	US 12,135,316 B2
	Controlling the purification of a macromolecule solution via real-time multi-angle light scattering
Daniel I. Some, Atlit (IL)
Assigned to Wyatt Technology, LLC, Goleta, CA (US)
Filed by Wyatt Technology, LLC, Goleta, CA (US)
Filed on Jun. 30, 2022, as Appl. No. 17/855,791.
Claims priority of provisional application 63/217,274, filed on Jun. 30, 2021.
Prior Publication US 2023/0022034 A1, Jan. 26, 2023
Int. Cl. G01N 30/74 (2006.01); G01N 30/02 (2006.01); G01N 30/64 (2006.01)

CPC G01N 30/74 (2013.01) [G01N 30/64 (2013.01); G01N 2030/027 (2013.01)]

14 Claims

1. A computer implemented method comprising:

receiving, by a computer system, from a set of instruments baseline multi-angle light scattering (MALS) signal values and baseline ultraviolet (UV) signal values of a pure buffer flowed 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 values from the MALS instrument,

receiving, by the computer system, from the MALS instrument scattering intensity values of a sample solution flowed from the chromatographic purification system to the MALS instrument over a time series,

wherein the sample solution comprises 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 average of the baseline MALS signal values from the scattering intensity values,

resulting in an intensity time series of excess scattering intensity values, Iscatt_j;

in response to receiving the baseline UV signal values from the UV detector,

receiving, by the computer system, from the UV detector UV absorption values of the sample solution flowed from the chromatographic purification system to the UV detector over the time series,

calculating, by the computer system, an average of the baseline UV signal values,

executing, by the computer system, a set of logical operations applying UV absorption alignment corrections with respect to UV absorption alignment parameters and UV absorption band broadening corrections with respect to UV absorption band broadening parameters, to the UV absorption values over the time series relative to the UV detector and the MALS instrument, resulting in 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 absorption values,

resulting in a UV time series of excess UV absorption values;

calculating, by the computer system, concentration values of the sample solution, c_j, with respect to the UV time series of excess UV absorption values and an absorption coefficient of the sample solution according to Beer's Law;

receiving, by the computer system, pH values of the sample solution from a pH detector, over the time series, and conductivity values of the sample solution from a conductivity detector, over the time series,

wherein the set of instruments further comprises the pH detector and the conductivity detector;

executing, by the computer system, a set of logical operations applying pH alignment corrections with respect to pH alignment parameters to the pH values over the time series relative to the pH detector and the MALS instrument, resulting in corrected pH values over the time series, and applying conductivity alignment corrections with respect to conductivity alignment parameters to the conductivity values over the time series relative to the conductivity detector and the MALS instrument, resulting in corrected conductivity values over the time series,

resulting in a plurality of signals comprising the intensity time series of excess scattering intensity values, Iscatt_j, the concentration values of the sample solution, c_j, the corrected pH values, and the corrected conductivity values;

executing, by the computer system, a set of logical operations averaging each of the plurality of signals over time ranges, t₁, t₂, . . . t_n,

wherein the time series comprises the time ranges,

wherein the time ranges correspond to fractions of the sample solution collected by a fraction collector connected to the set of instruments,

resulting in average values, [lscatt, c, pH, cond]₁, [lscatt, c, pH, cond]₂, . . . [lscatt, c, pH, cond]_n;

receiving, by the computer system, aggregate content values, % agg₁, % agg₂, . . . % agg_n, and weight-average molar mass values, M₁, M₂, . . . M_n, of the fractions of the sample solution measured by a SEC-MALS instrument; and

storing, by the computer system, in a macromolecular characterization look-up table (MCLUT) the aggregate content values, % agg₁, % agg₂, . . . % agg_n, and the weight-average molar mass values, M₁, M₂, . . . M_n, versus the average values, [lscatt, c, pH, cond]₁, [lscatt, c, pH, cond]₂, . . . [lscatt, c, pH, cond]_n.