving preparative chromatography, particularly for large-scale or large-volume purification of therapeutic proteins, efficiency of the purification process is essential. The disclosed systems and methods may be used at any scale. However, they are particularly useful when large numbers of preparations are being processed and when a malfunctioning system (e.g., a chromatography column through which a valuable analyte passes through a separation composition when it should be captured) results in the loss of valuable product and valuable production time. The disclosed systems and methods predict and detect malfunctions in chromatography processes in real time, identifying the malfunctioning process and allowing for recovery of function and efficient and reliable purification before additional work is performed using the malfunctioning process.

[0032] The present disclosure encompasses any form of chromatography. As a technique, chromatography is a method for separating components from a mixture. Separation is achieved through the partitioning of a mobile phase and a stationary phase. As described herein, the starting composition, which includes the test analyte and is optionally also preparative, belongs to the mobile phase. As described herein, the separation composition, which includes the matrix, belongs to the stationary phase. Exemplary matrices of the present disclosure may include any material capable of separating elements of the mobile phase based on chemical properties (charge) and / or physical properties (size).

[0033] When used in some embodiments of the present disclosure, such as methods for preparing therapeutic proteins, chromatographic processes can include, but are not limited to, column and planar chromatography. In some preferred embodiments, chromatographic processes can include, but are not limited to, column chromatography.

[0034] Column chromatography The present disclosure encompasses any form of column chromatography, including wet and dry methods. In dry methods, the separation composition comprises a dry matrix, which is then contacted with a liquid starting composition, optimally wetting the entire matrix and allowing the matrix to not dry out until the end of the entire process. In wet methods, the separation composition comprises a suspension or slurry of matrix and eluent, which is introduced into a column and then contacted with the liquid starting composition.

[0035] Ion exchange chromatography In some embodiments of the present disclosure, the separation composition may comprise any material capable of separating elements of a mobile phase based on charge. For example, the separation composition may comprise a matrix that reversibly binds charged protein analytes from the starting composition. Thus, the separation matrix may be suitable for anion or cation exchange chromatography, and may comprise a matrix having a net negative or positive charge, respectively. In some embodiments of the present disclosure, the separation composition comprises a matrix comprising Sepharose or Q Sepharose. In some embodiments of the present disclosure, the chromatography column is a Q column.

[0036] Affinity chromatography In some embodiments of the present disclosure, a separation composition may comprise any material capable of separating elements of a mobile phase based on specific interactions between binding partners, including, but not limited to, antigen / antibody binding partners, enzyme / substrate binding partners, receptor / ligand binding partners, protein / protein binding partners, protein / nucleic acid binding partners, and nucleic acid / nucleic acid binding partners. For example, a separation composition may comprise a matrix that reversibly binds one of the binding partners (e.g., an analyte from the starting composition) by including the other of the binding partners. By way of example, a separation composition may comprise a matrix containing an antigen to which an analyte from the starting composition specifically binds, selectively purifying the antibody analyte from the starting material. As a further example, a separation composition may comprise a matrix containing an epitope of the VEGF protein to which VEGF-Trap selectively binds, selectively purifying the VEGF-Trap analyte from the starting material.

[0037] Hydrophobic Interaction Chromatography (HIC) In some embodiments of the present disclosure, the separation composition may comprise any material capable of separating components of a mobile phase based on their relative hydrophobicity. HIC may be used to selectively purify protein analytes while maintaining their biological activity as a result of the use of conditions and matrices that operate under less denaturing conditions. In some embodiments, the analytes of the present disclosure, which contain hydrophobic and hydrophilic regions, are contacted with the HIC column in a high-salt buffer. The salt in the buffer reduces solvation of the analyte solutes in the starting material. As solvation decreases, the exposed hydrophobic regions are adsorbed by the medium. The more hydrophobic the molecules in the starting material, the less salt is required to promote binding. To increase hydrophobicity, the sample may be eluted from the column using a decreasing salt gradient. The elution buffer of the present disclosure may also contain a mild organic modifier or a mild detergent.

[0038] Size Exclusion Chromatography (SEC) In some embodiments of the present disclosure, the separation composition may comprise any material capable of separating components of a mobile phase based on their relative size, or in some cases, their relative molecular weight. SEC may be used to selectively purify large molecules or macromolecular complexes, such as proteins and polymers. In some embodiments, e.g., in which an aqueous solution is used to transport the mobile phase through the column, the technique is known as gel filtration chromatography. In some embodiments, e.g., in which an organic solvent is used as the mobile phase through the column, the technique is known as gel permeation chromatography. In some embodiments, the chromatography column is packed with fine, porous beads composed of, e.g., dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore size of these beads is used to estimate the size of the macromolecules. Gel filtration chromatography can be used to fractionate protein analytes and other water-soluble polymers, and gel permeation chromatography can be used to fractionate organic-soluble polymers based on their molecular weight distribution.

[0039] High-performance liquid chromatography (HPLC) In some embodiments of the present disclosure, the separation composition may include any material capable of separating elements of a mobile phase based on the interaction of the separation composition with a sorbent material, which causes different flow rates for different components, leading to their separation as they exit the column. Analytes in the mobile phase can be separated, for example, through hydrophobic, ionic, or dipole-dipole interactions. HPLC relies on a pump to pass a pressurized liquid solvent containing the mobile phase through a column packed with a solid sorbent material. In some embodiments, the active component of the column, the sorbent, comprises a granular material made from solid particles (e.g., silica or polymer). Exemplary particle sizes include particles between 2 and 50 microns, which can provide excellent resolving power for HPLC chromatography. In some embodiments, the pressurized mobile phase is a mixture of solvents such as water, acetonitrile, and / or methanol. In some embodiments, the HPLC pump mixes multiple solvents in ratios that vary over time to generate a gradient composition in the mobile phase. In some embodiments, the aqueous component of the mobile phase contains an acid (e.g., formic acid, phosphoric acid, or trifluoroacetic acid) or a salt to aid in the separation of sample components. In some embodiments, HPLC comprises partition chromatography, normal phase chromatography, displacement chromatography, reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, hydropobic interaction chromatography, or aqueous normal phase chromatography.

[0040] Fast Protein Liquid Chromatography (FPLC) In some embodiments of the present disclosure, the separation composition may comprise any material capable of separating components of a mobile phase based on their different affinities for the separation composition. Fast protein liquid chromatography (FPLC) is a form of liquid chromatography used to separate mixtures of protein analytes. In some embodiments, separation is possible because different components of the mobile phase have different affinities for the mobile phase and the stationary phase (separation composition). In some embodiments, the mobile phase is an aqueous solution or a buffer. The buffer flow rate can be controlled by a positive displacement pump. In some embodiments, the buffer flow rate is constant and the buffer composition is varied. In some embodiments, the stationary phase is a resin composed of beads, e.g., agarose beads. One skilled in the art can vary the bead size and the surface ligands of the stationary phase depending on the mobile phase and analyte of a particular application.

[0041] In some embodiments, FPLC involves ion exchange chromatography. For example, a stationary phase resin binds to protein analytes through charge interactions in a first buffer (running buffer) but dissociates in a second buffer (elution buffer). A mixture containing one or more protein analytes is dissolved in the first buffer and pumped onto the column. The proteins of interest bind to the resin, while other components are carried in the first buffer. The total buffer flow rate is kept constant. Over time, the percentage of elution buffer is gradually increased from 0% to 100% according to a programmed change in concentration to generate a gradient. At some point along the gradient, each of the bound protein analytes dissociates and appears in the eluate. The eluate then passes through a detector. An exemplary detector can measure the salt concentration of the eluate (via conductivity) and the protein concentration of the eluate (via ultraviolet light absorption).

[0042] Pressurization In some embodiments of the disclosed methods, the flow of the mobile phase through the stationary phase is controlled by gravity. In some embodiments, gravity chromatography is used for single-step purification, such as desalting and affinity applications. A typical gravity column includes an upper buffer reservoir element operably connected to a chromatography column with an outlet at the bottom. Flow is controlled using a stopcock or tube pincher between the buffer reservoir and the column. A flow adapter can be used to minimize sample loading variability. In some embodiments, gravity chromatography includes partition chromatography, normal-phase chromatography, displacement chromatography, reversed-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, or aqueous normal-phase chromatography.

[0043] In some embodiments of the disclosed method, the chromatography comprises low-pressure chromatography. In some embodiments, the low-pressure chromatography system is a system that operates at a pressure of less than 50 psi (about 3 bar). Low-pressure chromatography can be used in protein separations that do not require high resolution.

[0044] In some embodiments of the disclosed methods, the chromatography comprises medium-pressure chromatography. In some embodiments, the medium-pressure chromatography system is a system operating at pressures up to 3,500 psi (24 MPa). The medium-pressure chromatography system generates sufficient pressure to accommodate higher resolution stationary phase compositions, for example, using 5-15 μm beads. In some embodiments, the medium-pressure chromatography comprises partition chromatography, normal-phase chromatography, displacement chromatography, reversed-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, or aqueous normal-phase chromatography.

[0045] In some embodiments of the disclosed methods, the chromatography comprises high pressure chromatography, for example, HPLC.

[0046] mobile phase The present disclosure encompasses any form of mobile phase, including, but not limited to, elution solutions, solvents, sterile solvents, and pharmaceutically acceptable carriers. The elution compositions of the present disclosure may include a pharmaceutically acceptable carrier.

[0047] stationary phase The present disclosure encompasses any form of stationary phase, including, but not limited to, silica, alumina (Al2O3), cellulose, Sepharose, and Q Sepharose. Exemplary separation compositions of the present disclosure may comprise a matrix, a polymer, a resin, a protein, or a combination thereof. In some embodiments, the separation composition comprises Sepharose or Q Sepharose.

[0048] chromatogram As used herein, a chromatogram is the visual output of a chromatograph. Typically, the retention time and / or flow volume of the eluent is represented on the x-axis of the chromatogram, and the signal is plotted on the y-axis. Exemplary signals include, but are not limited to, electrical conductance, absorbance, and mass spectrometry, and correspond to the detector response to the analytes exiting the chromatograph. In some embodiments, the signal is proportional to the concentration of the specific analytes being separated by the chromatograph.

[0049] In one exemplary embodiment, protein concentration, as measured by light absorption, is plotted on the y-axis of the chromatogram. Many proteins in solution absorb light, e.g., ultraviolet light at a wavelength of 280 nm, which can be used to calculate the protein concentration in a sample. Depending on the protein, additional wavelengths of light are contemplated. For example, proteins containing heme groups or fluorophores can be detected using different wavelengths of light. One skilled in the art can select a suitable detection system for a particular analyte.

[0050] Biological samples In some embodiments of the disclosed methods, the analyte comprises a protein, a peptide, a nucleic acid, or a virus. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the virus is an adenovirus, an adeno-associated virus, or a lentivirus. [Example]

[0051] Example 1: Purification of VEGF Trap Using VEGF-Trap as an exemplary analyte for purification, Figures 1-8 show both exemplary reference chromatograms (Figures 1 and 2) as well as exemplary process chromatograms with atypical profiles (Figures 3-5).

[0052] Briefly, the chromatograms in Figures 1-8 were generated by contacting a starting composition containing multiple analytes (VEGF-Trap analytes as well as multiple contaminants (e.g., host cell proteins, DNA, and poorly sialylated proteins)) and a separation composition containing a matrix (in this example, Q Sepharose) under conditions sufficient for at least one VEGF-Trap protein of the starting composition to reversibly bind to the matrix, whereby a chromatography column containing the separation composition was generated, producing an analyte-bound column and a first waste composition. The analyte-bound column was then contacted with a first wash composition, whereby the VEGF-Trap protein of the starting composition remained bound to the matrix, producing a purified analyte-bound column and a second waste composition. The analyte-bound column was then contacted with a second wash composition, whereby the VEGF-Trap protein of the starting composition remained bound to the matrix, producing a purified analyte-bound column and a third waste composition, in reference chromatograms showing a typical profile. To generate a process chromatogram exhibiting an atypical profile, the analyte binding column and the second wash composition are contacted, and in the process chromatogram exhibiting the atypical profile, only the VEGF-Trap protein of the starting composition remains bound to the matrix, producing a purified analyte binding column and a third waste composition, which contains contaminants or VEGF-Trap protein. In both the reference chromatogram and the process chromatogram, the purified analyte binding column and the elution composition are contacted, and at least one analyte of the starting composition is released from the matrix into the elution composition, producing an eluted analyte composition. In both the reference chromatogram and the process chromatogram, the eluted analyte composition is collected. Simultaneously with each of the above, the first waste composition, the second waste composition, the third waste composition, and the eluted analyte composition are each contacted with ultraviolet (UV) light.The UV light absorbance of each of the first waste composition, the second waste composition, the third waste composition, and the eluted analyte composition is measured simultaneously or sequentially to generate an absorbance value for each of the first waste composition, the second waste composition, the third waste composition, and the eluted analyte composition. The absorbance values ​​for each of the first waste composition, the second waste composition, the third waste composition, and the eluted analyte composition are plotted as a function of the volume of liquid passing through the chromatography column to generate a chromatogram. The chromatogram is generated in real time with the chromatography process, and the presence of an atypical profile is determined when additional peaks appear on the chromatogram. The appearance of an atypical profile causes the detector and / or processor to send a warning signal to a processor operably linked to the chromatography column, which prevents further chromatography processing until the column malfunction can be resolved.

Claims

1. 1. A method for predicting or detecting malfunction in a chromatography process, comprising: (a) generating a reference chromatogram from a chromatography column; (b) generating a process chromatogram from the chromatography column; and (c) detecting atypical profiles in the process chromatogram; Including, the atypical profile is indicative of a potential or actual malfunction of the chromatography process; method.

2. 10. The method of claim 1, further comprising: (d) generating a warning signal.

3. 3. The method of claim 2, further comprising: (e) repacking the chromatography column.

4. The method of any one of claims 1 to 3, wherein the chromatography column is suitable for use in ion exchange chromatography.

5. The method of any one of claims 1 to 3, wherein the chromatography column is suitable for use in affinity chromatography.

6. The method of any one of claims 1 to 3, wherein the chromatography column is suitable for use in hydrophobic interaction chromatography.

7. The method of any one of claims 1 to 6, wherein the potential or actual malfunction of the chromatography process is caused by column degradation.

8. The method of claim 7, wherein the column deterioration comprises a break in the column bed.

9. 9. The method of any one of claims 1 to 8, wherein the atypical profile comprises additional peaks on the process chromatogram compared to equivalent positions on the reference chromatogram.

10. 10. The method of claim 9, wherein the additional peak occurs during a wash step in the chromatography process.

11. 11. The method of claim 10, wherein the washing step precedes an elution step in the chromatography process.

12. 12. The method of claim 11, wherein the additional peak occurs during the wash step and precedes the elution step in the chromatography process.

13. 13. The method of any one of claims 9 to 12, wherein the magnitude of the additional peak indicates the severity of the potential or actual malfunction of the chromatographic process.

14. 14. The method of claim 13, wherein an increase in the magnitude of the additional peak indicates an increase in the severity of the potential or actual malfunction of the chromatographic process.

15. The method according to any one of claims 9 to 14, wherein the malfunction of the chromatography process induces a loss of separation capacity of the chromatography column.

16. 9. The method of any one of claims 1 to 8, wherein the atypical profile comprises at least one peak or at least one dip on the process chromatogram that has a reduced sharpness or a reduced magnitude when compared to a peak or dip at an equivalent position on the reference chromatogram.

17. 17. The method of claim 16, wherein the at least one peak or the at least one dip on the process chromatogram having a reduced sharpness or a reduced magnitude occurs during the washing step.

18. 18. The method of claim 16 or 17, wherein the at least one peak or the at least one dip on the process chromatogram having a reduced sharpness or a reduced magnitude occurs after the elution step.

19. 9. The method of claim 1, wherein the atypical profile comprises a first peak or a first dip having a reduced sharpness or a reduced magnitude on the process chromatogram when compared to a peak or dip at an equivalent first position on the reference chromatogram, and a second peak or a second dip having a reduced sharpness or a reduced magnitude on the process chromatogram when compared to a peak or dip at an equivalent second position on the reference chromatogram.

20. 20. The method of claim 19, wherein the first peak or the first dip occurs after the elution step.

21. 21. The method of claim 20, wherein the first peak or the first dip corresponds to a dip occurring at the beginning of a recovery step.

22. 22. The method of any one of claims 19 to 21, wherein the second peak or the second depression occurs after a recovery step.

23. The method of claim 22 , wherein the second peak or the second depression comprises a strip peak.

24. The method of any one of claims 1 to 23, wherein the process chromatogram comprises measured absorbance intensities, maximum absorbance intensities, and overall volume windows.

25. 25. The method of claim 24, wherein the atypical profile comprises a measured absorbance intensity that exceeds a maximum absorbance intensity during an entire volume window.

26. 26. The method of claim 24 or 25, wherein the measured absorbance intensity is determined at a wavelength corresponding to ultraviolet (UV) light.

27. 27. The method of claim 26, wherein the measured absorbance intensity is determined at wavelengths between 260 nanometers (nm) and 280 nm, inclusive.

28. The measured absorbance intensity is determined at a wavelength of 260 nm, and A 260 27. The method of claim 26, further comprising providing a value.

29. The measured absorbance intensity is determined at a wavelength of 280 nm, and A 280 27. The method of claim 26, further comprising providing a value.

30. The atypical profile includes a measured absorbance intensity that exceeds a maximum absorbance intensity during an entire volume window, and the maximum absorbance intensity is greater than an A of at least 0.

05. 260 Value or A 280 30. The method of claim 28 or 29, having a value.

31. The typical profile includes a measured absorbance intensity that does not exceed a maximum absorbance intensity during the entire volume window, and the maximum absorbance intensity is less than or equal to A of 0.

05. 260 Value or A 280 The method of any one of claims 1 to 30, having a value.

32. 32. The method of any one of claims 1 to 31, wherein the global volume window occurs between 1000 L and 2500 L, inclusive of endpoints.

33. 33. The method of claim 32, wherein the global volume window occurs between 1250L and 2250L, inclusive.

34. 33. The method of claim 32, wherein the global volume window occurs between 1600L and 1800L inclusive.

35. 35. The method of any one of claims 1 to 34, wherein the chromatography column comprises Q Sepharose anion exchange resin.

36. (a) the step of generating the reference chromatogram comprises: (1) contacting a starting composition comprising a plurality of predetermined analytes and a separation composition comprising a matrix under conditions sufficient to reversibly bind at least one analyte of said starting composition to said matrix, said chromatography column comprising said separation composition, to produce an analyte-bound column and a first waste composition; (2) contacting the analyte-binding column of (1) with a wash composition, so that only the at least one analyte of the starting composition remains bound to the matrix, producing a purified analyte-binding column and a second waste composition; (3) contacting the purified analyte binding column of (2) and an elution composition, wherein the at least one analyte of the starting composition is released from the matrix into the elution composition to produce an eluted analyte composition; (4) recovering the eluted analyte composition; (5) simultaneously with each of steps (1) through (4), contacting each of the first waste composition, the second waste composition, and the eluted analyte composition with ultraviolet (UV) light; (6) measuring the absorbance of the UV light for each of the first waste composition, the second waste composition, and the eluted analyte composition to generate an absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition; and (7) generating a chromatogram by plotting the absorbance values ​​of each of the first waste composition, the second waste composition, and the eluted analyte composition as a function of the volume of liquid passed through the chromatography column; The method of any one of claims 1 to 35, comprising:

37. (b) generating the process chromatogram comprises: (1) contacting a starting composition comprising a plurality of test analytes and a separation composition comprising a matrix under conditions sufficient to reversibly bind at least one analyte of the starting composition to the matrix, wherein the chromatography column comprises the separation composition, producing an analyte-bound column and a first waste composition; (2) contacting the analyte-binding column of (1) with a wash composition, so that only the at least one analyte of the starting composition remains bound to the matrix, producing a purified analyte-binding column and a second waste composition; (3) contacting the purified analyte binding column of (2) and an elution composition, wherein the at least one analyte of the starting composition is released from the matrix into the elution composition to produce an eluted analyte composition; (4) recovering the eluted analyte composition; (5) simultaneously with each of steps (1) through (4), contacting each of the first waste composition, the second waste composition, and the eluted analyte composition with ultraviolet (UV) light; (6) measuring the absorbance of the UV light for each of the first waste composition, the second waste composition, and the eluted analyte composition to generate an absorbance value for each of the first waste composition, the second waste composition, and the eluted analyte composition; and (7) generating a chromatogram by plotting the absorbance values ​​of each of the first waste composition, the second waste composition, and the eluted analyte composition as a function of the volume of liquid passed through the chromatography column; The method of any one of claims 1 to 35, comprising:

38. 38. The method of claim 36 or 37, wherein the contacting step (5) and measuring step (6) are sequential.

39. The method of any one of claims 36 to 38, wherein the matrix comprises a resin.

40. The method of any one of claims 36 to 38, wherein the test analyte is a biological composition.

41. 41. The method of claim 40, wherein the biological composition is a therapeutic composition for use in a method of treating a human subject.

42. 42. The method of any one of claims 2 to 41, wherein the warning signal is transmitted electronically to a processor, and the processor is operably linked to the chromatography column.

43. 43. The method of claim 42, wherein the processor receives the warning signal and halts the chromatography process or prevents the initiation of further chromatography processes until the potential or actual malfunction of the chromatography column is resolved.

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