Controlling purification of macromolecular solutions via real-time multi-angle light scattering

Abstract

The present disclosure describes a computer-implemented method, system, and computer program product for controlling the purification of a macromolecular solution via real-time multi-angle light scattering.

Description

[0001] Related Applications

[0002] This application claims the benefit of the earlier filing date of U.S. patent application serial number 17 / 855,791, filed on June 30, 2022, and entitled “CONTROLLING THE PURIFICATION OF A MACROMOLECULE SOLUTION VIAREAL-TIME MULTI-ANGLE LIGHT SCATTERING,” the entire contents of which are incorporated herein by reference. Background Art

[0003] The present disclosure relates to multi-angle light scattering, and more particularly to controlled purification of macromolecular solutions via real-time multi-angle light scattering. Summary of the invention

[0004] The present disclosure describes a computer-implemented method, system, and computer program product for controlling the purification of a macromolecular solution via real-time multi-angle light scattering. In an exemplary embodiment, the computer-implemented method, system, and computer program product include (1) receiving, by a computer system, a baseline multi-angle light scattering (MALS) signal value and a baseline ultraviolet (UV) signal value of a pure buffer solution flowing from a chromatographic purification system to the set of instruments from a set of instruments, wherein the set of instruments includes a MALS instrument and a UV detector, (2) in response to receiving the baseline MALS signal value from the MALS instrument, (a) receiving, by the computer system, from the MALS instrument, scattering intensity values ​​of a sample solution flowing from the chromatographic purification system to the MALS instrument in a time series, wherein the sample solution contains at least one type of macromolecule, (b) calculating, by the computer system, an average value of the baseline MALS signal value, and (c) subtracting, by the computer system, the average value of the baseline MALS signal value from the scattering intensity value to obtain an intensity time series of excess scattering intensity values ​​Iscatt. j, (3) in response to receiving the baseline UV signal value from the UV detector, (a) receiving, by the computer system, UV absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in a time series, (b) calculating, by the computer system, an average value of the baseline UV signal value, (c) performing, by the computer system, a set of logical operations that applies a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​in the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​in the time series, and (d) subtracting, by the computer system, the average value of the baseline UV signal value from the corrected UV absorption values ​​to obtain a UV time series of excess UV absorption values, (4) calculating, by the computer system, a concentration value c of the sample solution relative to the UV time series of excess UV absorption values ​​according to Beer's law j and an absorption coefficient of the sample solution, (5) receiving, by a computer system, pH values ​​of the sample solution over a time series from a pH detector, and receiving conductivity values ​​of the sample solution over a time series from a conductivity detector, wherein the set of instruments further comprises a pH detector and a conductivity detector, (6) performing, by a computer system, a set of logical operations, the set of logical operations (a) applying a pH calibration correction relative to a pH calibration parameter to the pH values ​​over a time series associated with the pH detector and the MALS instrument to obtain corrected pH values ​​over a time series, and (b) applying a conductivity calibration correction relative to a conductivity calibration parameter to the conductivity values ​​over a time series associated with the conductivity detector and the MALS instrument to obtain corrected conductivity values ​​over a time series, obtaining an intensity time series Iscatt including excess scattering intensity values. j , the concentration value of the sample solution c j , a plurality of signals of the corrected pH value and the corrected conductivity value, (7) a set of logical operations is performed by the computer system, the set of logical operations being performed within the time range t 1 ,t 2 ,……t n averaging each of the plurality of signals, wherein the time series includes a time range, wherein the time range corresponds to a fraction of the sample solution collected by a fraction collector connected to the set of instruments, to obtain an average value [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n (8) receiving the aggregation content value % agg of the fraction of the sample solution measured by the SEC-MALS instrument by the computer system 1 、%agg 2 、……%agg nand weight-average molar mass value M 1 、M 2 , ...M n , and (9) the computer system converts the aggregate content value %agg 1 、%agg 2 、……%agg n and weight-average molar mass value M 1 、M 2 , ...M n Relative to the mean [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n Stored in the Macromolecular Characterization Lookup (MCLUT). BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1A An apparatus according to an embodiment is depicted.

[0006] Figure 1B An apparatus according to an embodiment is depicted.

[0007] Figure 1C An apparatus according to an embodiment is depicted.

[0008] Figure 2A A flow chart according to an exemplary embodiment is depicted.

[0009] Figure 2B A flow chart according to an exemplary embodiment is depicted.

[0010] Figure 2C A flow chart according to an exemplary embodiment is depicted.

[0011] Figure 3 Time according to the implementation scheme is depicted.

[0012] Figure 4 Time according to the implementation scheme is depicted.

[0013] Figure 5 A computer system according to an exemplary embodiment is depicted. DETAILED DESCRIPTION

[0014] The present disclosure describes a set of computer-implemented methods, systems, and computer program products for controlling the purification of macromolecular solutions via real-time multi-angle light scattering. In an exemplary embodiment, the computer-implemented methods, systems, and computer program products include (1) receiving, by a computer system, baseline multi-angle light scattering (MALS) signal values ​​and baseline ultraviolet (UV) signal values ​​of a pure buffer solution flowing from a chromatographic purification system to the set of instruments from a set of instruments, wherein the set of instruments includes a MALS instrument and a UV detector, (2) in response to receiving the baseline MALS signal values ​​from the MALS instrument, (a) receiving, by the computer system, scattering intensity values ​​of a sample solution flowing from the chromatographic purification system to the MALS instrument in a time series from the MALS instrument, wherein the sample solution contains at least one type of macromolecule, (b) calculating, by the computer system, an average value of the baseline MALS signal values, and (c) subtracting, by the computer system, the average value of the baseline MALS signal values ​​from the scattering intensity values ​​to obtain an intensity time series of excess scattering intensity values ​​Iscatt j , (3) in response to receiving the baseline UV signal value from the UV detector, (a) receiving, by the computer system, UV absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in a time series, (b) calculating, by the computer system, an average value of the baseline UV signal value, (c) performing, by the computer system, a set of logical operations that applies a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​in the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​in the time series, and (d) subtracting, by the computer system, the average value of the baseline UV signal value from the corrected UV absorption values ​​to obtain a UV time series of excess UV absorption values, (4) calculating, by the computer system, a concentration value c of the sample solution relative to the UV time series of excess UV absorption values ​​according to Beer's law j and an absorption coefficient of the sample solution, (5) receiving, by a computer system, pH values ​​of the sample solution over a time series from a pH detector, and receiving conductivity values ​​of the sample solution over a time series from a conductivity detector, wherein the set of instruments further comprises a pH detector and a conductivity detector, (6) performing, by a computer system, a set of logical operations, the set of logical operations (a) applying a pH calibration correction relative to a pH calibration parameter to the pH values ​​over a time series associated with the pH detector and the MALS instrument to obtain corrected pH values ​​over a time series, and (b) applying a conductivity calibration correction relative to a conductivity calibration parameter to the conductivity values ​​over a time series associated with the conductivity detector and the MALS instrument to obtain corrected conductivity values ​​over a time series, obtaining an intensity time series Iscatt including excess scattering intensity values. j , the concentration value of the sample solution c j, a plurality of signals of the corrected pH value and the corrected conductivity value, (7) a set of logical operations is performed by the computer system, the set of logical operations being performed within the time range t 1 ,t 2 ,……t n averaging each of the plurality of signals, wherein the time series includes a time range, wherein the time range corresponds to a fraction of the sample solution collected by a fraction collector connected to the set of instruments, to obtain an average value [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n (8) receiving the aggregation content value % agg of the fraction of the sample solution measured by the SEC-MALS instrument by the computer system 1 、%agg 2 、……%agg n and weight-average molar mass value M 1 、M 2 , ...M n , and (9) the computer system converts the aggregate content value %agg 1 、%agg 2 、……%agg n and weight-average molar mass value M 1 、M 2 , ...M n Relative to the mean [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n Stored in a Macromolecule Characterization Lookup Table (MCLUT). In one embodiment, the absorption coefficient of the sample solution is a property of the sample solution. In one embodiment, the absorption coefficient is received from a user input.

[0015] In one embodiment, the method, system and computer program product improves the chromatographic purification of monoclonal antibodies and other protein-based biopharmaceuticals with real-time MALS by overcoming the barrier of elution of identified aggregated proteins that must be separated from monomeric proteins. In one embodiment, the method, system and computer program product diverts process streams to a "sump" when aggregation levels are low enough and to a "waste" when aggregation levels are not low enough.

[0016] In one embodiment, the method, system and computer program product utilizes RT-MALS and additional real-time measurements to address the need for online and real-time determination of the presence of aggregates in the eluate in order to trigger optimal monomer collection in the pool. In one embodiment, the method, system and computer program product address the complexities and confounding factors caused by varying protein concentrations and buffer conditions.

[0017] definition

[0018] Particles

[0019] Particles can be components of aliquots of liquid samples. Such particles can be molecules, nanoparticles, virus-like particles, liposomes, emulsions, bacteria, and colloids of different types and sizes. The size range of these particles can be on the order of nanometers to micrometers.

[0020] Analysis of macromolecules or particulate matter in solution

[0021] Analysis of macromolecules or particulate matter in solution can be achieved by preparing the sample in an appropriate solvent and then injecting an aliquot of it into a separation system, such as a liquid chromatography (LC) column or a field flow fractionation (FFF) channel, where the different particulate matter contained in the sample is separated into their various components. Once separated, the sample can be analyzed by light scattering, refractive index, UV absorbance, electrophoretic mobility, and viscosity response, usually based on size, mass, or column affinity.

[0022] Light Scattering

[0023] Light scattering (LS) is a non-invasive technique used to characterize macromolecules and various particles in solution. Two types of light scattering assays commonly used to characterize macromolecules are static light scattering and dynamic light scattering.

[0024] Dynamic Light Scattering

[0025] Dynamic light scattering is also known as quasi-elastic light scattering (QELS) and photon correlation spectroscopy (PCS). In a DLS experiment, a fast photodetector is used to measure the time-varying fluctuations in the scattered light signal. DLS measurements determine the diffusion coefficients of molecules or particles, which in turn can be used to calculate their hydrodynamic radius.

[0026] Static Light Scattering

[0027] Static light scattering (SLS) includes a variety of techniques, such as single angle light scattering (SALS), dual angle light scattering (DALS), low angle light scattering (LALS), and multi-angle light scattering (MALS). SLS experiments typically involve measuring the absolute intensity of light scattered from a sample in solution, which is illuminated by a thin beam of light. Such measurements are typically used for particles / molecules of the appropriate class to determine the size and structure of the sample molecules or particles, and when combined with knowledge of the sample concentration, to determine the weight average molar mass. In addition, the nonlinearity of the scattered light intensity as a function of sample concentration can be used to measure particle-particle interactions and associations.

[0028] Multi-angle light scattering

[0029] Multi-angle light scattering (MALS) is an SLS technique for measuring light scattered from a sample into multiple angles. It is used to determine the absolute molar mass and average size of molecules in solution by detecting how they scatter light. Most commonly this is done with collimated light from a laser source, in which case the technique may be referred to as multi-angle laser light scattering (MALLS). The term "multi-angle" refers to detecting the scattered light at different discrete angles, such as by measuring with a single detector that moves over a range that includes a particular angle of choice, or with an array of detectors that are fixed at specific angular positions.

[0030] MALS measurements require a set of auxiliary elements. The most important of these is a collimated or focused light beam (usually from a laser source that produces a collimated beam of monochromatic light) that illuminates the sample area. The light beam is usually plane polarized light perpendicular to the measurement plane, but other polarizations can also be used, especially when studying anisotropic particles. Another required element is an optical cell to hold the sample to be measured. Alternatively, a cell combined with a device allowing the measurement of flowing samples can be used. If single particle scattering properties are to be measured, a method must be provided to introduce such particles one at a time via the light beam at points approximately equidistant from the surrounding detectors.

[0031] Although most MALS-based measurements are made in a plane containing a set of detectors, typically placed equidistant from a centrally located sample through which the illumination beam passes, three-dimensional versions have been developed in which the detectors are located on the surface of a sphere with the sample controlled to pass through its center where it intersects the path of the incident beam passing along the diameter of the sphere. MALS techniques typically collect multiplexed data sequentially from the outputs of a set of discrete detectors. MALS light scattering photometers typically have multiple detectors.

[0032] Because different detectors in a MALS detector (i) may have slightly different quantum efficiencies and different gains, and (ii) may look into different geometric scattering volumes, it may be necessary to normalize the signal captured at each angle by the photodetectors of the MALS detector. Without normalizing for these differences, the MALS detector results may be meaningless and may inappropriately weight different detector angles.

[0033] Concentration detector

[0034] Differential Refractive Index Detector

[0035] A differential refractive index detector (dRI) or differential refractometer or refractive index detector (RI or RID) is a detector that measures the refractive index of an analyte relative to a solvent. They are commonly used as detectors for high performance liquid chromatography and size exclusion chromatography. dRIs are considered universal detectors because they can detect any substance with a different refractive index than the solvent, but they have low sensitivity. When light leaves one material and enters another, it bends or refracts. The refractive index of a material is a measure of how much light bends when it enters.

[0036] A differential refractive index detector includes a flow cell having two sections: one for the sample; and one for the reference solvent. dRI measures the refractive index of two components. When only the solvent passes through the sample component, the measured refractive index of the two components is the same, but when the analyte passes through the flow cell, the two measured refractive indices are different. The difference appears as a peak in the chromatogram. A differential refractive index detector is commonly used to analyze polymer samples in size exclusion chromatography. dRI can output a concentration detector signal value corresponding to the concentration value of the sample.

[0037] UV-Vis spectroscopy

[0038] UV-Vis spectroscopy or UV-Vis spectrophotometry (UV-Vis or UV / Vis) refers to absorption or reflectance spectroscopy in the UV-Visible spectral region. A UV-Vis detector / UV-Vis spectrophotometer uses light in the visible and adjacent ranges, where absorption or reflection in the visible range directly affects the perceived color of the chemical in question, where atoms and molecules undergo electronic transitions in this region of the electromagnetic spectrum. This type of absorption spectroscopy measures transitions from a ground state to an excited state. A UV-Vis detector / UV-Vis spectrophotometer measures the intensity of light that passes through a sample (I) and compares it to the intensity of the light before it passed through the sample (I o ) are compared, where the ratio I / I o It is called transmittance and is usually expressed as a percentage (%T). The absorbance A is based on the transmittance according to the formula

[0039] A = -log (%T / 100%).

[0040] The UV-Visible spectrophotometer can also be configured to measure reflectance, where the spectrophotometer measures the intensity of light reflected from a sample (I) and compares it to the intensity of light reflected from a reference material (I o ) is compared to the intensity of the reflected light, where the ratio I / I o It is called reflectance and is usually expressed as a percentage (%R). The UV absorption detector can output a concentration detector signal value corresponding to the concentration value of the sample.

[0041] Current Technology

[0042] In the process of producing therapeutic proteins such as monoclonal antibodies (mAb), proteins are produced by fermenting cells in bioreactors. Although a large amount of purification from lysates, nucleic acids and most host cell proteins is easy to achieve, it is necessary to further purify the protein in pure monomeric form from undesirable aggregates. Usually this type of purification is completed by chromatography, such as size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography or membrane chromatography. In the chromatographic purification process, the desired and undesirable components are dissolved or suspended in a solution, and either flow through a stationary phase such as a post or a film, or first load onto a post or a film under a buffer condition (loading buffer), then eluted from the post or the film under one or more buffer conditions (elution buffer) that are different from the loading buffer.

[0043] As a result of flowing through the stationary phase, various components such as monomers and aggregates are separated and eluted at different times. However, in a typical production process, some aggregates elute with pure monomers in a ratio that varies with time and / or elution conditions (such as buffer salt concentration). Assuming that a particular fraction of the eluted material contains the desired component (in this case, pure monomer) and a minimal mixture of undesirable components, a particular fraction of the eluted material is collected and pooled. Maintaining the correct cutoff points that optimize the collection of monomers and reduce aggregate components to acceptable levels is a major challenge that affects yield and profitability.

[0044] To characterize the purification process, multiple test fractions are obtained and analyzed on separate offline analytical instruments to determine the type and amount of solution components in each fraction. These characteristics are related to process parameters, such as elution time and signal level from a concentration detector (such as a UV absorbance detector). Then, during the actual purification run, the material pool is collected according to the time and signal level corresponding to the time and signal level that the test fraction produces the best results in terms of purity and quantity. The final pool can be considered pure enough or can be processed through additional purification steps to further remove impurities. However, the information obtained online during the purification process is not sufficient to determine whether aggregates are actually present in the pool or their amount; additional offline testing must be performed to verify that the correct fractions are collected. Unexpected changes in elution conditions, column aging, and other factors may change the monomer-aggregate co-elution characteristics associated with the initial characterization of the test fraction, resulting in unacceptable levels of aggregates in the pool (needing to reject and repurify or discard the batch) or loss of valuable monomers that should have been collected but were not collected.

[0045] Various methods have been proposed and tested to meet this need, including dual wavelength UV light, Raman scattering, and static light scattering (the latter including MALS). Most of these measurement techniques have generally been found to be insufficiently sensitive to the presence of aggregates to meet industrial needs. MALS provides a weight-average molar mass, M, of the protein in solution. w The signal related to the product of the concentration c is, in principle, sufficiently sensitive because the presence of aggregates disproportionately increases M w , thereby increasing the light scattering amplitude. However, under typical purification conditions—high protein concentrations that vary with elution, and / or changing pH and salt concentrations—these parameters may have a greater impact on the light scattering amplitude than the desired sensitivity to aggregates, reducing the suitability of MALS for your application.

[0046] It would therefore be advantageous to implement an instrument and method for determining on-line and in real time whether aggregates are present in the eluate and their concentration, so as to specify with certain knowledge the optimal fractions to be collected when they elute.There is therefore a need to control the purification of macromolecular solutions via real-time multi-angle light scattering.

[0047] In one embodiment, Figure 1A , Figure 1B and Figure 1C The hardware / apparatus configuration of the method, system, and computer product is described. In addition, in one embodiment, Figure 3 The times t1, t2, t3, t4, and t5 of the method, system, and computer product are depicted. In one embodiment, Figure 4 Times t1 , t2 , t3 , t4 , and t5 of the method, system, and computer product are depicted.

[0048] 2 , in an exemplary embodiment, the computer-implemented method, system, and computer program product are configured to perform the following operations: operation 110, receiving, by a computer system, baseline multi-angle light scattering (MALS) signal values ​​and baseline ultraviolet (UV) signal values ​​of a pure buffer solution flowing from a chromatography purification system to the group of instruments from a set of instruments, wherein the group of instruments includes a MALS instrument and a UV detector; operation 112, in response to receiving the baseline MALS signal values ​​from the MALS instrument, (a) receiving, by the computer system, scattering intensity values ​​of a sample solution flowing from the chromatography purification system to the MALS instrument in a time series from the MALS instrument, wherein the sample solution includes at least one type of macromolecule, (b) calculating, by the computer system, an average value of the baseline MALS signal values, and (c) subtracting, by the computer system, the average value of the baseline MALS signal values ​​from the scattering intensity values ​​to obtain an intensity time series of excess scattering intensity values ​​Iscatt j ; Operation 114, in response to receiving the baseline UV signal value from the UV detector, (a) receiving, by the computer system, ultraviolet absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in a time series from the UV detector, (b) calculating, by the computer system, an average value of the baseline UV signal value, (c) performing, by the computer system, a set of logical operations, the set of logical operations applying a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​in the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​in the time series, and (d) subtracting, by the computer system, the average value of the baseline UV signal value from the corrected UV absorption values ​​to obtain a UV time series of excess UV absorption values; Operation 116, calculating, by the computer system, a concentration value c of the sample solution relative to the UV time series of excess UV absorption values ​​according to Beer's law j and the absorption coefficient of the sample solution; operation 118, receiving, by the computer system, the pH value of the sample solution in a time series from the pH detector, and receiving the conductivity value of the sample solution in a time series from the conductivity detector, wherein the set of instruments also includes a pH detector and a conductivity detector; operation 120, performing, by the computer system, a set of logical operations, the set of logical operations (a) applying a pH calibration correction relative to the pH calibration parameter to the pH value in the time series associated with the pH detector and the MALS instrument to obtain a corrected pH value in the time series, and (b) applying a conductivity calibration correction relative to the conductivity calibration parameter to the conductivity value in the time series associated with the conductivity detector and the MALS instrument to obtain a corrected conductivity value in the time series, and obtaining an intensity time series Iscatt including excess scattering intensity values. j , the concentration value of the sample solution cj , a plurality of signals of the corrected pH value and the corrected conductivity value; Operation 122, a set of logical operations is performed by the computer system, the set of logical operations is performed within the time range t 1 ,t 2 ,……t n averaging each of the plurality of signals, wherein the time series includes a time range, wherein the time range corresponds to a fraction of the sample solution collected by a fraction collector connected to the set of instruments, to obtain an average value [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n Operation 124, receiving the aggregation content value % agg of the fraction of the sample solution measured by the SEC-MALS instrument by the computer system 1 、%agg 2 、……%agg n and weight-average molar mass value M 1 、M 2 , ...M n ; and operation 126, by the computer system aggregate content value% agg 1 、%agg 2 、……%agg n and weight-average molar mass value M 1 、M 2 , ...M n Relative to the mean [lscatt,c,pH,cond] 1 、[lscatt,c,pH,cond] 2 , ...[lscatt,c,pH,cond] n Stored in the Macromolecular Characterization Lookup Table (MCLUT).

[0049] In an exemplary embodiment, the computer system is a stand-alone computer system such as Figure 5 A computer system 500 shown in FIG. 5 ; a distributed computer network in which at least some of the computers are computer systems such as Figure 5 The computer system 500 shown in FIG. 5 ; or a cloud computing node server, such as Figure 5 In one embodiment, the computer system is as shown in FIG. Figure 5 The computer system 500 shown in FIG. 1 is configured to control the purification of a macromolecular solution via a real-time multi-angle light scattering script or computer software application that performs at least the operations of the method. In one embodiment, the computer system is configured to control the purification of a macromolecular solution via a real-time multi-angle light scattering script or computer software application that performs at least the operations of the method. Figure 5The computer system / server 512 shown in FIG. 1 is configured to control the purification of the macromolecular solution via a real-time multi-angle light scattering script or computer software application that performs at least the operations of the method. In one embodiment, the computer system is such as Figure 5 The processing unit 516 shown executes a purification of the macromolecular solution via a real-time multi-angle light scattering script or computer software application that performs at least the operations of the method. In one embodiment, the computer system is a machine learning computer software / program / algorithm that executes the purification of the macromolecular solution via real-time multi-angle light scattering.

[0050] In one embodiment, the computer system is Figure 5 500, which executes a computer system 500 for controlling the purification of a macromolecular solution via a real-time multi-angle light scattering script or computer software application, the script or application performing at least operations 110, 112, 114, 116, 118, 120, 122, and 124. In one embodiment, the computer system is as Figure 5 The computer system / server 512 shown in FIG. 1 is configured to execute a computer software application or script to control the purification of a macromolecular solution via real-time multi-angle light scattering, the script or application performing at least operations 110, 112, 114, 116, 118, 120, 122, and 124. In one embodiment, the computer system is a computer system such as Figure 5 A processing unit 516 is shown that executes a computer software application or script to control purification of a macromolecular solution via real-time multi-angle light scattering that performs at least operations 110 , 112 , 114 , 116 , 118 , 120 , 122 , and 124 .

[0051] Using MCLUT

[0052] In further embodiments, in one embodiment, the computer-implemented method, system, and computer program product further include (1) receiving, by the computer system, second baseline MALS signal values ​​and second baseline UV signal values ​​for a pure buffer solution flowing from a second chromatographic purification system to a second set of instruments, wherein the second set of instruments includes a second MALS instrument and a second UV detector, (2) in response to receiving the second baseline MALS signal values ​​from the second MALS instrument, (a) receiving, by the computer system, second scattering intensity values ​​for a second sample solution flowing from the second chromatographic purification system to the second MALS instrument over a second time series, wherein the second sample solution contains the at least one type of macromolecule, and (b) receiving, by the computer system, second scattering intensity values ​​for a second sample solution flowing from the second chromatographic purification system to the second MALS instrument over a second time series. The system calculates an average value of the second baseline MALS signal values, and (c) subtracts the average value of the second baseline MALS signal values ​​from the second scattering intensity values ​​by the computer system to obtain a second intensity time series of excess scattering intensity values ​​Iscatt2, (3) in response to receiving the second baseline UV signal values ​​from the second UV detector, (a) receiving, by the computer system, second UV absorbance values ​​of a second sample solution flowing from the second chromatographic purification system to the second UV detector over a second time series from the second UV detector, (b) calculating the average value of the second baseline UV signal values ​​by the computer system, and (c) performing, by the computer system, a set of logical operations that convert the second UV absorption calibration parameters relative to the second UV absorption calibration parameters and the second UV absorption calibration parameters relative to the second UV absorption calibration parameters. (d) the computer system subtracts an average value of the second baseline UV signal value from the corrected second UV absorption values ​​to obtain a second UV time series of excess UV absorption values; (4) the computer system calculates second concentration values ​​c2 of the second sample solution relative to the second UV time series of excess UV absorption values ​​and a second absorption coefficient of the second sample solution according to Beer's law; (5) the computer system receives second pH values ​​of the second sample solution in the time series from the second pH detector; and (6) the computer system receives second pH values ​​of the second sample solution in the time series from the second pH detector. The second conductivity detector receives second conductivity values ​​of the second sample solution over the time series, wherein the second set of instruments further includes a second pH detector and a second conductivity detector, (6) a set of logical operations is performed by the computer system, the set of logical operations (a) applying a second pH calibration correction relative to a second pH calibration parameter to the second pH values ​​over the second time series associated with the second pH detector and the second MALS instrument to obtain corrected second pH values ​​over the second time series, and (b) applying a second conductivity calibration correction relative to the second conductivity calibration parameter to the second conductivity values ​​over the second time series associated with the second conductivity detector and the second MALS instrument to obtain corrected second conductivity values ​​over the second time series,A plurality of second signals including a second intensity time series Iscatt2 of excess scattering intensity values, a second concentration value c2 of the second sample solution, a corrected second pH value, and a corrected second conductivity value are obtained, (7) the aggregation content value %agg and the weight-average molar mass M corresponding to the plurality of second signals are retrieved by the computer system from MCLUT via at least one of correlation, interpolation, and extrapolation, (8) a set of logical operations are performed by the computer system, the set of logical operations determining whether at least one property of the second sample solution has reached an aggregation content limit relative to at least one of the retrieved aggregation content value %agg, the retrieved weight-average molar mass M, and the second concentration value c2 of the second sample solution, and (9) in response to determining that the at least one property of the second sample solution has reached the aggregation content limit, the computer system sends a transfer to waste command to transfer the sample solution to a waste container. In one embodiment, the second absorption coefficient of the sample solution is a property of the sample solution. In one embodiment, the second absorption coefficient is received from a user input. ,

[0053] In one embodiment, the retrieval includes retrieving, by the computer system via interpolation, from the MCLUT the aggregate content value %agg and the weight-average molar mass M corresponding to the plurality of second signals when Iscatt2 / c2, the second pH value, and the second conductivity value are within the values ​​in the MCLUT. In one embodiment, the retrieval includes retrieving, by the computer system via extrapolation, from the MCLUT the aggregate content value %agg and the weight-average molar mass M corresponding to the plurality of second signals when Iscatt2 / c2, the second pH value, and the second conductivity value are outside the values ​​in the MCLUT.

[0054] Reaching the aggregation content limit

[0055] In one embodiment, determining that the at least one property of the second sample solution has reached an aggregation content limit comprises determining, by the computer system, that the retrieved aggregation content value %agg exceeds a maximum aggregation content value. In one embodiment, determining that the at least one property of the second sample solution has reached an aggregation content limit comprises determining, by the computer system, that the retrieved weight-average molar mass M exceeds a maximum weight-average molar mass value.

[0056] In one embodiment, determining that the at least one property of the second sample solution has reached an aggregation content limit comprises (a) determining, by the computer system, that the at least one property of the second sample solution has reached an aggregation content limit according to the following relative to the subset of aggregation content values ​​% agg retrieved from the MCLUT: k and a second subset of concentration values ​​%agg k To calculate the cumulative aggregation content value % agg 累积 :

[0057] %agg累积 =(Σ k %agg k ×c2 k ) / Σ k c2 k ,

[0058] where %agg k and c2 k is determined from the effective start of elution of the second chromatographic purification system, and (b) performing a set of logical operations by the computer system to determine the cumulative content value % agg 累积 In one embodiment, the effective start of elution from the second chromatographic purification system is when the % agg 累积 When the summation starts.

[0059] The beginning of elution

[0060] In one embodiment, the effective start of elution of the second sample solution from the second chromatography purification system corresponds to the time at which a trigger signal is received by the computer system from the second chromatography purification system. In one embodiment, the trigger signal is from at least one of user programming and an established method. For example, such a trigger signal can be used for bind-elute gradient ion exchange chromatography, where it is known a priori that a specific time difference between the start of the gradient and the elution of the product contains only the desired protein and at most a small amount of % agg.

[0061] In one embodiment, the effective start of elution of the second sample solution from the second chromatography purification system corresponds to the time at which the concentration value of the second sample solution has reached a minimum acceptable value. In one embodiment, the minimum acceptable value is user defined. For example, the minimum acceptable value can be used when the concentration accumulates in the second chromatography purification system. In addition, for example, the minimum acceptable value can be used in flow-through hydrophobic interaction chromatography as a polishing step, where it is known a priori that the pure monomer will be the first species to elute at an appreciable concentration.

[0062] In one embodiment, the effective start of eluting the second sample solution from the second chromatography purification system corresponds to the time when the weight-average molar mass of the second sample solution is within the acceptable range of molar mass values. In one embodiment, the acceptable range of molar mass values ​​is user-defined. For example, the acceptable range of molar mass values ​​can be used for ion exchange chromatography, where it is known a priori that the species initially eluted is mainly composed of undesirable low molar mass species, and therefore before the solution is transferred to the pool, it may be advantageous to wait until the weight-average molar mass of the solution is close to the weight-average molar mass of the monomer. In a specific embodiment, the second sample solution comprises protein, and the acceptable range of molar mass values ​​corresponds to the molar mass of the monomer protein in the second sample solution. For example, the molar mass of the monomer protein in the second sample solution can be used for size exclusion chromatography, where it is known a priori that the species initially eluted is mainly composed of undesirable aggregates, and therefore before the solution is transferred to the pool, it may be advantageous to wait until the weight-average molar mass of the solution is close to the weight-average molar mass of the monomer.

[0063] In one embodiment, the effective start of elution of the second sample solution from the second chromatography purification system corresponds to at least two of the following: (a) the time when a trigger signal is received by the computer system from the second chromatography purification system, (b) the time when the concentration value of the second sample solution has reached a minimum acceptable value, and (c) the time when the weight average molar mass of the second sample solution is within an acceptable range of molar mass values. For example, these two correspondences can be used in various chromatography purification processes by requiring that multiple conditions are met and thereby reducing the possibility of impurities being collected in the pool.

[0064] Single MALS signal and MCLUT adjustment

[0065] In further embodiments, the computer-implemented method, system, and computer program product further include (1) retrieving, by the computer system, an expected value of Iscatt, Iscatt_exp1, from the MCLUT based on measured values ​​of c2, conductivity, and pH of a particular portion of the second sample solution flowing from the second chromatographic purification system, (2) calculating, by the computer system, an average value Iscatt_meas1 of Iscatt2 measured for the particular portion of the second sample solution flowing from the second chromatographic purification system, and (3) multiplying, by the computer system, the second time series of excess scattering intensity values ​​Iscatt2 by the ratio of Iscatt_exp1 to Iscatt_meas1 to obtain an adjusted value of Iscatt2. In one embodiment, Iscatt2 is adjusted to account for calibration differences associated with the first MALS instrument.

[0066] In one embodiment, the second sample solution comprises protein, and the specific portion of the second sample solution flowing from the second chromatographic purification system corresponds to monomeric protein. For example, the specific portion corresponding to monomeric protein corresponds to the time when the expected % agg is equal to zero.

[0067] Computer Systems

[0068] In an exemplary embodiment, the computer system is Figure 5 The computer system 500 shown in FIG. 5 is only an example of a computer system and is not intended to impose any limitations on the scope of use or functionality of the embodiments of the present invention. In any case, the computer system 500 can be implemented to perform and / or be capable of performing any function / operation of the present invention.

[0069] The computer system 500 includes a computer system / server 512 that can operate with numerous other general or special purpose computing system environments or configurations. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with the computer system / server 512 include, but are not limited to, personal computer systems, server computer systems, thin clients, fat clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computer systems, and distributed cloud computing environments including any of the above systems or devices.

[0070] Computer system / server 512 may be described in the general context of computer system executable instructions (such as program modules) executed by a computer system. In general, program modules may include routines, programs, objects, components, logic and / or data structures that perform specific tasks or implement specific abstract data types. Computer system / server 512 may be practiced in a distributed cloud computing environment, where tasks are performed by remote processing devices linked through a communication network. In a distributed cloud computing environment, program modules may be located in both local and remote computer system storage media, including memory storage devices.

[0071] like Figure 5 As shown in FIG. 5 , the computer system / server 512 in the computer system 500 is shown in the form of a general-purpose computing device. The components of the computer system / server 512 may include, but are not limited to, one or more processors or processing units 516, a system memory 528, and a bus 518 that couples various system components including the system memory 528 to the processor 516.

[0072] Bus 518 represents any of one or more of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an accelerated graphics port, and a processor or local bus using any of a variety of bus architectures. By way of example and not limitation, such architectures include an Industry Standard Architecture (ISA) bus, a Micro Channel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, a Video Electronics Standards Association (VESA) local bus, and a Peripheral Component Interconnect (PCI) bus.

[0073] Computer system / server 512 typically includes a variety of computer system readable media. Such media can be any available media that can be accessed by computer system / server 512, and includes volatile and nonvolatile media, removable and non-removable media.

[0074] System memory 528 may include computer system readable media in the form of volatile memory, such as random access memory (RAM) 530 and / or cache memory 532. Computer system / server 512 may also include other removable / non-removable, volatile / non-volatile computer system storage media. By way of example only, storage system 534 may be provided for reading from and writing to a non-removable, non-volatile magnetic medium (not shown and commonly referred to as a "hard drive"). Although not shown, a disk drive for reading from and writing to a removable, non-volatile disk (e.g., a "floppy disk") may be provided, as well as an optical drive for reading from or writing to a removable, non-volatile optical disk (e.g., a CD-ROM, DVD-ROM, or other optical media). In such instances, each may be connected to bus 518 via one or more data media interfaces. As will be further depicted and described below, memory 528 may include at least one program product having a set (e.g., at least one) of program modules configured to perform the functions / operations of embodiments of the present invention.

[0075] By way of example and not limitation, a program / utility 540 having a set (at least one) of program modules 542 may be stored in memory 528. Exemplary program modules 542 may include an operating system, one or more application programs, other program modules, and program data. Each of the operating system, one or more application programs, other program modules, and program data, or some combination thereof, may include an implementation of a networking environment. Program modules 542 generally perform the functions and / or methods of embodiments of the present invention.

[0076] The computer system / server 512 may also communicate with one or more external devices 514, such as a keyboard, a pointing device, a display 524, one or more devices that enable a user to interact with the computer system / server 512, and / or any device that enables the computer system / server 512 to communicate with one or more other computing devices (e.g., a network card, a modem, etc.). Such communication may occur via an input / output (I / O) interface 522. In addition, the computer system / server 512 may communicate with one or more networks, such as a local area network (LAN), a general wide area network (WAN), and / or a public network (e.g., the Internet) via a network adapter 520. As shown, the network adapter 520 communicates with other components of the computer system / server 512 via a bus 518. It should be understood that, although not shown, other hardware and / or software components may be used in conjunction with the computer system / server 512. Examples include, but are not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data archive storage systems.

[0077] Computer program product

[0078] The present invention may be a system, method and / or computer program product. The computer program product may include a computer-readable storage medium (or media) having computer-readable program instructions thereon to enable a processor to perform various aspects of the present invention.

[0079] A computer-readable storage medium may be a tangible device that can retain and store instructions used by an instruction execution device. A computer-readable storage medium may be, for example, but not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of computer-readable storage media includes the following: a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disc (DVD), a memory stick, a floppy disk, a mechanical encoding device (such as a punched card or a raised structure in a groove on which instructions are recorded), and any suitable combination of the foregoing. As used herein, a computer-readable storage medium should not be understood as a transient signal itself, such as a radio wave or other freely propagating electromagnetic wave, an electromagnetic wave propagating through a waveguide or other transmission medium (e.g., a light pulse passing through a fiber optic cable), or an electrical signal transmitted by a wire.

[0080] The computer-readable program instructions described herein may be downloaded from a computer-readable storage medium to a corresponding computing / processing device, or downloaded to an external computer or external storage device via a network (e.g., the Internet, a local area network, a wide area network, and / or a wireless network). The network may include copper transmission cables, optical transmission fibers, wireless transmissions, routers, firewalls, switches, gateway computers, and / or edge servers. The network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards the computer-readable program instructions to be stored in a computer-readable storage medium within the corresponding computing / processing device.

[0081] The computer-readable program instructions for performing the operation of the present invention may be source code or object code written in any combination of assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, etc., and conventional process programming languages ​​such as "C" programming language or similar programming languages. The computer-readable program instructions may be executed completely on a user's computer, partially on a user's computer, as an independent software package, partially on a user's computer, and partially on a remote computer or completely on a remote computer or server. In the latter case, the remote computer may be connected to the user's computer via any type of network (including a local area network (LAN) or a wide area network (WAN)), or may be connected to an external computer (e.g., via the Internet using an Internet service provider). In some embodiments, an electronic circuit including, for example, a programmable logic circuit, a field programmable gate array (FPGA), or a programmable logic array (PLA) may execute a computer-readable program instruction to personalize the electronic circuit by utilizing the state information of the computer-readable program instructions, so as to perform various aspects of the present invention.

[0082] Various aspects of the present invention are described herein with reference to the flow diagrams and / or block diagrams of the methods, devices (systems) and computer program products according to embodiments of the present invention. It will be understood that each frame in the flow diagrams and / or block diagrams, and the combination of frames in the flow diagrams and / or block diagrams, can be implemented by computer-readable program instructions.

[0083] These computer-readable program instructions may be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device form a means for implementing the functions / actions specified in the flowchart and / or one or more block diagram blocks. These computer-readable program instructions may also be stored in a computer-readable storage medium, which may instruct a computer, a programmable data processing device, and / or other equipment to operate in a specific manner, so that the computer-readable storage medium having the instructions stored therein includes such an article of manufacture, which includes instructions for implementing various aspects of the functions / actions specified in the flowchart and / or one or more block diagram blocks.

[0084] Computer-readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device, so that a series of operating steps are performed on the computer, other programmable apparatus, or other device to produce a computer-implemented process, so that the instructions executed on the computer, other programmable apparatus, or other device implement the functions / actions specified in the flowchart and / or one or more block diagram blocks.

[0085] The flow chart and block diagram in the accompanying drawings illustrate the possible specific implementation architecture, function and operation of the system, method and computer program product according to various embodiments of the present invention.In this regard, each frame in the flow chart or block diagram may represent a module, section or part of an instruction, which includes one or more executable instructions for realizing a specified logical function.In some alternative specific implementations, the function mentioned in the frame may not occur in the order described in the accompanying drawings.For example, depending on the function involved, the two frames shown in succession may actually be performed substantially simultaneously, or the frame may sometimes be performed in reverse order.It should also be noted that each frame in the block diagram and / or the flow diagram and the combination of the frames in the block diagram and / or the flow diagram can be realized by a system based on dedicated hardware that performs a specified function or action or performs a combination of dedicated hardware and computer instructions.

[0086] The description of various embodiments of the present disclosure is for illustrative purposes only, but is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and substance of the described embodiments. The terms used herein are selected to explain the principles of the embodiments, practical applications or technical improvements over existing technologies on the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A computer-implemented method, the computer-implemented method comprising: receiving, by a computer system, from a set of instruments, a baseline multi-angle light scattering (MALS) signal value and a baseline ultraviolet (UV) signal value of a pure buffer solution flowing from a chromatographic purification system to the set of instruments, wherein the set of instruments comprises a MALS instrument and a UV detector; In response to receiving the baseline MALS signal value from the MALS instrument, receiving, by the computer system, from the MALS instrument, scattering intensity values ​​of sample solutions flowing from the chromatography purification system to the MALS instrument in a time series, wherein the sample solution contains at least one type of macromolecule, calculating, by the computer system, an average of the baseline MALS signal values, and subtracting, by the computer system, the mean of the baseline MALS signal values ​​from the scattered intensity values, Get the intensity time series of excess scattering intensity values ​​Iscatt j ; In response to receiving the baseline UV signal value from the UV detector, receiving, by the computer system, from the UV detector, UV absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in the time series, calculating, by the computer system, an average value of the baseline UV signal value, performing, by the computer system, a set of logical operations that applies a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​over the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​over the time series, and subtracting, by the computer system, the average of the baseline UV signal values ​​from the corrected UV absorbance values, Get the UV time series of excess UV absorption values; The computer system calculates the concentration value c of the UV time series of the sample solution relative to the excess UV absorption value according to Beer's law. j and an absorption coefficient of the sample solution; The computer system receives the pH value of the sample solution in the time series from a pH detector and receives the conductivity value of the sample solution in the time series from a conductivity detector, wherein the set of instruments further comprises the pH detector and the conductivity detector; performing, by the computer system, a set of logical operations that applies a pH calibration correction relative to a pH calibration parameter to the pH values ​​over the time series associated with the pH detector and the MALS instrument to obtain corrected pH values ​​over the time series, and applies a conductivity calibration correction relative to a conductivity calibration parameter to the conductivity values ​​over the time series associated with the conductivity detector and the MALS instrument to obtain corrected conductivity values ​​over the time series, The intensity time series Iscatt including the excess scattered intensity value is obtained j , the concentration value c of the sample solution j , a plurality of signals of said corrected pH value and said corrected conductivity value; A set of logical operations is executed by the computer system, and the set of logical operations is executed in the time range t1, t2, ... t n averaging each of the plurality of signals, wherein the time series includes the time range, wherein the time range corresponds to fractions of the sample solution collected by a fraction collector connected to the set of instruments, Get the average value [lscatt,c,pH,cond]1, [lscatt,c,pH,cond]2, ... [lscatt,c,pH,cond] n ; The computer system receives the aggregation content values ​​% agg1, ​​% agg2, ... % agg of the fractions of the sample solution measured by the SEC-MALS instrument. n and weight-average molar mass values ​​M1, M2, ...M n ;as well as The computer system converts the aggregate content values ​​%agg1, ​​%agg2, ... %agg n and the weight-average molar mass values ​​M1, M2, ... M n Relative to the average values ​​[lscatt,c,pH,cond]1, [lscatt,c,pH,cond]2, ... [lscatt,c,pH,cond] n Stored in the Macromolecular Characterization Lookup Table (MCLUT).

2. The method according to claim 1, further comprising: receiving, by the computer system, from the second set of instruments, a second baseline MALS signal value and a second baseline UV signal value of the pure buffer flowing from a second chromatographic purification system to the second set of instruments, wherein the second set of instruments comprises a second MALS instrument and a second UV detector; In response to receiving the second baseline MALS signal value from the second MALS instrument, receiving, by the computer system from the second MALS instrument, second scattering intensity values ​​of a second sample solution flowing from the second chromatographic purification system to the second MALS instrument over a second time series, wherein the second sample solution contains the at least one type of macromolecules, calculating, by the computer system, an average of the second baseline MALS signal values, and subtracting, by the computer system, the average of the second baseline MALS signal values ​​from the second scattered intensity values, Obtain a second intensity time series Iscatt2 of excess scattering intensity values; In response to receiving the second baseline UV signal value from the second UV detector, receiving, by the computer system, from the second UV detector, a second UV absorption value of the second sample solution flowing from the second chromatographic purification system to the second UV detector in the second time series, calculating, by the computer system, an average of the second baseline UV signal values, and performing, by the computer system, a set of logical operations that applies a second UV absorption calibration correction relative to a second UV absorption calibration parameter and a second UV absorption band broadening correction relative to a second UV absorption band broadening parameter to the second UV absorption values ​​over the second time series associated with the second UV detector and the second MALS instrument to obtain corrected second UV absorption values ​​over the second time series, and subtracting, by the computer system, the average of the second baseline UV signal values ​​from the corrected second UV absorbance values, Obtaining a second UV time series of excess UV absorption values; The computer system calculates, according to Beer's law, a second concentration value c2 of the second UV time series of the second sample solution relative to the excess UV absorption value and a second absorption coefficient of the second sample solution; The computer system receives a second pH value of the second sample solution in the time series from a second pH detector, and receives a second conductivity value of the second sample solution in the time series from a second conductivity detector, wherein the second set of instruments further comprises the second pH detector and the second conductivity detector; performing, by the computer system, a set of logical operations that applies a second pH calibration correction relative to a second pH calibration parameter to the second pH values ​​over the second time series associated with the second pH detector and the second MALS instrument to obtain corrected second pH values ​​over the second time series, and applies a second conductivity calibration correction relative to a second conductivity calibration parameter to the second conductivity values ​​over the second time series associated with the second conductivity detector and the second MALS instrument to obtain corrected second conductivity values ​​over the second time series, Obtaining a plurality of second signals including the second intensity time series Iscatt2 of excess scattering intensity values, the second concentration value c2 of the second sample solution, the corrected second pH value and the corrected second conductivity value; retrieving, by the computer system via at least one of correlation, interpolation, and extrapolation, aggregate content values ​​%agg and weight-average molar mass M corresponding to the plurality of second signals from the MCLUT; performing, by the computer system, a set of logical operations that determine whether at least one property of the second sample solution has reached an aggregation content limit relative to at least one of the retrieved aggregation content value %agg, the retrieved weight-average molar mass M, and the second concentration value c2 of the second sample solution; as well as In response to determining that the at least one property of the second sample solution has reached the aggregate content limit, a divert to waste command is sent by the computer system to divert the sample solution to a waste container.

3. The method of claim 2, wherein determining that the at least one property of the second sample solution has reached the aggregation content limit comprises It is determined by the computer system that the retrieved aggregation content value % agg exceeds a maximum aggregation content value.

4. The method of claim 2, wherein determining that the at least one property of the second sample solution has reached the aggregation content limit comprises It is determined by the computer system that the retrieved weight-average molar mass M exceeds a maximum weight-average molar mass value.

5. The method of claim 2, wherein determining that the at least one property of the second sample solution has reached the aggregation content limit comprises: Calculated by the computer system relative to a subset of aggregate content values ​​retrieved from the MCLUT % agg k and the second concentration value subset c2 k The cumulative aggregation content value % agg 累积 , where %agg k and c2 k is determined from the effective start of elution of the second chromatographic purification system; as well as A set of logical operations is performed by the computer system, which determines the cumulative content value %agg 累积 The maximum cumulative aggregate content value was exceeded.

6. The method of claim 5, wherein the effective start of eluting the second sample solution from the second chromatographic purification system corresponds to a time when a trigger signal is received by the computer system from the second chromatographic purification system.

7. The method of claim 5, wherein the effective start of eluting the second sample solution from the second chromatographic purification system corresponds to a time when a concentration value of the second sample solution has reached a minimum acceptable value.

8. The method of claim 5, wherein the effective start of eluting the second sample solution from the second chromatographic purification system corresponds to a time when the weight-average molar mass of the second sample solution is within an acceptable range of molar mass values.

9. The method according to claim 8, wherein the second sample solution comprises protein, and The acceptable range of molar mass values ​​corresponds to the molar mass of monomeric protein in the second sample solution.

10. The method of claim 5, wherein the effective start of eluting the second sample solution from the second chromatographic purification system corresponds to at least two of: (a) when a trigger signal is received by the computer system from the second chromatographic purification system, (b) the time at which the concentration value of the second sample solution has reached a minimum acceptable value, and (c) the time during which the weight average molar mass of the second sample solution is within an acceptable range of molar mass values.

11. The method according to claim 2, further comprising: Retrieving, by the computer system, an expected value Iscatt_exp1 of Iscatt from the MCLUT based on the measured values ​​of c2, conductivity, and pH of a specific portion of the second sample solution flowing from the second chromatographic purification system; calculating, by the computer system, an average value Iscatt_meas1 of Iscatt2 measured for the specific portion of the second sample solution flowing from the second chromatographic purification system; and multiplying said second time series of excess scatter intensity values ​​Iscatt2 by the ratio of Iscatt_exp1 to Iscatt_meas1 by said computer system, Get the adjusted value of Iscatt2.

12. The method according to claim 11, wherein the second sample solution comprises protein, and wherein the specific portion of the second sample solution flowing from the second chromatographic purification system corresponds to monomeric protein.

13. A computer-implemented method, the computer-implemented method comprising: receiving, by a computer system, from a group of instruments, a baseline MALS signal value and a baseline UV signal value of a pure buffer solution flowing from a chromatographic purification system to the group of instruments, wherein the set of instruments comprises a MALS instrument and a UV detector; In response to receiving the baseline MALS signal value from the MALS instrument, receiving, by the computer system, from the MALS instrument, scattering intensity values ​​of sample solutions flowing from the chromatography purification system to the MALS instrument in a time series, wherein the sample solution contains at least one type of macromolecule, calculating, by the computer system, an average of the baseline MALS signal values, and subtracting, by the computer system, the mean of the baseline MALS signal values ​​from the scattered intensity values, Get the intensity time series Iscatt of excess scattering intensity values; In response to receiving the baseline UV signal value from the UV detector, receiving, by the computer system, from the UV detector, UV absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in the time series, calculating, by the computer system, an average value of the baseline UV signal value, performing, by the computer system, a set of logical operations that applies a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​over the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​over the time series, and subtracting, by said computer system, said average of said baseline average UV signal values ​​from said corrected UV absorbance values, Get the UV time series of excess UV absorption values; The computer system calculates the concentration value c of the UV time series of the sample solution relative to the excess UV absorption value and the absorption coefficient of the sample solution according to Beer's law; The computer system receives the pH value of the sample solution in the time series from a pH detector and receives the conductivity value of the sample solution in the time series from a conductivity detector, wherein the set of instruments further comprises the pH detector and the conductivity detector; performing, by the computer system, a set of logical operations that applies a pH calibration correction relative to a pH calibration parameter to the pH values ​​over the time series associated with the pH detector and the MALS instrument to obtain corrected pH values ​​over the second time series, and applies a conductivity calibration correction relative to a conductivity calibration parameter to the conductivity values ​​over the time series associated with the conductivity detector and the MALS instrument to obtain corrected conductivity values ​​over the time series, Obtaining a plurality of signals including the intensity time series Iscatt of excess scattering intensity values, the concentration value c of the sample solution, the corrected pH value and the corrected conductivity value; retrieving, by the computer system via at least one of correlation, interpolation, and extrapolation, aggregation content values ​​%agg and weight-average molar mass M corresponding to the plurality of signals from a macromolecular characterization lookup table (MCLUT); performing, by the computer system, a set of logical operations that determine whether at least one property of the sample solution has reached an aggregation content limit relative to at least one of the retrieved aggregation content value %agg, the retrieved weight-average molar mass M, and the concentration value c of the second sample solution; as well as In response to determining that the at least one property of the sample solution has reached the aggregate content limit, a divert-to-waste command is sent by the computer system to divert the sample solution to a waste container.

14. A system, comprising: Memory; and a processor in communication with the memory, the processor being configured to perform a method comprising: receiving, from a set of instruments, a baseline multi-angle light scattering (MALS) signal value and a baseline ultraviolet (UV) signal value of a pure buffer flowing from a chromatographic purification system to the set of instruments, wherein the set of instruments includes a MALS instrument and a UV detector, in response to receiving the baseline MALS signal value from the MALS instrument, receiving from the MALS instrument scattering intensity values ​​of a sample solution flowing from the chromatographic purification system to the MALS instrument in a time series, wherein the sample solution contains at least one type of macromolecule, calculating, by the computer system, an average of the baseline MALS signal values, and subtracting, by the computer system, the mean of the baseline MALS signal values ​​from the scattered intensity values, Get the intensity time series of excess scattering intensity values ​​Iscatt j , In response to receiving the baseline UV signal value from the UV detector, receiving, by the computer system, from the UV detector, UV absorption values ​​of the sample solution flowing from the chromatography purification system to the UV detector in the time series, calculating, by the computer system, an average value of the baseline UV signal value, performing, by the computer system, a set of logical operations that applies a UV absorption calibration correction relative to a UV absorption calibration parameter and a UV absorption band broadening correction relative to a UV absorption band broadening parameter to the UV absorption values ​​over the time series associated with the UV detector and the MALS instrument to obtain corrected UV absorption values ​​over the time series, and subtracting, by the computer system, the average of the baseline UV signal values ​​from the corrected UV absorbance values, Get the UV time series of excess UV absorption values; The computer system calculates the concentration value c of the UV time series of the sample solution relative to the excess UV absorption value according to Beer's law. j and the absorption coefficient of the sample solution, The computer system receives the pH value of the sample solution in the time series from a pH detector and receives the conductivity value of the sample solution in the time series from a conductivity detector, wherein the set of instruments further comprises the pH detector and the conductivity detector, performing, by the computer system, a set of logical operations that applies a pH calibration correction relative to a pH calibration parameter to the pH values ​​over the time series associated with the pH detector and the MALS instrument to obtain corrected pH values ​​over the time series, and applies a conductivity calibration correction relative to a conductivity calibration parameter to the conductivity values ​​over the time series associated with the conductivity detector and the MALS instrument to obtain corrected conductivity values ​​over the time series, The intensity time series Iscatt including the excess scattering intensity value is obtained j , the concentration value c of the sample solution j , a plurality of signals of said corrected pH value and said corrected conductivity value, A set of logical operations is executed by the computer system, and the set of logical operations is executed in the time range t1, t2, ... t n averaging each of the plurality of signals, wherein the time series includes the time range, wherein the time range corresponds to the fractions of the sample solution collected by the fraction collector connected to the set of instruments, to obtain average values ​​[lscatt,c,pH,cond]1, [lscatt,c,pH,cond]2, ... [lscatt,c,pH,cond] n , The computer system receives the aggregation content values ​​% agg1, ​​% agg2, ... % agg of the fractions of the sample solution measured by the SEC-MALS instrument. n and weight-average molar mass values ​​M1, M2, ...M n ,as well as The computer system converts the aggregate content values ​​%agg1, ​​%agg2, ... %agg n and weight-average molar mass values ​​M1, M2, ...M n Relative to the average values ​​[lscatt,c,pH,cond]1, [lscatt,c,pH,cond]2, ... [lscatt,c,pH,cond] n Stored in the Macromolecular Characterization Lookup Table (MCLUT).

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