# US 12,000,775 B2 · System suitability method for use with protein concentration determination by slope

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- Number: US 12,000,775 B2
- Document ID: US12000775B2
- Status: Granted
- Kind: B2
- Application number: 17/312,773
- Filed: 2019-12-13
- Published: 2022-05-12
- Granted: 2024-06-04
- Earliest claimed: 2018-12-14
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Robert John Duff; Christian Gutierrez
- Web page: https://patentgrep.com/patent/US12000775B2

## CPC

- G01N 21/33: using ultraviolet light (G01N21/39 takes precedence)
- G01N 21/274: {Calibration, base line adjustment, drift correction}
- G01N 21/278: {Constitution of standards}
- G01N 21/31: Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry {(G01N21/72 takes precedence)}
- G01N 2201/0668: Multiple paths; optimisable path length
- 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)

## Abstract

Disclosed are methods of determining the suitability of a variable-length spectrophotometer using Patent Blue dye or AMG Blue dye. Also disclosed herein are methods of determining the suitability of a fixed path length spectrophotometer for determining protein concentration of a protein sample. AMG Blue dye may also be used to determine the suitability of fixed path length spectrophotometers.

## Claims

### Claim 1

A method of determining the suitability of a variable-length spectrophotometer for determining protein concentration of a protein sample, comprising measuring the absorbance of Patent Blue dye (PBD) or AMG Blue Dye (ABD) at least two wavelengths, the first wavelength of 280 nm and the subsequent wavelength(s) selected from the group consisting of 310 nm, 412 nm, 510 nm and 615 nm.

### Claim 2 (depends on 1)

The method of claim 1, wherein at least a third wavelength is used, wherein the third wavelength is different than the second wavelength and selected from the group consisting of 310 nm, 412 nm, 510 nm and 615 nm.

### Claim 3 (depends on 2)

The method of claim 2, wherein the variable-length spectrophotometer is considered suitable if the absorbance readings of the PBD or ABD are equal to or less than 10% of those values supplied in a certificate of analysis supplied with the PBD or ABD.

### Claim 4 (depends on 1)

The method of claim 1, wherein the PBD or ABD is measured three times for each wavelength before measuring the protein concentration of the protein sample, and wherein the PBD or ABD is measured three times for each wavelength after measuring the protein concentration of the protein samples.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first wavelength is 280 nm and the second wavelength is 310 nm.

### Claim 6 (depends on 1)

The method of claim 1, wherein the variable-length spectrophotometer or fixed path length spectrophotometer is considered suitable if the absorbance readings of the PBD or ABD are at least equal to or less than 5% of those supplied in a certificate of analysis supplied with the PBD or ABD.

### Claim 7 (depends on 1)

The method of claim 1, wherein the readings have a relative standard deviation (RSD) percentage of ≤5%.

### Claim 8 (depends on 1)

The method of claim 1, wherein the protein sample comprises a therapeutic protein.

### Claim 9 (depends on 8)

The method of claim 8, wherein the therapeutic polypeptide is selected from the group consisting of infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, a glycoprotein, CD polypeptide, a HER receptor polypeptide, a cell adhesion polypeptide, a growth factor polypeptide, an insulin polypeptide, an insulin-related polypeptide, a coagulation polypeptide, a coagulation-related polypeptide, albumin, IgE, a blood group antigen, a colony stimulating factor, a receptor, a neurotrophic factor, an interferon, an interleukin, a viral antigen, a lipoprotein, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, mouse gonadotropin-associated peptide, DNAse, inhibin, activing, an integrin, protein A, protein D, a rheumatoid factor, an immunotoxin, a bone morphogenetic protein, a superoxide dismutase, a surface membrane polypeptide, a decay accelerating factor, an AIDS envelope, a transport polypeptide, a homing receptor, an addressin, a regulatory polypeptide, an immunoadhesin, a myostatin, a TALL polypeptide, an amyloid polypeptide, a thymic stromal lymphopoietin, a RANK ligand, a c-kit polypeptide, a TNF receptor, and an angiopoietin, the antibodies shown in Table 1 and biologically active fragments, analogs or variants thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the protein sample comprises an antigen binding protein, an antibody, a bi-specific antibody, a tri-specific antibody, a BiTE molecule, or a fragment or derivative thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the variable length spectrophotometer is a SoloVPE spectrophotometer (C Technologies, Inc.; Bridgewater, NJ).

### Claim 12

A method of determining the suitability of a fixed path length spectrophotometer for determining protein concentration of a protein sample, comprising measuring the absorbance of ABD at least two wavelengths, the first wavelength of 280 nm and the subsequent wavelength(s) selected from the group consisting of 310 nm, 412 nm, 510 nm and 615 nm.

### Claim 13 (depends on 12)

The method of claim 12, wherein at least a third wavelength is used, wherein the third wavelength is different than the second wavelength and selected from the group consisting of 310 nm, 412 nm, 510 nm and 615 nm.

### Claim 14 (depends on 12)

The method of claim 12, wherein the PBD or ABD is measured three times for each wavelength before measuring the protein concentration of the protein sample, and wherein the PBD or ABD is measured three times for each wavelength after measuring the protein concentration of the protein samples.

### Claim 15 (depends on 12)

The method of claim 12, wherein the first wavelength is 280 nm and the second wavelength is 310 nm.

### Claim 16 (depends on 12)

The method of claim 12, wherein the variable-length spectrophotometer or fixed path length spectrophotometer is considered suitable if the absorbance readings of the PBD or ABD are at least equal to or less than 5% of those supplied in a certificate of analysis supplied with the PBD or ABD.

### Claim 17 (depends on 12)

The method of claim 12, wherein the protein sample comprises a therapeutic protein.