Development of high-throughput assay for identification of al3+ traces in biological drug products

Abstract

The present invention provides the use of a compound of formula (I) as defined herein for detecting aluminum ion concentration in a biological product such as, for example, an aqueous antibody composition.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480068912.4 (22) Application Date 2024.11.01 (30) Priority Data 23207576.2 2023.11.03 EP (85) PCT International Application Entering National Phase Date 2026.04.27 (86) PCT International Application Application Data PCT / EP2024 / 080912 2024.11.01 (87) PCT International Application Publication Data WO2025 / 093726 EN 2025.05.08 (71) Applicant: Hofmeister Roche Ltd. Address: Switzerland (72) Inventors: A. Almendinger, S. Elozabrech, L. Dietler, K. Gregoriza, S.K.K. Raouli, T. Strom (74) Patent Agency: Beijing Zhongzi Law Firm, 11247 Patent Attorneys: Shen Xiaoshu, Huang Gesheng (51) Int.Cl. G01N 33 / 52 (2006.01) G01N 33 / 84 (2006.01) (54) Invention Title: Development of a high-throughput assay for identifying trace amounts of Al3+ in biopharmaceutical products (57) Abstract: This invention provides the use of compounds of formula (I) as defined herein for detecting the concentration of aluminum ions in biopharmaceutical products such as, for example, aqueous antibody compositions. Claims 2 pages, Description 11 pages, Drawings 5 ​​pages, CN 122095249 A 2026.05.26 CN 1 22 09 52 49 A 1. Use of Formula (I) and Compound (I) in the diagnostic determination of aluminum ions (Al3+) in quantitative biological products. 2. The use according to claim 1, wherein the quantification is performed by fluorescence spectroscopy. 3. The use according to claim 2, wherein a fixed excitation wavelength of 374 nm is used to record fluorescence spectra at 400 nm to 500 nm (in increments of 1 nm). 4. The use according to claim 2, wherein fluorescence emission is recorded at 452 nm (excited at 374 nm). 5. The use according to any one of claims 2 to 4, wherein the sample solution is transferred to a black 96-well plate, and the wells are homogenized by shaking the plate for 120 seconds before each measurement. 6. The use according to any one of claims 1 to 6, wherein it is used for the quantification of aluminum ions in the ppb range, preferably up to 500 ppm, more preferably up to 300 ppm. 7. The use according to claim 6, wherein it is used for the quantification of aluminum ions in the range of 0.006 ppm to 300 ppm.8. For the quantification of aluminum ions in the range of 5 ppm to 300 ppm; or for the quantification of aluminum ions in the range of 5 ppm to 300 ppm. 9. The use according to any one of claims 1 to 7, wherein the biological product is an aqueous antibody composition optionally comprising additional pharmaceutically acceptable excipients, such as buffers, stabilizers, antioxidants, and surfactants. 10. The use according to claim 8, wherein the antibody is present in the aqueous composition at a concentration providing the desired pharmaceutical activity and acceptable safety characteristics of the antibody. 11. The use according to claim 9, wherein the antibody is present in the following concentration ranges: 1 mg / ml to 220 mg / ml; or 5 mg / ml to 180 mg / ml; or 5 to 100 mg / ml; or 5 mg / ml to 25 mg / ml. 12. The use according to any one of claims 8 to 10, wherein the biological product comprises a histidine buffer, methionine, and a surfactant, wherein the histidine buffer has a pH of 5.0 to 6.0, preferably 5.0 to 5.5, and the surfactant is selected from the polysorbate or poloxamer class. 12. The use according to any one of claims 8 to 11, wherein the surfactant is selected from PS20 (Croda International, Snaith, UK), PS80 (polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Px188 (poloxam 188; BASF, Ludwigshafen, D). 13. The use according to any one of claims 8 to 12, wherein the surfactant is present at a concentration of 0.001 mg / ml to 1.0 mg / ml; or at a concentration of 0.01 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 0.6 mg / ml. 14. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is PS2O at a concentration of about 0.006 mg / ml to 0.6 mg / ml. 15. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is at a concentration of about 0.006 mg / ml to 0.6 mg / ml, preferably 0.06 mg / ml.16. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is Px188 at a concentration up to 0.6 mg / ml. 17. The use according to any one of claims 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20. 18. The use according to any one of claims 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 230 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP), and 0.05% PS20. 19. A method for detecting the amount of aluminum ions in a biological product, wherein the method comprises any one of the uses according to claims 1 to 18. 20. The method according to claim 19, wherein the method is an automated high-throughput screening method that uses standard equipment for sample uptake (e.g., a 96-well plate) and a software-equipped fluorescence reader to automatically analyze each well, detect, and display the analytical results, such as the Al3+ concentration in a sample of a biological product or such product. Claims 2 / 2 Page 3 CN 122095249 A Development of a High-Throughput Determination Technology for Identifying Trace Aluminum 3+ in Biopharmaceutical Products

[0001] The present invention relates to the field of quantifying trace metals in aqueous pharmaceutical protein (e.g., antibody) formulations containing polysorbate 20, polysorbate 80, and poloxamer 188 as surfactants. Therefore, the present invention helps stabilize said formulations to prevent the formation of visible particles. Background Art

[0002] Surfactants are key excipients in protein formulations because they protect unstable proteins from interfacial stresses that can lead to protein aggregation. Proteins such as monoclonal antibodies (mAbs) are administered parenterally,This limits the choice of surfactants, including the most commonly used surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80). PS20 may degrade during the shelf life of pharmaceutical products via oxidative degradation or hydrolytic degradation, which is typically catalyzed by host cell proteins. In particular, hydrolytic degradation produces free fatty acids (FFA) as degradation products, which precipitate in solution once their solubility limits are exceeded and subsequently form sub-visible and visible particles. Under conditions commonly found in biopharmaceutical formulations, FFA may even precipitate below their solubility limits, depending on various factors. Previous studies have demonstrated that the primary cause of premature particle formation is the presence of trivalent cations, such as aluminum ions, which act as nucleating seeds by complexing negatively charged FFA. These trivalent cations may originate from purification processes or leach from the primary packaging material over time. Even trace amounts (in the ppb range) of these trivalent cations have been shown to efficiently form metal-FFA complexes, leading to premature FFA particle formation below the solubility limits of FFA [1,2].

[0003] Traditionally, various analytical methods, such as inductively coupled plasma mass spectrometry and atomic absorption spectrometry, have been used for the detection and quantification of trace levels (down to parts per quadrillion (ppq)) of metallic impurities in biofluids. While these techniques offer excellent sensitivity and accuracy, they also have drawbacks such as high material consumption (in the mL range), high cost, labor-intensive, high skill requirements for staff, and slow analysis time. Therefore, the limited sample throughput of these methods poses a significant challenge for analyzing large batches of biopharmaceutical products [3-5].

[0004] The present invention addresses these problems by providing a high-throughput determination of aluminum ions in bioproducts in the ppb range using a conventional fluorescence plate reader. For this purpose, a fluorescence “on” type chemical sensor based on a thieno-Schiff base (i.e., compound of formula (I)) is used, which exhibits high sensitivity and Al3+ selectivity in water [6]. Figure Descriptions

[0005] Figure 1: The fluorophore molecule “thieno-Schiff base derivative” (TSB) and its proposed binding mechanism with aluminum ions.

[0006] Figure 2: Fluorescence intensity (FI) spectra of TSB in buffer solutions (20 mM His / His-HCl, 240 mM sucrose, 10 mM methionine, pH 5.5), with Al3+ concentrations ranging from 0 ppb to 150 ppb (374 nm excitation, 400 nm to 500 nm emission, 1 nm measurement step).

[0007] Figure 3: 0 ppb in different histidine buffer systems supplemented with 10 µM TSBFluorescence intensity (FI) curve of Al3+ up to 150 ppb at 452 nm (excitation 374 nm, emission 452 nm, cutoff 435 nm). Buffer contained 20 mM His / His-HCl, 240 mM sucrose, and 10 mM methionine. A: pH 5.0 to 6.5; B: 0 mg / mL to 0.6 mg / mL PS20, pH 5.5; C: 0 mg / mL to 0.6 mg / mL PS80, pH 5.5; D: 0 mg / mL to 0.6 mg / mL Px188, pH 5.5. FI was corrected for EDTA blank and plotted as the average of three replicates.

[0008] Figure 4: Fluorescence intensity (FI) curves at pH 5.5 with high (A: 0 ppb to 300 ppb) and low Al3+ concentrations (B: 0.05 ppb to 10 ppb, x-axis plotted on a logarithmic scale) of 20 mM His / His-HCl, 240 mM sucrose, and 10 mM methionine supplemented with 10 µM TSB. FI was corrected for EDTA blank and plotted as the average of three replicates.

[0009] Figure 5: Fluorescence intensity (FI) curves of 0 ppb to 150 ppb Al3+ spiked in different mAbs supplemented with 10 µM TSB at 452 nm (solid circles represent fully prepared mAbs, and hollow circles depict mAbs in 20 mM histidine-chloride buffer) (374 nm excitation, 452 nm emission, 435 nm cutoff). FI was corrected for EDTA blank, plotted as the average of three replicates, and fitted with a quadratic polynomial. Detailed Description

[0010] In one embodiment, the present invention provides the use of compound (I)

[0011] (I)

[0012] in the diagnostic assay of aluminum ions (Al3+) in quantitative biological products. In some embodiments, the diagnostic assay is an in vitro assay. In this document, compound (I) is sometimes also referred to as a “thiophene Schiff base derivative” (TSB).

[0013] In another embodiment, the present invention provides the use of compounds of formula (I) as defined above for the quantification of aluminum ions (Al3+) in biological products, wherein quantification is performed by fluorescence spectroscopy. Fluorescence measurements can be performed using standard instruments known to those skilled in the art. In one embodiment, a SpectraMax® M2 Molecular Devices is used.Quantification is performed. In one embodiment, fluorescence spectra are recorded at 400 nm to 500 nm (in 1 nm increments) using a fixed excitation wavelength of 374 nm. In another embodiment, fluorescence emission is recorded at 452 nm (excited at 374 nm). In another embodiment, the sample solution is transferred to a black 96-well plate, and the wells are homogenized by shaking the plate for 120 seconds before each measurement.

[0014] As used herein, the term “quantification” means the detection of the quantity (amount) of aluminum ions in the said bioproduct. In one embodiment, the aluminum ions can be quantified in the ppb range. In another embodiment, the amount of aluminum ions is at most 500 ppm. In another embodiment, the amount of aluminum ions is at most 300 ppm. In another embodiment, the amount of aluminum ions is in the range of 0.006 ppm to 300 ppm. In another embodiment, the amount of aluminum ions is in the range of 5 ppm to 300 ppm. In another embodiment, the amount of aluminum ions is in the range of 10 ppm to 150 ppm. Those skilled in the art will understand that calibration is required to quantify the amount of aluminum ions in a sample. According to the invention, such calibration can be performed on a placebo sample (i.e., a sample without antibodies). In some aspects, the calibration can also be a standard additive calibration method using an antibody solution instead of a placebo. This method is described, for example, in Example 2.

[0015] As used herein, the term "biological product" means an aqueous composition comprising one or more nitrogen-containing large organic compounds consisting of long chains of one or more amino acid residues as an active ingredient. In one embodiment, a biological product as defined herein comprises a protein as an active ingredient. In one embodiment, a biological product as defined herein comprises a hormone, enzyme, or antibody as an active ingredient.

[0016] The term “antibody” is used in the broadest sense herein and covers a variety of antibody classes or structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody-cytokine fusion proteins (fpMabs), and antibody fragments, provided they exhibit the desired antigen-binding activity. In one embodiment, the fusion protein in an antibody-cytokine fusion protein is IL-2. In one embodiment, the term “antibody” as used herein refers to a polyprotein, preferably a pentameric protein.

[0017] In one embodiment of the invention, “antibody” is a monoclonal antibody. The term “monoclonal antibody” is known to those skilled in the art. In one embodiment, the term “monoclonal antibody” refers to a protein derived from a single clone (including any true monoclonal antibody).This refers to antibodies (nuclear, prokaryotic, or phage clones), not to methods of antibody production. As used herein, "monoclonal antibody" can be monospecific or bispecific. In one aspect, "monoclonal antibody" is a bispecific monoclonal antibody in a 1+1 or 2+1 format. Such formats and methods of preparation are known to those skilled in the art and are used, for example, as so-called T-cell conjugates or T-cell bispecific antibodies in cancer therapy. The antibodies used in the working examples were supplied by F. Hoffmann-La Roche AG, Basel, Switzerland.

[0018] In another aspect, the antibody is an "antibody product" selected from the following: alemtuzumab (LEMTRADA®), atezolizumab (TECENTRIQ®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (PERJETA®, 2C4, Omnitarg), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), and adalimumab (HUMIRA®). Apolizumab, acelizumab, atlizumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, belimumab (BENLYSTA®), briankinumab, canakinumab (ILARIS®), cedelizumab, certolizumab pegol (CIMZIA®), cidfusituzumab, cidtuzumab, cixutumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®) ), dalotuzumab, denosumabPROLIA®, XGEVA®, eculizumab (SOLIRIS®), efalizumab, epratuzumab, erlizumab, emicizumab (HEMLIBRA®), felvizumab, fontolizumab, gantenerumab, glofitamab (COLUMVI®), golimumab (SIMPONI®), ipilimumab, imgatuzumab, infliximab (REMICADE®) Labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucarumumab, pegol, lumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, motovizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®), nimotuzumab (THERACIM®), nolovizumab, numavizumab, obinutuzumab (GAZYVA®), olokizumab, omalizumab (XOLAIR®), onartuzumab (also known as MetMAb), pallizumab (SYNAGIS®), pascolizumab, pecfusituzumab, pectuzumab, pembrolizumabPembrolizumab (KEYTRUDA®), pexelizumab, priliximab, ralivizumab, ranibizumab (LUCENTIS®), reslivizumab, reslizumab, resyvizumab, robatumumab, rontalizumab, rovelizumab, ruplizumab, sarilumab, secukinumab, seribantumab, sifalimumab, sibrotuzumab, siltuximab (SYLVANT®) ), siplizumab, sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab (ACTEMRA®), toralizumab, tucusituzumab, umavizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), visilizumab, zanolimumab, zalutumumab.

[0019] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0020] The “class” of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, andIgM antibodies, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some aspects, the antibody is an IgG1 isotype. In some aspects, the antibody is an IgG1 isotype containing P329G, L234A, and L235A mutations to reduce the function of the Fc region effector. In other aspects, the antibody is an IgG2 isotype. In some aspects, the antibody is an IgG4 isotype containing an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively referred to as a, d, e, g, and m. The light chain of the antibody, based on the amino acid sequence of its constant domain, can be classified into one of two types, referred to as Kappa (κ) and Lambda (λ). In one embodiment, the antibody according to the invention is an IgG1 and / or IgG4 antibody.

[0021] In one embodiment, any of the antibodies according to the invention is human or humanized. A “human antibody” is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from the amino acid sequence of a non-human antibody using a complete library of human antibodies or other antibody-encoded sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations formed through random or site-specific mutations in vitro or introduced through somatic mutations in vivo).

[0022] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR as described on page 4 / 11 of the specification, CN 122095249 A. In some aspects, a humanized antibody will substantially comprise at least one, typically two, variable domains, wherein all or substantially all CDRs correspond to the CDRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. Optionally, a humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. "Humanized form" antibodies, such as non-human antibodies, refer to antibodies that have undergone humanization.

[0023] In one embodiment, the present invention provides a biological product, which is an aqueous composition comprising an antibody, preferably a monoclonal antibody. In one embodiment, the antibody is present in the aqueous composition at a concentration that provides its desired pharmaceutical activity and acceptable safety characteristics. In another embodiment, the present invention provides a composition as defined herein, wherein the antibody is present at a concentration of 1 mg / ml to 220 mg / ml, preferably 5 mg / ml to 180 mg / ml, or 5 mg / ml to 100 mg / ml, or 5 mg / ml to 25 mg / ml.The concentration range is present.

[0024] This biological product may contain additional excipients, such as buffers, stabilizers, antioxidants and / or surfactants known to those skilled in the art, for example. In one embodiment, the buffer is a histidine buffer with a pH of 5.0 to 6.0, preferably 5.0 to 5.5, the stabilizer is sucrose, and the antioxidant is methionine. In one embodiment, the surfactant according to the invention is any surfactant from the polysorbate or poloxamer class. In another embodiment, the surfactant according to the invention is selected from PS20 (Croda International, Snaith, UK), PS80 (polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Px188 (poloxamer 188; BASF, Ludwigshafen, D).

[0025] The excipients are present in amounts typically used for aqueous antibody compositions. In one embodiment, the present invention provides a biological product as defined herein, such as an aqueous antibody composition, wherein the surfactant is present at a concentration of 0.001 mg / ml to 1.0 mg / ml, or 0.01 mg / ml to 1.0 mg / ml, or 0.06 mg / ml to 1.0 mg / ml, or 0.06 mg / ml to 0.6 mg / ml. In one embodiment, the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is PS20, with a concentration below 0.6 mg / ml or in the range of about 0.006 mg / ml to 0.6 mg / ml. In another embodiment, the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is PS80, with a concentration below 0.06 mg / ml or in the range of about 0.006 mg / ml to 0.6 mg / ml or in the range of about 0.006 mg / ml to 0.06 mg / ml. In yet another embodiment, the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is Px188 at a concentration of up to 0.6 mg / ml.

[0026] In one embodiment, the biological product according to the invention comprises a 20 mM His-HCl buffer (Ajinomoto, JP) formulated at pH 5.5 containing 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP), and 0.05% PS20.

[0027] In another embodiment, the biological product according to the invention comprises a monoclonal antibody formulated at pH 5.5 in a 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) containing 230 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP), and 0.05% PS20;

[0028] In another embodiment, the invention provides a method for detecting the amount of aluminum ions in a biological product as defined herein, wherein the method includes any of the uses of a compound of formula (I) as defined herein.

[0029] In yet another embodiment, the invention provides a method and use as defined herein for fluorescence-based high-throughput assays. In one aspect, the assay can be transferred to an automated screening process using standard equipment for sample intake (e.g., 96-well plates), using a fluorescence reader equipped with software to automatically analyze each well, detect, and display the analytical results, such as the Al3+ concentration in a sample of a biological product or such product. Specification 5 / 11 pages 8 CN 122095249 A

[0030] The use and methods of the present invention are suitable for preventing the formation of visible particles in aqueous antibody compositions. In one embodiment, the formation of visible particles occurs during storage of the aqueous antibody composition. As used herein, the term “storage” means maintaining an aqueous pharmaceutical preparation under conditions known to those skilled in the art or indicated, for example, as indicated in the package insert of a comparable commercially available pharmaceutical product. In one aspect, the storage involves a period of up to 6 months, or 12 months, or 18 months, or 24 months, or 30 months. In another aspect, the storage involves storing the liquid pharmaceutical composition under conditions (such as temperature) also approved by a regulatory agency for up to the shelf life approved by that regulatory agency. On the one hand, the shelf life and storage conditions can be found, for example, in the packaging inserts accompanying approved protein-based drugs.

[0031] A set of terms defining the invention and its preferred aspects and embodiments is as follows:

[0032] 1. Compound of formula (I)

[0033] (I)

[0034] Use in the diagnostic determination of aluminum ions (Al3+) in a quantitative biological product.

[0035] 2. The use according to clause 1, wherein the quantification is performed by fluorescence spectroscopy.

[0036] 3. The use according to clause 2, wherein fluorescence emission is recorded at 400 nm to 500 nm (in increments of 1 nm) using a fixed excitation wavelength of 374 nm.

[0037] 4. The use according to Clause 2, wherein fluorescence emission is recorded at 452 nm using a fixed excitation wavelength of 374 nm.

[0038] 5. The use according to any one of Clauses 2 to 4, wherein the sample solution is transferred to a black 96-well plate and the wells are homogenized by shaking the plate for 120 seconds before each measurement.

[0039] 6. The use according to any one of Clauses 1 to 6, for the quantification of aluminum ions in the ppb range, preferably up to 500 ppm, more preferably up to 300 ppm.

[0040] 7. The use according to Clause 6, for the quantification of aluminum ions in the range of 0.006 ppm to 300 ppm; or for the quantification of aluminum ions in the range of 5 ppm to 300 ppm.

[0041] 8. The use according to any one of Clauses 1 to 7, wherein the biological product is an aqueous antibody composition optionally comprising additional pharmaceutically acceptable excipients, such as buffers, stabilizers, antioxidants, and surfactants.

[0042] 9. The use according to Clause 8, wherein the antibody is present in the aqueous composition at a concentration that provides the desired pharmaceutical activity and acceptable safety characteristics of the antibody.

[0043] 10. The use according to Clause 9, wherein the antibody is present in a concentration range of: 1 mg / ml to 220 mg / ml; or 5 mg / ml to 180 mg / ml; or 5 to 100 mg / ml; or 5 mg / ml to 25 mg / ml. Instructions for Use, Page 6 / 11, CN 122095249 A

[0044] 11. The use according to any one of Clauses 8 to 10, wherein the biological product comprises a histidine buffer, methionine, and a surfactant, wherein the histidine buffer has a pH of 5.0 to 6.0, preferably 5.0 to 5.5, and wherein the surfactant is selected from polysorbates or poloxamers.

[0045] 12. The use according to any one of Clauses 8 to 11, wherein the surfactant is selected from PS20 (Croda International, Snaith, UK), PS80 (polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Px188 (poloxamer 188; BASF, Ludwigshafen, D).

[0046] 13. The use according to any one of Clauses 8 to 12, wherein the surfactant is present at a concentration of 0.001 mg / ml to 1.0 mg / ml; or at a concentration ofThe histidine buffer solution is present at a concentration of 0.01 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 0.6 mg / ml.

[0047] 14. The use according to any one of claims 8 to 13, wherein the pH of the histidine buffer solution is 5.0 or 5.5, and the surfactant is PS20 at a concentration of about 0.006 mg / ml to 0.6 mg / ml.

[0048] 15. The use according to any one of claims 8 to 13, wherein the pH of the histidine buffer solution is 5.0 or 5.5, and the surfactant is PS80 at a concentration of about 0.006 mg / ml to 0.6 mg / ml, preferably 0.06 mg / ml to 0.6 mg / ml.

[0049] 16. The use according to any one of clauses 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is Px188 at a concentration of up to 0.6 mg / ml.

[0050] 17. The use according to any one of Clauses 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20;

[0051] 18. The use according to any one of Clauses 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 230 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20;

[0052] 19. A method for detecting the amount of aluminum ions in a biological product, wherein the method comprises any of the uses described in claims 1 to 18.

[0053] 20. The method according to claim 19, wherein the method is an automated high-throughput screening method that uses standard equipment for sample uptake (e.g., 96...(Well plate), using a fluorescent reader equipped with software to automatically analyze each well, detect and display the analytical results, such as the Al3+ concentration in a sample of a biological product or such product.

[0054] The invention will now be further illustrated by the following non-limiting working examples

[0055] Examples

[0056] Materials and Methods

[0057] Materials

[0058] The following model monoclonal antibody (mAb, Mab) used in this study was provided by F. Hoffmann-La Roche (Basel, CH):

[0059] Mab1 was prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20; this is a bispecific (2+1) antibody. Instructions for Use, Page 7 / 11, CN 122095249 A

[0060] Mab2 is prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 230 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20; this is a bispecific (2+1) antibody.

[0061] Mab3 is prepared at pH 5.5 in 20 mM His-Ac buffer (Ajinomoto, Tokyo, JP) with 130 mM arginine-chloride (Ajinomoto, Tokyo, JP), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.06% PS20; this is a monospecific IgG1 antibody.

[0062] Mab4 was prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) (in buffer);

[0063] 240 mM sucrose (Pfanstiehl Inc., Illinois, US) and 10 mM methionine were prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP).Mab4 (fully prepared) in 0.05% PS20 at pH 6.0 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.04% PS20; this is a bispecific (1+1) antibody.

[0065] Mab6 (in buffer) in 10 mM His-HCl buffer (Ajinomoto, Tokyo, JP) at pH 5.5. This is a bispecific (2+1) antibody.

[0066] Mab7 was prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.04% PS20. This is a bispecific (2+1) antibody.

[0067] The fluorophore “thiophene Schiff base derivative” (TSB) was prepared internally without the use of a PEG linker, as instructed by Xu et al. (22).

[0068] Surfactants PS20 (Roche-specified polysorbate 20; Croda International, Snaith, UK), PS80 (polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Px188 (poloxamer 188; BASF, Ludwigshafen, D) were tested.

[0069] For calibration, an aluminum solution in 3% HNO3 (AAAL1, 1000 μg / mL; inorganic ventures, Christiansburg, US) was used.

[0070] All other reagents, such as ethylenediaminetetraacetic acid (EDTA; JT Baker; Deventer, NL) and ethanol (EtOH; Merck KGa, Darmstadt, Germany), were analytical grade.

[0071] Methods

[0072] Preparation of TSB Stock Solution

[0073] A 1 mM TSB stock solution was prepared in pure ethanol (≥ 99.5%, HPLC grade). The stock solution was further diluted with water for injection (WFI) to a working concentration of 0.1 mM to 0.2 mM for experimental use. The prepared solution was loaded into aliquots and stored at -20°C until use.

[0074] Preparation of Aluminum Calibration Solution

[0075] An aluminum stock solution (DO: 10 μg / mL, 10000 ppb) was prepared from AAAL1 (1000 μg / mL) in WFI at pH 2.5 (adjusted with 1 M HCl). The stock solution was prepared fresh each time before the experiment. Then, the stock solutions were diluted with WFI at pH 2.5 to D1–D6 (D1 = 1500 ppb; D2 = 1000 ppb; D3 = 750 ppb; D4 = 500 ppb; D5 = 250 ppb; D6 = 100 ppb).

[0076] Fluorescence Measurement

[0077] Calibration buffers (buffer only, buffer + EDTA, buffer + 10 ppb to 150 ppb Al3+) and sample solutions (mAb only, mAb + EDTA, sample + 25 ppb Al3+) were transferred to black 96-well plates (half area). The composition of the calibration buffers must be matched to the mAb sample to be tested. Recovery of spiked samples (sample + 25 ppb Al3+) was used for system suitability testing for each experiment (FI(mAb + spike) = FI(mAb) + calibration (25 ppb); acceptance criteria: 80% to 120%). All solutions were prepared in triplicate. The sample solutions were homogenized by shaking the well plate for 120 seconds before each measurement. Fluorescence measurements were performed on a SpectraMax M2 fluorescence reader. Fluorescence spectra were recorded at 400 nm to 500 nm (in 1 nm increments) using a fixed excitation wavelength of 374 nm. Alternatively, fluorescence emission was recorded at 452 nm (excited at 374 nm).

[0078] Example 1: Compatibility of TBS with Formulation Matrix

[0079] Here, the interference of different excipients on TBS was evaluated. The ability of TSB to quantify aluminum ions has been demonstrated in previous studies [6], but only in water. In contrast, the first set of experiments here shows that, using 10 µm TSB, Al3+ can be effectively utilized in biopharmaceutical systems with various pH values ​​and surfactant concentrations.Quantification was performed within the ppb range (0.006 ppb to 500 ppb) (Figures 3A to 3D). Increased fluorescence signal was detected, depending on the aluminum concentration for all test conditions. The study in this invention demonstrates a cubic relationship between aluminum content and fluorescence intensity (R² > 0.98). Furthermore, fluorescence intensity (FI) was found to be independent of buffer salts, sucrose, or methionine (data not shown), but strongly dependent on pH (Figure 3A) and surfactant content (Figures 3B to 3D). Higher pH and higher surfactant content both resulted in decreased FI. The highest fluorescence intensity in histidine buffer was demonstrated to be reached at pH 5.0 and 5.5. PS20 at commonly used concentrations from 0.06 mg / mL to 0.6 mg / mL reduced FI by 20% to 50%. When PS20 was diluted to 0.006 mg / mL, the reduction in FI decreased to 10% to 20%. Using the corresponding PS80 concentration, FI decreased by approximately 25% to 50%. No significant change in FI was detected after the sample was diluted to 0.006 mg / mL PS80. Buffer solutions containing up to 0.6 mg / mL Px188 showed almost no decrease in FI. These values ​​show slight deviations (5% to 15%) from reference values ​​because the surfactant-free measurements were performed on different days. All values ​​are within the error tolerance.

[0080] The pH dependence can be explained by the pH-dependent hydrolysis of Al3+ in aqueous solutions [7]. Although free Al3+ is the dominant substance at acidic pH, aluminum gradually hydrolyzes to form insoluble Al(OH)3 and other hydroxyl substances that cannot be complexed by TSB.

[0081] The property of surfactants to form micelles above the critical micelle concentration is expected to cause interference with the determination of PS20 and PS80. Hydrophobic TSB is expected to be incorporated into the hydrophobic micelle core and therefore no longer usable for complexation with Al3+. Consequently, FI decreases with increasing surfactant concentration. Px188 behaves quite differently here and does not show any significant decrease in FI.

[0082] Nevertheless, even with the use of surfactants and at higher pH values, a consistent correlation is established between FI and Al3+ concentration, and therefore aluminum can be reliably quantified, provided that the same buffer composition is used for both the sample and calibration solutions.

[0083] It can be demonstrated that the quantification can cover the expected range of aluminum impurities in biopharmaceutical solutions. Tests were also conducted at higher (up to 300 ppb; Figure 4A) and lower Al3+ concentrations (down to 0.006 ppb; Figure 4B).FI is used to define the limits of determination. However, in the higher concentration range, the curve fit is similar to the standard determination concentration range (10–150 ppb Al3+). With 0.6 mg / mL PS20, a consistent correlation is established between FI and Al3+ concentrations up to 500 ppb Al3+. However, when higher concentrations are included, the slope of the curve shifts slightly. Therefore, it is important to adjust the calibration range to the expected Al3+ concentration if necessary.

[0084] The lowest Al3+ concentration that can be reliably quantified is 5 ppb, because at lower concentrations, background fluorescence and scattering become excessive. However, it has been shown that Al3+ concentrations can be quantified over a wide concentration range from 5 ppb to 500 ppb.

[0085] Example 2: Quantification of Al3+ in mAb formulations

[0086] After quantifying aluminum ions in different buffer systems, the next step is to quantify Al3+ in different mAb-containing formulations. For this purpose, fully prepared mAb solutions were used. Calibration samples were prepared using a matched placebo to compensate for matrix effects. Spiked samples (sample + 25 ppb Al3+) were prepared for system suitability testing.

[0087] For both mAb formulations tested, the effect of mAb on FI has been shown to be negligible (within the acceptance criteria: 80% to 120%) because the calibration standards were prepared using the same formulation buffer composition.

[0088] If the system suitability test (≠ 80% to 120%) is not passed, i.e., the acceptance criteria, it indicates that the matrix effect of the mAb is too significant. In this case, the mAb solution is used instead of the placebo for the standard addition calibration method. Samples containing a chelating agent (i.e., ethylenediaminetetraacetic acid, EDTA) were used as blank controls because no free Al3+ could be complexed with the fluorescent probe in the presence of EDTA. Blank-corrected FI was plotted against the spiked Al3+ concentration and fitted with a quadratic polynomial. The polynomial fit was extrapolated to Y=0. The absolute value of the fitted x-intercept corresponds to the Al3+ concentration in the unspiked sample. See Figure 5.

[0089] Therefore, the present invention provides a low-sample-consumption, fluorescence-based high-throughput assay for the quantification of trace (ppb range) Al3+ in biopharmaceutical API solutions.

[0090] Abbreviations

[0091] Al3+ or Al3+: aluminum ion

[0092] EDTA: ethylenediaminetetraacetic acid

[0093] FFA: free fatty acid

[0094] FI: fluorescence intensity

[0095] His: histidine base

[0096] His-HCl: Histidine chloride

[0097] mAb: Monoclonal antibody

[0098] ppb: Parts per billion

[0099] ppq: Parts per quadrillion

[0100] PS20: Polysorbate 20

[0101] PS80: Polysorbate 80

[0102] Px188: Poloxamer 188

[0103] TSB: Thiophene Schiff base derivative

[0104] WFI: Water for injection

[0105] References

[0106] [1] Allmendinger, A. et al., Glass Leachables as a Nucleation Factor for Free Fatty Acid Particle Formation in Biopharmaceutical Formulations. Journal of Pharmaceutical Sciences, 2021. 110(2): pp. 785-795.

[0107] [2] Gregoritza, K. et al., Metal-Induced Fatty Acid Particle Formation Resulting from Hydrolytic Polysorbate Degradation. J Pharm Sci, 2022.111(3): Specification 10 / 11 pages 13 CN 122095249 A Pages 743 to 751.

[0108] [3] Wilschefski, SC and MR Baxter, Inductively Coupled Plasma Mass Spectrometry: Introduction to Analytical Aspects. Clin. Biochem. Rev., 2019. 40(3): Pages 115 to 133.

[0109] [4] Sudhakar, P., P. Latha and P.V. Reddy, Chapter 17 – Analytical techniques, in Phenotyping Crop Plants for Physiological and Biochemical Traits, P. Sudhakar, P. Latha, and PV Reddy, eds. 2016, Academic Press. pp. 137–149.

[0110] [5] Svitkova, B. et al., Plate reader spectroscopy as an alternative to atomic absorption spectroscopy for the assessment of nanoparticle cellular uptake. Heliyon, 2022. 8(11): Page e11595. [6] Xu, Y. et al., A new water-soluble polymer fluorescent chemosensor with thiophene Schiff base site for selectively sensing Al3+ ions. Tetrahedron, 2021. 79: Page 131888. [7] Lekhlif, B. et al., Study of the electrocoagulation of electroplating industry wastewaters charged by nickel (II) and chromium (VI). J. Mater. Environ. Sci. 5, 2014. 5(1): pages 111 to 120. Instruction manual, page 11 / 11, 14 CN 122095249 A, Figure 1; Instruction manual drawing, page 1 / 5, 15 CN 122095249 A, Figure 2; Instruction manual drawing, page 2 / 5, 16 CN 122095249 A, Figure 3; Instruction manual drawing, page 3 / 5, 17 CN 122095249 A, Figure 4; Instruction manual drawing, page 4 / 5, 18 CN 122095249 A, Figure 5; Instruction manual drawing, page 5 / 5, 19 CN 122095249 A

Claims

1. Compound of formula (I) (I) Aluminum ions (Al) used in quantitative biological products 3+ Its use in diagnostic testing.

2. The use according to claim 1, wherein the quantification is performed by fluorescence spectroscopy.

3. The use according to claim 2, wherein a fixed excitation wavelength of 374 nm is used to record fluorescence spectra at 400 nm to 500 nm (in increments of 1 nm).

4. The use according to claim 2, wherein fluorescence emission is recorded at 452 nm (excited at 374 nm).

5. The use according to any one of claims 2 to 4, wherein the sample solution is transferred to a black 96-well plate, and the wells are homogenized by shaking the plate for 120 seconds before each measurement.

6. The use according to any one of claims 1 to 6, for the quantification of aluminum ions in the range of ppb, preferably up to 500 ppm, more preferably up to 300 ppm.

7. The use according to claim 6, for the quantification of aluminum ions in the range of 0.006 ppm to 300 ppm; or for the quantification of aluminum ions in the range of 5 ppm to 300 ppm.

8. The use according to any one of claims 1 to 7, wherein the biological product is an aqueous antibody composition optionally comprising additional pharmaceutically acceptable excipients, such as buffers, stabilizers, antioxidants and surfactants.

9. The use according to claim 8, wherein the antibody is present in the aqueous composition at a concentration that provides the desired pharmaceutical activity and acceptable safety characteristics of the antibody.

10. The use according to claim 9, wherein the antibody is present in the following concentration ranges: 1 mg / ml to 220 mg / ml; or 5 mg / ml to 180 mg / ml; or 5 to 100 mg / ml; or 5 mg / ml to 25 mg / ml.

11. The use according to any one of claims 8 to 10, wherein the biological product comprises a histidine buffer, methionine, and a surfactant, wherein the histidine buffer has a pH of 5.0 to 6.0, preferably 5.0 to 5.5, and wherein the surfactant is selected from the polysorbate or poloxamer class.

12. The use according to any one of claims 8 to 11, wherein the surfactant is selected from PS20 (Croda International, Snaith, UK), PS80 (polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Px188 (poloxam 188; BASF, Ludwigshafen, D).

13. The use according to any one of claims 8 to 12, wherein the surfactant is present at a concentration of 0.001 mg / ml to 1.0 mg / ml; or at a concentration of 0.01 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 1.0 mg / ml; or at a concentration of 0.06 mg / ml to 0.6 mg / ml.

14. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is PS20 at a concentration of about 0.006 mg / ml to 0.6 mg / ml.

15. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is PS80 at a concentration of about 0.006 mg / ml to 0.6 mg / ml, preferably 0.06 mg / ml to 0.6 mg / ml.

16. The use according to any one of claims 8 to 13, wherein the histidine buffer solution has a pH of 5.0 or 5.5, and the surfactant is Px188 at a concentration up to 0.6 mg / ml.

17. The use according to any one of claims 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 240 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20.

18. The use according to any one of claims 1 to 13, wherein the biological product is a monoclonal antibody prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) with 230 mM sucrose (Pfanstiehl Inc., Illinois, US), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.05% PS20.

19. A method for detecting the amount of aluminum ions in a biological product, wherein the method comprises any one of the uses described in claims 1 to 18.

20. The method of claim 19, wherein the method is an automated high-throughput screening method that uses standard equipment for sample uptake (e.g., a 96-well plate) and a fluorescence reader equipped with software to automatically analyze each well, detect, and display the analysis results, such as the Al3+ concentration in a sample of a biological product or such product.

Images