Development of high-throughput assays for identifying trace amounts of Al3+ in biopharmaceutical products

Abstract

This invention provides the use of compounds of formula (I) as defined herein for detecting the concentration of aluminum ions in biological products such as, for example, aqueous antibody compositions.

Description

Technical Field

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

[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, which limits the choice of surfactants, including the most commonly used polysorbate 20 (PS20) and polysorbate 80 (PS80). PS20 may degrade during the shelf life of the pharmaceutical product via oxidative degradation or hydrolytic degradation, typically catalyzed by host cell proteins. In particular, hydrolytic degradation produces free fatty acids (FFAs) as degradation products, which precipitate in solution once their solubility limits are exceeded, subsequently forming sub-visible and visible particles. Under conditions commonly found in biopharmaceutical formulations, FFAs 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 FFAs. 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 formation of FFA particles below the solubility limit 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 nature, 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] This invention addresses these problems by providing a high-throughput method for the 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]. Attached Figure Description

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

[0006] Figure 2: Fluorescence intensity (FI) spectra of TSB in buffer (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: Fluorescence intensity (FI) curves of Al3+ at 452 nm (excitation 374 nm, emission 452 nm, cutoff 435 nm) at different histidine buffer systems supplemented with 10 µM TSB, ranging from 0 ppb to 150 ppb. The buffers 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 supplementation of 10 µM TSB for 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 log(2)). 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 Implementation

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

[0011] (I)

[0012] Aluminum ions (Al) used in quantitative biological products 3+ Use in diagnostic assays of (I). In some embodiments, the diagnostic assay is an in vitro assay. In this document, the compound of formula (I) is sometimes also referred to as a "thiophene Schiff base derivative" (TSB).

[0013] In another embodiment, the present invention provides a compound of formula (I) as defined above for the quantification of aluminum ions (Al) in biological products. 3+ The application of this method involves quantification via fluorescence spectroscopy. Fluorescence measurements can be performed using standard instruments known to those skilled in the art. In one embodiment, quantification is performed using a SpectraMax® M2 Molecular Devices fluorescence reader. 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 yet 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 "quantitative" refers to the detection of the quantity (amount) of aluminum ions in the biological product. In one embodiment, the aluminum ions can be quantified in the range of ppb. 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 large nitrogen-containing organic compounds consisting of long chains of one or more amino acid residues as active ingredients. 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" as used herein is used in the broadest sense and encompasses a wide range 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 pentamer.

[0017] In one embodiment of the invention, an "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 an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), rather than to a method of producing the antibody. As used herein, a "monoclonal antibody" can be monospecific or bispecific. In one aspect, a "monoclonal antibody" is a bispecific monoclonal antibody in a 1+1 or 2+1 format. Such formats and methods of their 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 respect, 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, cixutumumab, clazakizumab, crenezumab, daclizumab ZENAPAX®, dalotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®), efalizumab, epratuzumab, erlizumab, emicizumab (HEMLIBRA®), felvizumab, fontolizumab, gantenerumab, glofitamab (COLUMVI®), golimumab (SIMPONI®), ipilimumab(ipilimumab), imgatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucarumumab, lulizumab 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), palivizumab (SYNAGIS®), pascolizumab, pecfusituzumab, pectuzumab, pembrolizumab (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®), totalizumab, 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 some antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0020] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some respects, antibodies are IgG1 isotypes. In some respects, antibodies are IgG1 isotypes containing P329G, L234A, and L235A mutations to reduce Fc region effector function. In other respects, antibodies are IgG2 isotypes. In some respects, antibodies are IgG4 isotypes containing S228P mutations in the hinge region to improve the stability of IgG4 antibodies. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chain of an antibody, based on the amino acid sequence of its constant structural domain, can be classified into one of two types, referred to as kappa (κ) and lamunda (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-encoding 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] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human CDR and amino acid residues from a human FR. In some respects, humanized antibodies will substantially contain at least one, typically two, variable domains, wherein all or substantially all CDRs correspond to the CDRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," 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 range 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.

[0024] This biological product may contain additional excipients, such as buffers, stabilizers, antioxidants, and / or surfactants known to those skilled in the art. 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 excipient is present in an amount typically used in aqueous antibody compositions. In one embodiment, the invention provides a biological product, such as an aqueous antibody composition as defined herein, 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 at a concentration of less than 0.06 mg / ml, or in the range of about 0.006 mg / ml to 0.6 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 at most 0.6 mg / ml.

[0026] In one 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 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 present 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 methods and uses for fluorescence-based high-throughput assays as defined herein. In one aspect, the assay can be performed using standard equipment transferred to an automated screening process for sample intake (e.g., a 96-well plate), using a software-equipped fluorescence reader to automatically analyze each well, detect, and display the analytical results, such as Al in samples of biological products or similar products. 3+ concentration.

[0030] The uses and methods of the present invention are adapted to prevent 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, for example, as indicated in the package insert of a comparable commercially available drug. On one hand, this storage involves a period of up to 6 months, or 12 months, or 18 months, or 24 months, or 30 months. On the other hand, this storage involves preserving 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 one hand, this shelf life and storage conditions can be found, for example, in the packaging insert accompanying an approved protein-based drug.

[0031] The following set of terms defines the present invention, its preferred aspects, and embodiments:

[0032] 1. Compound of formula (I)

[0033] (I)

[0034] Aluminum ions (Al) used in quantitative biological products 3+ Its use in diagnostic testing.

[0035] 2. As described in Clause 1, the quantification is performed by fluorescence spectroscopy.

[0036] 3. As described in 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 as described in 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 range of ppb, preferably up to 500 ppm, more preferably up to 300 ppm.

[0040] 7. As described in Clause 6, it is used 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 said biological product is an aqueous antibody composition that optionally contains additional pharmaceutically acceptable excipients, such as buffers, stabilizers, antioxidants and surfactants.

[0042] 9. The use as described in 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 as described in Clause 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.

[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 the polysorbate or poloxamer class.

[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 (poloxam 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 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 clauses 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.

[0048] 15. 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 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 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 includes any of the uses described in clauses 1 to 18.

[0053] 20. The method according to Clause 19, wherein the method is an automated high-throughput screening method that uses standard equipment for sample intake (e.g., a 96-well plate) and 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.

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

[0055] Example

[0056] Materials and methods

[0057] Material

[0058] The following model monoclonal antibodies (mAbs, Mab) used in this study were 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.

[0060] Mab2 was 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 was 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 (in buffer) was prepared at pH 5.5 in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP).

[0063] Mab4 (completely prepared) 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. Mab4 is a bispecific (1+1) antibody.

[0064] Mab5 was prepared 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) was prepared at pH 5.5 in 10 mM His-HCl buffer (Ajinomoto, Tokyo, JP). 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 connector, 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 (poloxam 188; BASF, Ludwigshafen, D) were tested.

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

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

[0071] method

[0072] Preparation of TSB stock solution

[0073] Prepare a 1 mM TSB stock solution in pure ethanol (≥ 99.5%, HPLC grade). Further dilute the stock solution to a working concentration of 0.1 mM to 0.2 mM using water for injection (WFI) for experimental use. Dilute the prepared solution into aliquots and store at -20°C until use.

[0074] Preparation of aluminum calibration solution

[0075] A stock solution of aluminum (D0: 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 freshly prepared directly before each experiment. The stock solution was then 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] Transfer calibration buffers (buffer only, buffer + EDTA, buffer + 10 ppb to 150 ppb Al3+) and sample solutions (mAb only, mAb + EDTA, sample + 25 ppb Al3+) to black 96-well plates (half area). The composition of the calibration buffers must be compatible with the mAb sample to be tested. Recovery of the spiked sample (sample + 25 ppb Al3+) is used for system suitability testing for each experiment (FI(mAb + spike) = FI(mAb) + calibration (25 ppb); acceptance criteria: 80% to 120%). All solutions are prepared in triplicate. Before each measurement, homogenize the sample solutions by shaking the plate for 120 seconds. Perform fluorescence measurements on a SpectraMax M2 fluorescence reader. Record fluorescence spectra 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, here it is shown in the first set of experiments that Al3+ can be quantified in a range of ppb (0.006 ppb to 500 ppb) in biopharmaceutical systems with a variety of pH and surfactant concentrations using TSB at a concentration of 10 µm (Figs. 3A to 3D). Increased fluorescence signal was detected depending on the aluminum concentration for all test conditions. The study in this invention shows a cubic relationship between aluminum content and fluorescence intensity (R2 > 0.98). Furthermore, it was found that fluorescence intensity (FI) was not dependent on buffer salt, sucrose, or methionine (data not shown), but rather strongly dependent on pH (Fig. 3A) and surfactant content (Figs. 3B to 3D). Higher pH and higher surfactant content both resulted in decreased FI. It can be demonstrated that the highest fluorescence intensity in histidine buffer was reached at pH 5.0 and 5.5. PS20 at commonly used concentrations ranging from 0.06 mg / mL to 0.6 mg / mL reduces FI by 20% to 50%. When PS20 is diluted to 0.006 mg / mL, the reduction in FI decreases to 10% to 20%. Using the corresponding PS80 concentration, FI is reduced by approximately 25% to 50%. No significant change in FI was detected after diluting the sample to 0.006 mg / mL PS80. Buffer solutions containing up to 0.6 mg / mL Px188 showed almost no reduction in FI. These values ​​show slight deviations (5% to 15%) from reference values ​​because surfactant-free measurements were performed on different days. All values ​​are within the error tolerance.

[0080] 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 surfactant's ability to form micelles above the critical micelle concentration is expected to interfere 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 was established between FI and Al3+ concentrations, and therefore aluminum can be reliably quantified, provided that the same buffer composition is used for both the sample and the calibration solution.

[0083] It can be demonstrated that quantitative determination can cover the expected range of aluminum impurities in biopharmaceutical solutions. FI was also tested at higher (up to 300 ppb; Figure 4A) and lower Al3+ concentrations (down to 0.006 ppb; Figure 4B) to limit the determination. However, in the higher concentration range, the curve fit was similar to the standard determination concentration range (10–150 ppb Al3+). With 0.6 mg / mL PS20, a consistent correlation was established between FI and Al3+ concentrations up to 500 ppb Al3+. However, the slope of the curve shifted slightly when higher concentrations were included. 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 demonstrated that Al3+ concentrations can be quantified over a wide concentration range from 5 ppb to 500 ppb.

[0085] Example 2: Al in mAb formulations 3+ Quantitative

[0086] After quantifying aluminum ions in different buffer systems, the next step is to quantify the aluminum ions in different mAb-containing formulations. 3+ 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 the mAb on FI has been shown to be negligible (within the acceptance range of 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 standard is met, it indicates that the matrix effect of the mAb is too significant. In this case, the mAb solution is used instead of a placebo for the standard addition calibration method. Samples containing a chelating agent (i.e., ethylenediaminetetraacetic acid, EDTA) are used as blank controls because no free Al3+ is available to complex with the fluorescent probe in the presence of EDTA. The blank-corrected FI is plotted against the spiked Al3+ concentration and fitted with a quadratic polynomial. The polynomial fit is extrapolated to Y=0. The absolute value of the x-intercept of the fit corresponds to the Al3+ concentration in the unspiked sample. See Figure 5.

[0089] Therefore, this invention provides a low-sample-consumption, fluorescence-based high-throughput assay that can be used for trace (ppb range) Al in biopharmaceutical API solutions. 3+ Quantitatively.

[0090] abbreviation

[0091] Al3+ or Al 3+ Aluminum ions

[0092] EDTA: Ethylenediaminetetraacetic acid

[0093] FFA: Free fatty acids

[0094] FI: Fluorescence intensity

[0095] His: Histidine base

[0096] His-HCl: Histidine chloride

[0097] mAb: Monoclonal antibody

[0098] ppb: one in a billion

[0099] ppq: one in a quadrillion

[0100] PS20: Polysorbate 20

[0101] PS80: Polysorbate 80

[0102] Px188: Polosham 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 to 795.

[0107] [2] Gregoritza, K. et al., Metal-Induced Fatty Acid Particle Formation Resulting from Hydrolytic Polysorbate Degradation. J Pharm Sci, 2022.111(3): 743-751.

[0108] [3] Wilschefski, SC and MR Baxter, Inductively coupled plasma massspectrometry: Introduction to analytical aspects. Clin. Biochem. Rev., 2019.40(3): pp. 115 to 133.

[0109] [4] Sudhakar, P., P. Latha and PV 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): p. e11595.

[0111] [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: p. 131888.

[0112] [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): pp. 111 to 120.

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