Quantitative determination of polysorbates in aqueous formulations.

Abstract

The present disclosure is directed to a method (a) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more (poly)peptides, comprising the following steps: (a.1) providing a sample of the aqueous formulation; (a.2) adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine to the sample of step (a.1) to obtain a fluorescent dye-containing sample to be tested in a fluorescent micelle assay; (a.3) optionally incubating the fluorescent dye-containing sample of step (a.2); (a.4) performing a fluorescent micelle assay with the fluorescent dye-containing sample obtained in step (a.2) or step (a.3); (a.5) determining the content of polysorbate present in the sample from the fluorescent micelle assay performed in step (a.4); provided that the method is performed without the use of an HPLC-system. The further method (b) of the present invention also allows high-throughput screening of a large number of samples. The presence of (poly)peptides(s) does not in any way interfere with the fluorescent micelle assay measurement.

Description

[Technical field]

[0001] The present invention relates to a method for the quantification of polysorbate in an aqueous formulation in the presence of one or more (poly)peptides.

[0002] prior art Nonionic surfactants have emerged as excipients of choice in aqueous biopharmaceutical formulations to stabilize proteins against interfacial stresses, saturate protein aggregation-prone regions, or help prevent adsorption. In that regard, nonionic surfactants help maintain the colloidal and conformational stability of active pharmaceutical ingredients (APIs) and thus the activity and efficacy of biotherapeutics. Due to their biocompatibility and low toxicity, the most widely used surfactant in aqueous biopharmaceutical formulations is polysorbate (PS), also known as Tween® [1]. However, polysorbates exhibit many degradation reactions, especially oxidation and (enzymatic) hydrolysis, which may lead to instability of aqueous formulations, as many degradation products are formed and functional properties are lost. Thus, the degradation of polysorbates may cause particle formation in aqueous formulations, which may be a major quality concern and potential risk factor [2].

[0003] For example, due to known degradation of polysorbates used for stabilization of biopharmaceutical formulations or biotherapeutic products, the presence and concentration of polysorbates must be carefully monitored during aqueous formulation development, manufacturing, and also during storage and shelf life.

[0004] Known methods for determining polysorbate content are based on, for example, mass spectrometry, evaporative light scattering detection, charged aerosol detection, nuclear magnetic resonance, and hybrid methods such as liquid chromatography with charged aerosol detection (LC-CAD) or LC-evaporative light scattering detection using mixed-mode on-line solid phase extraction (SPE) cartridges and acidified mobile phases. These methods require expensive equipment, are time- and personnel-intensive, and sometimes require complex sample preparation. Moreover, most of the methods are not compatible with the presence of proteins, so a protein removal step is required.

[0005] Thus, although a large number of different assays are known for the quantitative analysis of polysorbates in biopharmaceutical products, the HPLC techniques combined with charged aerosol detection (CAD) and the HPLC techniques combined with fluorescent micellar assay (FMA) belong to the most widely used techniques for the quantification of polysorbates [3].

[0006] The fluorescent micelle assay (FMA) is based on the partitioning of the hydrophobic dye N-phenyl-1-naphthylamine (NPN) into detergent micelles. NPN exhibits a low fluorescent signal in aqueous environments, which increases in more hydrophobic environments, such as the core of the micelles. Polysorbates, non-ionic detergents, form such micelles in aqueous solutions. Micelles form above the critical micelle concentration (CMC), which represents the concentration of detergent above which micelles form. For polysorbates, the CMC at 25°C ranges from 15 to 75 μM for polysorbate 20 (PS20 HP) and is much lower, ranging from 7 to 16 μM for polysorbate 80 (PS80 HP) [4]. Thus, the fluorescent micelle assay is applied to determine the polysorbate content in samples with polysorbate concentrations above the critical micelle concentration (CMC). In fact, the CMC of polysorbates is significantly lower than the concentration of interest in the desired aqueous formulation.

[0007] For a deeper understanding, the principle of the fluorescent micelle assay is illustrated in Figures 1 to 3 as follows:

[0008] In Figures 1A and 1B, the concept of fluorescent micelle assay is illustrated in schematic form. In Figure 1A, a polysorbate-containing sample 100a is depicted with water 125 as the solvent, so that a number of polysorbate molecules 130, namely 130.1, 130.2, 130.3, ..., are present at the air-liquid interface. The hydrophobic tails of the polysorbate molecules 130 extend away from the water surface, while the hydrophilic heads point into the water. In sample 100a, the concentration c of polysorbate (PS) is selected to be equal to or greater than the critical micelle concentration (c(PS) ≧ CMC), i.e., polysorbate forms micelles. In the aqueous solvent 125, three micelles 110a, 110b, and 110c are illustrated in schematic form, each micelle having a spherical shape. In each micelle 110a, 110b, or 110c, the hydrophilic head of each polysorbate molecule 130 is located on the outside of the sphere, and the hydrophobic tail of each polysorbate molecule 130 faces toward the inside of the sphere.

[0009] When the hydrophobic dye N-phenyl-1-naphthylamine (NPN) is added to sample 100a, the dye is "entrapped" in the non-polar core of polysorbate micelles 110a, 110b, and 110c (schematically represented by the NPN symbol in the center of each sphere), as shown diagrammatically in Figure 1B. Partitioning of the NPN dye into the non-polar core of the micelles results in a significant increase in the fluorescent signal.

[0010] In Figure 1B, the irradiated light is shown diagrammatically by arrow 150.1, and the emitted fluorescent light is shown diagrammatically by arrow 150.2, which is detected by fluorescence detector 165. This feature makes it possible to determine the micelle concentration, and thus the polysorbate concentration, by measuring the fluorescent signal of N-phenyl-1-naphthylamine (NPN) present in the micelles. This method was first reported by Brito et al. [5], where N-phenyl-1-naphthylamine was used to determine the critical micelle concentration (CMC) of various surfactants.

[0011] Figures 2A and 2B show the same embodiment as Figures 1A and 1B, but the polysorbate is polysorbate 20 (PS20). Polysorbate 20 forms fully intact micelles 110a, 110b, 110c, and 110d. In this embodiment, the fluorescent signal obtained by the fluorescent micelle assay is a very strong signal.

[0012] In Figures 3A and 3B, the polysorbate is partially degraded polysorbate 20 (PS20). As can be seen, micelles 120a and 120b may change properties such as destabilization of the initial micelle or decrease the micelle concentration. Degradation products 175 are formed from intact polysorbate from micelles 120a and 120b of polysorbate 20 and are present in the aqueous phase depending on their physicochemical properties such as increased polarity. In this embodiment, the fluorescent signal obtained by the fluorescent micelle assay is a decreased signal.

[0013] As a result, the fluorescent micelle assay can be used as a method for determining the content of polysorbate in aqueous formulations, is easy to perform, and can rapidly determine the polysorbate content.

[0014] One of the most widely applied methods to determine the concentration of polysorbates is the fluorescent micelle assay (FMA) performed on a high performance liquid chromatography (HPLC) system. In such cases, the fluorescent micelle assay is performed using an HPLC system connected to a fluorescence detector as a direct flow analysis of a fluorescent dye present in the mobile phase.

[0015] Figure 4 shows a simplified schematic diagram of a fluorescent micellar assay (FMA) performed in an HPLC system. The mobile phase 180 contains a fluorescent dye in the form of N-phenyl-1-naphthylamine (NPN), an organic solvent such as acetonitrile, a buffer such as tris(hydroxymethyl)aminomethane (Trizma base) or tris(hydroxymethyl)methylamine, and optionally suitable additives, such as salts such as NaCl, and surfactants, such as non-ionic surfactants such as polyalkylene glycol ethers (known brands are Brij®, Genapol®, Lutensol®). The mobile phase 180 is taken in through a capillary tube 182 controlled by a pump 185 with a suitable flow rate. A sample 187 containing polysorbate in aqueous solution is withdrawn through a capillary tube 188 and combined with the mobile phase 180. The polysorbate-containing mobile phase 180 is then directed through a reaction loop 190 and then delivered to a fluorescence detector 195, where the fluorescent signal is measured. The polysorbate-containing sample can be analyzed and then disposed of as waste 197. For quantification, a calibration curve is used to determine the peak area of ​​polysorbate [3, 6]. The fluorescent micelle assay can also be performed in an automated HPLC system, since most HPLC instruments are equipped with an autosampler.

[0016] Although this approach to determine polysorbate concentration seems straightforward and easy, the fluorescent micelle assay performed in an HPLC system has many problems, such as a long analysis time of about 2 min per sample, which contributes to an analytical bottleneck, especially for large sample sets. Furthermore, the nonspecific adsorption of the fluorescent dye N-phenyl-1-naphthylamine (NPN) to all accessible surfaces, especially the capillaries and valves of the chromatographic system, requires prolonged flushing with the mobile phase containing the dye to saturate all surfaces with NPN. This NPN adsorption requires a HPLC system that is specially adapted and permanently allocated to perform the fluorescent micelle assay. In addition, desorption of the dye NPN can also lead to false surfactant concentration measurements.

[0017] Furthermore, it is well known in the prior art that fluorescent micelle assays are not suitable for use in the presence of protein(s). The hydrophobic fluorescent dye NPN naturally interacts with other hydrophobic components in a biotherapeutic formulation, such as proteins. The known autofluorescence or autofluorescence of components in a biotherapeutic formulation, such as proteins, can excite NPN (350 nm), which can cause errors in quantification [7, 12]. Previous prior art was consistent in finding that fluorescent micelle assays are not possible in the presence of proteins [3, 6, 8, 9, 10, 11, 12]. Additional prior art searches confirmed that no fluorescent micelle assays have been successfully performed in the presence of proteins to date.

[0018] Even in 2020, a publication [9] showed that the fluorescent micelle assay could not quantify polysorbate 20 in the presence of proteins (see the paragraph covering p. 653 / 4). Thus, the most recent publication on the fluorescent micelle assay only reports data in the absence of proteins. As a result, a preparatory step to remove proteins seems to be necessary to perform the fluorescent micelle assay in aqueous formulations.

[0019] [3] also points out that the N-phenyl-1-naphthylamine (NPN) reaction does not react uniformly with species esterified with various polysorbates and therefore could lead to erroneous estimates of polysorbate content.

[0020] However, the present invention has been developed contrary to the widely or universally held opinion or preconception among experts in the field that fluorescent micelle assays in the presence of proteins would be impractical.

[0021] It is therefore an object of the present invention to provide a method for the quantification of poisolbate that avoids the problems of the prior art and overcomes the problems of fluorescent micelle assays performed on HPLC systems.

[0022] It is a further object to provide a method for quantifying polysorbate that may also be suitable for performing high-throughput analyses of samples containing polysorbate.

[0023] Summary of the Invention Surprisingly, it has been found that by performing a fluorescent micellar assay (FMA) using N-phenyl-1-naphthylamine (NPN) as a fluorescent dye, without using an HPLC system, it is possible to overcome the problems known from the prior art and to quantify the polysorbates contained in a liquid sample in the presence of proteins as well as one or more peptides or polypeptides.

[0024] Thus, a facile method is provided using a fluorescent micelle assay. Method (a) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more (poly)peptides comprises the following steps: (a.1) providing a sample of an aqueous formulation; (a.2) adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine to the sample of step (a.1) to obtain a fluorescent dye-containing sample to be tested in the fluorescent micelle assay; (a.3) optionally incubating the fluorescent dye-containing sample of step (a.2); (a.4) performing a fluorescent micelle assay using the fluorescent dye-containing sample obtained in step (a.2) or (a.3); (a.5) determining the content of polysorbate present in the sample from the fluorescent micelle assay performed in step (a.4); However, However, it is carried out without the use of an HPLC-system.

[0025] Also possible is a high-throughput quantitative analysis of polysorbate-containing samples which contain, in addition to proteins, one or more peptides or polypeptides.According to another embodiment, therefore, a method (b) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more (poly)peptides is provided, comprising the steps of: (b.1) providing multiple samples of an aqueous formulation; (b.2) adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine to each sample, respectively, to obtain a fluorescent dye-containing sample to be tested in the fluorescent micelle assay; (b.3) optionally incubating the fluorescent dye-containing sample of step (b.2); (b.4) performing a fluorescent micelle assay using the fluorescent dye-containing sample obtained in step (b.2) or (b.3); (b.5) determining the content of polysorbate present in each sample from the fluorescent micelle assay performed in step (b.4); However, However, a method (b) is provided which is carried out without the use of an HPLC-system.

[0026] It was unexpected that any kind of (poly)peptide, specifically proteins, present in the starting aqueous formulation would not interfere with the fluorescent micellar assay measurements, and it was further unexpected that impurities, additives and excipients would not interfere with the fluorescent micellar assay measurements.

[0027] Indeed, the method of the present invention has been found to reliably provide the content of polysorbate in aqueous formulations in which one or more peptides, polypeptides, or proteins are present at the same time. Moreover, for high-throughput quantitative analysis of polysorbate in samples containing peptide(s), polypeptide(s), or protein(s), quantification can be performed simultaneously for a set of tests.

[0028] The present invention is also directed to the use of the method according to the invention for quantifying polysorbate in an aqueous formulation, e.g. as a stability test of the aqueous formulation during its development, production, storage or storage, wherein the aqueous formulation contains polysorbate and one or more (poly)peptides as disclosed herein, with the proviso that the method is performed without the use of an HPLC-system and the fluorescent micellar assay is performed in the presence of one or more peptides and / or one or more polypeptides, including one or more proteins.

[0029] A further subject of the present invention is also the use of the fluorescent micelle assay in a method for quantifying polysorbate in an aqueous formulation comprising polysorbate and one or more (poly)peptides as disclosed herein, with the proviso that the method is carried out without the use of an HPLC-system and that the fluorescent micelle assay is carried out in the presence of one or more polypeptides, including one or more peptides and / or one or more proteins.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The prior art and embodiments of the present invention are described, by way of example, with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale, and therefore cannot assume precise geometric values ​​with respect to original size. The drawings of the present disclosure are incorporated in and constitute a part of this specification and illustrate embodiments of the present invention without being limited to the specific embodiments described. The drawings, together with the general description and the detailed description, serve to explain the principles of the present disclosure. In the drawings, like features are represented by like reference numerals. Shown as follows: [Brief description of the drawings]

[0031] [Figure 1A-1B] Schematic of the fluorescent micelle assay concept. [Figure 2A-2B] Schematic of a sample containing polysorbate 20 (PS20) before and after addition of the dye N-phenyl-1-naphthylamine, used in the fluorescent micelle assay, in which PS20 forms intact micelles. [Figure 3A-3B] Schematic diagram of a sample containing polysorbate 20 (PS20) before and after addition of the dye N-phenyl-1-naphthylamine used in the fluorescent micelle assay, in which PS20 is partially degraded and does not form intact micelles or forms micelles that are not intact. [Figure 4] Simplified schematic of the fluorescent micellar assay (FMA) performed on an HPLC system. [Diagram 5] 1 is a schematic diagram according to an embodiment of the present invention. [Figure 5A] Schematic diagram of an embodiment of a microplate. [Figure 6] 1 is a linearity plot of the fluorescent signal in a fluorescent micelle assay according to an embodiment of the present invention using increasing polysorbate concentrations. [Figure 7A-7F] FIG. 1 shows the determination of polysorbate concentration to investigate the specificity of a fluorescent micelle assay according to an embodiment of the present invention in the presence or absence of various (poly)peptides with and without polysorbate.

[0032] Figures 1A, 1B, 2A, 2B, 3A, 3B, and 4 have already been described in view of some embodiments of the prior art. Figure 5 will be described below in connection with embodiments of the present invention. Figures 6 and 7A-7F will be described in the experimental section.

[0033] Detailed Description of the Invention General definitions Terms not specifically defined herein should be given the meaning that would be given to such terms by one of ordinary skill in the art in light of the disclosure and the context.

[0034] The expression "quantification of polysorbate" shall be understood in the broadest sense and means determining the concentration or amount of polysorbate present in an aqueous formulation from which a sample is taken and examined. Quantification is achieved by using a fluorescent micellar assay (FMA).

[0035] The "Fluorescent Micelle Assay (FMA)" is a test method known to those skilled in the art, about which various literature [3, 5, 9] is available, so that the skilled artisan will understand the details of this procedure and will be familiar with, for example, the amount of sample that can be used, the appropriate solvents to be used, the characteristics of the fluorescent dye N-phenyl-1-naphthylamine, such as its light sensitivity to be taken into account, the composition of the fluorescent dye solution to be added to the sample, etc.

[0036] The term "polysorbate" is used interchangeably and synonymously to describe a class of emulsifiers or nonionic surfactants used in pharmaceuticals, cosmetics, and foods. It is often used to dissolve products that are not water-soluble in water. Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Well-known brand names are Kolliphor, Scattics, Alkest, Canarcel, and Tween. Commercially available polysorbates usually consist of a mixture of structurally related molecules such as non-esterified sorbitan and / or isosorbitan-polyethylene glycol (PEG) species, mono-, di-, tri-, tetra-esters, and fatty acids.

[0037] Exemplary representative polysorbates include: Polysorbate 20 (PS20) or polyoxyethylene (20) sorbitan monolaurate; Polysorbate 40 (PS40) or polyoxyethylene (20) sorbitan monopalmitate; Polysorbate 60 (PS60) or polyoxyethylene (20) sorbitan monostearate; and Polysorbate 80 (PS80) or Polyoxyethylene (20) Sorbitan Monooleate It is.

[0038] The numbers 20, 40, 60, and 80 following the term "polysorbate" refer to the predominant type of fatty acid attached to the polyoxyethylene sorbitan residue of the molecule; i.e., monolaurate is designated by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80.

[0039] The number 20 following the term "polyoxyethylene" refers to the total number of oxyethylene --(CH2CH2O)-- groups present in the molecule.

[0040] In this invention we focus on Polysorbate 20 and 80, which are primarily used in the pharmaceutical industry, while Polysorbate 40 and 60 are more commonly used in the food and cosmetic industries.

[0041] According to the European Pharmacopoeia (Ph.Eur.) standard, in terms of fatty acid content, laurate esters account for 40-60% of the esters in Polysorbate 20, with the remaining esters ranging from C8 to C18. Oleate esters account for 58-85% of the esters in Polysorbate 80, with the remainder ranging from C14 to C18, including stearates, linoleates, and linolenates.

[0042] The term "aqueous" is intended to mean that water is present in the formulation. For example, water may constitute a part of the solvent, e.g., a minor or major part of the solvent, or may be the only solvent present. In fact, any type of water may be used. Purified water may be preferred, but according to some embodiments, tap water may be used. The type of water selected will depend on the intended use of the aqueous formulation. Purified water used in accordance with the present invention is water that has been subjected to a purification process, such as distillation, reverse osmosis, carbon filtration, capacitive or electrolytic deionization, microfiltration or ultrafiltration, ultraviolet oxidation, etc., to remove impurities to make it suitable for use. A combination of these processes may be used to obtain water of high purity, e.g., ultrapure water, whose trace contaminants are measured in parts per billion (ppb) or parts per trillion (ppt). In one embodiment, the water used in the method of the present invention is ultrapure water, e.g., Type 1 ultrapure water (Milli-Q® water) according to ASTM D1193 or ISO 3696. In another embodiment, the water used may be sterile water suitable for administration to a subject, such as water for injection (WFI). Also, distilled, double-distilled or deionized water may be used.

[0043] The term "aqueous formulation" refers to a solution in which the solvent or one of the solvents is water. Solutions include true solutions, dispersions, suspensions, etc., unless otherwise specified. Aqueous formulations according to the present invention comprise a polysorbate, one or more (poly)peptides, and optionally excipient(s).

[0044] The term "sample" is understood in the broadest possible sense to mean a limited or small amount of an aqueous preparation taken for analysis. Sampling may be performed manually or by automated methods.

[0045] The expressions "(poly)peptide" or "(poly)peptides" grouped under the term "(poly)peptide(s)" include "peptide", "peptides", "polypeptides", "protein" and "proteins", whereby "protein" or "proteins" are usually considered to be included in "peptide(s)" or "polypeptide(s)". However, for a better understanding and to ensure that the term (poly)peptide(s) also includes proteins, in addition to peptides and polypeptides, proteins are listed herein. That is, "(poly)peptide" or "one or more (poly)peptides" means one or more peptides and / or one or more polypeptides, including one or more proteins. Thus, peptide(s), polypeptide(s) and protein(s), as well as variations of these terms, refer, respectively, to peptide(s), oligomer(s), such as oligopeptide(s), polypeptide(s), or protein(s), including fusion protein(s). As is generally known, peptides are organic compounds containing peptide bonds between amino acids. According to their number, a distinction is made between oligopeptides with a small number of amino acids and polypeptides with a large number of amino acids. For example, a peptide may contain at least two amino acids bound to each other, for example by a normal peptide bond or by a modified peptide bond, as in the case of isosteric peptides. For the sake of brevity, the terms "peptide", "polypeptide" or "protein" may be used interchangeably in this application, and the skilled person is familiar with the terms themselves and understands their meaning. A peptide, polypeptide or protein may be composed of L-amino acids and / or D-amino acids. Preferably, a peptide, polypeptide or protein is composed (entirely) of L-amino acids or (entirely) of D-amino acids, thereby forming a "retro-inverso peptide sequence".

[0046] The term "peptide", "polypeptide" or "protein" refers in particular to "classical" peptides, polypeptides or proteins, which are typically composed of amino acids selected from the 20 amino acids defined by the genetic code, linked together by peptide bonds. The term "peptide", "polypeptide" or "protein" may also include or be composed of amino acids other than the 20 defined by the genetic code in addition to these amino acids. In particular, peptides, polypeptides or proteins in the context of the present invention may equally be composed of amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes well known to those skilled in the art.

[0047] The terms "peptide", "polypeptide", and "protein" also include modified peptides, polypeptides, and proteins. For example, modifications of peptides, polypeptides, or proteins may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, senenylation, sulfation, amino acid addition such as arginylation, or ubiquitination. In particular, the terms "peptide", "polypeptide", or "protein" also include "peptidomimetics", which are defined as peptide analogs containing non-peptidic structural elements, which are capable of mimicking or antagonizing the biological action(s) of the natural parent peptide.

[0048] The peptides, polypeptides or proteins may be therapeutic peptides, therapeutic polypeptides or therapeutic proteins, which are used for the prevention or treatment of any disease or any disorder. (Poly)peptides, specifically peptides, polypeptides or proteins, may be "antigen-binding molecules" capable of binding to a target antigen, and include monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies or and antibody fragments (e.g., Fv, scFv, Fab, Fab', scFab, F(ab')2, Fab2, Fc and Fc' fragments), heavy and light immunoglobulin chains and their constant regions, variable regions or hypervariable regions as well as Fv and Fd fragments, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.), so long as they exhibit binding to the relevant target molecule(s) or mixtures thereof. According to the present invention, in addition to the protein in the form of one or more antibodies, the protein may also be in the form of one or more enzymes. Enzymes range in size from a monomer of 4-oxalocrotonate tautomerase (approximately 6.8 kDa with only 62 amino acid residues) to animal fatty acid synthase with over 2,500 residues (approximately 275 kDa). Enzymes belonging to protein groups such as peptidases, glycosidases, lipases, nucleases, and lactases are included here, but ribozymes, which are composed only of RNA, are not included.

[0049] Additionally, it should be noted that in this disclosure, the singular and plural forms are not used in a limiting sense, and thus, as used herein, the singular forms "a," "an," "one," and "the" refer to both the singular and the plural unless otherwise specified or clear from the context.

[0050] The words "comprising," "comprises," "comprised," "containing," "contain," or "contained" are intended to include the more restrictive term "consisting of" unless otherwise specified or clear from the context.

[0051] The term "about" or "approximately" means within 20%, specifically within 10%, and more specifically within 5% of a given value or an upper or lower value.

[0052] EMBODIMENTS OF THE PRESENT DISCLOSURE In the following, the multi-step method according to the present invention is described. The optimal process conditions and parameters for each individual step may vary depending on the specific aqueous formulation, optional additive(s), optional excipient(s), selected non-aqueous solvent(s), and (poly)peptide(s) present. Unless otherwise specified, the process conditions and parameters for each process step can be easily selected by the skilled artisan based on the present disclosure. Exemplary procedures are provided in the experimental section.

[0053] According to an embodiment of the present invention, a method (a) was developed for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more (poly)peptides:

[0054] In a first step (a.1) a sample of an aqueous formulation containing polysorbate and one or more (poly)peptides is provided.

[0055] According to an embodiment, the polysorbate is a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80, specifically polysorbate 20 or polysorbate 80.

[0056] According to an embodiment, the polysorbate may be intended for pharmaceutical use, specifically for use in drugs. In particular, the polysorbate 20 or polysorbate 80 may be pharma- ceutically acceptable and of pharmaceutical grade quality, such as European / US / Chinese Pharmacopoeia grade quality. For example, the polysorbate may be selected from polysorbate 20 or polysorbate 80 suitable for parenteral administration.

[0057] Polysorbate 20 may be selected from the following commercially available products: Super Refined™ PS20, Tween™ 20, Tween™ 20 HP, Tween™ 20 Pharmaceutical Grade, or PS20 HP available from Croda Europe Ltd., or PS20 China Grade available from Nanjing Well Pharmaceutical Co, Ltd. In view of Polysorbate 80, it may be selected from the following commercially available products: Super Refined™ PS80, Tween™ 80, Tween™ 80 HP, Tween™ 80 Pharmaceutical Grade, or PS80 HP available from Croda Europe Ltd., or PS80 China Grade available from Nanjing Well Pharmaceutical Co, Ltd.

[0058] The (poly)peptide(s) present are not particularly limited within the scope of the present invention, rather any peptide, polypeptide or protein known to the skilled artisan may be used, regardless of its type, characteristics and size, as already explained. Also, a mixture of two or more (poly)peptides, such as peptides, polypeptides, proteins, etc. may be present in the aqueous formulation.

[0059] Sampling can be performed by any procedure known to the expert, for example, manually by a person or by an automated device or system. Of course, sampling should be performed in such a way that it does not adversely affect or impair the following analytical procedure. The amount of sample taken from the aqueous formulation in which the polysorbate content is to be determined depends on several factors, such as the type and composition of the aqueous formulation, the type and size of the sample container in which the sample(s) are filled, the type and size of the fluorescent micelle assay test device, etc. It is also possible that the volume of the sample may be adjusted before performing the fluorescent micelle assay. For example, the sample may be diluted using water as a solvent or the sample may be concentrated. The change in sample volume must be taken into account when later quantifying the polysorbate in the starting aqueous formulation.

[0060] In the following step (a.2), a solution containing the fluorescent dye N-phenyl-1-naphthylamine is added to the sample obtained in step (a.1) in order to obtain a fluorescent dye-containing sample that is subsequently tested in the fluorescent micelle assay.

[0061] The fluorescent micelle assay method is based on the detection of the fluorescence emission intensity of the fluorescent dye N-phenyl-1-naphthylamine in the presence of polysorbate at or above the critical micelle concentration. 16 H 13 N) has the following chemical structure: [ka] And it is commercially available. Since it is hardly soluble in water, a solution containing an organic solvent is prepared or provided. The solution containing the dye in step (a.2) usually has the same components, specifically the same composition, as those used in the mobile phase in the combined FMA-HPLC method in the prior art (see, for example, reference [9], page 649, third paragraph: "The mobile phase contained 150 mM sodium chloride, 50 mM tris(hydroxymethyl)methylamine, pH 8.0, 5% (v / v) ACN, 15 ppm Brij35, and 5 mM NPN.").

[0062] According to an embodiment, such a solution containing a fluorescent dye in step (a.2) typically contains a hydrophobic dye in the range of 1 μM to 10 μM, specifically 5 μM, an organic solvent such as acetonitrile in the range of 1% (v / v) to 10% (v / v), specifically 5% (v / v), a buffer such as tris(hydroxymethyl)aminomethane (Trizma base) or tris(hydroxymethyl)methylamine in the range of 5 mM to 500 mM, specifically 50 mM, a salt such as NaCl in the range of 10 mM to 1000 mM, specifically 150 mM, and a surfactant such as a non-ionic surfactant in the range of 0.00015% to 0.015%, specifically 0.0015%, e.g. polyalkylene glycol ether (known brands are Brij®, Genapol®, Lutensol®). The solution is typically a buffer solution in which a component for solubilizing the fluorescent dye is present. Other compositions of the dye-containing solution are possible, so long as the subsequent fluorescent micelle assay can be performed.

[0063] It may be advantageous to mix the solution comprising the fluorescent dye N-phenyl-1-naphthylamine before adding it to the sample in step (a.2). Mixing may be performed, for example, by shaking or stirring. According to an embodiment, shaking, for example by using an orbital shaker, is employed to achieve particularly effective shaking in a very short time.

[0064] Such mixing of the solutions can eliminate any concentration gradients of the dye due to non-specific adsorption to the reservoir surface and contribute to obtaining a more homogeneous solution of the dye.

[0065] Method step (a.3) is an optional method step. However, in individual cases, incubation may be advantageous since it may improve the distribution of the fluorescent dye into the inner core of the polysorbate micelles. Thus, according to optional method step (a.3), the sample obtained in step (a.2) is incubated.

[0066] According to an embodiment, the incubation is carried out under shaking.

[0067] In a further embodiment, the incubation temperature is selected from the range of 10-60° C., specifically 30-40° C., more specifically 35° C. The incubation temperature can be readily selected from the ranges disclosed above.

[0068] The incubation time can be selected according to the knowledge of an average person skilled in the art, and can be omitted, shorter or longer, and a person skilled in the art can easily select suitable incubation temperatures and times.

[0069] In analytical procedures such as the fluorescent micelle assay, it is useful to prepare the samples as quickly as possible before the actual fluorescent micelle assay measurement, so that there is not a large time difference between the individual samples, which results in, for example, different contact times with the dye. Therefore, all samples are incubated as soon as possible immediately after adding the dye solution. In either case, it may be useful to protect the samples containing the dye from direct light exposure before performing the fluorescent micelle assay.

[0070] In method step (a.4), a known fluorescent micellar assay is performed on the sample obtained in step (a.2) or step (a.3). Step (a.3) is an optional step and is not always performed. If step (a.3) is omitted, a fluorescent micellar assay is performed on the sample obtained in step (a.2), and if step (a.3) is employed, a fluorescent micellar assay is performed on the sample obtained in step (a.3). In the fluorescent micellar assay, light with a wavelength of 350 nm is used to excite a fluorescent dye, which then emits fluorescence at a wavelength of 420 nm. A spectrofluorometer is used to perform the fluorescence measurement. Also known sample containers such as quartz cuvettes can be used for the measurement. It is advantageous if the sample containers or receptacles used in steps (a.1), (a.2) and (a.3) are the same ones that are also used for the fluorescence measurement in step (a.4).

[0071] Thus, method (a) is carried out using a fluorescence detector and no HPLC system or equipment is used.

[0072] In step (a.5), the content of polysorbate present in the sample is determined using a fluorescent micelle assay. For quantification, the fluorescent signal can be evaluated using a calibration curve, blank values, and suitable software that can be used to determine the amount of polysorbate.

[0073] Thus, method (a) can be applied to determine the polysorbate content in (poly)peptide(s)-containing aqueous formulations without the need for a prior (poly)peptide(s) removal step.

[0074] According to a further embodiment of the invention, the method steps (a.1), (a.2), (a.3), (a.4) and (a.5) are carried out one by one in the mentioned order, e.g. immediately one by one. It may be useful that there are no intermediate steps between the individual method steps other than in the embodiment already described.

[0075] If several samples have to be evaluated, it is expedient to be able to process the samples in parallel or simultaneously.

[0076] Furthermore, method (a) enables the analysis time per sample to be significantly reduced from 120 seconds for the FMA-HPLC method to approximately 15 to 20 seconds for the fluorescent micelle assay, thereby reducing the analysis time by at least a factor of six.

[0077] It is also possible that some or all of the method steps may be chosen to be automated.

[0078] In another embodiment, method (b) can be used for quantitative screening of polysorbates in aqueous formulations of multiple samples simultaneously. The method can be adapted to be performed using a liquid microbatch format combined with several liquid handling steps for high throughput analysis.

[0079] Thus, in step (b.1), several, in particular multiple, samples of an aqueous formulation containing polysorbate and one or more (poly)peptides are provided.

[0080] The number of samples that can be provided in step (b.1) depends on several factors, such as the type of sampling, the size and type of sampling device used, the desired sample throughput, the size and type of fluorescent micelle assay device, etc. By way of example, the number of samples can be specified to be at least 2, 5, 10, 20, 50, 96, 100, 200, 384, 500, or 1000. Other numbers of samples can also be provided in step (b.1).

[0081] In method step (b.1), samples of the aqueous formulation to be tested may be provided together (simultaneously) or the samples to be tested may be provided one by one. If the samples are provided simultaneously, throughput may be significantly increased.

[0082] According to an embodiment, the method (b) of the invention is carried out in a microplate format. The expression "microplate format" is understood to mean that a quantified number of samples are provided in a microplate (also known as a microtiter plate), each sample being provided in one well of the microplate. A microplate is a well-known laboratory device for investigating biological or physical properties. A roughly rectangular microplate is usually made of plastic and contains a large number of wells separated from each other in rows and columns. A microplate usually has 6, 12, 24, 48, 96, 384 or 1536 sample wells, with microplates with 3456 or 9600 wells also being known. Each well of a microplate can usually receive and accommodate a liquid sample with a volume ranging from a few tens of nanoliters to a few milliliters.

[0083] Such microplates are particularly suitable for high throughput screening in pharmaceutical research. Thus, method (b) of the invention can be adapted for use in a "microplate format", since method (b) can be carried out using a microplate or several microplates.

[0084] In order to achieve a high throughput method, some or all of the method steps of method (b) may be chosen to be automated.

[0085] According to an embodiment, step (b.1) is automated. Thus, according to an embodiment, each sample to be tested is transferred to a well of a microplate by an automated device or system. For example, multiple samples can be processed in parallel and simultaneously. The number of samples that can be processed simultaneously is not particularly limited, and the number of samples to be tested may be limited only by the type and size of the automated processing station used or the size of the microplate used.

[0086] Thus, for example, liquid handling devices or stations can be used that can process most or all samples simultaneously in parallel. In such cases, microplates with a defined number of wells are used in connection with robotic workstations that have the facilities to automatically pick up and process analytical samples, such as liquid flexible channel arms, liquid handling arms with disposable or fixed tips for multi-pipetting, robotic gripper arms, etc. Such robotic workstations usually also have an integrated control system, such as liquid level detection or similar functions, to ensure accurate processing of samples.

[0087] Such automation has great advantages, since the sample volumes of all samples analyzed are exactly the same, the method steps themselves and the combination of method steps are carried out as quickly as possible, and many more samples can be processed simultaneously and in parallel.

[0088] In step (b.2), a solution containing the fluorescent dye N-phenyl-1-naphthylamine is added to each sample respectively to obtain fluorescent dye-containing samples to be tested in the subsequent fluorescent micellar assay. According to an embodiment, the solution containing the fluorescent dye N-phenyl-1-naphthylamine is added simultaneously to the samples present, for example, in a microplate, i.e. each sample is placed in a well of a microplate and then the fluorescent dye N-phenyl-1-naphthylamine is simultaneously present in each sample. Thus, different samples can be processed simultaneously. It is also possible to add the fluorescent dye N-phenyl-1-naphthylamine one by one to each sample. Otherwise, the explanations for step (a.2) apply here as well. In particular, according to an embodiment, the solution with the dye in step (b.2) can have the same composition as that used for the mobile phase in the combined FMA-HPLC method described in the prior art (see [9] cited passage).

[0089] It is advantageous if step (b.2) is automated as this ensures that all samples are treated equally.

[0090] Before adding the solution containing the fluorescent dye N-phenyl-1-naphthylamine to the sample, it may be useful to mix the solution, e.g., by shaking the solution automatically on a shaker, to avoid non-uniform concentrations of the dye due to non-specific adsorption to the reservoir surface.

[0091] Then, in method step (b.3), the samples obtained in step (b.2) may be incubated. This is an optional method step. The incubation may be performed under shaking. For example, the incubation in step (b.3) can be performed with or without shaking, which is achieved for the samples simultaneously or one by one.

[0092] According to an embodiment, the incubation temperature is selected in the range of 10-60° C., in particular 30-40° C., more particularly 35° C. Incubation steps, especially under shaking, may have the advantage that the partitioning of the fluorescent dye into the inner core of the polysorbate micelles may be increased.

[0093] The incubation time can be easily selected by those skilled in the art. However, in the present invention, as already explained, the incubation time can be adjusted to be very short. It is also possible to make the incubation time shorter or longer. It is easily possible for those skilled in the art to select a suitable incubation temperature within the above range and to select the incubation time according to their own knowledge.

[0094] According to an embodiment, optional step (b.3) can also be automated. If step (b.3) is automated, it is advantageous to ensure that the incubation temperature and time of all samples are equivalent, and that the shaking performance and time are equivalent.

[0095] Shaking during incubation can be performed, for example, with an incubator shaker, which is a known temperature-controlled device that combines the functions of incubation and shaking.

[0096] It is not necessary to bring the samples to room temperature after incubation and before taking quantitative fluorescence measurements.

[0097] The fluorescence intensity of the sample obtained in method step (b.2) (if no incubation step is performed) or obtained in method step (b.3) (if an incubation step is performed) is then measured spectrophotometrically in a fluorescent micelle assay in step (b.4) using an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0098] According to an embodiment, step (b.4) can also be automated, which is advantageous since it allows the fluorescent micelle assay of different samples to be performed as quickly as possible.

[0099] The fluorescent micelle assay is used to determine the content of polysorbate present in each sample in step (b.5) by using a calibration curve. According to an embodiment, step (b.5) can also be automated.

[0100] Method (b) is carried out using a fluorescence detector and no HPLC system or instrument is used.

[0101] According to an embodiment, method steps (b.1) to (b.5) can be performed in a fully automated manner, whereby some or all samples are processed in parallel. A fully automated high-throughput fluorescent micelle assay can then be performed according to the method (b) of the invention, for example performed in a microplate format using a liquid handling station. This approach allows a significant reduction in the analysis time per sample from 120 seconds for the FMA-HPLC method to about 15 to 20 seconds for the fluorescent micelle assay performed according to method (b), resulting in at least a 6-fold reduction in analysis time.

[0102] FIG. 5 shows a schematic diagram of the method (b) according to steps (b.1) to (b.5) according to an embodiment of the present invention, and is described below:

[0103] In step (b.1), multiple samples 210.1, 210.2, 210.3, and 210.4 of an aqueous formulation containing polysorbate and one or more (poly)peptides, such as one or more peptides, polypeptides, or proteins, are provided. The concentration of polysorbate is equal to or greater than the critical micelle concentration (c(PS)≧CMC), since polysorbate forms micelles 220a, 220b, and 220c, depicted as black spheres. In the illustrated exemplary embodiment, samples 210.1, 210.2, 210.3, and 210.4 are present in wells of a microplate, i.e., each sample is present in one well of the microplate, respectively. That is, samples 210.1, 210.2, 210.3, and 210.4 in step (b.1) have already been transferred onto the microplate by an automated device or system, such as a liquid handling device or station. A schematic diagram of an embodiment of a microplate 400 is shown in FIG. 5A. Other embodiments of the microplate are possible.

[0104] In step (b.2) of this exemplary embodiment, a solution containing the fluorescent dye N-phenyl-1-naphthylamine is added simultaneously to each of the samples 210.1, 210.2, 210.3, and 210.4, respectively, by an automated procedure, e.g., the solutions are pipetted simultaneously by the automated system or device 300 using suitable delivery systems 310.1, 310.2, 310.3, 310.4.

[0105] Before adding the solution containing the dye, the solution may be automatically mixed (not shown), for example shaken, to avoid uneven concentration of the dye.

[0106] In the illustrated exemplary embodiment, optional method step (b.3) is performed and the samples are incubated with or without shaking. If shaking is selected, this may be performed, for example, by using an incubator shaker. Such an incubator shaker can simultaneously hold and process one or more microplates, each containing a different sample. In the illustrated exemplary embodiment, the incubation temperature is selected to be 35° C. for 60 seconds, so that the distribution of the fluorescent dye to the inner cores of the polysorbate micelles 220a, 220b, and 220c can be improved. Other incubation times, as well as no incubation, are contemplated and possible. A person skilled in the art can easily select the incubation time. The incubation temperature is selected from the ranges disclosed herein.

[0107] After incubation with or without shaking, the fluorescent micelle assay of step (b.4) of the sample obtained in method step (b.3) is carried out in a spectrophotometer with an excitation wavelength of 350 nm (represented by arrow 350.1) and an emission wavelength of 420 nm (represented by arrow 350.2). In step (b.5), the fluorescent micelle assay is used to determine the polysorbate content present in the sample, for example using suitable software.

[0108] It has been found to be particularly advantageous to provide and test samples in microplates, as microplates are also suitable for high throughput screening in a spectrophotometer.Accordingly, the methods of the invention involving the testing of samples can be carried out in a partially or fully automated, e.g. liquid handling station, and therefore the use of a microplate format is advantageous.

[0109] In the following, the term "process" refers to both the process (a) according to steps (a.1) to (a.5) and the process (b) according to steps (b.1) to (b.5), unless otherwise specified.

[0110] According to an embodiment, the method is performed using disposable labware, such as disposable microplates, which prevents carryover from one sample to another and eliminates the issue of cleaning the equipment.

[0111] In another embodiment, the method is carried out using disposable labware with a non-binding, low-binding, or medium-binding surface. To minimize the binding of the fluorescent dye N-phenyl-1-naphthylamine, it may be useful to use a non-binding, low-binding, or medium-binding surface. For example, several types of polystyrene microplates are known that have a non-binding, low-binding, or medium-binding surface for the above-mentioned fluorescent dye.

[0112] Such specific, non-binding, disposable labware can overcome the above-mentioned problems of fluorescent micelle assays performed in HPLC systems, such as the dedication of analytical equipment adapted for specific applications, and can also avoid the fluctuations in the fluorescent signal caused by the adsorption and desorption events of the hydrophobic dye N-phenyl-1-naphthylamine in the HPLC system.

[0113] The concentration of polysorbate in the sample can be easily determined, so that the method of the present invention allows almost all kinds of aqueous formulations to be analyzed. When determining the concentration of polysorbate in samples with very low or very high concentrations of polysorbate, it may be useful to adjust the sample volume of these samples, for example by additional dilution steps or by concentrating the sample. It may also be necessary to adjust the sample volume before analysis so as to fall within the linear range of the calibration curve. The linear range of the calibration curve may be influenced by various experimental parameters, for example the batch-by-batch composition of the polysorbate used. However, a person skilled in the art can easily calibrate with a calibration sample suitable for each polysorbate used, determine the linear range, and take the result into account in the measurement based on his / her own knowledge and the technical teachings disclosed herein.

[0114] Therefore, the results of the calibration samples are suitable for use in assessing the polysorbate content of a series of subsequent measurement samples for a given linearity.

[0115] As will be explained in detail below, the method is also highly suitable for biopharmaceutical samples and shows accurate and specific results for determining polysorbate content. The high accuracy of the method found, both when screening different samples and in view of the automated liquid handling station, demonstrates the absence of random errors that may result from the use of some liquid handling steps.

[0116] The method was found to be highly selective and to show no or very little interference due to hydrophobic components potentially present in the aqueous formulations evaluated. Even (poly)peptides such as peptides, polypeptides or proteins with different hydrophobic properties do not impair the method in any way. This is unexpected.

[0117] Moreover, the polysorbate quantification method of the present invention is applied for the first time to analyze samples containing peptides, polypeptides or proteins. Indeed, the presence of (poly)peptides, especially proteins, in the aqueous formulation does not adversely affect the method of the present invention. The fluorescent micelle assay carried out according to the method of the present invention can be used in the presence of (poly)peptides(s), especially peptide(s), polypeptide(s) or protein(s). The hydrophobic fluorescent dye N-phenyl-1-naphthylamine has not been found to interact with other hydrophobic components in the aqueous formulation, such as peptides, polypeptides or proteins. Moreover, it was found that no undesired excitation of N-phenyl-1-naphthylamine (at a wavelength of 350 nm) occurred due to the known autofluorescence or autofluorescence of components in the aqueous formulation, such as peptides, polypeptides or proteins, and no errors in the quantification of polysorbate due to such interactions could be found. Thus, the well-established finding in the prior art that a fluorescent micelle assay should be impossible in the presence of proteins is incorrect and does not match the experiments by the inventors described below.

[0118] Furthermore, no heterogeneity could be observed in the interaction of N-phenyl-1-naphthylamine with the various polysorbates, so that erroneous estimation of the polysorbate content did not occur.

[0119] Thus, the polysorbate quantification method of the present invention can be unexpectedly applied to the analysis of samples containing peptide(s), polypeptide(s) or protein(s); in other words, the fluorescent micellar assay performed in the method of the present invention allows the quantification of polysorbate in the presence of peptide(s), polypeptide(s) or protein(s). As a result, no preliminary step of removing the peptide, polypeptide or protein is necessary.

[0120] The method of the present invention can be performed on one or a few samples, or multiple samples can be evaluated. The method can be performed manually or by an automated system. The method steps can be performed one by one or simultaneously in parallel to save time.

[0121] Furthermore, the methods of the invention, particularly method (b), based on a fluorescent micellar assay with the dye N-phenyl-1-naphthylamine, can be performed in a microplate format and are therefore suitable for high-throughput concentration screening.

[0122] The method of the present invention, which does not use an HPLC system, can reduce the analysis time per sample from 2 minutes per sample when performing the fluorescent micelle assay on an HPLC system to about 15-20 seconds per sample with the method of the present invention, thereby increasing the sample throughput in method (b). Since alternative polysorbate quantification methods, such as UPLC-QDa (ultra-performance liquid chromatography with quadrupole Dalton detection), require an analysis time of 10 minutes per sample

[13] , mixed-mode HPLC-CAD (high-performance liquid chromatography with charged aerosol detection) requires an analysis time of 8 minutes per sample, and reversed-phase (RP) UHPLC-CAD (ultra-performance liquid chromatography with charged aerosol detection) requires an analysis time of 36 minutes [3], the method of the present invention is clearly superior to the current state-of-the-art methods in terms of the required analysis time.

[0123] Thus, by transferring the assay from an FMA-HPLC system to a microplate format, for example, performed in a liquid handling station, particularly according to method (b), the analysis time per sample can be reduced by at least a factor of six. This reduction in analysis time is highly advantageous for large sample sets and makes the method according to the invention, particularly method (b), superior to other polysorbate analysis methods that have longer analysis times. In particular, such a reduction in analysis time is highly valuable on an industrial scale, for example during the development of biotherapeutic products.

[0124] Thus, a simple fluorescence-based method is provided for the rapid determination of polysorbate content in aqueous formulations that may also be highly suitable on an industrial scale.

[0125] Indeed, the described method (b) allows for the screening of a much larger number of samples, thereby providing more robust and reliable data on polysorbate content and possible polysorbate degradation. The high throughput also allows for the analysis of replicate samples, and therefore the statistical interpretation of the collected data.

[0126] There is no particular limitation regarding the sample volume to be checked, and the sample volume can be very small and is comparable to the prior art combined fluorescent micelle assay (FMA)-HPLC setup. For example, a sample volume of 10 μL can be tested with the method of the present invention.

[0127] The accuracy and reliability of the quantification of polysorbates by the fluorescent micelle assay has been questioned or even doubted in some publications, and in any case the fluorescent micelle assay as an analytical method needs to be investigated in detail in this regard.

[0128] To verify whether the fluorescent micelle assay performed in a liquid handling station in a microplate format is a reliable test method, the assay was investigated in terms of linearity, specificity, accuracy, robustness and precision according to the literature [14, 15]. Detailed experiments are described in the experimental section. The method of the present invention was evaluated in terms of these parameters for the determination of the surfactant concentration in a sample in the presence or absence of (poly)peptides, in particular peptides, polypeptides or proteins. The essence of these tests is summarized below.

[0129] linearity The ICH Q2(R1) guideline was followed

[14] , according to which the linearity of an analytical method indicates the ability to give results that are directly proportional to the analyte concentration in the sample within a certain range. To test the linearity between the fluorescence signal and polysorbate concentration, the fluorescence was measured for samples with different sets of polysorbate concentrations (see Experimental Section). The linearity of the fluorescence signal of the fluorescent micelle assay (FMA) performed in microplates was demonstrated at concentrations up to 0.6 mg / mL PS20 HP (Polysorbate 20 High Purity), up to 0.8 mg / mL Polysorbate 20 China Grade (PS20 China Grade), and up to 0.3 mg / mL PS80 HP (Polysorbate 80 High Purity).

[0130] Typically, biopharmaceutical formulations contain polysorbate concentrations in the ranges described above and can therefore be measured directly using the methods of the invention. For samples containing higher or lower concentrations of polysorbate, the sample volume can be adjusted as necessary, e.g., a dilution or concentration step can be performed prior to carrying out the methods of the invention.

[0131] specificity Specificity refers to the ability to unambiguously determine the analyte in the presence of expected components such as impurities, degradation products, matrix, etc. (see reference

[17] ). In fluorescent micellar assays, this means that the fluorescent signal is only due to the polysorbate, and there is no fluorescent signal from the peptide, polypeptide, or protein, buffer, or added excipients. Regarding specificity, various monoclonal antibodies (mAbs) have been studied, but most of them did not show any fluorescent signal, so no problem was faced regarding the specificity of the fluorescent signal from polysorbate in the presence of these peptides, polypeptides, or proteins.

[0132] Another protein in the form of a monoclonal antibody (mAb) showed an intrinsic fluorescent signal observed at the wavelengths used in the fluorescent micelle assay. In the absence of polysorbate, the fluorescent signal of this protein itself was referred to as the "apparent" polysorbate concentration. This "apparent" polysorbate concentration of the protein (in the absence of polysorbate) showed only a small fraction of the intensity of the fluorescent signal of polysorbate determined in another experiment. In the experiment shown in the experimental section, the protein showed a fluorescent signal equivalent to 15% of the total fluorescent signal of the polysorbate concentration of the formulated sample. Surprisingly, despite the presence of both protein and polysorbate components, only the protein showed a fluorescent signal in the fluorescent micelle assay and only polysorbate was selectively detected in the fluorescent micelle assay. There was no enhancement of the signal originating from the protein.

[0133] This means that even if peptides, polypeptides and especially proteins in a sample show an intrinsic fluorescent signal in the absence of polysorbate, the fluorescent micelle assay can selectively detect the signal arising from polysorbate when both components are present.

[0134] The high specificity of the assay was also shown in relation to a widely used standard protein, namely alpha-lactalbumin, which has a molecular weight of about 10% of the mAb and a significantly lower structural complexity. In contrast to other mAbs examined so far, alpha-lactalbumin has an acidic pI (isoelectric point) and therefore an overall negative surface charge under the experimental conditions used, whereas the other mAbs used had a positive net charge under the applied experimental conditions. In control samples consisting of pure buffer and pure formulation buffer with no added polysorbate, no interfering signals originating from buffer components and added excipients were observed. These results support our findings that the fluorescent micelle assay is highly specific for the analysis of polysorbate, independent of the physicochemical properties of peptides, polypeptides or proteins present in the solution, buffers or excipient substances.

[0135] The high specificity of the assay was also shown in the context of a widely used enzyme, namely lysozyme. Lysozyme is a protein and enzyme consisting of 129 amino acids and has a molecular weight of approximately 14-15 kDa. Lysozyme has an alkaline pI (isoelectric point) and therefore a positive surface charge under the experimental conditions used, as does the mAb used. These results also support our findings that the fluorescent micelle assay is highly specific for the analysis of polysorbates, regardless of the physicochemical properties of peptides, polypeptides or proteins, buffers or excipient substances present in the solution.

[0136] Accuracy The accuracy of an analytical procedure is expressed as the degree of agreement between the found value and a value that is either a traditional true value or a recognized reference value

[14] . In terms of accuracy, as shown in the experimental section, for PS20 HP (Polysorbate 20 High Purity) spiked into protein-free buffer, the calculated spike recoveries of polysorbate ranged from 80% to 117%. For samples containing protein, buffer substances, and either PS20 HP (Polysorbate 20 High Purity) or PS80 HP (Polysorbate 80 High Purity), the accuracy of the investigated formulations containing PS20 HP (Polysorbate 20 High Purity) ranged from 73% to 117%, and the accuracy of the formulations containing PS80 HP (Polysorbate 80 High Purity) ranged from 79% to 118%.

[0137] Based on our experience, this range is comparable to other analytical methods and can be considered acceptable for the analysis of samples, especially biopharmaceutical samples.

[0138] At concentrations as low as 0.1 mg / mL of PS20 HP (Polysorbate 20 High Purity), the fluorescent micelle assay may tend to determine values ​​slightly lower than the target concentration. This finding may be caused by a nonlinear correlation between polysorbate concentration and fluorescent signal at polysorbate concentrations in the range close to the critical micelle concentration (CMC).

[0139] accuracy The precision of an analytical procedure represents the degree of agreement (degree of variation) between a series of measurements obtained by multiple sampling of the same homogenous sample under given conditions

[17] . In the fluorescent micellar assay performed in a liquid handling station, the precision was tested for five different mAbs in experiments under each formulation condition containing the target polysorbate of high purity (PS HP) quality. All samples were analyzed multiple times as four technical replicates each. To investigate the precision, the average %CV (coefficient of variation, also known as relative standard deviation) of all four technical replicates analyzed multiple times ranges from 2.2% to 3.6%. The maximum %CV obtained during the precision analysis of the five different mAbs ranged from 3.5% to 6.8%. The highest average %CV and maximum %CV were observed for samples containing mAb and PS80 HP (polysorbate 80 high purity). This finding may indicate a low precision of the fluorescent micellar assay for PS80 HP.

[0140] Overall, the results of the parameters assessing the performance of the fluorescent micelle assay indicate that the high-throughput fluorescent micelle assay is specific for the analyte polysorbate and exhibits high precision and, together with acceptable accuracy, is highly suitable for determining the concentrations of polysorbate 20 and polysorbate 80 present in samples containing (poly)peptide(s) or placebo.

[0141] LOD and LOQ For fluorescent micelle assays, especially those performed in a microbatch format, the limits of detection (LOD) and quantification (LOQ) were determined according to

[17] . These terms are used to describe the lowest concentration of analyte, here the polysorbate concentration, that can be reliably measured with an analytical procedure. Typically, a test cannot simply measure the concentration of analyte precisely down to zero. A sufficient analyte concentration must be present to generate an analytical signal that can be reliably distinguished from the "analytical noise", which is the signal that occurs in the absence of analyte. Various analytical specifications can be applied to ensure that the LOD is meaningful and can be clearly distinguished from negative or blank samples. See also the experimental section for more information.

[0142] The following LODs and LOQs were found: The limit of detection (LOD) was determined to be 0.023 mg / mL PS20 HP (Polysorbate 20 High Purity) and the limit of quantification (LOQ) was determined to be 0.069 mg / mL PS20 HP (Polysorbate 20 High Purity).

[0143] In contrast to PS20 HP (Polysorbate 20 High Purity), the LOD of PS20 China Grade (Polysorbate 20 China Grade) was determined to be 0.009 mg / mL and the LOQ was 0.028 mg / mL, respectively. In the case of PS80 HP (Polysorbate 80 High Purity), the determined values ​​were 0.001 mg / mL PS80 HP (Polysorbate 80 High Purity) for LOD and 0.003 mg / mL PS80 HP (Polysorbate 80 High Purity) for LOQ, respectively.

[0144] Comparing the determined LOD and LOQ of the method of the present invention with the prior art method using HPLC-FMA, especially in the context of the automated microplate-based fluorescent micelle assay, the LOD / LOQ of PS20 HP (Polysorbate 20 High Purity) was in the same concentration range in both assay settings. For PS80 HP (Polysorbate 80 High Purity), the LOD and LOQ for the automated microplate-based fluorescent micelle assay (FMA) were significantly lower. However, Fekete et al.

[16] determined the LOD and LOQ of Polysorbate 80 (PS80) to be 0.005 mg / mL and 0.01 mg / mL, respectively, in the quantification of PS80 HP (Polysorbate 80 High Purity) via RP-HPLC coupled with a CAD detector (CAD: Charged Aerosol Detection). These values ​​were compared with the LOD / LOQ obtained with the method of the present invention, indicating that the LOD / LOQ are in a similar range and that the fluorescent micelle assay also has the potential to determine polysorbate 80 (PS80) concentrations in a range below the polysorbate 80 concentrations typically used in biopharmaceutical formulations.

[0145] Combining the linearity results with the LOD / LOQ determination, the application range of the method of the present invention can be set in the range of 0.1 mg / mL-0.6 mg / mL for samples containing PS20 HP (Polysorbate 20 High Purity), 0.1 mg / mL-0.8 mg / mL for samples containing PS20 China Grade (Polysorbate 20 China Grade), and 0.05 mg / mL-0.3 mg / mL for samples containing PS80 HP (Polysorbate 80 High Purity), for example, in the case of a microplate-based fluorescent micelle assay.

[0146] Thus, the present invention provides a selective and easy to perform method for quantifying pharma- ceutical relevant amounts of polysorbate 20 and polysorbate 80 in the presence of, for example, peptide(s), polypeptide(s) or protein(s) and without interference from components or excipients, without time-consuming sample preparation. Additionally, a high-throughput improvement of method (b) is also provided.

[0147] The present invention is also directed to the use of the method according to the present invention for quantifying polysorbate in an aqueous formulation, e.g. as a stability test of the aqueous formulation during its development, manufacture, storage or storage, wherein the aqueous formulation contains polysorbate and one or more (poly)peptides as disclosed herein.

[0148] A further subject of the present invention is also the use of the fluorescent micellar assay, in particular as disclosed herein, in a method for quantifying polysorbate in an aqueous formulation comprising polysorbate and one or more (poly)peptides.

[0149] The advantages of the present invention are manifold:

[0150] The method of the present invention was developed to overcome the problems of the prior art, particularly fluorescent micelle assays performed in HPLC systems, which have many drawbacks. It has been successful.

[0151] The polysorbate quantification method of the present invention is applied for the first time to the analysis of samples containing (poly)peptide(s). The method is also highly suitable for biopharmaceutical samples and gives accurate and specific results for the determined polysorbate content even in the presence of one or more (poly)peptides.

[0152] The method of the present invention allows the analysis of almost all kinds of aqueous samples, even biopharmaceutical samples, since the concentration of polysorbate in aqueous samples can be easily determined. In case of constraints in view of samples with very low or very high concentrations, adjustment of the sample volume, such as additional dilution or concentration steps, is possible for these samples. The high accuracy of the method found, even when screening various samples, proves the absence of random errors that may be due to the use of some liquid handling steps, even when using an automated liquid handling station.

[0153] According to an embodiment, the method is performed using disposable labware, which prevents carryover from one sample to another and eliminates the issue of cleaning the equipment.

[0154] In another embodiment, the method is carried out using disposable non-binding, low-binding or medium-binding labware in which the binding of the fluorescent dye N-phenyl-1-naphthylamine is minimized. The use of such specific non-binding disposable labware avoids the problems of fluorescent micelle assays carried out in HPLC systems, in particular the specially adapted analytical equipment, and the fluctuations in the fluorescent signal caused by the adsorption and desorption events of the hydrophobic dye N-phenyl-1-naphthylamine in the HPLC system.

[0155] The method of the invention can be performed on one or a few samples, and can also evaluate multiple samples. The method is very flexible and can be performed manually or by automated systems. According to an embodiment, the method (b) of the invention allows to perform a fully automated high-throughput fluorescent micelle assay, for example performed in a microplate format using a liquid handling station.

[0156] The method steps may be carried out one by one or simultaneously in parallel to save time. The method of the invention allows a clear reduction in the analysis time to about 15 to about 20 seconds per sample, compared to 120 seconds when performing the fluorescent micelle assay on an HPLC system, thus reducing the analysis time by at least a factor of 6. This reduction in analysis time is highly advantageous, especially for large sample sets, and makes the method of the invention superior to other polysorbate analysis methods in terms of analysis time. In particular, the use of the method steps (b.1) to (b.5) of the invention carried out on an industrial scale is extremely beneficial during the development of aqueous formulations, such as, for example, biotherapeutic preparations or products.

[0157] Some or all of the method steps may be automated, and in particular by transferring the method to a microbatch format and combining this with the advantage of many easy to implement liquid handling steps, the entire procedure can be carried out in a convenient and fully automated manner, even for large sample numbers.

[0158] The sample volumes used are typically very small and comparable to prior art combined FMA-HPLC setups, eg sample volumes of 10 μL can be examined.

[0159] The sample preparation is not complicated and no further pretreatment is required other than adding the dye to the sample. The incubation step is optional. Especially in high-throughput methods using multi-well plates, the interaction between the dye and polysorbate can be used in a simple way to perform quantification.

[0160] When testing a series of samples simultaneously, liquid handling stations can be used that are capable of performing parallelized, automated high-throughput analyses on a small scale

[17] .

[0161] In particular, method (b) allows for the screening of a much larger number of samples, thereby providing more robust and reliable data on polysorbate content and possible polysorbate degradation. The higher throughput also allows for the analysis of replicate samples, thus allowing for statistical interpretation of the collected data.

[0162] The methods of the present invention provided are sensitive, selective, and rapid, do not require expensive or delicate instrumentation, and are amenable to high throughput, especially in microplate format.

[0163] The determined LOD / LOQ allows the analysis of a wide range of samples, since polysorbate concentrations in aqueous samples are usually many times higher than these values. Also, the linear range of the fluorescent micellar assay allows the analysis of almost all biopharmaceutical samples. Potential limitations may occur for formulations with PS80 HP content >0.3 mg / mL. For these samples, sample volume adjustments, such as additional dilution steps, may be necessary.

[0164] The methods provided are highly suitable for the quantification of polysorbates in aqueous formulations, as demonstrated by determining the linearity, specificity, accuracy, robustness, and precision of several embodiments of the present invention.

[0165] The results also demonstrate that the developed method is highly suitable for use as a high-throughput method performed in a microplate format for screening large sample sets for the quantification of polysorbates even in the presence of (poly)peptides and common excipients used in aqueous formulations. The experimental results confirm that the method of the present invention is highly specific for the analysis of polysorbates, independent of the physicochemical properties of the (poly)peptides, buffers or excipient substances present in the solution.

[0166] [Table 1]

[0167] Experiments carried out are given below: Features and advantages of the present invention will become apparent from the following detailed experiments which illustrate some principles of the invention by way of example without limiting its scope.

[0168] experiment 1. Materials and Methods 1.1 Materials and Reagents All experiments were performed using PS20 HP (Polysorbate 20 High Purity) and PS80 HP (Polysorbate 80 High Purity) from Croda Europe Ltd (Snaith, United Kingdom), and PS20 China Grade (Polysorbate 20 China Grade) (Nanjing Well Pharmaceutical Co., Ltd, Nanjing, China).

[0169] A polysorbate stock solution with a concentration of 100 mg / mL was prepared by adding the surfactant to Milli-Q® water and then homogenizing with a magnetic stirrer. Milli-Q water® is water provided by a water treatment system that produces both ultrapure water (type 1) and pure water (type 2) of the highest quality directly from tap water. Type 1 water was used in this experiment. If necessary, for example to prepare calibration samples, the polysorbate stock solution was further diluted with Milli-Q® water.

[0170] The buffers used usually have the composition required for protein reconstitution and further processing methods such as UF / DF (ultrafiltration / diafiltration) steps.

[0171] Some proteins in the form of monoclonal antibodies (mAbs) were kindly provided by Boehringer Ingelheim Pharma GmbH & Co. KG. Alpha-lactalbumin ≥ 85% from bovine milk type III and lysozyme were purchased from Merck KGaA (Darmstadt, Germany). The molecular properties of the proteins investigated in these experiments are listed in Table 1.

[0172] All experiments were performed in black 96-well FLUOTRAC™ 200 polystyrene microplates (Greiner Bio-One GmbH, Frickenhausen, Germany).

[0173] Reagent grade (Sigma-Aldrich Corp., St. Louis, USA) hydrophobic dye N-phenyl-1-naphthylamine (NPN) was prepared at a concentration of 5 μM in a buffer containing 5% (v / v) acetonitrile HPLC Plus grade ≧99.9% (Sigma-Aldrich), 150 mM analytical sodium chloride EMSURE® (Merck KGaA, Darmstadt, Germany), 50 mM Trizma® base ≧99.9% (Sigma-Aldrich), and 0.0015% Brij-35 MQ200 quality (Sigma-Aldrich) at pH 8. After preparation, the buffer was filtered through a 0.22 μm Steritop® PES filter (Merck) and stored protected from light at ambient temperature for a maximum of 7 days.

[0174] 1.2 Protocol for FMA in liquid handling stations FMA was performed on a Tecan Fluent® 1080 liquid handling station (Tecan Group, Mannedorf, Switzerland). The platform was equipped with an 8-tip flexible channel arm (FCA), a 96-channel liquid handling arm, a robotic gripper arm, a BioShake 3000 Elm orbital shaker (Quantifoil Instruments GmbH, Jena, Germany), an incubator shaker DWP (INHECO GmbH, Martinsried, Germany), and an Infinite® M Nano+ dual-mode plate reader. All pipetting steps were performed with disposable tips (Tecan Group).

[0175] 10 μL of each sample was transferred to a 96-well microplate by FCA using the liquid level detection function. 240 μL of FMA buffer was added with a 96-channel liquid handling arm to ensure equal incubation times for all samples. Before adding FMA buffer to the samples, the buffer reservoirs were automatically shaken at 300 rpm for 60 s at room temperature in an orbital shaker to avoid non-uniform concentrations of NPN due to non-specific adsorption to the reservoir surface. The plate was then incubated at 1000 rpm and 35 °C for 60 s to allow better distribution of the fluorescent dye into the inner core of the PS micelles. After incubation, the fluorescence intensity was measured spectrophotometrically at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. All samples were analyzed as 4 technical replicates with the current experimental setup. Technical replicates refer to repeated measurements of the same sample to evaluate random noise associated with the protocol or equipment.

[0176] 2.Results 2.1. Development of general law By transferring FMA from an HPLC system to a microplate format, the analysis time per sample could be reduced from 2 min to about 15–20 s, thereby allowing for increased sample throughput. Polysorbate quantification methods require a significant increase in analysis time per sample (e.g., UPLC-QDa: 10 min per sample

[13] , mixed-mode HPLC-CAD: 8 min per sample, and reversed-phase (RP) UHPLC-CAD: 36 min per sample [3]).

[0177] 2.2. Analysis of polysorbate content The analytical method of the present invention was evaluated for the determination of the content of PS20 HP, PS20 China grade, and PS80 HP in aqueous solutions and samples including biopharmaceutical protein solutions and formulations. The parameters evaluated were linearity, specificity, accuracy, and precision. Furthermore, the LOD and LOQ were determined.

[0178] 2.2.1 Linearity The linearity of the present method was assessed according to the ICH Q2(R1) guideline

[14] , according to which the linearity of an analytical procedure is the ability to obtain a result (within a given range) that is directly proportional to the concentration of the analyte in the sample.

[0179] To test the linearity between the fluorescence signal and polysorbate concentration, the fluorescence was measured for samples with different sets of polysorbate concentrations ranging from 0.05 mg / mL to 0.8 mg / mL polysorbate. As a criterion for linearity, the R 2 ≥ 0.99 was defined. The excitation wavelength was set at 350 nm, and the emission was measured at 420 nm. Figure 6 shows the linearity results of FMA, i.e., the linearity of the fluorescence signal with increasing concentrations of PS20 HP, PS20 China grade, and PS80 HP. R 2 With the above constraints, the fluorescent signal was linear up to a content of 0.6 mg / mL PS20 HP and 0.8 mg / mL PS20 China grade. For PS80 HP, linearity was observed at concentrations of 0.3 mg / mL or less. The linear range of the calibration curve may be affected by various experimental parameters, such as the batch-by-batch composition of polysorbate used. However, a person skilled in the art can easily perform the calibration with a calibration sample suitable for each polysorbate used, determine the linear range, and take the results into account in the measurement based on his / her own knowledge and the technical teachings disclosed herein.

[0180] Therefore, the results of the calibration samples for a given linearity are suitable for use in assessing the polysorbate content of a series of subsequent measurement samples.

[0181] 2.2.2 Specificity Specificity refers to the ability to unambiguously evaluate an analyte in the presence of components that might be expected to be present. Typically, these can include impurities, degradants, matrix, etc.

[14] . In the case of fluorescent micellar assays, this means the absence of fluorescent signal emanating from proteins, buffer substances, or added excipients, except for the polysorbate present.

[0182] In this experiment, five different mAbs with different physicochemical characteristics were used, and the standard protein alpha-lactalbumin was used to examine the specificity of FMA. The detailed characteristics of these proteins are listed in Table 1. mAb1, mAb2, and mAb3 are IgG of IgG1 subclass, while mAb4 and mAb5 belong to IgG4 subclass. At the wavelengths used in FMA, only mAb2 showed a specific fluorescence signal, and all other mAbs showed no specific fluorescence at their respective wavelengths. In terms of protein hydrophobicity, there was a 4.61-fold difference between the proteins examined, with mAb4 being the least hydrophobic and mAb2 being the molecule with the most pronounced hydrophobic characteristics.

[0183] [Table 2]

[0184] -mAb1 (IgG1) In this experiment, the method of the present invention was carried out using purified mAb1. mAb1 was used in an acetate buffer system at pH 5.5 and a formulation buffer system containing acetate, sucrose, and L-arginine at concentrations typical of those used in biopharmaceutical formulations at pH 5.5. The target mAb1 concentration in the formulation was 150 mg / mL. PS20 HP (Polysorbate 20 High Purity) was used as the polysorbate.

[0185] Figure 7A shows the results of the specificity assessment of the FMA. The samples tested were as follows: - 176mg / mL mAb1 in polysorbate-free acetate buffer; - 150mg / mL mAb1 in polysorbate-free acetate buffer - Pure acetate buffer without polysorbate - Pure formulation buffers without polysorbate - Formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity), and - 150mg / mL mAb1 in formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity).

[0186] There was no significant background fluorescence signal, and therefore the results demonstrated that there was no effect on the required polysorbate concentration due to the components of the protein, acetate buffer (UF / DF buffer: ultrafiltration / diafiltration buffer), and the formulation buffer that does not contain polysorbate, e.g., the acetate buffer is a buffer used in the UF / DF step, a well-known procedure in protein processing.

[0187] In samples containing formulation buffer + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP concentration was determined to be 0.403 ± 0.005 mg / mL PS20 HP.

[0188] For a sample containing 150 mg / mL mAb1 + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity) in formulation buffer, the PS20 HP concentration was determined to be 0.394 ± 0.018 mg / mL PS20 HP. The target PS20 HP concentration was 0.4 mg / mL PS20 HP.

[0189] -mAb2 (IgG1) In this experiment, the method of the present invention was carried out using purified mAb2. The purified mAb2 was present in a formulation buffer system containing a histidine buffer system at pH 6.0 and histidine, sucrose, and mannitol at concentrations typical of those used in biopharmaceutical formulations at pH 6.0. The target mAb2 concentration in the formulation was 65 mg / mL. PS20 HP (Polysorbate 20 High Purity) was used as the polysorbate.

[0190] FIG. 7B shows the results of specificity assessment of the FMA performed with the following samples: - 92mg / mL mAb2 in polysorbate-free histidine buffer - 65mg / mL mAb2 in polysorbate-free formulation buffer; - Pure histidine buffer without polysorbate - Pure formulation buffers without polysorbate - Formulation buffer + 0.2mg / mL PS20 HP (Polysorbate 20 High Purity), and - 65mg / mL mAb2 + 0.2mg / mL PS20 HP (Polysorbate 20 High Purity) in formulation buffer.

[0191] The results demonstrated that there was no significant background fluorescence signal and therefore no effect on the required polysorbate concentration due to the histidine buffer (UF / DF buffer: ultrafiltration / diafiltration buffer) and the components of the formulation buffer that did not contain polysorbate. In contrast to mAb1, mAb2 showed an intrinsic fluorescence signal in the FMA with an "apparent" PS20 HP of 0.057 mg / mL at a mAb2 concentration of 92 mg / mL (no polysorbate was present) and an "apparent" PS20 HP of 0.03 mg / mL at a mAb2 concentration of 65 mg / mL (no polysorbate was present). Thus, this "apparent" PS20 HP concentration under the later formulation conditions of 65 mg / mL mAb2 in buffer without added PS20 HP was solely due to mAb2. This constituted approximately 15% of the signal of the later mAb2 formulation in buffer with 0.2 mg / mL PS20 HP.

[0192] In samples containing formulation buffer + 0.2 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP concentration was determined to be 0.198 ± 0.003 mg / mL PS20 HP.

[0193] For samples containing 65 mg / mL mAb2 in final formulation buffer + 0.2 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP concentration was determined to be 0.202 ± 0.005 mg / mL PS20 HP. The target PS20 HP concentration was 0.2 mg / mL.

[0194] -mAb3 (IgG1) The mAb3 molecule is also in IgG1 format and was formulated in an acetate buffer system containing the excipient trehalose with a surfactant concentration of 0.5 mg / mL PS80 HP at a high protein concentration of 100 mg / mL at pH 5.5. As the PS80 HP concentration was above the upper limit of the linear range (see 2.2.1 Linearity: 0.3 mg / mL PS80 HP), the samples were diluted 1:2 with Milli-Q water before analysis. As polysorbate, PS80 HP (Polysorbate 80 High Purity) was used.

[0195] FIG. 7C shows the results of specificity assessment of the FMA performed with the following samples: - 128mg / mL mAb3 in polysorbate-free acetate buffer - 100mg / mL mAb3 in polysorbate-free acetate buffer; - Pure acetate buffer without polysorbate - Pure formulation buffers without polysorbate - Formulation buffer + 0.5mg / mL PS80 HP (Polysorbate 80 High Purity), and - 100mg / mL mAb3 in formulation buffer + 0.5mg / mL PS80 HP (Polysorbate 80 High Purity).

[0196] The FMA did not give any significant fluorescent signal due to mAb3 in samples without polysorbate present, i.e. pure acetate buffer (ultrafiltration / diafiltration buffer), pure formulation buffer, or acetate buffer with protein concentrations of 128 mg / mL or 100 mg / mL, respectively. This means that the components present in the samples without polysorbate did not show any signal due to the components present. The formulation buffer containing 0.5 mg / mL PS80 HP was analyzed, and the PS80 HP concentration was determined to be 0.48 ± 0.01 mg / mL PS80 HP. For the final formulation consisting of 100 mg / mL mAb3 in formulation buffer, the PS80 HP content in the FMA was determined to be 0.48 ± 0.03 mg / mL PS80 HP. The target PS80 HP concentration was 0.5 mg / mL.

[0197] -mAb4 (IgG4) The mAb4 molecule is in IgG4 format. As polysorbate, PS20 HP (Polysorbate 20 High Purity) was used.

[0198] FIG. 7D shows the results of specificity evaluation of the FMA performed with the following samples: - 54mg / mL mAb4 in polysorbate-free acetate buffer - 20mg / mL mAb4 in polysorbate-free acetate buffer; - Pure acetate buffer without polysorbate - Pure formulation buffers without polysorbate - Formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity), and - 20mg / mL mAb4 in formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity).

[0199] Specificity testing of the FMA for this molecule showed no interfering signals from mAb4 at concentrations of 54 mg / mL and 20 mg / mL in pure acetate ultrafiltration / diafiltration (UF / DF) buffer and acetate buffer, respectively, in samples without polysorbate. Acetate buffer is the buffer used, for example, in the UF / DF step, a well-known procedure in protein processing. For other formulations, there were no signals from formulation buffer components.

[0200] In samples containing formulation buffer + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP content was determined to be 0.393 ± 0.014 mg / mL PS20 HP.

[0201] For samples containing 20 mg / mL mAb4 in formulation buffer + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP content was determined to be 0.404 ± 0.013 mg / mL PS20 HP. The target PS20 HP concentration was 0.4 mg / mL.

[0202] -mAb5 (IgG4) mAb5 was also in IgG4 format and was present in the same formulation as mAb4. As polysorbate, PS20 HP (Polysorbate 20 High Purity) was used.

[0203] FIG. 7E shows the results of specificity evaluation of the FMA performed with the following samples: - 26mg / mL mAb5 in polysorbate-free acetate buffer - 20mg / mL mAb5 in polysorbate-free acetate buffer; - Pure acetate buffer without polysorbate - Pure formulation buffers without polysorbate - Formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity), and - 20mg / mL mAb5 in formulation buffer + 0.4mg / mL PS20 HP (Polysorbate 20 High Purity).

[0204] The FMA results of the specificity test showed no signal in acetate ultrafiltration / diafiltration (UF / DF) buffer and acetate buffer for samples without polysorbate for mAb5 at concentrations of 26 mg / mL and 20 mg / mL, respectively, and no signal was generated from the formulation buffer without added PS20 HP.

[0205] In samples containing formulation buffer + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP content was determined to be 0.402 ± 0.014 mg / mL PS20 HP.

[0206] For samples containing 20 mg / mL mAb5 in formulation buffer + 0.4 mg / mL PS20 HP (Polysorbate 20 High Purity), the PS20 HP content was determined to be 0.409 ± 0.013 mg / mL PS20 HP. The target PS20 HP concentration was 0.4 mg / mL.

[0207] -Alpha-lactalbumin The specificity of the standard protein alpha-lactalbumin in FMA was also examined. Due to its low solubility, a concentration of 0.5 mg / mL was selected. PS20 HP (Polysorbate 20 High Purity) was used as the polysorbate.

[0208] FIG. 7F shows the results of specificity assessment of the FMA performed with the following samples: - 0.5mg / mL alpha-lactalbumin in mixed buffer, pH 7, polysorbate free - Pure mixed buffer, pH 7, polysorbate free - Mixed buffer, pH 7 + 0.2mg / mL PS20 HP (high purity polysorbate 20), and - 0.5mg / mL alpha-lactalbumin in mixed buffer, pH 7 + 0.2mg / mL PS20 HP (Polysorbate 20 High Purity).

[0209] Alpha-lactalbumin in the sample without polysorbate and the buffer system showed no signal in the FMA. The FMA of the sample containing mixed buffer spiked with 0.2 mg / mL PS20 HP gave 0.194 ± 0.004 mg / mL PS20 HP, and the FMA of the sample containing protein dissolved at a concentration of 0.5 mg / mL in this buffer spiked with 0.2 mg / mL PS20 HP gave 0.197 ± 0.006 mg / mL PS20 HP. The target PS20 HP concentration was 0.2 mg / mL.

[0210] 2.2.3 Accuracy The accuracy of an analytical procedure describes the degree of agreement between the found value and values ​​accepted as either traditional true values ​​or accepted reference values ​​[6]. The accuracy of the FMA was first assessed by spiking known amounts of different PS20 HP concentrations into acetate buffer. The PS20 HP spike concentrations investigated were 0.1, 0.3, and 0.6 mg / mL PS20 HP. The spike recoveries were calculated according to Equation I:

[0211]

number

[0212] The target PS20 HP concentration was 0.4 mg / mL PS20 HP. Accuracy was tested at 25%, 75%, and 150% of the target PS20 HP concentration. The results of the accuracy tests are summarized in Table 2 below:

[0213] [Table 3]

[0214] As can be seen from Table 2, for samples containing PS20 HP in acetate buffer, spike recoveries ranged from 80 to 104% for samples containing 25% of the target PS20 HP content, 99 to 117% for samples containing 75% of the PS20 HP target, and 97 to 108% for samples containing 150% of the target PS20 HP content, respectively.

[0215] -mAb1 (IgG1) The next step was to evaluate the accuracy of FMA in mAb1 formulations when the target PS20 HP concentration was 0.4 mg / mL PS20 HP. Accuracy was tested at 25%, 75%, and 150% of the target PS20 HP concentration. The results of the accuracy studies are summarized in Table 3:

[0216] [Table 4]

[0217] As can be seen from Table 3, for samples containing PS20 HP and 150 mg / mL mAb1 in acetate buffer, spike recoveries were determined to be in the range of 73-94% for samples containing 25% of the target PS20 HP content, 99-117% for samples containing 75% of the PS20 HP target, and 97-105% for 150% of the target PS20 HP concentration.

[0218] -mAb2 (IgG1) The accuracy of FMA was evaluated in mAb2 formulations at a target PS20 HP concentration of 0.4 mg / mL PS20 HP. Accuracy was tested at 25%, 50%, 75%, and 100% of the target PS20 HP concentration. The results of the accuracy studies are summarized in Table 4:

[0219] [Table 5]

[0220] As can be seen from Table 4, for samples containing PS20 HP and 65 mg / mL mAb2 in histidine buffer, the spike recoveries were determined to be in the ranges of 83-110% for samples containing 25% of the target PS20 HP content, 94-110% for samples containing 50% of the target PS20 HP content, 89-105% for samples containing 75% of the PS20 HP target, and 92-106% for samples containing 100% of the target PS20 HP concentration.

[0221] -mAb3 (IgG1) The accuracy of FMA was evaluated in mAb3 formulations with a target PS80 HP concentration of 0.5 mg / mL PS80 HP. Accuracy was tested at 20%, 40%, 60%, and 120% of the target PS80 HP concentration. The results of the accuracy studies are summarized in Table 5:

[0222] [Table 6]

[0223] As can be seen from Table 5, for samples containing PS80 HP and 100 mg / mL mAb3 in acetate buffer, spike recoveries were determined to be in the range of 79-111% for samples containing 20% ​​of the target PS80 HP content, 107-118% for samples containing 40% of the target PS80 HP content, 108-113% for samples containing 60% of the PS80 HP target, and 81-98% for samples containing 120% of the target PS80 HP content.

[0224] -mAb4 (IgG4) The accuracy of FMA was evaluated in mAb4 formulations with a target PS20 HP concentration of 0.4 mg / mL PS20 HP. Accuracy was tested at 25%, 75%, and 150% of the target PS20 HP concentration. The results of the accuracy studies are summarized in Table 6:

[0225] [Table 7]

[0226] As can be seen from Table 6, for samples containing PS20 HP and 20 mg / mL mAb4 in acetate buffer, spike recoveries were determined to be in the range of 85-106% for samples containing 25% of the target PS20 HP content, 98-109% for samples containing 75% of the PS20 HP target, and 88-100% for 150% of the target PS20 HP concentration.

[0227] -mAb5 (IgG4) The accuracy of FMA was evaluated in mAb5 formulations with a target PS20 HP concentration of 0.4 mg / mL PS20 HP. Accuracy was tested at 25%, 50%, 100%, and 150% of the target PS20 HP concentration. The results of the accuracy studies are summarized in Table 7:

[0228] [Table 8]

[0229] As can be seen from Table 7, for samples containing PS20 HP and 20 mg / mL mAb5 in acetate buffer, spike recoveries were determined to be in the range of 95-115% for samples containing 25% of the target PS20 HP content, 98-114% for samples containing 50% of the target PS20 HP content, 103-109% for samples containing 100% of the PS20 HP target, and 92-102% for samples containing 150% of the target PS20 HP concentration.

[0230] -Lysozyme The accuracy of FMA was evaluated in lysozyme formulations with a target PS80 HP concentration of 0.2 mg / mL PS80 HP. Accuracy was tested at 50%, 100%, and 150% of the target PS80 HP concentration. Lysozyme is a protein and an enzyme consisting of 129 amino acids. The results of the accuracy tests are summarized in Table 8:

[0231] [Table 9]

[0232] As can be seen from Table 8, for samples containing PS80 HP and 10 mg / mL lysozyme in PBS buffer, the spike recoveries were determined to be in the range of 86-103% for samples containing 50% of the target PS80 HP content, 92-102% for samples containing 100% of the target PS80 HP content, and 85-98% for samples containing 150% of the target PS80 HP concentration.

[0233] 2.2.4 Accuracy Analytical precision describes the degree of agreement (degree of variability) between a series of measurements obtained from multiple samplings of the same homogenous sample under given conditions [6]. Precision was tested for all mAbs under each formulation condition containing the target PS HP concentration for FMAs performed at a liquid handling station. All samples were analyzed multiple times, each with four technical replicates. Results for mAb1 are listed in Table 8. For this protein, the %CV (coefficient of variation, also known as relative standard deviation) for each quadruplicate was determined to be up to 3.6%, and the intra-assay variation (average %CV) was determined to be 2.2% for mAb1.

[0234] [Table 10]

[0235] Precision was also examined for the other mAbs in this study. Results showed a mean %CV of 2.5% (max 3.5%, 6 replicates, n=4 each) for mAb2, 3.6% (max 6.8%, 8 replicates, n=4 each) for mAb3, 2.6% (max 3.5%, 6 replicates, n=4 each) for mAb4, and 2.2% (max 5.0%, 8 replicates, n=4 each) for mAb5. All results are summarized in Table 9:

[0236] [Table 11]

[0237] 2.2.5 Determination of LOD and LOQ For FMA performed in microbatch format, the limits of detection (LOD) and limits of quantification (LOQ) were determined according to

[14] . The determined values ​​for PS20 HP were LOD 0.023 mg / mL and LOQ 0.069 mg / mL. In contrast to PS20 HP, the LOD for PS20 China grade was 0.009 mg / mL and LOQ 0.028 mg / mL. For PS80 HP, the determined values ​​were LOD 0.001 mg / mL and LOQ 0.003 mg / mL. The LODs and LOQs for PS20 HP, PS20 China grade, and PS80 HP are listed in Table 10 below.

[0238] [Table 12]

[0239] [Table 13]

[0240] References [Table 14] TIFF2024536083000018.tif216165

Claims

1. 1. A method (a) or method (b) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more polypeptides, comprising: Method (a) is: (a.1) providing a sample of an aqueous formulation; (a.2) adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine to the sample of step (a.1) to obtain a fluorescent dye-containing sample to be tested in the fluorescent micelle assay; (a.3) performing a fluorescent micelle assay using the fluorescent dye-containing sample obtained in step (a.2); (a.4) determining the content of polysorbate present in the sample from the fluorescent micelle assay performed in step (a.3); and Method (b) is: (b.1) providing multiple samples of an aqueous formulation; (b.2) adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine to each sample, respectively, to obtain fluorescent dye-containing samples to be tested in the fluorescent micelle assay; (b.3) performing a fluorescent micelle assay using the fluorescent dye-containing sample obtained in step (b.2); (b.4) determining the content of polysorbate present in each sample from the fluorescent micelle assay performed in step (b.3); Although 2. A method according to claim 1, wherein each of methods (a) and (b) is carried out without the use of an HPLC-system.

2. The method (a) further comprises a step (a.2') of incubating the fluorescent dye-containing sample of step (a.2), wherein step (a.2') is performed after step (a.2) and before step (a.3), and step (a.3) is performed using the fluorescent dye-containing sample obtained in step (a.2'); and The method of claim 1, wherein the method (b) further comprises a step (b.2') of incubating the fluorescent dye-containing sample of step (b.2), wherein step (b.2') is performed after step (b.2) and before step (b.3), and wherein step (b.3) is performed using the fluorescent dye-containing sample obtained in step (b.2').

3. The method of claim 1 or 2, which is method (a) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more polypeptides.

4. The method of claim 1 or 2, which is method (b) for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more polypeptides.

5. The following conditions: - the incubation of step (a.2') is carried out under shaking; - the incubation of step (b.2') is carried out under shaking; - providing samples of the aqueous formulation of step (b.1) simultaneously or one by one; - adding a solution containing the fluorescent dye N-phenyl-1-naphthylamine of step (b.2) to each sample simultaneously or one by one; - the incubation of step (b.2') is carried out for the samples simultaneously or one by one; The incubation of step (b.2') is carried out under shaking and is carried out for the samples simultaneously or one by one. - the incubation of step (a.2') is carried out at a temperature selected from the range of 10 to 60°C; - the incubation of step (b.2') is carried out at a temperature selected from the range of 10 to 60°C; and / or - Methods (a) and / or (b) are carried out using a fluorescence detector 3. The method according to claim 1, wherein one, two, three or more of the following conditions are satisfied:

6. 3. The method of claim 1 or 2, characterized in that the solution containing the fluorescent dye N-phenyl-1-naphthylamine is mixed before adding in step (a.2) or before adding in step (b.2) to remove any concentration gradient of said dye, said mixing being performed by shaking or stirring.

7. 3. The method according to claim 1 or 2, characterized in that the solution containing the fluorescent dye of step (a.2) or (b.2) has the same components as those used for the mobile phase in the combined FMA-HPLC method for the determination of polysorbates.

8. The method of claim 1 or 2, wherein the solution containing the fluorescent dye in step (a.2) or (b.2) contains a hydrophobic dye in the range of 1 μM to 10 μM, an organic solvent in the range of 1% (v / v) to 10% (v / v), a buffer in the range of 5 mM to 500 mM, a salt in the range of 10 mM to 1000 mM, and a surfactant in the range of 0.00015% to 0.015%.

9. Polysorbate is - intended for pharmaceutical use; and / or selected from Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80; and / or - Method according to claim 1 or 2, characterized in that it is selected from polysorbate 20 or polysorbate 80, suitable for parenteral administration.

10. 3. The method of claim 1 or 2, characterized in that it is carried out using disposable labware having a non-binding, low-binding, or medium-binding surface.

11. 3. The method according to claim 1 or 2, characterized in that the method steps (b.1) to (b.4) are carried out on a microplate.

12. some or all of the method steps (a.1) to (a.4) are automated; and / or 3. The method according to claim 1, wherein some or all of the method steps (b.1) to (b.4) are automated.

13. The method of claim 1 or 2, wherein the polypeptide is selected from monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies, or antibody fragments thereof, Fv, scFv, Fab, Fab', scFab, F(ab')2, Fab2, Fc, and Fc' fragments, heavy and light immunoglobulin chains and their constant regions, variable regions, or hypervariable regions as well as Fv and Fd fragments, diabodies, triabodies, scFv-Fc, minibodies, or single domain antibodies, and one or more enzymes, or mixtures thereof.

14. The method of claim 1 or 2, wherein the polypeptide is selected from therapeutic polypeptides used in the prevention or treatment of a disease or disorder.

15. 3. The method according to claim 1 or 2, characterized in that a biopharmaceutical formulation is used as the aqueous formulation.

16. The method of claim 1 or 2, wherein the method is used during the development, manufacture, storage, or preservation of an aqueous formulation, and the aqueous formulation contains polysorbate and one or more polypeptides.

17. Use of a fluorescent micelle assay in a method for quantifying polysorbate in an aqueous formulation containing polysorbate and one or more polypeptides.

18. 3. Use of a fluorescent micelle assay in a method for quantifying polysorbate in an aqueous formulation containing the polysorbate of claim 1 or 2 and one or more polypeptides.

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