Measurement of quality attributes of viral samples

Abstract

The present disclosure describes a computer-implemented method, system, and computer program product for measuring quality attributes of a viral sample. In one embodiment, the method, system, and computer program product include: (1) receiving light scattering (LS) data, dRI data, and UV data from a light scattering detector that analyzes an isolate of a viral sample, (2) receiving a molecular weight M protein (expected) of a protein component of the sample, a refractive index increment (dn / dc) protein of the protein component, a refractive index increment (dn / dc) DNA of a DNA component of the sample, and an extinction coefficient [epsilon] protein of the protein component, (3) calculating the protein fraction x protein of the sample relative to LS data, dRI data, M protein (expected), (dn / dc) protein, (dn / dc) DNA and optical constant K, (4) calculating the DNA extinction coefficient epsilon DNA of the sample, and (5) calculating the quality attribute value of the sample.

Description

[0001] priority

[0002] This application claims the benefit of an earlier filing date of U.S. patent application serial number 17 / 939,950, filed on September 7, 2022, entitled “Measuring Quality Attributes of a Virus Sample,” the entire contents of which are incorporated herein by reference. Background Art

[0003] The present disclosure relates to samples, and more particularly to measuring quality attributes of viral samples. Summary of the invention

[0004] The present disclosure describes a computer-implemented method, system, and computer program product for measuring the quality attributes of a virus sample. In an exemplary embodiment, the computer-implemented method, system, and computer program product include: (1) receiving, by a computer system, light scattering data from a light scattering detector of an isolated substance of an analyzed virus sample, differential refractive index (dRI) data from a differential refractometer of the isolated substance, and UV absorbance data A at at least one wavelength of the isolated substance from an ultraviolet (UV) detector, (2) receiving, by the computer system, molecular weights M of protein components of the sample from a data source, 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 , (3) The protein fraction x of the sample is calculated by the computer system relative to the following terms 蛋白质 :Light scattering data, differential refractive index data, molecular weight M of protein components 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA and optical constant K, (4) the DNA extinction coefficient ε of the sample at at least one wavelength is calculated by the computer system relative to the following terms DNA : The calculated protein fraction x 蛋白质 , differential refractive index data, UV absorbance data A, refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient of the protein component at at least one wavelength ε 蛋白质 and the path length L of the cell in the UV detector, and (5) the DNA extinction coefficient ε calculated by the computer system relative to DNATo calculate the quality attribute value of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1A A flow chart according to an exemplary embodiment is depicted.

[0006] Figure 1B A block diagram is depicted according to an exemplary embodiment.

[0007] Figure 2 A flow chart according to an embodiment is depicted.

[0008] Figure 3A Depicted is a graph according to an embodiment.

[0009] Figure 3B Depicted is a graph according to an embodiment.

[0010] Figure 4 An apparatus according to an embodiment is depicted.

[0011] Figure 5 A computer system according to an exemplary embodiment is depicted. DETAILED DESCRIPTION

[0012] The present disclosure describes a computer-implemented method, system, and computer program product for measuring the quality attributes of a virus sample. In an exemplary embodiment, the computer-implemented method, system, and computer program product include: (1) receiving, by a computer system, light scattering data from a light scattering detector of an isolated substance of an analyzed virus sample, differential refractive index (dRI) data from a differential refractometer of the isolated substance, and UV absorbance data A at at least one wavelength of the isolated substance from an ultraviolet (UV) detector, (2) receiving, by the computer system, molecular weights M of protein components of the sample from a data source, 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 , (3) The protein fraction x of the sample is calculated by the computer system relative to the following terms 蛋白质 :Light scattering data, differential refractive index data, molecular weight M of protein components 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA and optical constant K, (4) the DNA extinction coefficient ε of the sample at at least one wavelength is calculated by the computer system relative to the following terms DNA : The calculated protein fraction x 蛋白质, differential refractive index data, UV absorbance data A, refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient of the protein component at at least one wavelength ε 蛋白质 and the path length L of the cell in the UV detector, and (5) the DNA extinction coefficient ε calculated by the computer system relative to DNA To calculate the quality attribute value of the sample. In one embodiment, the light scattering data includes static light scattering (SLS) data. For example, the light scattering data is static light scattering (SLS) data. In one embodiment, the light scattering data also includes dynamic light scattering (DLS) data. For example, the light scattering data is also dynamic light scattering (DLS) data.

[0013] In one embodiment, the virus sample comprises a virus having a radius of less than 30 nm. For example, the virus is small-sized and non-enveloped). For example, the virus has a radius of 14 nm. In a specific embodiment, the virus sample comprises an adeno-associated virus (AAV). For example, the virus sample is an adeno-associated virus (AAV). In one embodiment, the at least one wavelength is 260 nm. In one embodiment, the at least one wavelength is 280 nm. In one embodiment, the protein component comprises a protein capsid. For example, the protein component is a protein capsid. In one embodiment, the data source comprises at least one of a user input and a database.

[0014] In an exemplary embodiment, the computer-implemented method, system, and computer program product include: (1) receiving, by a computer system, light scattering data from a light scattering detector for analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer for analyzing the isolate, and UV absorbance data A at at least one wavelength of the isolate from an ultraviolet (UV) detector, (2) receiving, by the computer system, molecular weights M of protein components of the sample from a data source. 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 , (3) calculating, by a computer system, the DNA extinction coefficient εDNA of the sample at at least one wavelength relative to the following terms: protein fraction x 蛋白质 , differential refractive index data, UV absorbance data A, refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient of the protein component at at least one wavelength ε 蛋白质and the path length L of the cell in the UV detector, and (4) the DNA extinction coefficient ε calculated by the computer system relative to DNA In another embodiment, the method, system and computer program product further comprises calculating, by the computer system, the protein fraction x of the sample relative to: 蛋白质 :Light scattering data, differential refractive index data, molecular weight M of protein components 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA and the optical constant K.

[0015] In one embodiment, the computer implemented methods, systems, and computer program products allow for accurate calculation of DNA extinction coefficients at 260 nm and 280 nm, which in turn allows for accurate AAV quality attribute measurements.

[0016] definition

[0017] Particles

[0018] 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.

[0019] Analysis of macromolecules or particulate matter in solution

[0020] 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.

[0021] Light Scattering

[0022] 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.

[0023] Dynamic Light Scattering

[0024] 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.

[0025] Static Light Scattering

[0026] 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.

[0027] Multi-angle light scattering

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Concentration detector

[0033] Differential Refractive Index Detector

[0034] 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.

[0035] A differential refractive index detector includes a flow cell having two sections: one section for the sample; and one section for a 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.

[0036] UV-Vis spectroscopy

[0037] 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. Such absorption spectroscopy measures transitions from a ground state to an excited state. A UV-Vis detector / UV-Vis spectrophotometer measures the intensity of light passing through a sample (I) and compares it to the intensity of light passing through the sample (I). o ) is compared with the intensity of the light before, where the ratio I / I o It is called transmittance and is usually expressed as a percentage (%T). The absorbance A is calculated from the transmittance using the formula

[0038] A = -log (%T / 100%).

[0039] 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 light reflected, 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.

[0040] Current Technology

[0041] Current techniques for measuring AAV quality attributes require the following sample constants: the corresponding dn / dc values ​​and extinction coefficients of the AAV protein capsid and encapsulated DNA. Although the dn / dc values ​​are well known, the extinction coefficients of the protein capsid at 260nm and 280nm can be estimated from the amino acid sequence of the capsid protein and can also be determined experimentally using software. However, the encapsulated DNA extinction coefficient is not well known. Current techniques reverse-calculate these values ​​assuming that the "full" AAV sample is 100% full. However, in the real world, 100% full AAV samples are often not available, and the encapsulated DNA extinction coefficient can vary with DNA length. Therefore, there is a need to more accurately measure the quality attributes of viral samples.

[0042] See also Figure 1AIn an exemplary embodiment, the computer-implemented method, system, and computer program product are configured to perform operation 110: receiving, by the computer system, light scattering (LS) data from a light scattering detector of an isolated object of an analyzed virus sample, differential refractive index (dRI) data from a differential refractometer of the analyzed isolated object, and UV absorbance data A at at least one wavelength of the analyzed isolated object from an ultraviolet (UV) detector; operation 112: receiving, by the computer system, a molecular weight M of a protein component of the sample from a data source; 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 ; Operation 114: Calculate, by the computer system, the protein fraction x of the sample relative to 蛋白质 :Light scattering data, differential refractive index data, molecular weight M of protein components 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA and optical constant K; Operation 116: Calculate, by the computer system, the DNA extinction coefficient ε of the sample at at least one wavelength relative to DNA : The calculated protein fraction x 蛋白质 , differential refractive index data, UV absorbance data A, refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient of the protein component at at least one wavelength 蛋白质 and the path length L of the cell in the UV detector; and operation 118: calculating by the computer system relative to the calculated DNA extinction coefficient ε DNA To calculate the quality attribute value of the sample.

[0043] In an exemplary embodiment, the computer system is a stand-alone computer system such as Figure 5 The computer system 500 shown is 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 a computer system that executes a virus sample script or a computer software application that performs at least the operations of method 100. In one embodiment, the computer system is as follows: Figure 5The computer system / server 512 shown in FIG. 1 is a computer system / server 512 that executes a virus sample script or a computer software application that performs at least the operations of method 100. In one embodiment, the computer system is as shown in FIG. Figure 5 The processing unit 516 is shown, which executes a measured quality attribute of a virus sample script or a computer software application that performs at least the operations of method 100. In one embodiment, the computer system is a machine learning computer software / program / algorithm that executes a measured quality attribute of a virus sample script or a computer software application that performs at least the operations of method 100.

[0044] In one embodiment, the computer system is Figure 5 The computer system 500 shown in FIG. 1 is a computer system that executes a virus sample script or a computer software application for measuring quality attributes, the virus sample script or computer application performing at least operations 110, 112, 114, 116, and 118. In one embodiment, the computer system is as follows Figure 5 The computer system / server 512 shown executes a virus sample script or a computer software application to measure quality attributes, the virus sample script or computer application performing at least operations 110, 112, 114, 116, and 118. In one embodiment, the computer system is as shown in FIG. Figure 5 A processing unit 516 is shown executing a measured quality attribute of a virus sample script or a computer software application that performs at least operations 110 , 112 , 114 , 116 , and 118 .

[0045] See also Figure 1B In an exemplary embodiment, the computer-implemented method, system, and computer program product include a receiver 120 and a computer 122. In one embodiment, the receiver 120 is configured to receive light scattering (LS) data 130 from a light scattering detector 140 analyzing an isolate of a virus sample 150, differential refractive index (dRI) data 132 from a differential refractometer 142 analyzing the isolate 150, and UV absorbance data A 134 at at least one wavelength of the analyzed isolate 150 from an ultraviolet (UV) detector 144. In one embodiment, the receiver 120 includes a computer system that performs operation 110, such as Figure 5 In one embodiment, receiver 120 includes a computer system that performs operation 110, such as Figure 5 The computer system / server 512 is shown. In one embodiment, the receiver 120 includes a computer system that performs operation 110, such as Figure 5 In one embodiment, the receiver 120 is implemented as a processor in a computer system such as Figure 5 10. In one embodiment, receiver 120 is implemented as computer software executed on a computer system (such as a computer system 500 shown in FIG. 10 ) to cause the computer system to perform operation 110. Figure 5 1, the receiver 120 is implemented as computer software executed on a computer system (such as a computer system / server 512) to cause the computer system to perform operation 110. In one embodiment, the receiver 120 is implemented as a computer system (such as a computer system / server 512) to perform operation 110. Figure 5 1. In one embodiment, receiver 120 performs operation 110 as computer software executed on a processor of receiver 120.

[0046] In one embodiment, the receiver 120 is configured to receive the molecular weight M of the protein component of the sample from the data source 146. 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 In one embodiment, receiver 122 includes a computer system that performs operation 112, such as Figure 5 In one embodiment, receiver 120 includes a computer system that performs operation 112, such as Figure 5 The computer system / server 512 is shown. In one embodiment, the receiver 120 includes a computer system that performs operation 112, such as Figure 5 In one embodiment, the receiver 120 is implemented as a processor in a computer system such as Figure 5 110. In one embodiment, receiver 120 is implemented as computer software executed on a computer system (such as a computer system 500) to cause the computer system to perform operation 112. Figure 5 112) to cause the computer system to perform operation 112. In one embodiment, receiver 120 is implemented as computer software executed on a computer system (such as Figure 5 1. In one embodiment, receiver 120 performs operation 112 as computer software executed on a processor of receiver 120.

[0047] In one embodiment, calculator 122 is configured to calculate the protein fraction x of a sample relative to 蛋白 160: Light scattering (LS) data 130, differential refractive index (dRI) data 132, molecular weight M of protein components 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA and the optical constant K. In one embodiment, the calculator 122 includes a computer system that performs operation 114, such as the computer system 800 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 114, such as the computer system / server 812 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 114, such as the processing unit 816 shown in FIG. 8. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system 800 shown in FIG. 8), so that the computer system performs operation 114. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system / server 812 shown in FIG. 8), so that the computer system performs operation 114. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the processing unit 816 shown in FIG. 8), so that the computer system performs operation 114. In one embodiment, the calculator 122 performs operation 114 as computer software executed on a processor of the calculator 122.

[0048] In one embodiment, calculator 122 is configured to calculate the DNA extinction coefficient ε of the sample at at least one wavelength relative to DNA 162: Calculated protein fraction x 蛋白质 160. Differential refractive index (dRI) data 132. Ultraviolet (UV) absorbance data A 134. Refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质and the path length L of the cell in the UV detector. In one embodiment, the calculator 122 includes a computer system that performs operation 116, such as the computer system 800 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 116, such as the computer system / server 812 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 116, such as the processing unit 816 shown in FIG. 8. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system 800 shown in FIG. 8) so that the computer system performs operation 116. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system / server 812 shown in FIG. 8) so that the computer system performs operation 116. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the processing unit 816 shown in FIG. 8) so that the computer system performs operation 116. In one embodiment, the calculator 122 performs operation 116 as computer software executed on a processor of the calculator 122.

[0049] In one embodiment, calculator 122 is configured to calculate the DNA extinction coefficient ε DNA 162 to calculate the quality attribute value 164 of the sample. In one embodiment, the calculator 122 includes a computer system that performs operation 118, such as the computer system 800 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 118, such as the computer system / server 812 shown in FIG. 8. In one embodiment, the calculator 122 includes a computer system that performs operation 118, such as the processing unit 816 shown in FIG. 8. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system 800 shown in FIG. 8), causing the computer system to perform operation 118. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the computer system / server 812 shown in FIG. 8), causing the computer system to perform operation 118. In one embodiment, the calculator 122 is implemented as computer software executed on a computer system (such as the processing unit 816 shown in FIG. 8), causing the computer system to perform operation 118. In one embodiment, the calculator 122 performs operation 118 as computer software executed on a processor of the calculator 122.

[0050] See also Figure 2In an exemplary embodiment, the computer-implemented method, system, and computer program product are configured to perform operation 210: receiving, by the computer system, light scattering data from a light scattering detector for analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer for analyzing the isolate, and UV absorbance data A at at least one wavelength of the analyzer isolate from an ultraviolet (UV) detector; operation 212: receiving, by the computer system, a molecular weight M of a protein component of the sample from a data source 蛋白质(预期的) , Refractive index increment of protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at at least one wavelength 蛋白质 Operation 214: Calculate the DNA extinction coefficient ε of the sample at at least one wavelength by the computer system relative to the following terms DNA : Protein fraction x 蛋白质 , differential refractive index data, UV absorbance data A, refractive index increment of protein components (dn / dc) 蛋白质 , Refractive index increment of DNA component (dn / dc) DNA , the extinction coefficient of the protein component at at least one wavelength ε 蛋白质 and the path length L of the cell in the UV detector; and operation 216: calculating by the computer system relative to the calculated DNA extinction coefficient ε DNA To calculate the quality attribute value of the sample.

[0051] In one embodiment, the computer system is Figure 5 The computer system 500 shown in FIG. 1 is a computer system that executes a virus sample script or a computer software application that performs at least the operations of method 200. In one embodiment, the computer system is as follows: Figure 5 The computer system / server 512 shown in FIG. 5 is a computer system / server 512 that executes a virus sample script or a computer software application that performs at least the operations of method 200. In one embodiment, the computer system is as shown in FIG. Figure 5 The processing unit 516 is shown, which executes a virus sample script or a computer software application that measures quality attributes, and the virus sample script or computer application performs at least the operations of method 200. In one embodiment, the computer system is a machine learning computer software / program / algorithm that executes a virus sample script or a computer software application that measures quality attributes, and the virus sample script or computer application performs at least the operations of method 200.

[0052] In one embodiment, the computer system is Figure 5The computer system 500 shown in FIG. 1 is a computer system that executes a virus sample script or a computer software application for measuring quality attributes, the virus sample script or computer application performing at least operations 210, 212, 214, and 216. In one embodiment, the computer system is as follows Figure 5 The computer system / server 512 shown executes a virus sample script or a computer software application for measuring quality attributes, the virus sample script or computer application performing at least operations 210, 212, 214, and 216. In one embodiment, the computer system is as follows Figure 5 A processing unit 516 is shown that executes a measured quality attribute of a virus sample script or a computer software application that performs at least operations 210 , 212 , 214 , and 216 .

[0053] Calculation of protein fractions

[0054] In one embodiment, the protein fraction x of a sample is calculated 蛋白质 The computer system calculates the protein fraction x of the sample according to the following formula: 蛋白质

[0055]

[0056] Where R θ is the hyper-Rayleigh ratio derived from the light scattering data, and dRI is the differential refractive index (dRI) data. In another embodiment, the protein fraction x of the sample is calculated 蛋白质 Also included is the calculation by the computer system of the optical constant K with respect to the solvent refractive index n0, the wavelength λ of the light scattering detector, and the Avogrado number N A In one embodiment, calculating the optical constant K comprises calculating the optical constant K by a computer system according to the following formula:

[0057]

[0058] Calculation of DNA extinction coefficient

[0059] In one embodiment, the DNA extinction coefficient ε of the sample is calculated DNA The DNA extinction coefficient ε of the sample is calculated according to the following formula: DNA :

[0060]

[0061] Calculating Mass Property Values

[0062] In one embodiment, calculating the quality attribute value of the sample includes calculating, by the computer system, the value of the quality attribute value relative to the calculated DNA extinction coefficient εDNA To calculate the ratio of whole capsid to total capsid of the sample V g / C p In one embodiment, calculating the quality attribute value of the sample includes calculating, by the computer system, the value of the quality attribute of the sample relative to the calculated DNA extinction coefficient ε DNA To calculate the particle concentration value C of the sample p In one embodiment, calculating the quality attribute value of the sample includes calculating, by the computer system, the value of the quality attribute of the sample relative to the calculated DNA extinction coefficient ε DNA To calculate the molar mass value M of the sample.

[0063] Example

[0064] In one embodiment, Figure 3A and Figure 3B The method, system and computer product are described to measure the whole capsid to total capsid ratio value V of a sample g / C p It is worth noting that Figure 3A Depicts the measured V g / C p Values ​​and expected V g / C p There is a high correlation between the values. Figure 3A , Figure 3B and Table 1 present examples of a series of AAV samples for which the optimized extinction coefficient of encapsulated DNA was collected at 260 nm, at 280 nm, or at both 260 nm and 280 nm (if UV data was available at both wavelengths) using the method, system, and computer product.

[0065] AAV samples were prepared by mixing known amounts of "full" and "empty" AAV of the same serotype. Mixtures ranged from 3% full (Vg / Cp=0.03) to 97% full (Vg / Cp=0.97), and data for 100% empty and 100% full were also collected. Each sample was analyzed in duplicate. For each measurement, particle concentration, molar mass values, and full to total ratio (Vg / Cp) were calculated by using (i) non-optimized "seed values" and (ii) optimized coefficients obtained according to the method, system, and computer product. The input parameters are summarized in Table 1.

[0066]

[0067] Table 1

[0068] Although the optimized coefficients are not much different from the seed values, their impact on the measured Vg / Cp is significant. Figure 3AThe Vg / Cp measured using the optimized extinction coefficient is shown compared to the seed value. In mixtures with a low percentage of full AAV (low Vg / Cp), there is minimal effect of the improved extinction coefficient, however at higher Vg / Cp typical of purified samples, the difference becomes significant, as shown in Figure 3A and Figure 3B shown.

[0069] In the case of an unoptimized extinction coefficient, the error of Vg / Cp gradually gets worse as Vg / Cp increases, e.g. Figure 3B As shown. Figure 3B As shown, in the worst case, with the unoptimized coefficients, a sample known to be 97% full was measured to be only 88% full, whereas the error of Vg / Cp using the optimized coefficients was within 0.04, with most of the data falling within ±0.02 of the actual value.

[0070] In one embodiment, Figure 4 A typical hardware / instrument configuration of the method, system, and computer product is depicted. For example, a typical hardware / instrument configuration may include a degasser 410 , a pump 412 , an autosampler 414 , a UV detector 416 , an SLS instrument 418 , and a dRI detector 420 .

[0071] Computer Systems

[0072] In an exemplary embodiment, the computer system is Figure 5 The computer system 500 shown. The computer system 500 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.

[0073] 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.

[0074] 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.

[0075] like Figure 5 As shown, 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Computer program product

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Various aspects of the present invention are described herein with reference to the flow diagram and / or block diagram of the method, device (system) and computer program product according to embodiments of the present invention. It will be understood that each frame in the flow diagram and / or block diagram, and the combination of frames in the flow diagram and / or block diagram can be implemented by computer-readable program instructions.

[0087] 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 the computer, programmable data processing device, and / or other device 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.

[0088] 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 boxes.

[0089] 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.

[0090] 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, light scattering data from a light scattering detector analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer analyzing the isolate, and UV absorbance data A from an ultraviolet (UV) detector at at least one wavelength analyzing the isolate; The computer system receives the molecular weight M of the protein component of the sample from a data source. 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 ; The protein fraction x of the sample is calculated by the computer system relative to 蛋白质 :The light scattering data, the differential refractive index data, the molecular weight M of the protein component 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA and the optical constant K; The DNA extinction coefficient ε of the sample at the at least one wavelength is calculated by the computer system relative to DNA : The calculated protein fraction x 蛋白质 , the differential refractive index data, the ultraviolet absorbance data A, the refractive index increment (dn / dc) of the protein component 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 and the path length L of the cell in the UV detector; as well as The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the quality attribute value of the sample.

2. The method of claim 1, wherein the light scattering data comprises static light scattering (SLS) data.

3. The method of claim 2, wherein the light scattering data further comprises dynamic light scattering (DLS) data. The method according to claim 1 , wherein the virus sample comprises viruses having a radius less than 30 nm.

5. The method of claim 4, wherein the viral sample comprises adeno-associated virus (AAV). The method of claim 1 , wherein the at least one wavelength is 260 nm. The method of claim 1 , wherein the at least one wavelength is 280 nm.

8. The method according to claim 1, wherein the protein fraction x of the sample is calculated 蛋白质 Also includes: The optical constant K is calculated by the computer system with respect to: the solvent refractive index n0, the wavelength λ of the light scattering detector and the Avogrado number N A .

9. The method of claim 1 , wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the ratio of the whole capsid to the total capsid of the sample V g / C p .

10. The method of claim 1, wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the particle concentration value C of the sample p .

11. The method of claim 1 , wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the molar mass value M of the sample.

12. A computer-implemented method, the computer-implemented method comprising: Receiving, by a computer system, light scattering data from a light scattering detector analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer analyzing the isolate, and UV absorbance data A from an ultraviolet (UV) detector at at least one wavelength analyzing the isolate; The computer system receives the molecular weight M of the protein component of the sample from a data source. 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 , the protein fraction x of the sample 蛋白质 ; The DNA extinction coefficient ε of the sample at the at least one wavelength is calculated by the computer system relative to DNA : The protein fraction x 蛋白质 , the differential refractive index data, the ultraviolet absorbance data A, the refractive index increment (dn / dc) of the protein component 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 and the path length L of the cell in the UV detector; as well as The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the quality attribute value of the sample.

13. The method according to claim 12, further comprising: The protein fraction x of the sample is calculated by the computer system relative to 蛋白质 :The light scattering data, the differential refractive index data, the molecular weight M of the protein component 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA and the optical constant K.

14. The method according to claim 13, wherein the protein fraction x of the sample is calculated 蛋白质 , also includes: The optical constant K is calculated by the computer system with respect to the solvent refractive index n0, the wavelength λ of the light scattering detector and the Avogrado number N A .

15. The method of claim 12, wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the ratio of the whole capsid to the total capsid of the sample V g / C p .

16. The method of claim 12, wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the particle concentration value C of the sample p .

17. The method of claim 12, wherein calculating the quality attribute value of the sample comprises: The DNA extinction coefficient ε calculated by the computer system is DNA To calculate the molar mass value M of the sample.

18. A system, comprising: Memory; and a processor in communication with the memory, the processor being configured to perform a method comprising: receiving light scattering data from a light scattering detector analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer analyzing the isolate, and UV absorbance data A from an ultraviolet (UV) detector at at least one wavelength analyzing the isolate, Receive the molecular weight M of the protein component of the sample from the data source 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 , The protein fraction x of the sample was calculated relative to 蛋白质 :The light scattering data, the differential refractive index data, the molecular weight M of the protein component 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA and the optical constant K, The DNA extinction coefficient ε of the sample at the at least one wavelength is calculated relative to DNA : The calculated protein fraction x 蛋白质 , the differential refractive index data, the ultraviolet absorbance data A, the refractive index increment (dn / dc) of the protein component 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 and the path length L of the cell in the UV detector, and Relative to the calculated DNA extinction coefficient ε DNA To calculate the quality attribute value of the sample.

19. A computer program product, the computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions being executable by a processor to cause the processor to perform a method comprising: receiving light scattering data from a light scattering detector analyzing an isolate of a virus sample, differential refractive index data from a differential refractometer analyzing the isolate, and UV absorbance data A from an ultraviolet (UV) detector at at least one wavelength analyzing the isolate; Receive the molecular weight M of the protein component of the sample from the data source 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component of the sample (dn / dc) DNA and the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 ; The protein fraction x of the sample was calculated relative to 蛋白质 :The light scattering data, the differential refractive index data, the molecular weight M of the protein component 蛋白质(预期的) , the refractive index increment of the protein component (dn / dc) 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA and the optical constant K; The DNA extinction coefficient ε of the sample at the at least one wavelength is calculated relative to DNA : The calculated protein fraction x 蛋白质 , the differential refractive index data, the ultraviolet absorbance data A, the refractive index increment (dn / dc) of the protein component 蛋白质 , the refractive index increment of the DNA component (dn / dc) DNA , the extinction coefficient ε of the protein component at the at least one wavelength 蛋白质 and the path length L of the cell in the UV detector; as well as Relative to the calculated DNA extinction coefficient ε DNA To calculate the quality attribute value of the sample.

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