# US 12,181,404 B2 · Systems and methods for determining protein concentrations of unknown protein samples based on automated multi-wavelength calibration

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- Number: US 12,181,404 B2
- Document ID: US12181404B2
- Status: Granted
- Kind: B2
- Application number: 17/632,886
- Filed: 2020-08-06
- Published: 2022-09-01
- Granted: 2024-12-31
- Earliest claimed: 2019-08-06
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Shang Zeng; Gang Xue
- Web page: https://patentgrep.com/patent/US12181404B2

## CPC

- G01N 21/33: using ultraviolet light (G01N21/39 takes precedence)
- G01J 3/2823: {Imaging spectrometer}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01J 2003/283: {computer-interfaced}
- G01J 2003/2833: {and memorised spectra collection}
- G01N 2201/127: Calibration; base line adjustment; drift compensation
- G01N 2201/129: Using chemometrical methods
- G01N 2201/1296: using neural networks
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G01J: MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY (light sources F21, H01J, H01K, H05B; investigating properties of materials by optical means G01N)

## Abstract

ultraviolet (UV) based imaging method for determining protein concentrations of unknown protein samples based on automated multi-wavelength calibration. In various embodiments, a processor receives each of a standard set of wavelength data and an unknown set of wavelength data as recorded by a detector. Each standard set of wavelength data and unknown set of wavelength data defines a series of absorbance-to-wavelength value pairs across a first range of wavelengths selected from a range of a single-wavelength light beams of a UV spectra. The processor generates a multi-wavelength calibration model based on each of a first series of first absorbance-to-wavelength value pairs of the standard set of wavelength data. The processor implements the multi-wavelength calibration model to determine, for each unknown protein sample of the given unknown protein samples, a plurality of protein concentration values.

## Claims

### Claim 1

An ultraviolet (UV) based imaging system configured to determine protein concentrations of unknown protein samples based on automated multi-wavelength calibration, the UV based imaging system comprising:
a light source configured to project a multi-wavelength light beam;
a monochromator configured to receive the multi-wavelength light beam and to project, based on the multi-wavelength light beam, a range of single-wavelength light beams of a UV spectra;
a sample holder structure positioned for receiving a protein sample, the protein sample being either of (1) a standard protein sample selected from a set of standard protein samples, or (2) an unknown protein sample selected from a set of unknown protein samples, the unknown protein sample having an unknown protein concentration, wherein the sample holder is positioned to allow the range of single-wavelength light beams to pass through at least a portion of the protein sample;
a light-sensitive detector device configured to detect the range of single-wavelength light beams passing through the at least the portion of the protein sample, wherein the light-sensitive detector device is configured to determine absorbance values by measuring a variance between the projected range of single-wavelength light beams by the monochromator and the single-wavelength light beams passed through the at least the portion of the protein sample;
a memory storing program instructions and configured to store as wavelength data the range of single-wavelength light beams that pass through the at least the portion of the standard sample and the at least the portion of the unknown sample; and
a processor communicatively coupled to the memory, wherein the processor is configured to execute the program instructions to cause the processor to:
receive, from the memory, a standard set of wavelength data as recorded by the detector for each standard protein sample of the set of standard protein samples, wherein the standard set of wavelength data includes, for each standard protein sample, a first series of first absorbance-to-wavelength value pairs across a first range of wavelengths selected from the range of the single-wavelength light beams, each first absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value,
generate a multi-wavelength calibration model based on each of the first series of first absorbance-to-wavelength value pairs of the standard set of wavelength data,
receive, from the memory, an unknown set of wavelength data as recorded by the detector for each unknown protein sample of the set of unknown protein samples, wherein the unknown set of wavelength data includes, for each unknown protein sample, a second series of second absorbance-to-wavelength value pairs across a second range of wavelengths selected from the range of the single-wavelength light beams, each second absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value, and
determine with the multi-wavelength calibration model, for each unknown protein sample of the set of unknown protein samples, a plurality of protein concentration values, each protein concentration value of the plurality of protein concentration values corresponding to the second range of wavelengths selected from the range of the single-wavelength light beams.

### Claim 2 (depends on 1)

The UV based imaging system of claim 1, wherein the light source, the monochromator, the sample holder, and the detector form a portion of a spectrophotometer device.

### Claim 3 (depends on 1)

The UV based imaging system of claim 1, wherein the processor is communicatively coupled to the detector.

### Claim 4 (depends on 1)

The UV based imaging system of claim 1, wherein the memory or the processor is remote to the detector.

### Claim 5 (depends on 1)

The UV based imaging system of claim 1, wherein the set of standard protein samples comprises a plurality of standard protein samples, and wherein the set of unknown protein samples comprises a single protein sample having the unknown protein concentration.

### Claim 6 (depends on 1)

The UV based imaging system of claim 1, wherein the set of standard protein samples are protein standard solutions in the range of 0.1 to 220 milligrams (mg) per milliliter (ml).

### Claim 7 (depends on 1)

The UV based imaging system of claim 1, wherein the range of single-wavelength light beams comprises single-wavelength light beams in the UV spectra from 200 nanometers (nm) to 400 nanometers (nm).

### Claim 8 (depends on 1)

The UV based imaging system of claim 1, wherein generating the multi-wavelength calibration model includes generating a single-wavelength calibration model for each of the first range of wavelengths selected from the range of the single-wavelength light beams, each single-wavelength calibration model comprising a slope value and a y-intercept value corresponding to UV absorbance of particular single wavelength.

### Claim 9 (depends on 1)

The UV based imaging system of claim 1, wherein the multi-wavelength calibration model is trained as a machine-learning model.

### Claim 10 (depends on 1)

The UV based imaging system of claim 1, further comprising a standard wavelength data filter, the processor further configured to execute the program instructions to cause the processor to implement the standard wavelength data filter against the range of the single-wavelength light beams to select the first range of wavelengths.

### Claim 11 (depends on 1)

The UV based imaging system of claim 1, further comprising an unknown wavelength data filter, the processor further configured to execute the program instructions to cause the processor to implement the unknown wavelength data filter against the range of the single-wavelength light beams to select the second range of wavelengths.

### Claim 12 (depends on 7)

The UV based imaging system of claim 7, wherein each single-wavelength light beam in the UV spectra is separated by a one nanometer interval.

### Claim 13 (depends on 10)

The UV based imaging system of claim 10, wherein the processor is further configured to execute the program instructions to cause the processor to implement the standard wavelength data filter to limit each first series of first absorbance-to-wavelength value pair based on UV absorbance values within a first filter range of UV absorbance values.

### Claim 14 (depends on 11)

The UV based imaging system of claim 11, wherein the processor is configured to execute the program instructions to cause the processor to implement the unknown wavelength data filter to limit each second series of second absorbance-to-wavelength value pair based on UV absorbance values within a second filter range of UV absorbance values.

### Claim 15

An ultraviolet (UV) based imaging method for determining protein concentrations of unknown protein samples based on automated multi-wavelength calibration, the UV based imaging method comprising:
projecting a multi-wavelength light beam toward a monochromator;
projecting a range of single-wavelength light beams based on the multi-wavelength light beam toward a protein sample, wherein the protein sample includes either an unknown protein sample or a standard protein sample;
detecting a range of single-wavelength light beams that pass through at least a portion of the protein sample;
recording the range of single-wavelength light beams as wavelength data;
receiving, at a processor, a standard set of wavelength data as recorded by a detector for each standard protein sample of a set of standard protein samples, wherein the standard set of wavelength data includes, for each standard protein sample, a first series of first absorbance-to-wavelength value pairs across a first range of wavelengths selected from a range of a single-wavelength light beams of a UV spectra, each first absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value;
generating, with the processor, a multi-wavelength calibration model based on each of the first series of first absorbance-to-wavelength value pairs of the standard set of wavelength data;
receiving, at the processor, an unknown set of wavelength data as recorded by the detector for each unknown protein sample of a set of unknown protein samples, wherein the unknown set of wavelength data includes, for each unknown protein sample, a second series of second absorbance-to-wavelength value pairs across a second range of wavelengths selected from the range of the single-wavelength light beams, each second absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value; and
determining, with the multi-wavelength calibration model as implemented on the processor, for each unknown protein sample of the set of unknown protein samples, a plurality of protein concentration values, each protein concentration value of the plurality of protein concentration values corresponding to the second range of wavelengths selected from the range of the single-wavelength light beams.

### Claim 16 (depends on 15)

The UV based imaging method of claim 15, wherein the processor is further configured to generate an enhanced prediction based on the plurality of protein concentration values.

### Claim 17 (depends on 15)

The UV based imaging method of claim 15, wherein at least one unknown protein sample of the set of unknown protein samples comprises a protein therapeutic.

### Claim 18 (depends on 16)

The UV based imaging method of claim 16, wherein the generating of the enhanced prediction comprises averaging each protein concentration value of the plurality of protein concentration values corresponding to the second range of wavelengths.

### Claim 19 (depends on 17)

The UV based imaging method of claim 17, wherein the protein therapeutic is selected from the group consisting of an antibody, an antigen-binding antibody fragment, an antibody protein product, a Bi-specific T cell engager molecule, a bispecific antibody, a trispecific antibody, an Fc fusion protein, a recombinant protein, and an active fragment of a recombinant protein.

### Claim 20

A tangible, non-transitory computer-readable medium storing instructions for determining protein concentrations of unknown protein samples based on automated multi-wavelength calibration, that when executed by one or more processors of a computing device, cause the computing device to:
receive, at a processor, a standard set of wavelength data as recorded by a detector for each standard protein sample of a set of standard protein samples, wherein the standard set of wavelength data includes, for each standard protein sample, a first series of first absorbance-to-wavelength value pairs across a first range of wavelengths selected from a range of a single-wavelength light beams of a UV spectra, each first absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value;
generate, with the processor, a multi-wavelength calibration model based on each of the first series of first absorbance-to-wavelength value pairs of the standard set of wavelength data;
receive, at the processor, an unknown set of wavelength data as recorded by the detector for each unknown protein sample of a set of unknown protein samples, wherein the unknown set of wavelength data includes, for each unknown protein sample, a second series of second absorbance-to-wavelength value pairs across a second range of wavelengths selected from the range of the single-wavelength light beams, each second absorbance-to-wavelength value pair comprising a UV absorbance value and a wavelength value; and
determine, with the multi-wavelength calibration model as implemented on the processor, for each unknown protein sample of the set of unknown protein samples, a plurality of protein concentration values, each protein concentration value of the plurality of protein concentration values corresponding to the second range of wavelengths selected from the range of the single-wavelength light beams.