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

Abstract

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

Description

[0001] Development of high-throughput assay for identification of A13+ traces in biological drug products

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of quantification of metal traces in aqueous pharmaceutical protein (e.g. antibody) formulations comprising polysorbate 20, polysorbate 80, and poloxamer 188 as surfactants. The present invention thus helps stabilizing said formulations against the formation of visible particles.

[0004] BACKGROUND OF THE INVENTION

[0005] Surfactants are crucial excipients in protein formulations as they protect the labile protein from interfacial stress that may lead to protein aggregation. Proteins, such as monoclonal antibodies (mAb), are administered parenterally, which limits the choice of the surfactant, including the most commonly used surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80). PS20 can degrade over the shelf-life of a drug product either by oxidative degradation or by hydrolytic degradation, which is often catalyzed by host cell proteins. In particular, the latter yields free fatty acids (FFA) as degradation products, which can precipitate in solution once their solubility limit is exceeded and subsequently form sub-visible and visible particles. Under conditions typically found in biopharmaceutical formulations, FFA can precipitate even below their solubility limit dependent on different factors. Previous studies have shown that a major cause of premature particle formation is the presence of trivalent cations, such as aluminum ions, which act as a nucleation seed by complexing negatively charged FFAs. These trivalent cations can derive from the purification process, or leach out of the primary packaging material over time. Even trace amounts (ppb range) of these trivalent cations have been shown to efficiently form metal-FFA complexes, resulting in the premature formation of FFA particles below the FFA solubility limit [1,2].

[0006] Traditionally, various analytical methods, such as inductively coupled plasma-mass spectrometry and atomic absorption spectroscopy, have been employed for detection and quantification of metal impurity analysis at trace levels (as low as per quadrillion (ppq)) in biological fluids. While these techniques offer excellent sensitivity and accuracy, they suffer from drawbacks like high material consumption (in the mL range), high costs, labor- intensiveness, high staff expertise, and slow analysis time. As a result, the limited sample throughput of these methods constitutes a major challenge for analyzing large numbers of biological drug product batches [3-5],

[0007] The present invention solves these problems by providing an assay for quantifying aluminum ions in biological products in the ppb range in a high-throughput mode using a conventional fluorescent plate reader. For this purpose, a thiophene Schiff base-based fluorescent “turn-on” chemosensor (i.e. the compound of formula (I)) was used that has shown high sensitivity and A13+-selectivity in water [6],

[0008] BRIEF DESCRIPTION OF THE FIGURES

[0009] Fig. 1: Fluorophore molecule “Thiophene Schiff base derivate” (TSB) and its proposed binding mechanism for aluminum ions.

[0010] Fig. 2: Fluorescence intensity (FI) spectra of TSB in buffer (20 mM His / His-HCl, 240 mM sucrose, 10 mM methionine, pH 5.5) with A13+ concentrations from 0-150 ppb (374 nm excitation, emission 400-500 nm, 1 nm measure step).

[0011] Fig- 3 : Fluorescence intensity (FI) curve at 452 nm for 0-150 ppb A13+ in different histidine buffer systems supplemented with 10 pM TSB (374 nm excitation, emission 452 nm, cut-off 435 nm). Buffers comprised 20 mM His / His-HCl, 240 mM sucrose, 10 mM methionine. A: pH 5.0-6.5; B: 0-0.6 mg / mL PS20 at pH 5.5, C: 0-0.6 mg / mL PS80 at pH 5.5, D: 0-0.6 mg / mL Pxl88 at pH 5.5. FI are corrected by EDTA blank and plotted as average from triplicates.

[0012] Fig. 4: Fluorescence intensity (FI) curve for high (A: 0 to 300 ppb) and low A13+ concentrations (B: 0.05 to 10 ppb, plotted against a log(2) x axis) in 20 mM His / His-HCl, 240 mM sucrose, 10 mM methionine, pH 5.5 supplemented with 10 pM TSB. FI are corrected by EDTA blank and plotted as average from triplicates.

[0013] Fig. 5: Fluorescence intensity (FI) curve at 452 nm for 0-150 ppb A13+ spiked in different mAbs (filled circles represent fully formulated mAbs and open circles depict mAb in 20 mM histidinechloride buffer) supplemented with 10 pM TSB (374 nm excitation, emission 452 nm, cut-off 435 nm). FI are corrected by EDTA blank, plotted as average from triplicates, and fitted with a second-degree polynomial. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one embodiment, the present invention provides the use of the compound of formula (I) in a diagnostic assay for quantifying aluminum ions (Al3+) in biological products. In one embodiment, said diagnostic assay is an in vitro assay. Herein, the compound of formula (I) is sometimes also referred to as “Thiophene Schiff base derivate” (TSB).

[0015] In another embodiment, the present invention provides the use of the compound of formula (I) as defined above for quantifying aluminum ions (Al3+) in biological products, wherein the quantification is carried out by fluorescence spectroscopy. Fluorescence measurements can be carried out by standard instruments known to the skilled person. In one embodiment, the quantification is carried out using a SpectraMax® M2 fluorescence plate reader (Molecular Devices). In one embodiment, fluorescence spectra were recorded at 400-500 nm (in 1 nm increments), using a fixed excitation wavelength of 374 nm. In still another embodiment, fluorescence emission was recorded at 452 nm (excitation at 374 nm). In still another embodiment, sample solutions are transferred to a black 96 well plate, and the wells were homogenized by shaking of the well plate for 120 sec prior to each measurement.

[0016] The term “quantifying” as used herein means detecting the amount (quantity) of aluminium ions in said biological product. In one embodiment, said aluminum ions can be quantified in the ppb range. In another embodiment, the quantity of aluminum ions is up to 500 ppm. In another embodiment, the quantity of aluminum ions is up to 300 ppm. In another embodiment, the quantity of aluminum ions is in the range of 0.006 ppm to 300 ppm. In another embodiment, the quantity of aluminum ions is in the range of 5 ppm to 300 ppm. In another embodiment, the quantity of aluminum ions is in the range of 10 ppm to 150 ppm. The skilled person understands that, in order to quantify the amount of aluminium ions in a sample, a calibration needs to be carried out. In accordance with the present invention such calibration can be carried out against placebo samples, i.e. samples which do not contain the antibody. In some aspects, said calibration can also be a standard addition calibration method using the antibody solution instead of placebo. This method is for example described in Example 2.

[0017] The term “biological product” as used herein means an aqueous composition comprising, as an active ingredient, one or several nitrogenous large organic compound(s) being composed of one or more long chains of amino acid residues. 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.

[0018] The term “antibody” herein is used in the broadest sense and encompasses various antibody classes or structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody-cytokine fusion proteins (fpMab’s), and antibody fragments so long as they exhibit the desired antigen-binding activity. In one embodiment the fusion protein in the antibody-cytokine fusion proteins is IL-2. In one embodiment, the term “antibody” as used herein refers to multimeric proteins, preferably pentameric proteins.

[0019] In one embodiment in accordance with the present invention, the “antibody” is a monoclonal antibody. The term “monoclonal antibody” is known to a person of skill in the art. In one embodiment, the term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A “monoclonal antibody” as used herein can be mono- or bispecific. In one aspect the “monoclonal antibody” is bispecific in the format 1+1 or 2+1. Such formats, and methods for making them, are known to a person of skill in the art and are, for example, used as so-called T-cell engagers or T-cell bispecific antibodies in the treatment of cancer. The antibodies used in the working examples are provided by F. Hoffmann-La Roche AG, Basel, Switzerland.

[0020] In one aspect, the antibody is an “antibody product” selected from alemtuzumab (LEMTRADA®), atezolizumab (TECENTRIQ®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (PERJETA®, 2C4, Omnitarg), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), adalimumab (HUMIRA®), apolizumab, aselizumab, 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, imgatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab, 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®), toralizumab, tucusituzumab, umavizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), visilizumab, zanolimumab, zalutumumab.

[0021] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0022] The “class” of an antibody 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 several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In certain aspects, the antibody is of the IgGl isotype. In certain aspects, the antibody is of the IgGl isotype with the P329G, L234A and L235A mutation to reduce Fc- region effector function. In other aspects, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. In one embodiment, the antibody in accordance with the present invention is an IgG 1 and / or IgG4 antibody.

[0023] In one embodiment, any of the antibodies in accordance with the present invention is human or humanized. A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a nonhuman source that utilizes human antibody repertoires or other human antibody-encoding sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0024] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0025] 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, said antibody is present in said aqueous composition at a concentration which provides its desired pharmaceutical activity and an acceptable safety profile. In another embodiment, the present invention provides the compositions as defined herein, wherein the antibody is present at a concentration range from 1 to 220 mg / ml, preferably 5 to 180 mg / ml, or 5 to 100, or 5 to 25 mg / ml.

[0026] The present biological products can comprise further excipients such as, for example, buffers, stabilizers, antioxidants and / or surfactants which are known to a person of skill in the art. In one embodiment, the buffer is a histidine buffer at a pH of 5.0-6.0, preferably 5.0 to 5.5, the stabilizer is sucrose, and the antioxidant is methionine. In one embodiment, a surfactant in accordance with the present invention is any surfactant from the class of polysorbates, or a poloxamer. In another embodiment, a surfactant in accordance with the present invention is selected from PS20 (Croda International, Snaith, UK), PS80 (Polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Pxl88 (Poloxamer 188; BASF, Ludwigshafen, D).

[0027] The excipients are present in amounts typically used in aqueous antibody compositions. In one embodiment, the present invention provides the biological products as defined herein, e.g. aqueous antibody compositions, wherein the surfactant is present at a concentration of 0.001 to l.O mg / ml; or 0.01 to l.O mg / ml, or 0.06 to l.O mg / ml; or 0.06 to 0.6 mg / ml. In one embodiment, the pH of the histidine buffer solution is 5.0 or 5.5 and the surfactant is PS20 at a concentration below 0.6 mg / ml; or at a concentration in the range of about 0.006 to 0.6 mg / ml. In another embodiment, the pH of the histidine buffer solution is 5.0 or 5.5 and the surfactant is PS80 at a concentration below 0.06 mg / ml; or at a concentration in the range of about 0.006 to 0.6 mg / ml; or at a concentration in the range of about 0.006 to 0.06 mg / ml. In yet another embodiment, the pH of the histidine buffer solution is 5.0 or 5.5 and the surfactant is Pxl88 at a concentration up to 0.6 mg / ml.

[0028] In one embodiment, biological products in accordance with the present invention comprise a monoclonal antibody formulated 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 at pH 5.5;

[0029] In another embodiment, biological products in accordance with the present invention comprise a monoclonal antibody formulated 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 at pH 5.5;

[0030] In another embodiment, the present invention provides a method to detect the quantity of aluminum ions in a biological product as defined herein, wherein said method comprises any of the uses of the compound of formula (I) as defined herein.

[0031] In yet another embodiment, the present invention provides the methods and uses defined herein on a fluorescence based high-throughput assay. In one aspect, that assay is transferable to an automated screening process using conventional equipment for sample uptake (e.g. 96 well plates), fluorescence readers equipped with software to automatically analyze each well, detect and display the analytical results, e.g. the Al3+concentration in a biological product or samples of such product. The uses and methods of the present invention are suitable to prevent the formation of visible particles in an aqueous antibody compositions. In one embodiment, the formation of visible particles occurs upon storage of said aqueous antibody compositions. The term “storage” as used herein means keeping an aqueous pharmaceutical preparation under conditions known to a person of skill in the art, or as for example indicated in the package inserts of comparable commercially available drugs. In one aspect said storage involves a time of up to 6 months, or 12 months, or 18 months, or 24 months, or 30 months. In another aspect said storage involves keeping said liquid pharmaceutical composition up to its shelf life as approved by regulatory authorities under conditions (such as e.g. temperature) as also approved by such regulatory authority. In one aspect such shelf life and storage conditions can, for example, be found in the package insert accompanying an approved protein based drug.

[0032] A set of clauses defining the invention and its preferred aspects and embodiments is as follows:

[0033] 1. The use of the compound of formula (I) in a diagnostic assay for quantifying aluminum ions (Al3+) in biological products.

[0034] 2. The use according to clause 1, wherein the quantification is carried out by fluorescence spectroscopy.

[0035] 3. The use according to clause 2, wherein fluorescence emission is recorded at 400-500 nm (in 1 nm increments), using a fixed excitation wavelength of 374 nm.

[0036] 4. The use according to clause 2, wherein fluorescence emission is recorded at 452 nm, using a fixed excitation wavelength of 374 nm. 5. The use according to any one of clauses 2 to 4, wherein the sample solutions are transferred to a black 96 well plate, and the wells were homogenized by shaking of the well plate for 120 sec prior to each measurement.

[0037] 6. The use according to any one of clauses 1 to 6 for quantification of aluminum ions in the ppb range, preferably in any range up to 500 ppm, more preferably in any range up to 300 ppm.

[0038] 7. The use according to clause 6, for quantification of aluminum ions in the range of 0.006 ppm to 300 ppm; or in the range of 5 ppm to 300 ppm.

[0039] 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.

[0040] 9. The use according to clause 8, wherein the antibody is present in said aqueous composition at a concentration which provides its desired pharmaceutical activity and an acceptable safety profile.

[0041] 10. The use according to clause 9, wherein the antibody is present at a concentration range from 1 to 220 mg / ml; or from 5 to 180 mg / ml, or from 5 to 100, or from 5 to 25 mg / ml.

[0042] 11. The use according to any one of clauses 8 to 10, wherein the biological product comprises a histidine buffer at a pH of 5.0-6.0, preferably 5.0 to 5.5, methionine and a surfactant selected from the class of polysorbates or poloxamers.

[0043] 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 Pxl88 (Poloxamer 188; BASF, Ludwigshafen, D).

[0044] 13. The use according to any one of clauses 8 to 12, wherein the surfactant is present at a concentration of 0.001 to 1.0 mg / ml; or 0.01 to 1.0 mg / ml; or 0.06 to 1.0 mg / ml; or 0.06 to 0.6 mg / ml.

[0045] 14. The use according to any one of clauses 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 to 0.6 mg / ml. 15. The use according to any one of clauses 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 to 0.6 mg / ml, preferably 0.06 to 0.6 mg / ml.

[0046] 16. The use according to any one of clauses 8 to 13, wherein the pH of the histidine buffer solution is 5.0 or 5.5 and the surfactant is Pxl88 at a concentration up to 0.6 mg / ml.

[0047] 17. The use according to any one of clauses 1 to 13, wherein the biological product is a monoclonal antibody formulated 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 at pH 5.5;

[0048] 18. The use according to any one of clauses 1 to 13, wherein the biological product is a monoclonal antibody formulated 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 at pH 5.5;

[0049] 19. A method to detect the quantity of aluminum ions in a biological product, wherein said method comprises any of the uses according to clauses 1 to 18.

[0050] 20. The method of clause 19, wherein said method is an automated, high-throughput screening method using conventional equipment for sample uptake (e.g. 96 well plates), fluorescence readers equipped with software to automatically analyze each well, detect and display the analytical results, e.g. the A13+ concentration in a biological product or samples of such product.

[0051] The invention will now be further illustrated by the following, non-limiting working examples

[0052] EXAMPLES

[0053] Materials and Methods

[0054] Materials

[0055] The model monoclonal antibodies (mAb, Mab) used for this study were provided by F. Hoffmann-La Roche (Basel, CH):

[0056] Mabl formulated 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 at pH 5.5; This is a bispecific (2+1) antibody.

[0057] Mab2 formulated 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 at pH 5.5; This is a bispecific (2+1) antibody.

[0058] Mab3 formulated in 20 mM His-Ac buffer (Ajinomoto, Tokyo, JP) with 130 mM argininechloride (Ajinomoto, Tokyo, JP), 10 mM methionine (Sekisui Medical, Tokyo, JP) and 0.06% PS20 at pH 5.5; This is a monospecific IgG 1 antibody.

[0059] Mab4 (in buffer) formulated in 20 mM His-HCl buffer (Ajinomoto, Tokyo, JP) at pH 5.5;

[0060] Mab4 (fully formulated) formulated 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 at pH 5.5; Mab4 is a bispecific (1+1) antibody.

[0061] Mab5 formulated 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 at pH 6.0; This is a bispecific (1+1) antibody.

[0062] Mab6 (in buffer) formulated in 10 mM His-HCl buffer (Ajinomoto, Tokyo, JP) at pH 5.5. This is a bispecific (2+1) antibody.

[0063] Mab7 formulated 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 at pH 5.5. This is a bispecific (2+1) antibody. The fluorophore “thiophene Schiff base derivative” (TSB) was prepared in-house according to the instruction of Xu et al (22) without PEG linker.

[0064] The surfactants PS20 (Roche specified Polysorbate 20; Croda International, Snaith, UK), PS80 (Polysorbate 80 HX2; NOF Corporation, Shibuya-ku, JPN) and Pxl88 (Poloxamer 188; BASF, Ludwigshafen, D) were tested.

[0065] For calibration, an aluminum solution (AAAL1, 1000 pg / mL; inorganic ventures, Christiansburg, US) in 3% HNO was used.

[0066] All other reagents like ethylenediaminetetraacetic acid (EDTA; J. T. Baker; Deventer, NL) and ethanol (EtOH; Merck KGa, Darmstadt, Germany), were of analytical grade.

[0067] Methods

[0068] Preparation of TSB stock solution

[0069] A 1 mM stock solution of TSB was prepared in pure ethanol (> 99.5 %, HPLC grade). The stock solution was further diluted to a working concentration of 0.1-0.2 mM using water for injection (WFI) for experimental use. The prepared solutions were filled in aliquots and stored at -20 °C until use.

[0070] Preparation of aluminum calibration solutions

[0071] A stock solution of aluminum (DO: 10 pg / ml, 10000 ppb) was prepared from AAAL1 (1000 pg / mL) in WFI at pH 2.5, adjusted with 1 M HC1. The stock solution was prepared freshly each time directly prior to the experiment. Then, the stock solution was separately diluted with WFI at pH 2.5 to D1-D6 (DI = 1500 ppb; D2 = 1000 ppb; D3 = 750 ppb; D4 = 500 ppb; D5 = 250 ppb; D6 = 100 ppb).

[0072] Fluorescence measurements

[0073] Calibration buffers (buffer only, buffer + EDTA, buffer + 10-150 ppb A13+) and sample solutions (mAb only, mAb + EDTA, sample + 25 ppb A13+) were transferred into a black 96 well plate (half area). The composition of the calibration buffer has to match the mAb sample to be tested. Recovery of a spiked sample (sample + 25 ppb A13+) was used for system suitability testing for each experiment (FI(mAb+spike)=FI(mAb)+Calib(25ppb); acceptance criteria: 80-120%). All solutions were prepared as triplicates. Sample solutions were homogenized by shaking of the well plate for 120 sec prior to each measurement. Fluorescence measurements were performed on a SpectraMax M2 fluorescence plate reader. Fluorescence spectra were recorded at 400-500 nm (in 1 nm increments), using a fixed excitation wavelength of 374 nm. Alternatively, fluorescence emission was recorded at 452 nm (excitation at 374 nm).

[0074] Example 1: Compatibility of TBS with formulation matrix

[0075] Here, the interference of different excipients with TBS was assessed. The capability of TSB to quantify aluminum ions has been shown in previous studies [6], however only in water. In contrast, it is shown here in a first set of experiment that A13+ can be quantified in the ppb range (0.006-500 ppb) in biological formulation systems of various pH and surfactant concentrations using TSB at a concentration of 10 pm (Figure 3 A-D). Increasing fluorescence signals have been detected dependent on Aluminum concentration for all conditions tested. The studies in the presented invention show that there is a cubic relationship between aluminum content and fluorescence intensity (R2 > 0.98). In addition, fluorescence intensity (FI) was found to be not dependent on buffer salts, sucrose or methionine (data not shown), but was strongly dependent on pH (Fig. 3A) and surfactant content (Fig. 3B-D). Both, higher pH and high surfactant content, resulted in a reduced FI. It could be shown that the highest fluorescence intensities in histidine buffer were reached at pH 5.0 and 5.5. PS20 at a commonly used concentration range of 0.06-0.6 mg / mL diminished FI by 20-50%. When PS20 was diluted to 0.006 mg / mL, the decrease in FI was reduced to 10-20%. Using corresponding PS80 concentrations, the FI decrease was around 25-50%. After sample dilution to 0.006 mg / mL PS80, no major change of FI could be detected. Buffer solutions containing up to 0.6 mg / mL Pxl88 hardly show any reduction in FI. The values showed a slight deviation from the reference (5-15%), since the measurement without surfactant was performed on a different day. All values were within the error tolerance.

[0076] The pH dependence can be explained by the pH-dependent hydrolysis of A13+ in aqueous solutions [7], While free A13+ is the main species at acidic pH, aluminum is progressively hydrolyzed, forming insoluble Al(OH)s and other hydroxo species which cannot be complexed by TSB.

[0077] The characteristic of surfactants to form micelles above the critical micelle concentration is expected to cause the assay interference with PS20 and PS80. The hydrophobic TSB is expected to be incorporated into the hydrophobic micelle core and is, therefore, no longer available for the complexation with A13+. As a result, the FI decreases with increasing surfactant concentration. Pxl88 behaves rather differently here and does not show any major reduction of the FI.

[0078] Nevertheless, even with surfactants and at higher pH values, a consistent correlation between FI and A13+ concentration was established and thus, aluminum can be reliably quantified, provided that the same buffer composition was used for sample and calibration solutions.

[0079] It could be demonstrated that the expected range of aluminum impurities in biopharmaceutical drug product solutions can be covered by the quantification assay. The FI at higher (up to 300 ppb; Fig. 4A) and lower A13+ concentrations (down to 0.006 ppb; Fig. 4B) was also tested to define the limits of the assay. However, in higher concentration range, the curve fit was similar compared to the standard assay concentration range (10-150 ppb A13+). With 0.6 mg / mL PS20, a consistent correlation between FI and A13+ concentration was established up to 500 ppb A13+. However, the slope of the curve was slightly shifted when higher concentrations were included. Therefore, it is important to adjust the calibration range to the expected A13+ concentration if necessary.

[0080] The lowest A13+ concentration that was reliably quantified was 5 ppb since the background fluorescence and the scattering becomes too large at lower concentrations. Nevertheless, it is shown that A13+ concentrations can be quantified in a broad concentration range of 5 ppb to 500 ppb.

[0081] Example 2: Al3+quantification in mAb formulation

[0082] After quantifying aluminum ions in different buffer systems, the next step was to quantify Al3+in different mAb -containing formulations. For this purpose, fully formulated mAb solutions were used. Calibration samples were prepared using matching placebos to compensate for matrix effects. Spiked sample (sample + 25 ppb A13+) was prepared for system suitability testing.

[0083] For two tested mAb formulations, it has been shown that the influence of the mAb on the FI is negligibly low (within the range of acceptance criteria: 80-120%), given that the calibration standards are prepared with the same formulation buffer composition.

[0084] If the system suitability test, i.e. the acceptance criteria, was not passed (^80-120%), it indicates that the matrix effect of the mAb is excessively noticeable. In this case, a standard addition calibration method is performed using the mAb solution instead of placebo. Samples containing a chelator (i.e. Ethylenediaminetetraacetic acid, EDTA) were used as blank controls as no free A13+ is available for complexation with the fluorescent probe in the presence of EDTA. The blank-corrected FIs are plotted against the spiked A13+ concentration and fitted with a second- degree polynomial. The polynomial fit is extrapolated to Y=0. The absolute value of the x- intercept of the fit corresponds to the A13+ concentration in the unspiked sample. See Fig. 5.

[0085] The invention thus provides a low-sample consuming, fluorescence based high-throughput assay which can be used for the quantification of Al3+in trace amounts (ppb range) in biopharmaceutical API solutions.

[0086] Abbreviations

[0087] A13+ or Al3+: aluminum ions

[0088] EDTA: ethylenediaminetetraacetic acid

[0089] FFA: free fatty acids FI: Fluorescence Intensity

[0090] His: Histidine base

[0091] His-HCl: Histidine Chloride mAh: monoclonal antibodies ppb: parts per billion ppq: parts per quadrillion

[0092] PS20: Polysorbate 20

[0093] PS80: Polysorbate 80

[0094] Pxl88: Poloxamer 188

[0095] TSB: thiophene Schiff base derivative WFI: water for injections

[0096] References

[0097] [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): p. 785-795.

[0098] [2] Gregoritza, K., et al., Metal-Induced Fatty Acid Particle Formation Resulting from Hydrolytic Polysorbate Degradation. J Pharm Sci, 2022. 111(3): p. 743-751.

[0099] [3] Wilschefski, S.C. and M.R. Baxter, Inductively coupled plasma mass spectrometry: Introduction to analytical aspects. Clin. Biochem. Rev., 2019. 40(3): p. 115-133.

[0100] [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 P.V. Reddy, Editors. 2016, Academic Press, p. 137-149.

[0101] [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. e! 1595.

[0102] [6] Xu, Y., et al., A new water-soluble polymer fluorescent chemosensor with thiophene Schiff base site for selectively sensing A13+ ions. Tetrahedron, 2021. 79: p. 131888.

[0103] [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): p. 111-120

Claims

Claims1. The use of the compound of formula (I)in a diagnostic assay for quantifying aluminum ions (Al3+) in biological products.

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

3. The use according to claim 2, wherein fluorescence spectra are recorded at 400-500 nm (in 1 nm increments), using a fixed excitation wavelength of 374 nm.

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

5. The use according to any one of claims 2 to 4, wherein the sample solutions are transferred to a black 96 well plate, and the wells were homogenized by shaking of the well plate for 120 sec prior to each measurement.

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

7. The use according to claim 6, for quantification of aluminum ions in the range of 0.006 ppm to 300 ppm; or 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 said aqueous composition at a concentration which provides its desired pharmaceutical activity and an acceptable safety profile.

10. The use according to claim 9, wherein the antibody is present at a concentration range from 1 to 220 mg / ml; or from 5 to 180 mg / ml, or from 5 to 100, or from 5 to 25 mg / ml.

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

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 Pxl88 (Poloxamer 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 to 1.0 mg / ml; or 0.01 to 1.0 mg / ml; or 0.06 to 1.0 mg / ml; or 0.06 to 0.6 mg / ml.

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 to 0.6 mg / ml.

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 to 0.6 mg / ml, preferably 0.06 to 0.6 mg / ml.

16. 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 Pxl88 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 formulated 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 at pH 5.5;18. The use according to any one of claims 1 to 13, wherein the biological product is a monoclonal antibody formulated 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 at pH 5.5;19. A method to detect the quantity of aluminum ions in a biological product, wherein said method comprises any of the uses according to claims 1 to 18.

20. The method of claim 19, wherein said method is an automated, high-throughput screening method using standard equipment for sample uptake (e.g. 96 well plates), fluorescence readers equipped with software to automatically analyze each well, detect and display the analytical results, e.g. the A13+ concentration in a biological product or samples of such product.***

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